The Interactive Fly
Zygotically transcribed genes
Genes coding for adherens junction proteins
Genes coding for septate junction proteins
Analysis has been carried out on the pattern and development of cellular junctions in the different tissues of the Drosophila embryo from the blastoderm stage
until hatching. The cellular junctions found include: gap junctions, two types of septate junctions, and several
types of cell-cell and cell-substrate adherens junctions. During early and mid embryogenesis (stages 4 to 13) only spot
adherens junctions, gap junctions, and zonulae adherentes (circumferential adherens junctions) prevail. Scattered spot adherens junctions are already formed at the
blastoderm stage. During and shortly after gastrulation, spot adherens junctions become concentrated at the apical pole and fuse
into continuous zonulae adherentes in the posterior endoderm and the ectoderm. In addition to the zonulae adherentes,
ectodermally derived epithelia possess scattered gap junctions and form pleated septate junctions and hemiadherens junctions
during late embryogenesis (stages 14 to 17). Hemiadherens junctions (HAJ) are junctions connecting muscles directly to epidermal cells and are characterized either by the presence of an intervening extracellular electron-dense material (connecting HAJ) or by the presence of an intervening tendon (tendon HAJ) (Tepass, 1994 and Prokop, 1998).
Mesenchymal tissues (i.e., all nonepithelial tissues of the embryo, including the
neural primordium and, transiently, the mesoderm and endoderm) possess both spot adherens junctions and gap junctions at a
low frequency. Initially, the midgut epithelium does not establish a junctional complex and possesses only gap junctions and spot
adherens junctions. Only late in development does a circumferential smooth septate junction develop; zonulae adherentes are
missing. The various derivatives of the mesoderm express spot adherens junctions, hemiadherens junctions, and gap
junctions, but never zonulae adherentes or septate junctions. After organogenesis, several different types of tissue-specific
adherens junctions are formed, among them connecting hemiadherens junctions (between gut epithelium and visceral muscle
and early during the formation of the muscle tendon junction); muscle tendon junctions (between somatic muscle and tendon
cells); fasciae adherentes (between the cells of both the visceral muscle and the dorsal vessel), and autocellular nephrocyte
junctions (in nephrocytes). Interesting exceptions to the general pattern of junctional development are provided by the outer
epithelial layer of the proventriculus and the Malpighian tubules. Both tissues develop as typical ectodermal epithelia and
possess zonulae adherentes. During late embryogenesis, both epithelia lose the zonulae adherentes and form smooth rather than
pleated septate junctions, thereby expressing a junctional complex similar to that of the endodermally derived midgut
epithelium (Tepass, 1994).
The distribution of proteins in the apico-lateral cell junctions has been examined in Drosophila imaginal discs.
Antibodies to phosphotyrosine (PY), Armadillo (Arm) and Drosophila E-cadherin (DE-cad) as well as FITC phalloidin (which marks
filamentous actin) labels the site of the adherens junction, whereas antibodies to Discs large (Dlg), Fasciclin 3 (Fas3) and Coracle
(Cor) label the more basal septate junction. The junctional proteins labeled by these antibodies undergo specific changes in
distribution during the cell cycle. A loss-of-function dlg mutation, which causes neoplastic imaginal
disc overgrowth, leads to loss of the septate junctions and the formation of what appear to be ectopic adherens junctions. Based on staining with PY and Dlg antibodies, the apico-lateral junctional complexes appear normal in tissue from the
hyperplastic overgrowth mutants fat facets, discs overgrown, lethal (2) giant discs and warts. However, imaginal disc tissue from the neoplastic overgrowth mutants dlg and
lethal giant larvae show abnormal distribution of the junctional markers, including a complete loss of apico-basal polarity in loss-of-function dlg
mutations. These results support the idea that some of the proteins of apico-lateral junctions are required both for apico-basal cell
polarity and for the signaling mechanisms controlling cell proliferation, whereas others are required more specifically in cell-cell
signaling (Woods, 1997).
The role of integrins was examined in the formation of the cell junctions that connect muscles to
epidermis (muscle attachments) and muscles to neurons (neuromuscular junctions). At the ultrastructural level two types of muscle attachments can be distinguished: direct and indirect. At the direct muscle attachments, single muscles (such as the transverse muscles) attach to epidermal cells directly such that the hemiadherens junctions (HAJs) in opposing cells are separated by only 30-40nm, with a thin line of extracellular electron-dense material in between. These closed paired HAJs are referred to as connecting HAJs. Indirect muscle attachments occur at the segmental border, where the ends of multiple muscles attach at the same epidermal site, and contain extensive extracellular matrix consisting of fuzzy electron dense fibers, separated by up to several micrometers. This is referred to as tendon matrix because, like the vertebrate tendons, it is an extracellular matrix used to attach the muscles. Since HAJs at indirect muscle attachments are not closely paired but connected to the tendon matrix, they are referred to as tendon HAJs. Both types of muscle attachments have a common molecular basis: both contain PS integrins; both are sites were large secreted proteins Tiggrin and Masquerade accumulate; the intracellular appearance of connecting HAJs and tendon HAJs looks similar; connecting HAJs and tendon HAJs can appear together at the same site; they both appear to arise from short connecting HAJs; and both HAJs are separated from the extracellular electron dense matrix by a translucent gap of a few nanometers (Prokop, 1998).
Muscle attachments and neuromuscular junctions were examined ultrastructurally in single or double
mutant Drosophila embryos lacking PS1 integrin (alphaPS1betaPS), PS2 integrin (alphaPS2betaPS),
and/or their potential extracellular ligand Laminin A. At the muscle attachments PS integrins are
essential for the adhesion of hemiadherens junctions to extracellular matrix, but not for their
intracellular link to the cytoskeleton. The intracellular electron-dense material of connecting HAJs and tendon HAJs connects to microfilaments in the muscles, and to microtubules in the epidermis. The epidermal microtubules are anchored at the other end to apical focal HAJs that connect to the cuticle (Prokop, 1998).
The PS2 integrin is only expressed in the muscles, but it is
essential for the adhesion of muscle and epidermal HAJs to electron dense extracellular matrix. PS2 integrin is
also required for adhesion of muscle HAJs to a less electron dense form of extracellular matrix, the
basement membrane. The PS1 integrin is expressed in epidermal cells and can mediate adhesion of the
epidermal HAJs to the basement membrane. The ligands involved in adhesion mediated by both PS
integrins seem distinct because adhesion mediated by PS1 appears to require the extracellular matrix
component Laminin A, while adhesion mediated by PS2 integrin does not (Prokop, 1998).
At neuromuscular junctions (NMJs)
the formation of functional synapses occurs normally in embryos lacking PS integrins and/or Laminin A, but the extent of contact between neuronal and muscle surfaces is altered significantly in embryos lacking laminin A. It is suggested
that neuromuscular contact does not require laminin A directly at its point of contact, but requires basement membrane adhesion to the general muscle
surface, and this form of adhesion is completely abolished in the absence of Laminin A. In contrast, loss of PS integrin function causes the boutons to make a more extensive contact with the muscle surface. Since no PS integrins are found at neuromuscular contacts it seems likely that the boutons can adhere to more muscle area because the muscle surfaces are more relaxed (allowing them to bend around the bouton) in the severely detached muscles of embryos lacking both PS integrins functions. Adhesion molecules expressed at Drosophila NMJs, like Fasciclin II, Fasciclin III or Connectin, are unlikely to mediate adhesion at the mature embryonic NMJ because they either fade during stage 16 or show no phenotype when mutated. Instead, mutant analysis reveals the existence of yet unknown embryonic adhesion factors downstream of mef2 regulation. Such factors might include laminin receptors that promote adhesion, or other receptors that displace the basement synaptic cell junction. Identification of mef2-dependent receptors might be aided by the use of lamA mutation as a sensitized background (Prokop, 1998).
Morphogenesis of epithelial tissues relies on the interplay between cell division, differentiation and regulated changes in cell shape, intercalation and sorting. These processes are often studied individually in relatively simple epithelia that lack the complexity found during organogenesis when these processes might all coexist simultaneously. To address this issue, this study makes use of the developing fly retinal neuroepithelium. Retinal morphogenesis relies on a coordinated sequence of interdependent morphogenetic events that includes apical cell constriction, localized alignment of groups of cells and ommatidia morphogenesis coupled to neurogenesis. Live imaging was used to document the sequence of adherens junction (AJ) remodelling events required to generate the fly ommatidium. In this context, it was demonstrated that the kinases Rok and Drak function redundantly during Myosin II-dependent cell constriction, subsequent multicellular alignment and AJ remodelling. In addition, it was shown that early multicellular patterning characterized by cell alignment is promoted by the conserved transcription factor Atonal (Ato). Further ommatidium patterning requires the epidermal growth factor receptor (EGFR) signalling pathway, which transcriptionally governs Rho-kinase (rok) and Death-associated protein kinase related (Drak)-dependent AJ remodelling while also promoting neurogenesis. In conclusion, this work reveals an important role for Drak in regulating AJ remodelling during retinal morphogenesis. It also sheds new light on the interplay between Ato, EGFR-dependent transcription and AJ remodelling in a system in which neurogenesis is coupled with cell shape changes and regulated steps of cell intercalation (Robertson, 2013).
In Drosophila, Rok seems to be the main kinase responsible for phosphorylating the Myosin regulatory light chain (Sqh) during epithelial patterning and apical cell constriction. This is the case for the activation of MyoII during intercalation as germband extension proceeds, but also during various instances of compartment boundary formation and cell sorting situations in the embryo and in the wing imaginal disc. The current work reveals that in the constricting cells of the MF, Rok functions redundantly with Drak, a kinase recently shown to phosphorylate Sqh both in vitro and in vivo (Neubueser, 2010). It is noteworthy that previous work has shown that RhoGEF2 is not required for cell constriction in the MF, suggesting that perhaps another guanine exchange factor (GEF) might function redundantly with RhoGEF2 to promote cell constriction. These data on Drak reinforce the idea that redundancies exist in this context. Because the RhoA (Rho1 -- FlyBase) loss of function abolishes this cell response entirely, it would be expected that Drak function is regulated by RhoA. In addition, the current data indicate that Drak acts redundantly with Rok during MyoII-dependent multicellular alignment and AJ remodelling during ommatidia patterning. It will be interesting to test whether Drak functions in other instances of epithelial cell constriction or MyoII-dependent steps of AJ remodelling in other developmental contexts in Drosophila (Robertson, 2013).
This study demonstrates a two-tiered mechanism regulating the planar polarization of MyoII and Baz. In the constricting cells in the posterior compartment, MyoII and Baz are segregated from one another and this is exacerbated by the wave of cell constriction in the MF. Upon Ato-dependent transcription in the MF cells, this segregated pattern of expression is harnessed and these factors become planar polarized at the posterior margin of the MF. This is independent of the core planar polarity pathway including the Fz receptor and is accompanied by a striking step of multicellular alignment. Previous work has demonstrated that Ato upregulates E-Cad transcription at the posterior boundary of the MF. In addition, apical constriction leads to an increase in E-Cad density at the ZA. The current data are therefore consistent with both hh-dependent constriction and ato-dependent transcriptional upregulation of E-Cad promoting differential adhesion, thus leading to a situation in which the ato+ cells maximize AJ contacts between themselves and minimize contact with the flanking cells that express much less E-Cad at their ZA. This typically leads to a preferential accumulation of cortical MyoII at the corresponding interface. Such actomyosin cables are correlated with increased interfacial tension, and it is proposed that this is in turn responsible for promoting cell alignment. Unfortunately, the very small diameter of these constricted cells precludes direct measurements of the AJ-associated tension using laser ablation experiments (Robertson, 2013).
Supra-cellular cables of MyoII have been previously associated with cell alignment in various epithelia and have also been observed at the boundary of sorted clones, whereby cells align at a MyoII-enriched interface. Interestingly, this study found that the actomyosin cable defining the posterior boundary of the MF is also preferentially enriched for Rok, a component of the T1, MyoII-positive AJ in the ventral epidermis (Simoes, 2010). This indicates an important commonality between actomyosin cable formation during cell sorting and the process of cell intercalation. However, unlike during intercalation, this study found that in the developing retina baz is largely dispensable for directing the pattern of E-Cad and actomyosin planar polarization. Further work will therefore be required to understand better the relationship between Baz and E-Cad at the ZA during ommatidia morphogenesis. It is speculated that the creation of a high E-Cad versus low E-Cad boundary in the wake of the MF might be sufficient to promote Rok and MyoII enrichment at the posterior AJs. This posterior Rok and MyoII enrichment might perhaps prevent E-Cad accumulation by promoting E-Cad endocytosis, as has been recently shown in the fly embryo (Robertson, 2013).
This study has used live imaging to define a conserved step of ommatidia patterning that consists of the coalescence of the ommatidial cells' AJs into a central vertex to form a 6-cell rosette. The corresponding steps of AJ remodelling require Rok, Drak, Baz and MyoII, a situation compatible with mechanisms previously identified during cell intercalation in the developing fly embryo. The steps of AJ remodelling required to transform lines of cells into 5-cell pre-clusters are transcriptionally regulated downstream of EGFR in a ligand-dependent manner. Interestingly, in the eye EGFR signalling is activated in the cells that form lines and type1-arcs in the wake of the MF and, thus, are undergoing AJ remodelling. Previous work examining tracheal morphogenesis in the fly has demonstrated that interfaces between cells with low levels versus high levels of EGFR signalling correlate with MyoII-dependent AJ remodelling in the tracheal placode. This situation resembles that which is described in this study in the wake of the MF. In the eye, however, it was found that EGFR signalling is not required to initiate cell alignment. Nevertheless, taken together with work in the tracheal placode and previous studies related to multicellular patterning in the developing eye, this work indicates a conserved function for the EGFR signalling pathway in promoting MyoII-dependent AJ remodelling. This leaves open several interesting questions; for example, it is not presently clear how EGFR signalling can promote discrete AJ suppression and elongation. It is, however, tempting to speculate that previously described links between EGFR signalling and the expression of E-Cad or Rho1 might play a role during this process (Robertson, 2013).
The molecular mechanisms that contribute to the morphogenesis and homeostasis of the epithelium remain elusive. This study reports a novel role for the cylindromatosis (CYLD) tumor suppressor in these events. The results show that CYLD depletion disrupts epithelial organization in both Drosophila egg chambers and mouse skin and intestinal epithelia. Microscopic analysis of proliferating cells in mouse epithelial tissues and cultured organoids reveals that loss of CYLD synergizes with tumor-promoting agents to cause the misorientation of the mitotic spindle. Mechanistic studies show that CYLD accumulates at the cell cortex in epithelial tissues and cultured cells, where it promotes the formation of epithelial adherens junctions through the modulation of microtubule dynamics. These data suggest that CYLD controls epithelial morphogenesis and homeostasis by modulating the assembly of adherens junctions and ensuring proper orientation of the mitotic spindle. These findings thus provide novel insight into the role of CYLD in development, tissue homeostasis, and tumorigenesis (Xie, 2017).
Under conditions of homeostasis, dynamic changes in the length of individual adherens junctions (AJs) provide epithelia with the fluidity required to maintain tissue integrity in the face of intrinsic and extrinsic forces. While the contribution of AJ remodeling to developmental morphogenesis has been intensively studied, less is known about AJ dynamics in other circumstances. AJ dynamics were studied in an epithelium that undergoes a gradual increase in packing order, without concomitant large-scale changes in tissue size or shape. Neighbor exchange events were found to be driven by stochastic fluctuations in junction length, regulated in part by junctional actomyosin. In this context, the developmental increase of isotropic junctional actomyosin reduces the rate of neighbor exchange, contributing to tissue order. A model is proposed in which the local variance in tension between junctions determines whether actomyosin-based forces will inhibit or drive the topological transitions that either refine or deform a tissue (Curran, 2017).
This study finds that ectopic expression of transcription factors that specify cell fates causes abnormal epithelial cysts in Drosophila imaginal discs. Cysts do not form cell autonomously but result from the juxtaposition of two cell populations with divergent fates. Juxtaposition of wild-type and aberrantly specified cells induces enrichment of actomyosin at their entire shared interface, both at adherens junctions as well as along basolateral interfaces. Experimental validation of 3D vertex model simulations demonstrates that enhanced interface contractility is sufficient to explain many morphogenetic behaviors, which depend on cell cluster size. These range from cyst formation by intermediate-sized clusters to segregation of large cell populations by formation of smooth boundaries or apical constriction in small groups of cells. In addition, single cells experiencing lateral interface contractility are eliminated from tissues by apoptosis. Cysts, which disrupt epithelial continuity, form when elimination of single, aberrantly specified cells fails and cells proliferate to intermediate cell cluster sizes. Thus, increased interface contractility functions as error correction mechanism eliminating single aberrant cells from tissues, but failure leads to the formation of large, potentially disease-promoting cysts. These results provide a novel perspective on morphogenetic mechanisms, which arise from cell-fate heterogeneities within tissues and maintain or disrupt epithelial homeostasis (Bielmeier, 2016).
Lipid phosphate phosphatases (LPPs) are integral membrane enzymes that regulate the levels of bioactive lipids such as sphingosine 1-phosphate and lysophosphatidic acid. The Drosophila LPPs Wunen (Wun) and Wunen-2 (Wun2) have a well-established role in regulating the survival and migration of germ cells. This study now shows that wun has an essential tissue-autonomous role in development of the trachea: the catalytic activity of Wun is required to maintain septate junction (SJ) paracellular barrier function, loss of which causes failure to accumulate crucial luminal components, suggesting a role for phospholipids in SJ function. The integrity of the blood-brain barrier is also lost in wun mutants, indicating that loss of SJ function is not restricted to the tracheal system. Furthermore, by comparing the rescue ability of different LPP homologs it was shown that wun function in the trachea is distinct from its role in germ cell migration (Ile, 2012).
This study demonstrates a role for an LPP in development of the trachea. In the absence of wun function, the trachea suffers from breaks in the dorsal trunk (DT), non-uniform lumen diameter, and loss of luminal components resulting from ineffective paracellular barrier function. wun functions tissue autonomously and Wun activity can be replaced by that of the close paralog Wun2 and two mouse homologs, but not by a catalytically dead LPP (Ile, 2012).
Defects are only seen in the trachea when wun is removed both maternally and zygotically and this is likely to explain why wun has not been previously uncovered in screens performed to identify genes required for tracheal development. The genetic data suggest that maternally provided Wun protein or the product of the reaction it catalyses lasts at least until the start of tracheal system formation and that zygotically expressed Wun in tracheal cells is sufficient to provide this activity. (Ile, 2012).
In germ cells the Wunens function redundantly: the germ cell death caused by loss of both proteins from germ cells can be rescued by expression of either protein alone, indicating that the two proteins have overlapping substrate specificities. In the trachea the situation is similar in that overexpression of wun2 in the trachea is able to substitute for loss of wun. To determine whether the roles of Wunens in germ cell migration and tracheal development are identical two mammalian LPPs with different activities were used. mLPP3 is able to substitute for Wunens in germ cell migration and survival assays, whereas mLPP2 is not, in spite of both proteins being highly expressed and localizing to the cell surface. As expected, it was found that mLPP3-GFP is able to rescue the tracheal phenotypes of wun wun2 M-Z− embryos; however, mLPP2-GFP is also able to do so. Thus, mLPP2 lacks an activity required for germ cell migration but possesses activity sufficient for tracheal development. It is concluded that the crucial LPP substrate or substrates for germ cell and tracheal development are different. Wunens and mLPP3 show relatively little substrate specificity and can dephosphorylate the lipid essential for both germ cell and tracheal development. mLPP2, by contrast, shows more restrictive specificity and can only dephosphorylate the lipid that is crucial for tracheal development. (Ile, 2012).
The localization of Wun-GFP to particular regions of the tracheal cell plasma membrane is intriguing. Mammalian LPPs have been demonstrated to localize to specific plasma membrane domains. Human (h) LPP1 (PPAP2A -- Human Genome Nomenclature Committee) sorts apically, whereas hLPP3 colocalizes with E-cadherin in Madin-Darby canine kidney (MDCK) cells and the C-terminal domain of hLPP3 has been shown to bind the AJ protein p120 catenin (catenin δ1). However, it remains to be confirmed whether specific Wun-GFP localization is crucial for activity. Wun2-myc can also rescue and, although the protein shows no specific localization, there might be sufficient present at SJs or apical membranes to fulfill the requirement for LPP activity. What is clear is that the LPPs, just as in germ cell migration, are playing more than a structural role because Wun2-H326K showed no rescue ability (Ile, 2012).
How does loss of Wun affect the tracheal epithelial cells? Overall, the polarity of these cells is unaffected, but AJ proteins are weaker at the apical surface. In this respect the tracheal phenotype is reminiscent of weak alleles of shotgun, which encodes Drosophila E-cadherin. Such mutants exhibit incomplete fusion of the DT and uneven luminal diameter. However, no genetic interaction between wun wun2 and shotgun is seen, suggesting that reduced DE-cadherin is not the critical factor in causing the tracheal defects in wun wun2 mutants. (Ile, 2012).
SJs were also affected in the wun wun2 M-Z− mutants: SJ components were not confined to the subapical region, as in wild type, and paracellular barrier function was lost. However, mutants for all essential SJ components reported to date display an abnormally elongated and convoluted DT. This phenotype is not seen in wun wun2 M-Z− mutants, indicating that although barrier activity may be lost, SJs must still be present and indeed they are seen by EM. This situation is similar to that of yrt M-Z− embryos, which also show compromised paracellular barrier function despite a normal complement of septa when examined ultrastructurally (Ile, 2012).
The lack of luminal accumulation of Serp and Verm in the wun wun2 M-Z animals is striking. Embryos mutant for serp and verm also display trachea with an abnormally elongated and convoluted DT. As this is not seen in wun wun2 M-Z animals, it is suspected that the loss of luminal Serp and Verm is not absolute. Indeed, extremely weak luminal staining id occasionally seen, but mostly Serp and Verm are detected in the hemolymph of late embryos. Although the possibility cannot be excluded that Serp and Verm are incorrectly secreted at the basolateral surface, the possibility is favored that Serp and Verm are apically secreted but owing to defects in the SJ-mediated paracellular barrier they diffuse from the lumen into the hemolymph. (Ile, 2012).
The differential accumulation of Serp and Verm versus the 2A12 antigen in the tracheal lumen in various mutant backgrounds has been interpreted to suggest that multiple secretory pathways exist. The first is actin dependent and is based on the observation that in dia mutants the 2A12 antigen is not present in the tracheal lumen whereas Verm is. The second is SJ dependent and is based on the fact that in mutants for the α subunit of the Na/K-ATPase, the 2A12 antigen accumulates in the lumen but Verm, which at stage 15 can been seen both in the lumen and in the tracheal cells, is undetectable in the lumen and tracheal cells by stage 16. Similar results have been obtained with mutants for other SJ components, including those encoded by sinuous, Lachesin, varicose, coracle and kune-kune. Based on these data, it is likely that the failure in Serp and Verm accumulation results, at least in part, from their diffusion out of the trachea. The difference in behavior between Serp, Verm and ANF-GFP versus the 2A12 antigen might depend more on the strength of their interaction with luminal components than on differences in their secretion. (Ile, 2012).
A model is proposed in which Wun expression at the cell surface leads to changes in intracellular lipid levels, which affects both AJs and SJs. These changes result in paracellular barrier defects and prevent particular luminal components from accumulating. Although the identification of which lipid or lipids are being affected and how changes in their levels and/or localization result in defects in specific tissues is ongoing, one potential Wun substrate, S1P, is known to increase barrier function in HUVEC cells via an S1P1-dependent pathway (Ile, 2012).
What is particularly striking is that the role of Wun in barrier function for the trachea and ventral nerve cord in Drosophila appears to be representative of a more conserved aspect of LPP function. mLPP3 has an essential embryonic role in establishing vascular endothelial cell interactions during early development. In addition, mice with postnatal inactivation of Lpp3 specifically in the vascular endothelium are viable but have impaired vascular endothelial barrier function leading to vascular leakage, particularly in the lungs . Thus, it appears that mLPP3 is required in both the establishment and maintenance of vascular integrity in a tissue-autonomous fashion (Ile, 2012).
Recent studies have demonstrated crucial roles for lipids in establishing or maintaining epithelial cell plasma membrane identity. For example, phosphoinositides are central to establishing the apical surface during lumen formation in MDCK cells and glycosphingolipids are needed to maintain apicobasal domain identity in C. elegans intestinal cells. Modulation of lipid levels coupled with cell biological analyses in a developmental context will be invaluable in exploring this fascinating field further (Ile, 2012).
Epithelial sheets play essential roles as selective barriers insulating the body from the environment and establishing distinct chemical compartments within it. In invertebrate epithelia, septate junctions (SJs) consist of large multi-protein complexes that localize at the apicolateral membrane and mediate barrier function. This study reports the identification of two novel SJ components, Pasiflora1 (CG7713) and Pasiflora2 (CG8121), through a genome-wide glial RNAi screen in Drosophila. Pasiflora mutants show permeable blood-brain and tracheal barriers, overelongated tracheal tubes and mislocalization of SJ proteins. Consistent with the observed phenotypes, the genes are co-expressed in embryonic epithelia and glia and are required cell-autonomously to exert their function. Pasiflora1 and Pasiflora2 belong to a previously uncharacterized family of tetraspan membrane proteins conserved across the protostome-deuterostome divide. Both proteins localize at SJs and their apicolateral membrane accumulation depends on other complex components. In fluorescence recovery after photobleaching experiments, pasiflora proteins were found to be core SJ components as they are required for complex formation and exhibit restricted mobility within the membrane of wild-type epithelial cells, but rapid diffusion in cells with disrupted SJs. Taken together, these results show that Pasiflora1 and Pasiflora2 are novel integral components of the SJ and implicate a new family of tetraspan proteins in the function of these ancient and crucial cell junctions (Deligiannaki, 2015).
The generation of distinct chemical milieus within the body is essential for metazoan development. This compartmentalization is accomplished by epithelia that impede paracellular diffusion and selectively transport substances via membrane channels and transporters. To provide a barrier, epithelia have a narrow intercellular space, which is sealed by specialized junctions, including tight junctions (TJs) in vertebrates and septate junctions (SJs) in invertebrates. SJs are the ancestral sealing junctions and are found in all invertebrates from sponges to arthropods but are also present in vertebrates. In electron micrographs, SJs appear as an array of regularly spaced septa, which operate by extending the travel distance for solutes through the paracellular route. SJs are found in both primary epithelia, such as epidermis, trachea and hindgut, and secondary epithelia, which develop through mesenchymal-epithelial transition, such as the blood-brain barrier (BBB) and midgut. The BBB ensheaths the nervous system and is required to maintain its homeostasis. Owing to the high potassium content of the hemolymph, animals with a defective BBB die of paralysis. In Drosophila, the BBB is a squamous epithelium established late in embryogenesis by SJ-forming subperineurial glia (SPG). In addition to providing a paracellular barrier, SJs also serve as a fence for the diffusion of proteins across the lateral membrane. Molecularly and functionally homologous SJs are found in vertebrates at the node of Ranvier, where they form the paranodal junction between axons and myelinating glia (Deligiannaki, 2015).
The SJ consists of a large multi-protein complex. In Drosophila, more than 20 proteins have been characterized that when missing lead to disruption of SJs and loss of barrier integrity. Most of these are transmembrane (TM) and lipid-anchored proteins that localize at the SJ, such as the claudins Sinuous (Sinu), Megatrachea (Mega) and Kune-kune (Kune), the cell adhesion molecules Neurexin IV (Nrx-IV), Contactin (Cont), Neuroglian (Nrg), Lachesin (Lac) and Fasciclin III, the sodium pump with its two subunits ATP&alpha and Nervana 2 (Nrv2), Melanotransferrin (Transferrin 2) and Macroglobulin complement-related (Mcr). The complex also includes the intracellular scaffold proteins Coracle (Cora) and Varicose (Vari) that interact with the cytoplasmic tails of membrane proteins and connect them to the actin cytoskeleton. A hallmark of SJ proteins is that they are interdependent for localization, and removal of one component is sufficient to destabilize the whole complex. In addition, half of the known SJ proteins can be co-immunoprecipitated from tissue extracts and detected by mass spectrometry (MS), further suggesting that they function together in a multi-protein complex. Fluorescence recovery after photobleaching (FRAP) experiments have been instrumental in classifying most SJ proteins as core components based on their limited mobility after photobleaching and the observation that upon loss of function other SJ proteins diffuse rapidly into the bleached region due to impaired complex formation (Deligiannaki, 2015).
Accompanying epithelial morphogenesis, SJs are remodeled into mature junctions. At embryonic stage 12, SJ proteins accumulate along the lateral membrane of columnar epithelial cells. Subsequently, they gradually localize at more apical compartments and by stage 15 are restricted to the apicolateral membrane, basal to adherens junctions. The Ly-6 proteins Crooked (Crok), Crimpled (Crim) and Coiled (Cold) are required for SJ formation; however, they do not reside at SJs and instead localize to cytoplasmic puncta. In Ly-6 mutants, the FRAP kinetics of SJ proteins mirrors that of core complex mutants and therefore Ly-6 proteins are thought to be involved in the assembly of SJ (sub)complexes in an intracellular compartment. The subsequent relocalization of SJs requires endocytosis from the basolateral membrane and recycling to the apicolateral compartment. Gliotactin (Gli) and Discs-large (Dlg) localize at SJs but, in contrast to core components and Ly-6 proteins, upon their loss of function the complex is properly formed and SJ proteins, although mislocalized, retain their restricted mobility. Together with a lack of physical interactions with SJ components, this result suggests that Gli and Dlg are required for complex localization rather than its assembly (Deligiannaki, 2015).
In contrast to SJs, TJs localize apically of the zonula adherens and in electron microscopy appear as a series of fusions of adjacent membranes. Although the set of proteins that composes the TJ is different from that of the SJ, the two complexes share a key molecular component, the claudins. Claudins are a tetraspan membrane family of 20-34kDa proteins with intracellular N- and C-termini and constitute a main component of TJs. The larger first extracellular loop contains a claudin family signature motif and bears critical residues that define TJ charge and size selectivity in a tissue-specific manner. Claudins are part of a large protein clan, comprising the PMP22/EMP/MP20/Claudin, MARVEL, tetraspanin, connexin and innexin families, which share the same overall topology but differ in size and motif composition of extracellular and intracellular domains. Many members of this clan can form homo- and heterotypic oligomers on the same and neighboring membranes and play essential roles in junctional complexes, including TJs, gap junctions and the casparian strip of plants, as well as in membrane traffic and fusion events. Claudins have been shown to interact with other tetraspan proteins such as occludins, tetraspanins and MARVEL, as well as cell adhesion proteins and receptors. Similarly, tetraspanins form microdomains in the plasma membrane, in which cell adhesion proteins, TM receptors and their signaling components are enriched and, thereby, are thought to be modulated in their activity (Deligiannaki, 2015).
This study identifies and characterizes two new core components of the SJ, Pasiflora1 and Pasiflora2, which are part of a novel tetraspan protein family that is conserved across the prostostome-deuterostome divide and is characterized by specific sequence features. Both proteins localize at SJs, show interdependence for localization and restricted mobility with known SJ members and are required for the integrity of epithelial barriers. This work provides new insight into the composition of the SJ and implicates a second family of tetraspan proteins in the development of these crucial cell junctions (Deligiannaki, 2015).
This study has identified two previously uncharacterized proteins, Pasiflora1 and Pasiflora2, as novel components of the Drosophila SJ. Several lines of evidence support this notion. First, pasiflora1 and pasiflora2 mutants exhibit all the characteristic phenotypes associated with disrupted SJs: breakdown of blood-brain and tracheal barriers, overelongated dorsal trunks, and SJ mislocalization in a variety of tissues. In the BBB, SJs appear severely disorganized and in columnar epithelia SJ proteins fail to localize at the apicolateral membrane and instead spread basolaterally. Second, the genes are co-expressed in embryonic epithelia that rely on SJs for their function and the proteins overlap with Cora at the apicolateral membrane. Similar to known SJ proteins, pasiflora localization depends on other complex members, as they spread basolaterally in SJ mutant backgrounds. Finally, using FRAP it was demonstrated that pasiflora proteins are core SJ components. In stage 15 epidermal cells, Nrg-GFP displays limited lateral mobility after photobleaching owing to its incorporation in the large multi-protein complex. By contrast, in pasiflora mutants, Nrg-GFP diffuses rapidly, indicating that SJ complex formation is compromised. Overexpressed pasiflora proteins also move slowly within the membrane of wt cells, but diffuse rapidly in cells with disrupted SJs, showing that they are themselves associated with the SJ complex (Deligiannaki, 2015).
An emerging idea is that not all SJ proteins are as interdependent as previously thought and that distinct subcomplexes exist within the large, highly ordered, multi-protein complex. The current observations and those of others indicating that in SJ mutants the localization of other complex members is differentially affected and that the fluorescence of GFP-tagged SJ proteins does not fully recover after photobleaching support this notion (Deligiannaki, 2015).
Pasiflora proteins are conserved in arthropods and beyond and share the global topological features of the tetraspan superfamily, with short conserved sequence motifs. The ability of different tetraspan families to form ribbons based on homo- and heterotypic interactions in cis within the plasma membrane suggests that pasifloras, together with claudins, are involved in forming the highly regularly spaced septa of the SJ. Freeze-fracture experiments have shown that SJs form ribbons, with an apparent size of a single septum of 10 nm and a regular spacing of 15-20 nm. Depending on the tissue, these ribbons are either highly aligned with each other (mature ectoderm) or meandering (developing wing disc). In the SJ, the plasma membranes of neighboring cells are not fused but closely juxtaposed at a distance of 15 nm and there is no evidence in invertebrates that different tissues have distinct paracellular permeability. Claudins and pasifloras are therefore unlikely to create pores in trans with specific size and charge selectivity. This suggests that the small claudins and pasifloras act only in cis to form ribbons, while the single-pass membrane proteins of the complex mediate the trans interaction with the neighboring cell via their large extracellular adhesive domains. To date, the structural basis for the intermolecular interaction between tetraspan proteins has not been resolved. The pasiflora proteins belong to a larger family with nine members in Drosophila. Pasiflora1 and Pasiflora2 are expressed in embryonic epithelia and glia and act non-redundantly during SJ formation. Little is known about the other family members: Fire exit is expressed in exit and peripheral glia, which also form SJs; CG15098 is expressed in the midgut, which forms structurally different, smooth SJs (Deligiannaki, 2015).
This study reveals that the composition of the SJ complex strongly resembles that of other junctional and TM protein complexes, where adhesive or signaling receptors are embedded in a complex environment of hydrophobic tetraspan proteins of different types, in this case three different claudins and two different members of the novel pasiflora family. Membrane complexes such as the SJ are particularly refractory to biochemical and structural analysis owing to their hydrophobicity and large size. However, due to their crucial function in all invertebrates and the vertebrate paranode, it is possible, by genetic means, to identify and study the structural core components as well as the biogenesis of the complex. Given the medical importance of the paranodal SJ in particular and of tetraspan proteins in general, this discovery of pasiflora proteins opens the possibility of studying these proteins and their interactions in a highly accessible and sensitive paradigm (Deligiannaki, 2015).
Septate junctions (SJs) are membrane specializations that restrict the free diffusion of solutes via the paracellular pathway in invertebrate epithelia. In arthropods, two morphologically different types of SJs are observed: pleated SJs (pSJs) and smooth SJs (sSJs), which are present in ectodermally- and endodermally-derived epithelia, respectively. Recent identification of sSJ-specific proteins, Mesh and Snakeskin (Ssk), in Drosophila indicates that the molecular compositions of sSJs and pSJs differ. A deficiency screen based on immunolocalization of Mesh, identified a tetraspanin family protein, Tetraspanin 2A (Tsp2A), as a novel protein involved in sSJ formation in Drosophila. Tsp2A specifically localizes at sSJs in the midgut and Malpighian tubules. Compromised (Tsp2A) expression caused by RNAi or the CRISPR/Cas9 system is associated with defects in the ultrastructure of sSJs, changes localization of other sSJ proteins, and impairs barrier function of the midgut. In most Tsp2A-mutant cells, Mesh fails to localize to sSJs and is distributed through the cytoplasm. Tsp2A forms a complex with Mesh and Ssk and these proteins are mutually interdependent for their localization. These observations suggest that Tsp2A cooperates with Mesh and Ssk to organize sSJs (Izumi, 2016).
Epithelia separate distinct fluid compartments within the bodies of metazoans. For this epithelial function, specialized intercellular junctions, designated as occluding junctions, regulate the free diffusion of solutes through the paracellular pathway. In vertebrates, tight junctions act as occluding junctions, whereas, in invertebrates, septate junctions (SJs) are the functional counterparts of tight junctions. SJs form circumferential belts around the apicolateral regions of epithelial cells. In transmission electron microscopy, SJs are observed between the parallel plasma membranes of adjacent cells, with ladder-like septa spanning the intermembrane space. SJs are subdivided into several morphological types that differ among different animal phyla, and several phyla possess multiple types of SJs that vary among different types of epithelia (Izumi, 2016).
In arthropods, two types of SJs exist: pleated SJs (pSJs) and smooth SJs (sSJs). pSJs are found in ectodermally-derived epithelia and surface glia surrounding the nerve cord, while sSJs are found mainly in endodermally-derived epithelia, such as the midgut and the gastric caeca. The outer epithelial layer of the proventriculus (OELP) and the Malpighian tubules also possess sSJs, although these epithelia are ectodermal derivatives. The criteria distinguishing these two types of SJs are the arrangement of the septa. In oblique sections of lanthanum-treated preparations, the septa of pSJs are visualized as regular undulating rows but those in sSJs are observed as regularly spaced parallel lines. In freeze-fracture images, the rows of intramembrane particles in pSJs are separated from one another, whereas those in sSJs are fused into ridges. To date, more than 20 pSJ-related proteins, including pSJ components and regulatory proteins involved in pSJ assembly, have been identified and characterized in Drosophila. In contrast, few genetic and molecular analyses have been carried out on sSJs. Recently, two sSJ-specific membrane proteins, Ssk and Mesh, have been identified and characterized. Ssk consists of 162 amino acids and has four membrane-spanning domains, two short extracellular loops, cytoplasmic N- and C-terminal domains, and a cytoplasmic loop. Mesh has a single-pass transmembrane domain and a large extracellular region containing a NIDO domain, an Ig-like E set domain, an AMOP domain, a vWD domain, and a sushi domain. Mesh transcripts are predicted to be translated into three isoforms of which the longest isoform consists of 1,454 amino acids. In Western blot studies, Mesh is detected as a main ~90 kDa band and a minor ~200 kDa band. Compromised expression of ssk or mesh causes defects in the ultrastructure of sSJs and in the barrier function of the midgut against a 10-kDa fluorescent tracer. Ssk and Mesh physically interact with each other and are mutually dependent for their sSJ localization. Thus, Mesh and Ssk play crucial roles in the formation and barrier function of sSJs (Izumi, 2016).
Tetraspanins are a family of integral membrane proteins in metazoans with four transmembrane domains, N- and C-terminal short intracellular domains, two extracellular loops and one short intracellular turn. Among several protein families with four transmembrane domains, tetraspanins are characterized especially by the structure of the second extracellular loop. It contains a highly conserved cysteine-cysteine-glycine (CCG) motif and 2 to 4 other cysteine residues. These cysteines form 2 or 3 disulfide bonds within the loop. Tetraspanins are believed to play a role in membrane compartmentalization and are involved in many biological processes, including cell migration, cell fusion and lymphocyte activation, as well as viral and parasitic infections. Several tetraspanins regulate cell-cell adhesion but none are known to be involved in the formation of epithelial occluding junctions. In the Drosophila genome, there are 37 tetraspanin family members, and some have been characterized by genetic analyses. Lbm, CG10106 and CG12143 participate in synapse formation. Sun associates with light-dependent retinal degeneration. TspanC8 subfamily members, including Tsp3A, Tsp86D and Tsp26D, are involved in the Notch-dependent developmental processes via the regulation of a transmembrane metalloprotease, ADAM10. However, the functions of most other Drosophila tetraspanins remain obscure (Izumi, 2016).
This study identified a tetraspanin family protein, Tsp2A, as a novel molecular component of sSJs in Drosophila. Tsp2A is required for sSJ formation and for the barrier function of Drosophila midgut. Tsp2A and two other sSJ-specific membrane proteins Mesh and Ssk show mutually dependent localizations at sSJs and form a complex with each other. Therefore, it is concluded that Tsp2A cooperates with Mesh and Ssk to organize sSJs (Izumi, 2016).
Of the sSJ-specific components, Mesh is a membrane-spanning protein and has an ability to induce cell-cell adhesion, implying that it is a cell adhesion molecule and may be one of the components of the electron-dense ladder-like structures in sSJs. In contrast, both Ssk and Tsp2A are unlikely to act as cell adhesion molecules in sSJs because each of the two extracellular loops of Ssk (25 and 22 amino acids, respectively) appear to be too short to bridge the 15-20-nm intercellular space of sSJs. Furthermore, overexpression of EGFP-Tsp2A in Drosophila S2 cells did not induce cell aggregation, which is a criterion for cell adhesion activity (Izumi, 2016).
Several observations in Tsp2A-mutants may provide clues for understanding the role of Tsp2A in sSJ formation. In most Tsp2A-mutant midgut epithelial cells, Mesh fails to localize to the apicolateral membranes but was distributed in the cytoplasm, possibly to specific intracellular membrane compartments. To further examine where Mesh was localized in Tsp2A-mutant cells, the midgut was doublestained with the anti-Mesh antibody and the antibodies against typical markers of various intracellular membrane compartments, including the Golgi apparatus (anti-GM130), early endosomes (anti-Rab5), recycling endosomes (anti-Rab11) and lysosomes (anti-LAMP1). However, it was not possible to detect any overlap between staining by these markers and that of Mesh. The staining pattern in Tsp2A-mutant midgut epithelial cells produced with the anti-KDEL antibody, which labels endoplasmic reticulum, was similar, although not identical with that produced by the anti-Mesh antibody (Izumi, 2016).
Interestingly, some tetraspanins are known to control the intracellular trafficking of their partners. For instance, a mammalian tetraspanin, CD81 is necessary for normal trafficking or for surface membrane stability of a phosphoglycoprotein, CD19, in lymphoid B cells. The TspanC8 subgroup proteins, which all possess eight cysteine residues in their large extracellular domain, regulate the exit of a metalloproteinase, ADAM10, from the ER and differentially control its targeting to either late endosomes or to the plasma membrane. Consequently, TspanC8 proteins regulate Notch signaling via the activation of ADAM10 in mammals, Drosophila and Caenorhabditis elegans. If Mesh is retained in the trafficking pathway from endoplasmic reticulum to plasma membrane in Tsp2A-mutant cells, Tsp2A may have an ability to promote the intracellular trafficking of Mesh in the secretory pathway. To clarify the role of Tsp2A in sSJ formation, it will be necessary to determine the intracellular membrane compartment where Mesh was localized in Tsp2A-mutant cells (Izumi, 2016).
Tsp2A, Mesh and Ssk are mutually dependent for their localization at sSJs. Consistent with this intimate relationship, the co-immunoprecipitation experiment revealed that Tsp2A physically interacts with Mesh and Ssk in vivo. However, the amount of Ssk observed in the co-immunoprecipitation with EGFP-Tsp2A was barely enriched relative to that in the extracts of embryos expressing EGFP-Tsp2A. This was particularly striking in comparison to the degree of enrichment of Mesh in the co-immunoprecipitation with EGFP-Tsp2A. To interpret these results, the detailed manner of the interaction between Tsp2A, Mesh and Ssk proteins needs to be further clarified. Many tetraspanin family proteins are known to interact with one another and with other integral membrane proteins to form a dynamic network of proteins in cellular membranes. Tetraspanins are also believed to have a role in membrane compartmentalization. Given such functional properties of tetraspanins, Tsp2A may determine the localization of sSJs at the apicolateral membrane region by membrane domain formation (Izumi, 2016).
In the Tsp2A-mutant midgut epithelial cells, Lgl was distributed throughout the basolateral membrane region, whereas it was localized in the apicolateral membrane region in the wild-type. In view of the role of Lgl in the formation of the apical-basal polarity of ectodermally-derived epithelial cells, it is of interest to consider whether this abnormal localization of Lgl in the Tsp2A-mutant affects epithelial polarity. However, in the Tsp2A-mutant midgut epithelial cells, Dlg still showed polarized concentration into the apicolateral membrane region and the Lgl never leaked into the apical membrane domain. These observations suggest that the lack of Tsp2A does not affect the gross apical-basal polarity of the midgut epithelial cells (Izumi, 2016).
Some tetraspanins have been reported to be involved in the regulation of cell-cell adhesion. A mammalian tetraspanin, CD151, regulates epithelial cell-cell adhesion through PKC- and Cdc42-dependent actin reorganization, or through complex formation with α3γ1 integrin. A mammalian tetraspanin, CD9, is concentrated in the axoglial paranodal region in the brain and in the peripheral nervous system, and CD9 knockout mice display defects in the formation of paranodal septate junctions and in the localization of paranodal proteins. Paranodal septate junctions have electron-dense ladder-like structures and their molecular organization is similar to that of pSJs but tetraspanins involved in pSJ formation have not been reported in Drosophila (Izumi, 2016).
Interactions between several tetraspanins and claudins, the key integral membrane proteins involved in the organization and function of tight junctions, are also known. Claudin-11 forms a complex with OAP-1/Tspan-3 and chemical crosslinking reveals a direct association between claudin-1 and CD9. Furthermore, the interaction between claudin-1 and CD81 is shown to be required for hepatitis C virus infectivity. To date, no tight junction defect has been reported in CD9 knockout mice, CD81 knockout mice, or CD9/CD81 double knockout mice. Further investigation is necessary to clarify whether the interactions between tetraspanins and tight junction proteins are involved in the formation and function of tight junctions (Izumi, 2016).
Septate junction (SJ) complex proteins act in unison to provide a paracellular barrier and maintain structural integrity. This study has identified a non-barrier role of two individual SJ proteins, Coracle (Cora) and Kune-kune (Kune). Reactive oxygen species (ROS)-p38 MAPK signaling in non-myocytic pericardial cells (PCs) is important for maintaining normal cardiac physiology in Drosophila. However, the underlying mechanisms remain unknown. This study has found that in PCs, Cora and Kune are altered in abundance in response to manipulations of ROS-p38 signaling. Genetic analyses establish Cora and Kune as key effectors of ROS-p38 signaling in PCs on proper heart function. It was further determined that Cora regulates normal Kune levels in PCs, which in turn modulates normal Kune levels in the cardiomyocytes essential for proper heart function. These results thereby reveal select SJ proteins Cora and Kune as signaling mediators of the PC-derived ROS regulation of cardiac physiology (Lim, 2019).
Cell-cell interaction is typically maintained and regulated by various multi-protein complexes such as tight junctions, adherens junctions (AJs), and gap junctions. Invertebrate septate junctions (SJs), which have functional and molecular similarity to vertebrate tight junctions (TJs), are specialized, multi-protein junctional complexes that reside between the apposed plasma membranes of adjacent epithelial cells. In Drosophila, more than 20 molecular constituents of the SJ have been identified, and characterization of these proteins reveals their canonical role in in sealing neighboring cells and restricting the free diffusion of solutes between adjacent cells, thereby providing a paracellular permeability barrier. The SJ protein complex is also involved in the coordinated changes in cell shape and rearrangement during tissue morphogenesis at a stage when the SJ structure has not yet formed or matured to become optically visible. For instance, mutations in all tested SJ genes cause defects in head involution, dorsal closure, and salivary gland elongation during early embryonic development before a clear SJ structure has been formed. Mutations in all tested SJ genes also cause cell-cell dissociation in the Drosophila embryonic heart, a tissue that seemingly lacks discernable SJs. Although most studies on the SJ proteins are focused on their canonical barrier function, it has been known that subsets of SJ proteins may have a different, non-barrier role. For instance, the SJ proteins Neurexin-IV (Nrx-IV), the Na+K+ATPase β subunit Nervana 2 (Nrv2), Coracle (Cora), and Yurt form a group with a distinct role in promoting epithelial apical-basal polarity. SJ components have also recently been found to play a role in regulating Hippo signaling to control intestinal stem cell activity and hematopoiesis. Together, these findings support the emerging notion that SJ proteins could serve important roles beyond their canonical barrier function. However, the non-barrier functions of the SJ proteins and the individual SJ proteins that could be involved remain poorly understood (Lim, 2019).
The heart is a heterogeneous organ comprising the contractile cardiomyocytes (CMs) and non-myocytes, such as the epicardial cells and endocardial cells. The non-myocytes have important signaling roles that contribute to CM development, growth, and function. The Drosophila heart is a linear tube comprising two inner rows of contractile CMs closely flanked by two outer rows of non-myocytic pericardial cells (PCs). PCs have been characterized as nephrocytes that are analogous to the mammalian podocytes that function to filter toxins and proteins from the hemolymph, the equivalent of mammalian blood. The PC nephrocytes are characterized by an intricate cell shape that includes elongated infoldings of the plasma membrane to form foot processes and labyrinthine channels. The labyrinthine channels are sealed by the slit diaphragm, which is a highly organized structure composed of similar proteins as the slit diaphragm in mammals. The slit diaphragm serves as a filtration barrier to control the inflow of certain substances into the labyrinthine channels from the hemolymph. In addition, vesicular invaginations of the plasma membrane occur along the labyrinthine channels that are indicative of endocytosis of the sequestered materials from the hemolymph. Materials endocytosed into the nephrocytes, presumably toxic molecules from the hemolymph, are targeted for either degradation in the lysosome or recycling back to the hemolymph. Moreover, the CMs and PCs are separated by a basement membrane composed of extracellular matrix (ECM), which could serve as a filtration system for hemolymph content (Lim, 2019).
On the other hand, accumulating evidence is indicating an important secretory function of PC nephrocytes. An early observation of an increased synthesis of the bactericidal enzyme lysozyme in PCs following the experimental infection of the insect Calliphora erythrocephala with bacteria provided the first indication that PCs could manufacture proteins for release into the hemolymph. More recently, Drosophila PCs have been reported to secrete factors, such as the ECM components and hemolymph proteins that could directly control neighboring CM function. In addition, PCs have been reported to produce reactive oxygen species (ROS) under normal, non-stressed conditions. ROS belong to a group of reactive chemical species produced by the incomplete reduction of molecular oxygen and are now recognized to serve an important role in the regulation of various cardiac physiological processes. Physiological ROS produced in the PCs of the Drosophila heart control the production of downstream signals such as D-p38 MAPK in PCs that then act in a paracrine manner to regulate CM function and morphology. The phenomenon is apparently conserved, as a study on the zebrafish heart reported that injury-induced H2O2 in the epicardial cells promotes the regeneration of the neighboring myocardium through the activation of ERK1/2 MAPK signaling and likely the generation of soluble factors from the epicardial cells. Together, these findings support the notion that a conserved ROS-MAPK signaling axis operates in the epi- or pericardium to influence myocardial function. However, the molecular mechanisms underlying ROS-MAPK-mediated paracrine interactions are currently unknown (Lim, 2019).
This study found that among the SJ proteins tested in adult PCs, only Cora and Kune-kune (Kune) are altered in abundance by ROS-D-p38 signaling in PCs. The results further showed that pericardial ROS-D-p38 signaling regulates CM function and structure through Cora and Kune. It was also found that Cora controls Kune amount in PCs and that pericardial Kune in turn modulates myocardial Kune expression that is essential for normal cardiac physiology. This study thereby unravels an unexpected function of the select SJ proteins Cora and Kune as physiological signaling mediators in PCs, a role that is distinct from their common primary barrier function (Lim, 2019).
On the basis of the results of this study, a model is proposed for the ROS-mediated paracrine regulation of cardiac physiology. In PCs, physiological ROS-p38 level governs Cora amount, which in turn regulates the level of Kune in the cellular surface. Peripheral Kune then directs the abundance of Kune in the CMs, which is essential for proper myocardial function and morphology. As a result, lowering of ROS-p38 signaling to sub-physiological level in PCs reduces pericardial Cora level and heightens pericardial Kune level, thereby raising Kune in CMs to a level that is detrimental to normal cardiac function. Conversely, elevating ROS-p38 signaling to supra-physiological level in PCs increases pericardial Cora quantity and diminishes pericardial Kune content, thereby suppressing Kune in the CMs to a level that perturbs normal heart function (Lim, 2019).
The findings suggest that Cora and/or Kune serve dual roles as structural elements of the SJ complex and as downstream effectors of ROS signaling. Such a dual function of Cora or Kune is unexpected but perhaps not unprecedented. The signaling role of Cora and Kune as core SJ components appears analogous to that of Arm as a core AJ component. Within the AJ, Arm mediates cell-cell adhesion and anchoring of the actin cytoskeleton. However, upon activation by Wingless, the Drosophila homolog of Wnts, Arm accumulates in the cell and serves as a key effector of Wingless signal transduction. In the case of Cora and Kune in the PCs, in response to the ROS signal, p38 is activated which then regulates the abundance and/or activity of these two individual SJ proteins. It is therefore proposed that Cora and/or Kune in the SJ have parallel functions as Arm in the AJ in that they serve as a structural component of the junctional complex and as downstream effector of signaling pathways (Lim, 2019).
The results indicate that Kune level in the PC affects Kune level in the CM; however, the underlying mechanism is unclear. One possibility is that pericardial Kune and cardiomyocyte Kune homotypically interact. In this scenario, one would predict that Kune is likely localized at the cell-cell interface. This was not observed; however, it does not necessarily rule out the homotypic interaction hypothesis. It is possible that in addition to engaging in homotypic interaction to mediate ROS signaling, other obligations of Kune may cause Kune to become more evenly distributed across the cells. For instance, Kune might be involved in the nephrocytic activity of PCs, and hence localization of Kune all over the cell surface is essential to promote the uptake of materials from the hemolymph into PCs. In the myocardium, Kune might be involved in the synchronous contraction of the CMs, a process that could be facilitated by the uniform localization of Kune across the entire CM surface. Alternatively, Kune interaction between the pericardial and cardiac cells might not involve their direct homotypic interaction but rather be mediated by the basement membrane that resides between PCs and CMs, at least in certain regions of the fly heart. In addition, an aberrant change in the pericardial nephrocyte morphology caused by loss of pericardial Kune might also alter Kune level in the CM. Last but not least, paracrine factors could be released from the PC in a Kune-controlled manner, which then influence Kune level in the CM. Regardless of whether intercellular Kune interaction occurs via direct cell-cell contact or indirect mechanisms, the results have demonstrated an interesting phenomenon by which the maintenance of normal Kune abundance in CMs by its pericardial counterpart is essential for proper adult cardiac morphology and physiology. This further raises the question as to how Kune acts in CMs to control proper cardiac performance and morphology. One possibility is that Kune regulates ion channel level and/or activity in the CM plasma membrane, such as the transient receptor potential (TRP) family of Ca2+ channels. As such, alterations in the CM Kune level could perturb intracellular Ca2+ homeostasis, thereby disrupting proper cardiac contractility and structure. These possibilities remain to be investigated in future studies (Lim, 2019).
In summary, these findings reveal that select SJ proteins can act as signaling effectors and suggest that the SJ, like the AJ, could serve to organize signaling centers. This work also provides important insights into the essential mechanisms of ROS-mediated non-myocyte-myocyte signaling interactions, a process that appears to be conserved between invertebrates and vertebrates (Lim, 2019).
Tetraspanin-2A (Tsp2A) is an integral membrane protein of smooth septate junctions in Drosophila melanogaster. To elucidate its structural and functional roles in Malpighian tubules, this study used the GAL4/UAS system to selectively knockdown Tsp2A in principal cells of the tubule. Tsp2A localizes to smooth septate junctions (sSJ) in Malpighian tubules in a complex shared with partner proteins Snakeskin (Ssk), Mesh and Discs Large (Dlg). Knockdown of Tsp2A led to the intracellular retention of Tsp2A, Ssk, Mesh and Dlg, gaps and widening spaces in remaining sSJ, and tumorous and cystic tubules. Elevated protein levels in Malpighian tubules together with diminished V-type H(+)-ATPase activity is consistent with cell proliferation and reduced transport activity. Indeed, Malpighian tubules isolated from Tsp2A knockdown flies failed to secrete fluid in vitro. The absence of significant transepithelial voltages and resistances manifest an extremely leaky epithelium that allows secreted solutes and water to leak back to the peritubular side. The tubular failure to excrete fluid leads to extracellular volume expansion in the fly and to death within the first week of adult life. Expression of the c42-GAL4 driver begins in Malpighian tubules in the late embryo and progresses upstream to distal tubules in third instar larvae, which can explain why larvae survive Tsp2A knockdown and adults do not. Uncontrolled cell proliferation upon Tsp2A knockdown confirms the role of Tsp2A as tumor suppressor in addition to its role in sSJ structure and transepithelial transport (Beyenbach, 2020).
Epithelial tissues are fundamental for the establishment and maintenance of different body compartments in multicellular animals. To achieve this specific task epithelial sheets secrete an apical extracellular matrix for tissue strength and protection and they organize a transepithelial barrier function, which is mediated by tight junctions in vertebrates or septate junctions in invertebrates. This study shows that the bicistronic gene wurmchen (CG43780) is functionally expressed in epithelial tissues. CRISPR/Cas9-mediated mutations in both coding sequences reveal two essential polypeptides, Wurmchen1 and Wurmchen2, which are both necessary for normal epithelial tissue development. Wurmchen1 represents a genuine septate junction core component. It is required during embryogenesis for septate junction organization, the establishment of a transepithelial barrier function, distinct cellular transport processes and tracheal system morphogenesis. Wurmchen2 is localized in the apical membrane region of epithelial tissues and in a central core of the tracheal lumen during embryogenesis. It is essential during the later larval development (Konigsmann, 2020)
Protein components of the invertebrate occluding junction-known as the septate junction (SJ) - are required for morphogenetic developmental events during embryogenesis in Drosophila melanogaster. In order to determine whether SJ proteins are similarly required for morphogenesis during other developmental stages, this study investigated the localization and requirement of four representative SJ proteins during oogenesis: Contactin, Macroglobulin complement-related, Neurexin IV, and Coracle. A number of morphogenetic processes occur during oogenesis, including egg elongation, formation of dorsal appendages, and border cell migration. All four SJ proteins are expressed in egg chambers throughout oogenesis, with the highest and most sustained levels in the follicular epithelium (FE). In the FE, SJ proteins localize along the lateral membrane during early and mid-oogenesis, but become enriched in an apical-lateral domain (the presumptive SJ) by stage 10B. SJ protein relocalization requires the expression of other SJ proteins, as well as Rab5 and Rab11 in a manner similar to SJ biogenesis in the embryo. Knocking down the expression of these SJ proteins in follicle cells throughout oogenesis results in egg elongation defects and abnormal dorsal appendages. Similarly, reducing the expression of SJ genes in the border cell cluster results in border cell migration defects. Together, these results demonstrate an essential requirement for SJ genes in morphogenesis during oogenesis, and suggests that SJ proteins may have conserved functions in epithelial morphogenesis across developmental stages (Alhadyian, 2021).
Loss of ESCRT function in Drosophila imaginal discs is known to cause neoplastic overgrowth fuelled by mis-regulation of signalling pathways. Its impact on junctional integrity, however, remains obscure. To dissect the events leading to neoplasia, transmission electron microscopy (TEM)was used on wing imaginal discs temporally depleted of the ESCRT-III core component Shrub. A specific requirement for Shrub was found in maintaining Septate Junction (SJ) integrity by transporting the Claudin Megatrachea (Mega) to the SJ. In absence of Shrub function, Mega is lost from the SJ and becomes trapped on endosomes coated with the endosomal retrieval machinery Retromer. ESCRT function is required for apical localization and mobility of Retromer positive carrier vesicles, which mediate the biosynthetic delivery of Mega to the SJ. Accordingly, loss of Retromer function impairs the anterograde transport of several SJ core components, revealing a novel physiological role for this ancient endosomal agent (Pannen, 2020).
Developmental and physiological functions of epithelia rely on a set of cellular junctions, linking cells within the tissue to a functional unit. While E-cadherin-based adherens junctions (AJs) provide adhesion and mechanical properties, formation of the paracellular diffusion barrier depends on tight junctions (TJs). Proteins of the conserved claudin family play a key role in establishing and regulating TJ permeability in the intercellular space by homo- and heterophilic interactions with Claudins of neighboring cells. Arthropods, such as Drosophila, do not possess TJs but a functionally similar structure in ectoderm-derived epithelia termed pleated septate junction (pSJ, SJ hereafter), characterized by protein dense septa lining the intercellular space in electron micrographs. Structure and function of Drosophila SJs depend on a convoluted multiprotein complex containing at least a dozen components. Three claudins, among them Megatrachea (Mega), have been shown to be required for SJ formation and barrier function in flies. Besides claudins, several transmembrane proteins (TMPs) such as Neurexin-IV (NrxIV), Neuroglian (Nrg) or ATPα contribute to the formation of the stable SJ core complex, which is characterized by low mobility within the membrane. At the intracellular side of the junction, cytoplasmic proteins such as Coracle (Cora), Varicose (Vari), and Discs large (Dlg) associate with the transmembrane components, contributing to the formation of a stable fence-like scaffold . While junction formation during embryogenesis requires the SJ localized cytoplasmic protein Dlg, this basolateral cell polarity factor is not a structural part of the immobile junction core comple. This explains the functional separation of barrier formation and apicobasal polarity despite the close association of Dlg-complex components with the SJ. Albeit growing knowledge about the structural composition of SJs, the intracellular events required for assembly and maintenance of SJ complexes remain largely unknown. Specifically, how proliferative tissues, such as the imaginal disc epithelium, maintain SJ integrity is not well established (Pannen, 2020).
It was recently shown that newly synthesized SJ components integrate into the junction from the apical side (in between AJ and SJ) in a 'conveyor belt-like' fashion (Babatz, 2018; Daniel, 2018). In addition, SJ components are frequently associated with endosomal compartments, suggesting a role for the endosomal system in coordinating transport and turnover of SJ complexes. Consistently, endocytosis is required to concentrate SJ components at the junctional region during embryogenesis. This suggests that passage of SJ TMP components (or the whole SJ protein complex) through the endosomal system may be a requirement for SJ formation, with the underlying mechanisms remaining poorly characterized (Pannen, 2020).
The endosomal system fulfils a plethora of physiological functions by tightly regulating the intracellular transport of TMPs and membranes within the cell. Following endocytosis from the plasma membrane, TMPs enter the endosomal system where they undergo cargo specific sorting. This process provides separation of proteins destined for degradation from those that exit the endosomal system to be recycled. Two evolutionary conserved endosomal sorting machineries, the endosomal sorting complex required for transport (ESCRT) and the retromer complex, mediate cargo sorting into the degradative and recycling pathway, respectively. To coordinate these opposing transport activities, the endosomal system comprises a highly dynamic membrane network governing retromer-dependent tubulation for recycling and ESCRT-mediated generation of intraluminal vesicles (ILV) for degradation (Pannen, 2020).
Endocytosed proteins can evade ESCRT-dependent packaging into ILVs by exiting the maturing endosome (ME) through tubular retrieval domains induced by specialized recycling machineries such as retromer. Initially characterized as a regulator of endosome-to-Golgi cargo retrieval in yeast, this endosomal agent comprises two subcomplexes that cooperatively drive cargo sorting into tubular recycling carriers. Similar to the ESCRT machinery, cargo clustering and membrane deformation is performed by distinct functional units within the retromer pathway. Motif-based cargo recognition and aggregation is mediated by the endosomally localized Vps26:Vps29:Vps35 complex, which has been termed cargo-selective complex, considered to constitute the core functional component of retromer. Since the ancient CSC does not possess membrane bending activity, cooperation with tubulating factors such as proteins of the SNX-BAR (Sorting Nexin-Bin/Amphiphysin/Rvs) family is required for recycling carrier generation. Proteins containing the curved BAR-domain can assemble into regular helical coats on endosomes, thereby inducing cytoplasm faced tubulation. Concerted action of CSC stably complexed with SNX-BAR proteins to retrieve endosomal cargo was initially characterized as the classical retromer pathway in yeast. In metazoans however, retromer function is not restricted to SNX-BAR-dependent pathways. Specifically, cooperations of CSC with SNX3 or SNX27 (both lacking BAR-domains) emerged as alternative routes for endosomal retrieval. Proteomic data from mammalian cells suggest that surface levels of well over 100 TMPs depend on retromer and many of these proteins seemingly interact with CSC or SNX27. Recently, Drosophila has proven invaluable for assessing and confirming the physiological relevance of some of these putative retromer cargos in vivo (Pannen, 2020).
Cargo proteins within the endosomal system that do not undergo recycling can enter the degradative trafficking route starting with their sorting into ILVs. Generation of ILVs at the limiting membrane of MEs requires the canonical ESCRT function, which is performed by four in sequence acting complexes (ESCRT-0, -I, -II, III) and the ATPase Vps4. Ubiquitination of TMPs serves as the primary degradative sorting signal and sequestration of TMPs into ILVs is an essential prerequisite to complete lysosomal degradation. Several ESCRT components such as Vps27/Hrs (ESCRT-0) and Vps23/TSG101 (ESCRT-I) possess ubiquitin interacting motifs, which allow them to bind and cluster ubiquitinated TMPs. Consequently, local concentration of ubiquitinated cargo by ESCRT complexes establishes a degradative subdomain at the endosomal membrane that is spatially separated from the retrieval subdomain. While ESCRT-0-II complexes provide cargo recognition and clustering, the membrane-deforming activity required to bud and abscise ILVs into the endosomal lumen depends on ESCRT-III components, which polymerize into helical arrays at the endosomal membrane. The most abundant ESCRT-III component is the highly conserved yeast Snf7/Vps32, encoded by the gene shrub (shrb) in Drosophila. Unlike upstream ESCRT components, ESCRT-III proteins only transiently assemble into a heterooligomeric complex at the endosomal membrane. In consequence of ESCRT activity, the maturing endosome accumulates cargo-containing ILVs and is recognized in electron micrographs as a multi-vesicular body (MVB). The ESCRT/MVB pathway ends with Vps4-dependent dissociation of ESCRT-III components from the endosomal membrane. This step is required for the release of the nascent ILV and subsequent rounds of ILV formation. Loss of ESCRT function was initially studied in yeast cells in which it led to the emergence of an aberrant pre-vacuolar endosomal organelle, termed class E compartment. This defective endosomal structure is characterized by accumulation of degradative cargo and a failure to fuse with the vacuole/lysosome (Pannen, 2020).
The physiological relevance of ESCRT-mediated degradative TMP trafficking is particularly evident in Drosophila imaginal disc tissue. Here, loss of ESCRT function induces severe overgrowth, multilayering, apoptosis, and invasive behavior of the tissue; a phenotype attributed to mis-regulation of cellular signaling pathways, such as the Jak/Stat-, Jun-Kinase-, and Notch pathways. Consequently, ESCRT components were classified as endocytic neoplastic tumor suppressor genes (nTSG) in Drosophila. While induction of over-proliferation and apoptosis in nTSG mutants have been extensively characterized, the events leading to loss of cell polarity and ultimately neoplastic transformation of the tissue remain poorly understood (Pannen, 2020).
This study has analyzed the integrity of cellular junctions in an ESCRT-depleted wing imaginal disc epithelium to gain insight into the initial events leading to neoplastic transformation. Surprisingly, preceding neoplastic overgrowth, this study found a strong and specific reduction in the density of SJ. ESCRT and retromer functions are required for anterograde transport of SJ components. By dissecting the intracellular trafficking itinerary of the claudin Megatrachea, this study revealed that biosynthetic delivery of this core SJ component depends on a complex basal to apical transcytosis route relying on ESCRT and retromer functions (Pannen, 2020).
While transcytosis of SJ components has been shown to occur during the initial establishment of the SJ in the embryo, this study revealed that this mechanism is also continuously required during maintenance of the SJs in a rapidly proliferating epithelium. The data reveal that the retromer CSC functions downstream of ESCRT to export Mega from the endosome. A novel physiological role is proposed for the retromer CSC in regulating membrane levels of several SJ core components. While the data suggest that retromer fails to export Mega from aberrant endosomes induced by ESCRT depletion, the exact mechanism behind this remains to be determined (Pannen, 2020).
The data reveal a critical requirement for ESCRT in a transport pathway that depends on retromer-mediated transcytosis to deliver newly synthesized Mega to its apical destination. Defects in endosomal retrieval upon ESCRT inactivation have been previously described in other systems, such as yeast or mammalian cells and, thus, appear to represent a common feature of the pleiotropic ESCRT deficient phenotype. In yeast, the endosome-to-Golgi retrieval of the sorting receptor Vps10p and its cargo carboxypeptidase Y (CPY) depends on retromer function. ESCRT mutant strains accumulate CPY in class E compartments from which retrieval to the Golgi is blocked. Similarly, the mammalian retromer cargo mannose 6-phosphate receptor (M6PR) also failed to recycle from endosomes to the Golgi in HeLa cells depleted of TSG101/ESCRT-I function. In this study, it is suggested that generation of class E compartments occurs at the expense of endosomal tubules. Consistently, the retromer-associated tubulation factor SNX1 and its yeast homolog Vps5p were found on the rims of mammalian and yeast class E compartments, respectively. Together with the finding of CSC accumulation on Drosophila class E-like compartments, this suggests that ESCRT deficient endosomes remain coated with retromer components but fail to export specific cargo. (Pannen, 2020).
While the possibility cannot be ruled out of ESCRT components directly cooperating with retromer to form recycling tubules (note that SNX-BAR, Snx3, and Snx27 are not required for Mega transport), an indirect mechanism is favored linking ESCRT and retromer in this transport pathway. Analysis of the aberrant endosomal compartments induced upon Shrub depletion revealed that they are enriched in endosomal organizers such as Rab5 and Rab7, which could potentially interfere with retromer-dependent export when their activity at the limiting membrane is unrestrained. While the role of Rab7 in endosomal recruitment of the CSC is well established, the necessity for Rab7 GDP/GTP cycling during retromer-dependent carrier generation is still under debate. Rab7 and its GTPase-activating protein (GAP) Tbc1d5 are interaction partners of the CSC and can modulate its capability to retrieve endosomal cargo. For example, interfering with Rab7-GTP hydrolysis by Tbc1d5 depletion yielded defects in retromer-dependent transport in HeLa cells. Strikingly, under these conditions, retromer cargo was trapped in CSC-coated endosomes, paralleling the current observation of Mega subcellular localization upon ESCRT depletion. Similarly, by exposing the interplay between the CSC component Vps29, Tbc1d5, and Rab7 in adult Drosophila brains, the authors of a recent study reported the capability of endosomal Rab7 to interfere with retromer CSC function in vivo (Pannen, 2020).
While the exact mechanism rendering retromer dysfunctional at Drosophila class E compartments remains to be determined, the data support the mounting pool of evidence that ESCRT is required for multiple endosomal retrieval pathways. It is therefore likely that aspects of the pleiotropic ESCRT phenotype in metazoans stem from defective export of proteins from the endosomal system. For example, in Drosophila, leaky SJ could support ESCRT-mediated neoplastic transformation by permitting diffusion of signaling molecules within the imaginal disc tissue (Pannen, 2020).
This study found that biosynthetic delivery of Mega depends on a transcytosis-like mechanism from the basodistal to the apical plasma membrane. This long-distance transport required sequential action of endocytic (clathrin, dynamin, Rab5) and endosomal (ESCRT, retromer CSC) machineries. Importantly, the finding that overexpressed HA-Mega is unable to reach the SJ in absence of retromer and ESCRT function is in agreement with biosynthetic delivery of Mega relying on endosomal function. Therefore, although the possibility that Mega transiently passes the Golgi after endocytosis at the basodistal membrane cannot be excluded, the unconventional transcytosis model is favored. Strikingly, while retromer-dependent endosomal recycling has been extensively documented, only one mammalian cell culture study implicated retromer in transcytosis from one membrane domain to another. Thus, SJ delivery of Mega in imaginal discs represents a novel physiological role of retromer to study this process in vivo. The finding that CSC-mediated anterograde transport of Mega is independent of retromer-associated sorting nexins indicates that this transcytosis pathway is distinct from many established CSC-dependent routes and suggests that it may require unknown cofactors (or does not require endosomal tubulation) (Pannen, 2020).
Analysis of Vps35 clones in pupal wings or leg imaginal discs revealed that in these tissues, clones completely devoid of the SJ core component NrxIV occur frequently. Similarly, shrub mutant clones in the pupal notum were entirely lacking junctional ATPα (Roland Le Borgne, personal communication to Pannen, July 2020). This is in contrast to surface levels of SJ components in Vps35 mutant wing discs, which were consistently reduced by about 50%. This provokes the hypothesis that a parallel endosomal export pathway for SJ components may exist in wing discs that could partially compensate for loss of retromer. However, this is thought unlikely since overexpressed HA-Mega fails to reach the SJ not only upon Shrub but also upon Vps26 depletion. One has to keep in mind that this experiment specifically monitors delivery of newly synthesized HA-Mega while the Vps35 clonal analysis assesses the impact of retromer loss of function on pre-existing SJ. Thus, in a clonal situation, a 'thinning out' of junctions is expected with consecutive rounds of cell division, which could explain incomplete phenotypic expressivity in wing disc Vps35 clones. Nevertheless, it remains to be determined why in leg discs and pupal wings, SJ appear to be more sensitive toward CSC loss. During metamorphosis, wing imaginal disc cells undergo drastic morphogenetic changes to form the pupal wing epithelium, a process known to require AJ remodeling. It is therefore possible that analogously to AJ, SJ may also be actively remodeled during pupal wing formation. This could explain the strong requirement for retromer function in maintaining SJ integrity in a tissue undergoing morphogenetic changes (Pannen, 2020).
Paralleling the findings described in this paper, a previous study showed that embryonic SJ formation depends on endocytosis and subsequent redistribution of junction components from the lateral membrane to the SJ. Thus, transcytosis of SJ components is a mechanism likely required for both initial SJ formation as well as maintenance of SJ integrity in non-embryonic tissues in Drosophila. While the roles of ESCRT and retromer CSC in initial SJ formation were not assessed, it is conceivable that they are already required for transporting SJ components during embryogenesis. Consistently, shrub mutant embryos display a defective epithelial barrier function, suggesting they fail to form functional SJx (Pannen, 2020).
The reason for SJ maintenance to rely on such an elaborate trafficking of its components remains to be determined. It has been suggested that SJ components form stable complexes prior to integration into the junction. Potentially, essential post-translational modifications of certain SJ components required for complex formation may occur exclusively at the basodistal membrane or during the passage through the endosomal system. If transient localization of SJ components at the basodistal membrane is a prerequisite for efficient SJ core complex formation, depositing transcripts for structural components such as Mega in the basal cytoplasm would shorten the route individual SJ components need to pass prior to SJ complex formation at the basodistal membrane domain. Alternatively, the finding that Mega mRNA predominantly localizes in the basal cytoplasm provides the foundation for another hypothesis: It is widely accepted that apical and basolateral cargos undergo motif-based sorting leading to secretion toward the respective membrane domains. Basal subcellular localization of Mega transcripts could potentially reflect an apical/basolateral sorting divergence at the mRNA level. Accordingly, basal translation and exocytosis of Mega (induced by a putative basal/basolateral sorting signal) may lead to its targeting toward the basodistal membrane, despite the fact that the SJ resides apically in wing disc cells. Thus, transcytosis may serve as an adaptation for redistribution of cargos to their destined membrane domain when they are initially secreted to a different one due to early sorting signals. Since the columnar wing imaginal disc cells have a very elongated shape and possess Golgi stacks all along the apicobasal axis, it is conceivable that certain Golgi stacks residing at the apical and basal poles are specialized in secreting apical and basolateral cargos, respectively. Although this is highly hypothetical, systematic analysis of transcript localization for apical (e.g., E-cad) and basolateral (e.g., SJ components) cargos could reveal a potential spatial separation of distinct secretory routes already at the mRNA level (Pannen, 2020).
In wing imaginal discs, a similarly complex transcytosis route (albeit with an apical to basal direction) has been described for the signaling molecule Wingless, which is translated apically, transiently presented at the apical membrane and finally transcytosed toward the basal membrane where it is secreted (Yamazaki, 2016). Thus, distinct transcytosis pathways in the wing disc epithelium provide a mechanism for targeting certain proteins to their site of action, specifically when the protein is translated far away from its terminal destination (Pannen, 2020).
This study has unravel a novel physiological retromer function in regulating surface levels of a claudin and other structural SJ components (e.g., Nrg, ATPα, Lac, NrxIV, Cont) in several Drosophila tissues. Currently, it is not known how the SJ components are selected for this retromer-dependent pathway, and whether it requires physical interaction with CSC components. Since SJ proteins may traverse the endosomal system in complex, the vast number of different components brings about a plethora of possible interaction sites. Importantly, a mass-spectrometry-based study of the Mega interactome did not detect any retromer CSC components or associated factors but confidently found SJ core components as well as clathrin. While the interaction mode of CSC and SJ proteins remains to be determined, the data reflect the assumption that several SJ core components represent novel putative retromer cargos in Drosophila (Pannen, 2020).
Strikingly, among the proteins affected by retromer loss of function, many possess mammalian homologs (e.g., NrxIV/CNTNAP2, ATPα/ATP1A1, Nrg/NRCAM). This suggests they could represent a novel set of conserved retromer cargos. Indeed, several lines of evidence suggest that ESCRT/retromer-mediated transport of SJ components may be evolutionary conserved from Drosophila to mammals. Depletion of ESCRT-I component TSG101 in mammalian epithelial cells led to a reduction of trans-epithelial resistance (TER), indicating defects in TJ-mediated barrier function. Additionally, Claudin-1, an essential TJ component, continuously underwent endocytosis and recycling back to the plasma membrane in several mammalian cell lines in a process requiring ESCRT function (Pannen, 2020).
Importantly, the mechanism behind reduced recycling and entrapment of Claudin-1 in ubiquitin-positive aberrant endosomes upon interference with ESCRT function remained elusive. Thus, it is unknown how the export of Claudin-1 from the endosomal system in mammalian cells is achieved. By revealing an ESCRT-dependent function of the retromer CSC in claudin endosomal export in Drosophila, the data may provide an explanation for a possibly conserved trafficking pathway of claudins. In support of this, Claudin-1 and Claudin-4 membrane levels were significantly reduced in a mass spectrometry-based surface proteome study of Vps35-depleted human cells. Furthermore, the TJ protein Zonula occludens-2 (ZO-2) was strongly enriched in a Vps26 interactome, suggesting that a presumptive retromer function in TJ maintenance in mammalian cells may not be limited to claudins, similar to the findings in Drosophila presented in this study. It remains to be determined whether a physiological role of retromer in mammalian TJ maintenance occurs also in vivo. An increasing amount of tools, such as conditional Vps35 knockout mice, will enable analysis of this putatively conserved retromer function in mammalian systems and reveal any possible implications in development and/or disease (Pannen, 2020).
The vertebrate tight junction is a critical claudin-based cell-cell junction that functions to prevent free paracellular diffusion between epithelial cells. In Drosophila, this barrier is provided by the septate junction, which, despite being ultrastructurally distinct from the vertebrate tight junction, also contains the claudin-family proteins Megatrachea and Sinuous. This study identified a third Drosophila claudin, Kune-kune, that localizes to septate junctions and is required for junction organization and paracellular barrier function, but not for apical-basal polarity. In the tracheal system, septate junctions have a barrier-independent function that promotes lumenal secretion of Vermiform and Serpentine, extracellular matrix modifier proteins that are required to restrict tube length. As with Sinuous and Megatrachea, loss of Kune-kune prevents this secretion and results in overly elongated tubes. Embryos lacking all three characterized claudins have tracheal phenotypes similar to any single mutant, indicating that these claudins act in the same pathway controlling tracheal tube length. However, there are distinct requirements for these claudins in epithelial septate junction formation. Megatrachea is predominantly required for correct localization of septate junction components, while Sinuous is predominantly required for maintaining normal levels of septate junction proteins. Kune-kune is required for both localization and levels. Double- and triple-mutant combinations of Sinuous and Megatrachea with Kune-kune resemble the Kune-kune single mutant, suggesting that Kune-kune has a more central role in septate junction formation than either Sinuous or Megatrachea (Nelson, 2010).
The Drosophila genome encodes seven predicted claudin-family molecules. Two of these, Mega and Sinu, have previously been characterized and were shown to be required for SJ organization and function. Sequence comparisons indicated that, although all seven claudin-like molecules show a large sequence divergence, CG1298 is more closely related to Sinu and Mega than the other Drosophila claudin-family members. Therefore, although many Drosophila claudin-family members are not required for barrier function, it was reasoned that CG1298 may play a role in SJ paracellular barrier formation. Accordingly, a detailed analysis of CG1298, which was named kune-kune (Japanese for 'wiggling like a snake,' pronounced koon-eh koon-eh and abbreviated kune) was characterized for its tracheal phenotype (Nelson, 2010).
The kune locus contains a single exon that codes for a protein of 264 amino acids. As is characteristic for claudins, the TMpred transmembrane algorith predicts Kune to have four transmembrane domains with intracellular N and C termini, a large initial extracellular loop, and two smaller loops. Kune also contains a W-GLW-C-C motif in the large extracellular loop and a C-terminal PDZ-binding motif, features that are found in almost all claudin family members. Notably, the PDZ-binding motif in Kune is a better match to consensus PDZ-binding motifs than the motif in Sinuous. Furthermore, in contrast to Mega and Sinu, whose N termini are 28 and 38 aa respectively, Kune has a short N terminus of 9 aa that is more typical of vertebrate claudins. Thus, Kune has features that more closely resemble vertebrate claudins than do the so far characterized Drosophila claudins Sinu and Mega (Nelson, 2010).
To determine the expression pattern of Kune, anti-Kune sera were generated and wild-type (WT) embryos were immunized. As with Mega and Sinu, Kune is highly expressed in ectodermally derived tissues, including the epidermis, salivary gland, trachea, hindgut, and foregut beginning at embryonic stage 13. In these tissues, Kune colocalizes with the SJ protein Coracle (Cor) and localizes basal to the adherens junction marker, DE-cadherin (E-cad), suggesting that Kune is a SJ protein. As with many other SJ proteins, Kune is also expressed in glial cells (Nelson, 2010).
Since most SJ proteins show interdependence for correct localization and junction function, it was asked whether localization of Kune depends on the presence of other SJ proteins. Indeed, Kune is mislocalized to more basal positions in the primary epithelia of mega, sinu, cor, and Atpα null mutants, providing strong evidence that Kune is a SJ protein (Nelson, 2010).
To directly assess the function of Kune during development, a PiggyBac insertion, PBac{3HPy}C309, in the 5'-untranslated region (UTR) of kune was identified as a putative null mutation. Embryos homozygous for the kuneC309 chromosome fail to hatch and completely lack Kune protein as assessed by immunohistochemistry. Expression of a UAS-kune construct using the ubiquitous da-Gal4 driver at 28° rescued the embryonic lethality of kuneC309 embryos, demonstrating that lethality was due to loss of Kune. Further, embryos trans-heterozygous for kuneC309 and Df(2R)BSC696 (which deletes the kune locus and also eliminates Kune staining) or homozygous for Df(2R)BSC696 fail to hatch and display tracheal and septate junction phenotypes that are indistinguishable from kuneC309 homozygotes. These results indicate that kune is an essential gene and that kuneC309 is a null or strong loss-of-function allele of kune (Nelson, 2010).
To determine if Kune is required for SJ organization and function, the subcellular localization of several SJ proteins was examined in kune mutant epithelia. As is seen in other SJ mutants, kune epithelia show a reduction and/or mislocalization of the SJ components Cor, Mega, Sinu, Atpα, Discs large (Dlg), and NeurexinIV (Nrx) to more basal locations in all primary epithelia. This phenotype is also seen in animals that express kune-RNAi using the ubiquitous da-Gal4 driver, although the phenotype is less severe, presumably due to incomplete knockdown. Consistent with the immunohistological evidence of SJ defects, a 10-kDa fluorescent dye injected into the body cavity of kune animals readily diffused into the lumen of the trachea and salivary gland, indicating a loss of the paracellular barrier. Expression of the UAS-kune construct with da-Gal4 rescued Cor localization and improved the barrier function of kune mutants. Thus, Kune is an essential component of SJs in primary epithelia (Nelson, 2010).
In addition to their roles in epithelial tissues, SJs are also required to establish the blood-brain barrier in flies. In the central nervous system (CNS), surface glial cells completely ensheath the ventral nerve cord and form SJs at glia-glia contacts. This generates a tight paracellular seal that separates the K+-rich hemolymph from neural cells, which is essential for generation of action potentials. This study found that Kune is expressed in glial cells, which is most clearly seen at the central midline. Dye injections revealed that Kune, like Sinu and Mega, is required for the CNS glial barrier, since the dye penetrated into the nerve cord of kune but not WT embryos. Taken together, the above results all identify Kune as a critical SJ component in multiple tissue types (Nelson, 2010).
Since almost all characterized SJ proteins are required for tracheal tube size control, the tracheal system of kune embryos was examined. Staining with the 2A12 lumenal marker demonstrated that the length of the DT of stage 16 kune embryos was significantly increased over WT controls and appeared tortuous (thus the name kune-kune). This phenotype was identical in both kune/Df(2R)BSC696 embryos and embryos expressing kune-RNAi using the da-Gal4 driver (Nelson, 2010).
It has been established that Sinu, Mega, and other SJ proteins are required for apical secretion of the putative chitin deacetylases Verm and Serp, which restrict tracheal tube length. Therefore lumenal accumulation of Verm and Serp and the organization of the chitin-based lumenal matrix were examined at embryonic stage 16. As is typical for a SJ component, kune mutant embryos and embryos expressing kune-RNAi do not accumulate Verm or Serp in the tracheal lumen. This secretion defect could be largely rescued by expression of UAS-kune with the da-Gal4 driver, although not to WT levels. Additionally, staining with a fluorescent chitin binding probe showed that, while the lumen of WT trachea contains a dense, fibrilar chitin cable, kune trachea have a diffuse, disorganized lumenal matrix. kune trachea also lack the gap between the chitin cable and the apical surface of the cells that is found in WT trachea (Nelson, 2010).
Since Kune is closely related to both Mega and Sinu and all three localize to the SJ, it was asked if these claudins are partially redundant in junction organization or tube size control. To test this, the trachea of mega, kune, and sinu single, double, and triple mutants were examined. If the claudins have redundant functions in tube size control, the phenotypes should be worse when multiple claudins are missing. However, the tracheal length defects of mega; kune and kune; sinu embryos appear no more severe than in any of the single mutants. Strikingly, even embryos lacking all three Kune-related claudins do not appear to have more severe tracheal length defects than single mutants. In contrast, previous work has shown that embryos containing mutations in both sinu and the SJ gene varicose (vari) have trachea that are more tortuous than either single mutant. These results suggest that, although some SJ proteins have redundant functions in restricting tracheal tube length, the Drosophila claudins Kune, Sinu, and Mega all function in the same pathway of tracheal tube size control (Nelson, 2010).
Given that tracheal tube length is only a limited readout of SJ function, the effects of single-, double-, and triple-mutant combinations of kune, sinu, and mega on SJ organization were compared using the subcellular localization of Cor as an assay. Focused was placed on the hindgut and salivary gland, since SJ organization is clearest in these columnar cells. Interestingly, the levels and localization of Cor are strikingly different between the three claudin mutations, suggesting that different claudins have unique functions. For example, Cor is completely mislocalized to basal positions in the hindgut of mega embryos, but the levels are not dramatically lower than in WT. On the other hand, the hindguts of sinu embryos show lower overall levels of Cor, but retain significant apicolateral enrichment where the SJ is normally found. In kune mutants, Cor is both reduced and completely mislocalized. Similar, more pronounced effects are seen in the salivary glands where loss of mega causes only slight basolateral mislocalization of Cor, loss of sinu causes almost no Cor mislocalization, and loss of kune strongly mislocalizes and reduces Cor staining. The localization and levels of the SJ markers Dlg and Atpα were also more severely disrupted in kune mutants than in sinu or mega mutants, indicating that the effects were not specific to Cor. Interestingly, the levels and localization of Cor are not obviously different between the kune single mutant and the double and triple mutants, indicating that the kune phenotype is the most severe. Together, these results suggest that Kune has a more central role in SJ organization than either Sinu or Mega. This possibility is particularly intriguing in light of the greater similarity of Kune to vertebrate claudins than either Sinu or Mega. Perhaps a more central role for Kune in barrier junction formation has constrained its evolution and thus Kune more closely resembles ancestral claudins than do Sinu and Mega, which may have evolved more specialized functions (Nelson, 2010).
It is curious that multiple nonredundant claudins are required for SJ organization and barrier function. The exact reason for this is unclear, but perhaps each claudin interacts independently with specific junctional molecules to establish a SJ scaffold. This would be consistent with their divergent protein sequences and the differences in their N and C termini. Importantly, vertebrate claudins also have nonredundant roles in TJ function. For example, paracellular barrier function is compromised in the epidermis of mice lacking claudin-1 despite the presence of claudin-4 at the TJ (Nelson, 2010).
Like other SJ components, Kune does not appear to be required for establishment of apical-basal polarity since the levels and localization of the apical marker, Crumbs (Crb), and the adherens junction marker, E-cad, were normal in kune embryos. However, it was recently shown that some SJ components have a role in a newly identified phase of Drosophila epithelial polarity that occurs between stages 11 and 13. Because of redundancy between SJ components involved in this polarity phase and the SJ component yrt, SJ proteins required for polarity can be identified only in a yrt zygotic mutant background. For example, single zygotic mutations in either the SJ gene Atpα or yrt show normal apical localization of Crb at stage 12. Atpα, yrt double mutants on the other hand show severe mislocalization of Crb, indicative of a loss of polarity. In contrast, neither kune single mutants nor kune; yrt double mutants display any obvious polarity defects, demonstrating that kune is not required for either establishment or maintenance of epithelial polarity (Nelson, 2010).
Previous work has shown that neither mega nor sinu are required for establishment of apical-basal polarity or for maintenance of epithelial polarity at mid-embryogenesis. Together with the findings in this article, the available evidence suggests that Drosophila claudins are not required for epithelial polarity. This parallels the situation in Caenorhabditis elegans where mutations in the claudin-like proteins CLC1-4 disrupt barrier function, but not epithelial polarity. Similarly, claudins do not appear to be required for epithelial polarity in mammalian epithelial cells, since Eph4 cells can establish normal polarity even when lacking claudin complexes and tight junction strands due to elimination of ZO-1 and ZO-2. The absence of a role for claudins in polarity in any characterized species is consistent with the proposal that barrier junctions arose after polarity during the evolution of metazoans (Nelson, 2010).
These results show that Kune is an essential claudin that is required in all examined tissues for the organization and function of SJs. Kune expression and localization overlaps with the Drosophila claudins Mega and Sinu, but it was found that all three claudins play unique roles in SJ organization. Importantly, Kune more closely resembles vertebrate claudins than either Mega or Sinu and appears to play a more central role in SJ organization. Further work is needed to establish the complete molecular organization of SJs, but such work will be facilitated by the presented characterization of Kune and its interaction with other SJ components (Nelson, 2010).
Smooth septate junctions (sSJs) regulate the paracellular transport in the intestinal tract in arthropods. In Drosophila, the organization and physiological function of sSJs are regulated by at least three sSJ-specific membrane proteins: Ssk, Mesh, and Tsp2A. This study reports a novel sSJ membrane protein Hoka, which has a single membrane-spanning segment with a short extracellular region, and a cytoplasmic region with the Tyr-Thr-Pro-Ala motifs. The larval midgut in hoka-mutants shows a defect in sSJ structure. Hoka forms a complex with Ssk, Mesh, and Tsp2A and is required for the correct localization of these proteins to sSJs. Knockdown of hoka in the adult midgut leads to intestinal barrier dysfunction, and stem cell overproliferation. In hoka-knockdown midguts, aPKC is up-regulated in the cytoplasm and the apical membrane of epithelial cells. The depletion of aPKC and yki in hoka-knockdown midguts results in reduced stem cell overproliferation. These findings indicate that Hoka cooperates with the sSJ-proteins Ssk, Mesh, and Tsp2A to organize sSJs, and is required for maintaining intestinal stem cell homeostasis through the regulation of aPKC and Yki activities in the Drosophila midgut (Izumi, 2021).
Epithelia separate distinct fluid compartments within the bodies of metazoans. For this epithelial function, occluding junctions act as barriers that control the free diffusion of solutes through the paracellular pathway. Septate junctions (SJs) are occluding junctions in invertebrates and form circumferential belts along the apicolateral region of epithelial cells. In transmission electron microscopy, SJs are observed between the parallel plasma membranes of adjacent cells, with ladder-like septa spanning the intermembrane space. Arthropods have two types of SJs: pleated SJs (pSJs) and smooth SJs (sSJs). pSJs are found in ectoderm-derived epithelia and surface glia surrounding the nerve cord, whereas sSJs are found mainly in the endoderm-derived epithelia, such as the midgut and gastric caeca. Despite being derived from the ectoderm, the outer epithelial layer of the proventriculus (OELP) and the Malpighian tubules also possess sSJs. Furthermore, pSJs and sSJs are distinguished by the arrangement of septa. For example, the septa of pSJs form regular undulating rows, whereas those in sSJs form regularly spaced parallel lines in the oblique sections in lanthanum-treated preparations. To date, more than 20 pSJ-related proteins have been identified and characterized in Drosophila. In contrast, only three membrane-spanning proteins, Ssk, Mesh and Tsp2A, have been reported as specific molecular constituents of sSJs (sSJ proteins) in Drosophila. Therefore, the mechanisms underlying sSJ organization and the functional properties of sSJs remain poorly understood compared with pSJs. Ssk has four membrane-spanning domains; two short extracellular loops, cytoplasmic N- and C-terminal domains, and a cytoplasmic loop. Mesh is a cell-cell adhesion molecule, which has a single-pass transmembrane domain and a large extracellular region containing a NIDO domain, an Ig-like E set domain, an AMOP domain, a vWD domain and a sushi domain. Tsp2A is a member of the tetraspanin family of integral membrane proteins in metazoans with four transmembrane domains, N- and C-terminal short intracellular domains, two extracellular loops and one short intracellular turn. The loss of ssk, mesh and Tsp2A causes defects in the ultrastructure of sSJs and the barrier function against a 10 kDa fluorescent tracer in the Drosophila larval midgut. Ssk, Mesh and Tsp2A interact physically and are mutually dependent for their sSJ localization. Thus, Ssk, Mesh and Tsp2A act together to regulate the formation and barrier function of sSJs. Furthermore, Ssk, Mesh and Tsp2A are localized in the epithelial cell-cell contact regions in the Drosophila Malpighian tubules in which sSJs are present. Recent studies have shown that the knockdown of mesh and Tsp2A in the epithelium of Malpighian tubules leads to defects in epithelial morphogenesis, tubule transepithelial fluid and ion transport, and paracellular macromolecule permeability in the tubules. Thus, sSJ proteins are involved in the development and maintenance of functional Malpighian tubules in Drosophila (Izumi, 2021).
The Drosophila adult midgut consists of a pseudostratified epithelium, which is composed of absorptive enterocytes (ECs), secretory enteroendocrine cells (EEs), intestinal stem cells (ISCs), EC progenitors (enteroblasts: EBs) and EE progenitors (enteroendocrine mother cells: EMCs). The sSJs are formed between adjacent ECs and between ECs and EEs. To maintain midgut homeostasis, ECs and EEs are continuously renewed by proliferation and differentiation of the ISC lineage through the production of intermediate differentiating cells, EBs and EMCs. Recently, it has been reported that the knockdown of sSJ proteins Ssk, Mesh and Tsp2A in the midgut causes intestinal hypertrophy accompanied by the overproliferation of ECs and ISC. These results indicate that sSJs play a crucial role in maintaining tissue homeostasis through the regulation of stem cell proliferation and enterocyte behavior in the Drosophila adult midgut. Furthermore, it has been reported that the loss of mesh and Tsp2A in adult midgut epithelial cells causes defects in cellular polarization, although no remarkable defects in epithelial polarity were found in the first-instar larval midgut cells of ssk, mesh and Tsp2A mutants. Thus, sSJs may contribute to the establishment of epithelial polarity in the adult midgut (Izumi, 2021).
During the regeneration of the Drosophila adult midgut epithelium, various signaling pathways are involved in the proliferation and differentiation of the ISC lineage. Atypical protein kinase C (aPKC) is an evolutionarily conserved key determinant of apical-basal epithelial polarity . Importantly, it has been reported that aPKC is dispensable for the establishment of epithelial cell polarity in the Drosophila adult midgut. It has been reported that aPKC is required for differentiation of the ISC linage in the midgut. The Hippo signaling pathway is involved in maintaining tissue homeostasis in various organs. In the Drosophila midgut, inhibition of the Hippo signaling pathway activates the transcriptional co-activator Yorkie (Yki), which results in accelerated ISC proliferation via the Unpaired (Upd)-Jak-Stat signaling pathway. Recent studies have shown that Yki is involved in ISC overproliferation caused by the depletion of sSJ proteins in the midgut. Furthermore, it has been shown that aPKC is activated in the Tsp2A-RNAi-treated midgut, leading to activation of its downstream target Yki and causing ISC overproliferation through the activation of the Upd-Jak-Stat signaling pathway. Thus, crosstalk between aPKC and the Hippo signaling pathways appears to be involved in ISC overproliferation caused by Tsp2A depletion (Izumi, 2021).
To further understand the molecular mechanisms underlying sSJ organization, a deficiency screen was performed for Mesh localization, and the integral membrane protein Hoka was identified as a novel component of Drosophila sSJs. Hoka consists of a short extracellular region and the characteristic repeating 4-amino acid motifs in the cytoplasmic region, and is required for the organization of sSJ structure in the midgut. Hoka and Ssk, Mesh, and Tsp2A show interdependent localization at sSJs and form a complex with each other. The knockdown of hoka in the adult midgut results in intestinal barrier dysfunction, aPKC- and Yki-dependent ISC overproliferation, and epithelial tumors. Thus, Hoka plays an important role in sSJ organization and in maintaining ISC homeostasis in the Drosophila midgut (Izumi, 2021).
The identification of Ssk, Mesh and Tsp2A has provided an experimental system to analyze the role of sSJs in the Drosophila midgut. Recent studies have shown that sSJs regulate the epithelial barrier function and also ISC proliferation and EC behavior in the midgut. Furthermore, sSJs are involved in epithelial morphogenesis, fluid transport and macromolecule permeability in the Malpighian tubules. This study reports the identification of a novel sSJ-associated membrane protein Hoka. Hoka is required for the efficient accumulation of other sSJ proteins at sSJs and the correct organization of sSJ structure. The knockdown of hoka in the adult midgut leads to intestinal barrier dysfunction, increased ISC proliferation mediated by aPKC and Yki activities, and epithelial tumors. Thus, Hoka contributes to sSJ organization and the maintenance of ISC homeostasis in the Drosophila midgut (Izumi, 2021).
Arthropod sSJs have been classified together based on their morphological similarity. The identification of sSJ proteins in Drosophila has provided an opportunity to investigate whether sSJs in various arthropod species share similarities at the molecular level. However, Hoka homolog proteins appear to be conserved only in insects upon a database search, suggesting compositional variations in arthropod sSJs (Izumi, 2021).
Interestingly, the cytoplasmic region of Hoka includes three YTPA motifs. The same or similar amino acid motifs are also present in the Hoka homologs of other holometabolous insects, such as other Drosophila species, the mosquito, beetle (YTPA motif), butterfly, ant, bee, sawfly, moth (YQPA motif) and flea (YTAA motif), although the number of these motif(s) vary (1 to 3 in Drosophila species, 1 in other holometabolous insects). In contrast, the motif is not present in hemimetabolous insects. The extensive conservation of the YTPA/YQPA/YTAA motif in holometabolous insects suggests that the motif was evolutionarily acquired and plays a critical role in the molecular function of Hoka. It would be interesting to investigate the role of the YTPA/YQPA/YTAA motif in sSJ organization of holometabolous insects (Izumi, 2021).
The extracellular region of Hoka appears to be composed of 13 amino acids alone after the cleavage of the signal peptide, which is too short to bridge the 15-20 nm intercellular space of sSJs. Thus, Hoka is unlikely to act as a cell adhesion molecule in sSJs. Indeed, the overexpression of Hoka-GFP in Drosophila S2 cells did not induce cell aggregation, which is a criterion for cell adhesion activity (Izumi, 2021).
The loss of an sSJ protein results in the mislocalization of other sSJ proteins, indicating that sSJ proteins are mutually dependent for their sSJ localization. In thessk -deficient midgut, Mesh and Tsp2A were distributed diffusely in the cytoplasm. In the mesh mutant midgut, Ssk was localized at the apical and lateral membranes, whereas Tsp2A was distributed diffusely in the cytoplasm. In the Tsp2A-mutant midgut, Ssk was localized at the apical and lateral membranes, whereas Mesh was distributed diffusely in the cytoplasm. Among these three mutants, the mislocalization of Ssk, Mesh or Tsp2A is consistent; Mesh and Tsp2A were distributed in the cytoplasm, whereas Ssk was localized at the apical and lateral membranes. However, in the hoka-mutant larval midgut, Mesh and Tsp2A were distributed along the lateral membrane, whereas Ssk was mislocalized to the apical and lateral membranes. Interestingly, in some hoka mutant midguts, Ssk, Mesh and Tsp2A were localized to the apicolateral region, as observed in the wild-type midgut. Differences in subcellular misdistribution of sSJ proteins between the hoka mutant and the ssk, mesh and Tsp2A-mutants indicate that the role of Hoka in the process of sSJ formation is different from that of Ssk, Mesh or Tsp2A. Ssk, Mesh and Tsp2A may form the core complex of sSJs, and these proteins are indispensable for the generation of sSJs, whereas Hoka facilitates the arrangement of the primordial sSJs at the correct position, i.e. the apicolateral region. This Hoka function may also be important for rapid paracellular barrier repair during the epithelial cell turnover in the adult midgut. Notably, during the sSJ formation process of the outer epithelial layer of the proventriculus (OELP, the sSJ targeting property of Hoka was similar to that of Mesh, implying that Hoka may have a close relationship with Mesh, rather than Ssk and Tsp2A during sSJ development (Izumi, 2021).
The knockdown of hoka in the adult midgut leads to a shortened lifespan in adult flies, intestinal barrier dysfunction, increased ISC proliferation and the accumulation of ECs. These results are consistent with the recent observation for ssk, mesh and Tsp2A-RNAi in the adult midgut. The intestinal barrier dysfunction caused by RNAi for sSJ proteins may permit the leakage of particular substances from the midgut lumen, which may induce particular cells to secrete cytokines and growth factors for ISC proliferation. Alternatively, sSJs or sSJ-associated proteins may be directly involved in the secretion of cytokines and growth factors through the regulation of intracellular signaling in the ECs. In the latter case, it has been shown that Tsp2A knockdown in ISCs/EBs or ECs hampers the endocytic degradation of aPKC, thereby activating the aPKC and Yki signaling pathways, leading to ISC overproliferation in the midgut. Therefore, it has been proposed that sSJs are directly involved in the regulation of aPKC and the Hippo pathway-mediated intracellular signaling for ISC proliferation. This study has shown that the expression of hoka-RNAi together with aPKC-RNAi or yki-RNAi in ECs significantly reduced ISC overproliferation caused by hoka-RNAi. Thus, aPKC- and Yki-mediated ISC overproliferation appears to commonly occur in sSJ protein-deficient midguts. However, the possibility that the leakage of particular substances through the paracellular route may be involved in ISC overproliferation in the sSJ proteins-deficient midgut cannot be excluded (Izumi, 2021).
It has been reported that apical aPKC staining is observed in ISCs but is barely detectable in ECs. This study found that the expression of hoka-RNAi in ECs increased aPKC staining in the midgut. Additionally, in the hoka-RNAi midgut, apical aPKC staining was observed in ISCs and in differentiated cells, including EC-like cells. Thus, apical and increased cytoplasmic aPKC may contribute to ISC overproliferation. Interestingly, EC-like cells in the hoka-RNAi midgut do not always localize aPKC to the apical regions. Apical aPKC staining was detected in EC-like cells mounted by other cells but was barely detectable in the lumen-facing EC-like cells. These mounted cells are thought to be newly generated cells after the induction of hoka-RNAi, which may not be able to exclude aPKC from the apical region in the crowded cellular environment. A recent study showed that aberrant sSJ formation caused by Tsp2A-depletion impairs aPKC endocytosis and increases aPKC localization in the membrane of cell borders. The sSJ proteins, including Hoka, may also regulate endocytosis to exclude aPKC from the apical membrane of ECs. The identification of molecules involved in aPKC-mediated ISC proliferation may provide a better understanding of the aPKC-mediated signaling pathway, as well as the mechanisms underlying the increased expression and apical targeting of aPKC in the ECs deficient for sSJ proteins (Izumi, 2021).
The blood-brain barrier (BBB) of Drosophila comprises a thin epithelial layer of subperineural glia (SPG), which ensheath the nerve cord and insulate it against the potassium-rich hemolymph by forming intercellular septate junctions (SJs). Previous work identified a novel Gi/Go protein-coupled receptor (GPCR), Moody, as a key factor in BBB formation at the embryonic stage. However, the molecular and cellular mechanisms of Moody signaling in BBB formation and maturation remain unclear. This study identified cAMP-dependent protein kinase A (PKA) as a crucial antagonistic Moody effector that is required for the formation, as well as for the continued SPG growth and BBB maintenance in the larva and adult stage. PKA is enriched at the basal side of the SPG cell, and this polarized activity of the Moody/PKA pathway finely tunes the enormous cell growth and BBB integrity. Moody/PKA signaling precisely regulates the actomyosin contractility, vesicle trafficking, and the proper SJ organization in a highly coordinated spatiotemporal manner. These effects are mediated in part by PKA's molecular targets MLCK and Rho1. Moreover, 3D reconstruction of SJ ultrastructure demonstrates that the continuity of individual SJ segments, and not their total length, is crucial for generating a proper paracellular seal. It is proposed that Moody/PKA signaling plays a central role in controlling the cell growth and maintaining BBB integrity (Li, 2021).
Previous studies implicated a novel GPCR signaling pathway in the formation of the Drosophila BBB in late embryos. This work also revealed that besides the GPCR Moody two heterotrimeric G proteins (Gαiβγ, Gαoβγ5) and the RGS Loco participate in this pathway. This study provides a comprehensive molecular and cellular analysis of the events downstream of G protein signaling using a candidate gene screening approach. New, more sensitive methods for phenotypic characterization are presented, and the analysis was extended to beyond the embryo into larval stages. This work identifies, together with some of its targets, as crucial antagonistic effectors in the continued cell growth of SPG and maintenance of the BBB sealing capacity. This role is critical to ensure proper neuronal function during BBB formation and maturation (Li, 2021).
Multiple lines of evidence demonstrate a role of PKA for proper sealing of the BBB: loss of PKA activity leads to BBB permeability defects, irregular growth of SPG during epithelium formation, reduced membrane overlap, and a narrower SJ belt at SPG cell-cell contacts. The role of PKA as an effector of the Moody signaling pathway is further supported by dominant genetic interaction experiments, which show that the dye penetration phenotype of PkaC1 heterozygous mutant embryos was partially rescued by removing one genomic copy of Gβ13F or loco. Moreover, the analysis of the larval phenotype with live SJ and cytoskeleton markers shows that PKA gain of function behaved similarly to Moody loss of function. Conversely, PKA loss of function resembled the overexpression of GαoGTP, which mimics Moody gain-of-function signaling (Li, 2021).
The results from modulating PKA activity suggest that the total cell contact and SJ areas are a major function of PKA activity: low levels of activity cause narrow contacts, and high levels give rise to broad contacts. Moreover, the analysis of various cellular markers (actin, microtubules, SJs, vesicles) indicates that the circumferential cytoskeleton and delivery of SJ components respond proportionately to PKA activity. This, in turn, promotes the changes in cell contact and junction areas coordinately at the lateral side of SPG. These experiments demonstrate that the modulation of the SPG membrane overlap by PKA proceeds, at least in part, through the regulation of actomyosin contractility, and that this involves the phosphorylation targets MLCK and Rho1. This suggests that crucial characteristics of PKA signaling are conserved across eukaryotic organisms (Li, 2021).
At the ultrastructural level, ssTEM analysis of the larval SPG epithelium clarifies the relationship between the inter-cell membrane overlaps and SJ organization and function. Across different PKA activity levels, the ratio of SJ areas to the total cell contact area remained constant at about 30%. This proportionality suggests a mechanism that couples cell contact with SJ formation. The primary role of Moody/PKA appears in this process to be the control of membrane contacting area between neighboring cells. This is consistent with the results of a temporal analysis of epithelium formation and SJ insertion in late embryos of WT and Moody pathway mutants, which shows that membrane contact precedes and is necessary for the appearance of SJs. The finding that the surface area that SJs occupy did not exceed a specific ratio, irrespective of the absolute area of cell contact, suggests an intrinsic, possibly steric limitation in how much junction can be fitted into a given cell contact space. While most phenotypic effects are indeed a major function of Moody and PKA activity, the discontinuity and shortening of individual SJ strands is not. It occurred with both increased and decreased signaling and appears to cause the leakiness of the BBB in both conditions. ssTEM-based 3D reconstruction thus demonstrates that the total area covered by SJs and the length of individual contiguous SJ segments are independent parameters. The latter appears to be critical for the paracellular seal, consistent with the idea that Moody plays a role in the formation of continuous SJ stands (Li, 2021).
The asymmetric localization of PKA that was observed sheds further light on the establishment and function of apical-basal polarity in the SPG epithelium. Prior to epithelium formation, contact with the basal lamina leads to the first sign of polarity. Moody becomes localized to the apical surface only after epithelial closure and SJ formation, suggesting that SJs are required as a diffusion barrier and that apical accumulation of Moody protein is the result of polarized exocytosis or endocytosis. This study now shows that the intracellular protein PKA catalytic subunit-PkaC1 accumulates on the basal side of SPG, and that this polarized accumulation requires (apical) Moody activity. Such an asymmetric, activity-dependent localization has not previously been described for PKA in endothelium, and while the underlying molecular mechanism is unknown, the finding underscores that generating polarized activity along the apical-basal axis of the SPG is a key element of Moody pathway function (Li, 2021).
An intriguing unresolved question is how increased SPG cell size and SJ length can keep up with the expanding brain without disrupting the BBB integrity during larva growth. The SJ was found to grow dramatically in length (0.57 ± 0.07 μm vs. 2.16 ± 0.14 μm, about 3.7-fold) from the late embryo to third instar larva, which matches the increased cell size of SPG (about fourfold). During the establishment of the SPG epithelium in the embryo, both increased and decreased Moody signaling resulted in asynchronous growth and cell contact formation along the circumference of SPG, which in turn led to irregular thickness of the SJ belt. Therefore, a similar relationship may exist during the continued growth of the SPG epithelium in larvae, with the loss of continuity of SJ segments in Moody/PKA mutants resulting from unsynchronized expansion of the cell contact area and an ensuing erratic insertion of SJ components. Since SJs form relatively static complexes, any irregularities in their delivery and insertion may linger for extended periods of time. The idea that shortened SJ segments are a secondary consequence of unsynchronized cell growth is strongly supported by the finding that disruption of actomyosin contractility in MLCK and Rho1 mutants compromises BBB permeability (Li, 2021).
Collectively, these data suggest the following model: polarized Moody/PKA signaling controls the cell growth and maintains BBB integrity during the continuous morphogenesis of the SPG secondary epithelium. On the apical side, Moody activity represses PKA activity (restricting local cAMP level within the apial-basal axis in SPG) and thereby promotes actomyosin contractility. On the basal side, which first adheres to the basal lamina and later to the PNG sheath, PKA activity suppresses actomyosin contractility via MLCK and Rho1 phosphorylation and repression. Throughout development, the SPG grow continuously while extending both their cell surface and expanding their cell contacts. The data suggest that the membrane extension occurs on the basolateral surface through insertion of plasma membrane and cell-adhesive proteins, with similar behavior in epithelial cell, but regulated by a distinct polarized Moody/PKA signaling in SPG. In analogy to motile cells, the basal side of the SPG would thus act as the 'leading edge' of the cell, while the apical side functions as the 'contractile rear'. According to this model, Moody/Rho1 regulate actomyosin to generate the contractile forces at the apical side to driving membrane contraction, which directs the basolateral insertion of new membrane material and SJs. In this way, differential contractility and membrane insertion act as a conveyor belt to move new formed membrane contacts and SJ from the basolateral to apical side. Loss of Moody signaling leads to symmetrical localization of PKA and to larger cell contact areas between SPG due to diminished apical constriction. Conversely, loss of PKA causes smaller cell contact areas due to increased basal constriction (Li, 2021).
These results may have important implications for the neuron-glia interaction in the nervous system and the development and maintenance of the BBB in vertebrates. SJs have several structural and functional components in common with paranodal junctions, which join myelinating glial cells to axons in the vertebrate nervous system, and they share similar regulation mechanisms. The vertebrate BBB consists of a secondary epithelium with interdigitations similar to the ones between the Drosophila SPG . While the sealing is performed by TJs, it will be interesting to investigate whether there are similarities in the underlying molecular and cellular mechanisms that mediate BBB function (Li, 2021).
Septate junctions (SJs) are located between epithelial cells and play crucial roles in epithelial barrier formation and epithelia cell homeostasis. Nevertheless, the molecular constituents, especially those related to smooth SJs (sSJs), have not been well explored in non-Drosophilid insects. A putative integral membrane protein Snakeskin (Ssk) was identified in a Coleoptera foliar pest Henosepilachna vigintioctopunctata. RNA interference-aided knockdown of Hvssk at the third-instar larval stage arrested larval development. Most resultant larvae failed to shed larval exuviae until their death. Silence of Hvssk at the fourth-instar larvae inhibited the growth and reduced foliage consumption. Dissection and microscopic observation revealed that compromised expression of Hvssk caused obvious phenotypic defects in the midgut. A great number of morphologically abnormal columnar epithelial cells accumulated throughout the midgut lumen. Moreover, numerous vesicles were observed in the malformed cells of the Malpighian tubules (Mt). All the Hvssk depleted larvae remained as prepupae; they gradually darkened and eventually died. Furthermore, depletion of Hvssk at the pupal stage suppressed adult feeding and shortened adult lifespan. These findings demonstrated that Ssk plays a vital role in the integrity and function of both midguts and Mt, and established the conservative roles of Ssk in the formation of epithelial barrier and the homeostasis of epithelial cells in H. vigintioctopunctata (Zhang, 2023).
Epithelial tissues separate body compartments with different compositions. Tight junctions (TJs) in vertebrates and septate junctions (SJs) in invertebrates control the paracellular flow of molecules between these compartments. This epithelial barrier function of TJs and SJs must be stably maintained in tissue morphogenesis during cell proliferation and cell movement. This study shows that Bark beetle (Bark), a putative transmembrane scavenger receptor-like protein, is essential for the maturation but not the establishment of SJs in Drosophila. Embryos that lack bark establish functional SJs, but due to rudimentary septae formation during subsequent embryonic development, these become non-functional. Furthermore, cell adhesion is impaired at the lateral cell membrane and the core protein complexes of SJs are mis-localised, but appear to form otherwise normally in such embryos. A model is proposed in which Bark acts as a scaffold protein that mediates cell adhesion and mounting of SJ core complexes during cell rearrangement in tissue morphogenesis (Hildebrandt, 2015).
This study describes the characterisation and functional analysis of the putative scavenger receptor protein Bark during epithelial morphogenesis. Bark was found to be required for the maturation but not the establishment of SJs. Furthermore, Bark is involved in epithelial cell adhesion during SJ maturation (Hildebrandt, 2015).
The initiation and establishment of SJ formation appear to be independent of bark activity. This conclusion is based on the observation that the ultrastructural analysis of developing wild-type and bark mutant SJs is similar during stage 15 and 16. At stage 15, single septae have already formed in tracheal and epithelial cells of wild-type and bark mutant embryos, even though the typical ladder-like septae structure of later stages is not yet established. At stage 16 electron-dense material appears in SJs of bark mutant embryos as found in wild-type embryos. Thus, the ultrastructural analysis suggests a normal formation of SJs in bark mutants until stage 16. In addition, integral SJ components are also normally localised in bark mutants at stage 15 suggesting correct assembly of the SJs. Furthermore, the exocytosis of Serpentine and Vermiform into the tracheal lumen mediated by SJs during stage 16 is not affected in bark mutants, suggesting normal SJ function independent of bark. Remarkable is the delay of tracheal dorsal trunk elongation in bark mutants. Embryos in which the initial SJ formation is affected as found in mega display dorsal trunk elongation about two hours earlier than observed in bark mutants. Thus, establishment of SJs seems to be normal in bark mutants, since dorsal trunk elongation is indicative for disrupted SJs. Taken together, these results suggest that Bark does not critically participate in the initial morphogenesis and functional properties of SJs (Hildebrandt, 2015).
In contrast, subsequent SJ maturation (stage 17) strongly depends on Bark. Rhodamine-dextran injection experiments revealed that the transepithelial barrier function of SJs is compromised in bark mutants. In such embryos the wild-type ladder-like SJ structure is disrupted, only rudimentary septae are formed and cell adhesion is impaired. This phenotype is distinct from the archetypal SJ phenotype observed in mutants of the SJ core complexes, e.g., in mega or Nrg mutant embryos. In these mutants the septae are either reduced in number or are absent, while cell adhesion seems not to be affected, i.e., the uniform spacing between the plasma membranes of adjacent epithelial cells is maintained. Individual bark mutant embryos develop a great diversity of septae ranging from no detectable septae in places where septae would normally form to rudimentary septae and up to wild-type like septae. Furthermore, such mutant embryos show an erratic spacing of the epithelial plasma membranes; in extreme cases the plasma membranes detach from each other resulting in gaps between the cells. Such cell adhesion defects of bark epithelial cells have also been observed in mutants of Gliotactin, a marker for tricellular junctions, but so far in no other mutant that affects SJs (Hildebrandt, 2015).
The phenotypic differences between bark and archetypal SJ lack-of-function mutant embryos are also observed in gain-of-function experiments. Overexpression of Bark does not interfere with normal development or the barrier function of SJs and rescues the bark mutant phenotype. In contrast, overexpression of other SJ components causes mis-localisation of the components and a disruption of the barrier function. Thus, it has been proposed that SJ components are functionally interdependent. The observation that the level of Bark is not critical for bark function supports the argument that bark mediates a distinct role during SJ maturation in late embryogenesis (Hildebrandt, 2015).
The detachment of lateral cell membranes, which occasionally deteriorate and form gaps between the epithelial cells of bark mutant embryos, indicates that Bark plays also a role in epithelial cell adhesion in addition to its function in SJ integrity. As Bark represents a large transmembrane protein, it may mediate the cell-cell adhesion through homophilic interactions. This possibility was tested, and putative homophilic Bark binding was analyzed in a cell aggregation assay, but no homophilic Bark binding was detected. However, homophilic binding in the embryo might not been detected, since posttranslational modifications may not occur in the cell culture system. Such modifications could include the attachment of sugar moieties via several potential glycosylation sites noted in the extracellular Bark domain and/or binding of sugar moieties to Bark via its lectin domain. Heterophilic Bark binding with an already identified SJ component is also not very likely, since the lack of such components has no effect on cell adhesion in the region of SJs. Thus, it is speculated that Bark mediates its cell adhesion function by homophilic binding, which depends on specific posttranslational modifications, or in conjunction with an unknown interaction partner (Hildebrandt, 2015).
The distinctive feature of SJs is that they must maintain their functional properties, in particular the control of the transepithelial barrier function, while cells rearrange during tissue morphogenesis. Thus, epithelial layers must be able to simultaneously alter cell-cell-contacts, shuffle SJ protein components and establish functional SJ structures, which create distinct fluid compartments. Given these features, the questions that come to mind are: What is the molecular basis of these distinct requirements and what is the role of Bark? (Hildebrandt, 2015).
The recent finding of a stable SJ multiprotein core complex is an important step in understanding SJ protein dynamics. The SJ core complex is preassembled intracellularly before its incorporation into the SJs at the plasma membrane. Interestingly, the core complex seems to be stable even in cells actively rearranging their contacts (Oshima, 2011). FRAP experiments indicate that the core complex is also stable in bark mutants, but the morphology and function of SJs are severely affected. The mis-localisation of SJ core components along the basolateral cell membrane in stage 17 bark mutants suggest that the SJ core complexes do not properly coalesce to assemble SJs in the apicolateral membrane region. This observation is consistent with previous binding studies suggesting a direct interaction of Bark with Mega or another SJ core protein (Jaspers, 2012). Thus, it is speculated that Bark provides a scaffold-like matrix serving as a platform for the SJ core complexes, which assemble into functional SJs. The potential Bark matrix is not necessary for establishment of SJs, but becomes essential during tissue morphogenesis, i.e., during SJ maturation to ensure that SJ core complexes remain well-ordered and able to sustain the epithelial barrier function. This role of Bark becomes particularly apparent during morphogenesis of the embryonic tracheal system, which is established by extensive cell shape changes and cell-cell rearrangements. Such a possible role would explain why a lack of Bark leads not only to cell adhesion defects and SJ failure, but also to a severely mis-shapen, convoluted tracheal system (Hildebrandt, 2015).
A scenario that would then allow for cell-cell rearrangement is that Bark is regulated in a way that it may detach from defined regions within the SJs and thereby reduce both cell adhesion and SJ integrity, which results in SJ core complex release. Such opened SJ sub-regions may in turn tolerate cell-cell rearrangements, while nearby sub-regions may still contain functional septae that establish the transepithelial barrier function. Consistent with this view is the observation that Bark localises to recycling endosomes within intracellular compartments. Thus, Bark may shuttle from sites of SJ breakdown to sites of SJ assembly via the recycling endosomal pathway. At the site of SJ assembly Bark might mediate dual functions in cell adhesion and providing anchor points for the SJ core complexes (Hildebrandt, 2015).
How permeability barrier function is maintained when epithelial cells divide is largely unknown. This study has investigated how the bicellular septate junctions (BSJs) and tricellular septate junctions (TSJs) are remodeled throughout completion of cytokinesis in Drosophila epithelia. Following cytokinetic ring constriction, the midbody assembles, matures within SJs, and is displaced basally in two phases. In a first slow phase, the neighboring cells remain connected to the dividing cells by means of SJ-containing membrane protrusions pointing to the maturing midbody. Fluorescence recovery after photobleaching (FRAP) experiments revealed that SJs within the membrane protrusions correspond to the old SJs that were present prior to cytokinesis. In contrast, new SJs are assembled below the adherens junctions and spread basally to build a new belt of SJs in a manner analogous to a conveyor belt. Loss of function of a core BSJ component, the Na+/K+-ATPase pump Nervana 2 subunit, revealed that the apical-to-basal spread of BSJs drives the basal displacement of the midbody. In contrast, loss of the TSJ protein Bark beetle indicated that remodeling of TSJs is rate limiting and slowed down midbody migration. In the second phase, once the belt of SJs is assembled, the basal displacement of the midbody is accelerated and ultimately leads to abscission. This last step is temporally uncoupled from the remodeling of SJs. It is proposed that cytokinesis in epithelia involves the coordinated polarized assembly and remodeling of SJs both in the dividing cell and its neighbors to ensure the maintenance of permeability barrier integrity in proliferative epithelia (Daniel, 2018).
How permeability barrier function is maintained when epithelial cells divide is largely unknown. This study has investigated how the bicellular septate junctions (BSJs) and tricellular septate junctions (TSJs) are remodeled throughout completion of cytokinesis in Drosophila epithelia. Following cytokinetic ring constriction, the midbody assembles, matures within SJs, and is displaced basally in two phases. In a first slow phase, the neighboring cells remain connected to the dividing cells by means of SJ-containing membrane protrusions pointing to the maturing midbody. Fluorescence recovery after photobleaching (FRAP) experiments revealed that SJs within the membrane protrusions correspond to the old SJs that were present prior to cytokinesis. In contrast, new SJs are assembled below the adherens junctions and spread basally to build a new belt of SJs in a manner analogous to a conveyor belt. Loss of function of a core BSJ component, the Na+/K+-ATPase pump Nervana 2 subunit, revealed that the apical-to-basal spread of BSJs drives the basal displacement of the midbody. In contrast, loss of the TSJ protein Bark beetle indicated that remodeling of TSJs is rate limiting and slowed down midbody migration. In the second phase, once the belt of SJs is assembled, the basal displacement of the midbody is accelerated and ultimately leads to abscission. This last step is temporally uncoupled from the remodeling of SJs. It is proposed that cytokinesis in epithelia involves the coordinated polarized assembly and remodeling of SJs both in the dividing cell and its neighbors to ensure the maintenance of permeability barrier integrity in proliferative epithelia (Daniel, 2018).
Epithelial growth requires the formation of cell-cell junctions and physical separation of daughters upon cell division.
This study has characterized a multicellular mechanism that coordinates completion of cytokinesis and de novo formation of SJ in both cuboidal and columnar epithelia: (1) following actomyosin ring constriction, the midbody forms just basal to the AJ within the SJ and matures into an intercellular bridge that is displaced basally. (2) Neighboring interphase cells maintain SJ contacts, the finger-like protrusions connecting to the maturing midbody of the dividing cells. At this position, the ménage à quatre formed contains pre-existing SJs and is stable. (3) Novel BSJs are assembled below the AJs and above the finger-like protrusions and spread toward the basal side, thereby driving the basal displacement of the finger-like protrusion plus midbody. Once the new BSJ is complete, the midbody is positioned outside the SJ belt and basal displacement accelerated. Overall, it is proposed that this multicellular process is to ensure the maintenance of the permeability barrier throughout cytokinesis (Daniel, 2018).
The data show that, as in embryos, newly synthesized SJ components assemble into stable protein complexes exhibiting a low rate of diffusion, endocytosis, and recycling. These stable complexes assemble below the AJs and then propagate toward the basal pole, in a manner analogous to a conveyor belt, both in interphase and during cytokinesis (Daniel, 2018).
Loss of the core SJ component Nrv2 prevents both assembly of stable BSJ complexes and basal displacement of the midbody. Thus, it is proposed that the apico-basal flux of newly assembled BSJs is the propelling force for the basal displacement of the finger-like protrusions connected to the midbody. The conveyor belt model first predicts that newly synthesized and/or slowly recycling SJ components are delivered apically in the vicinity of AJs. The model also predicts that SJ disassembly occurs at the basal rim of SJ belt. Future work will determine what regulates the polarized traffic of SJ components and the disassembly of SJs to control both the positioning and thickness of the SJ belt (Daniel, 2018).
This study sheds light on the remodeling of TSJs at cytokinesis. Bark and Gli are initially detected as puncta next to the midbody, prior to the pearl necklace distribution along the finger-like protrusions. These dotted structures could represent new TSJs, as, at this location, a new three-way contact is formed between the two daughter cells and a neighbor. However, FRAP analyses suggest that these punctae do not contain exchangeable components and are not moving laterally. Moreover, the appearing of Gli and Bark punctae in mitotic cells is faster (20 min post-anaphase) than the de novo assembly of TSJs in interphase cells (~80% recovery in 90 min). Analyses of the punctae by TEM will be necessary to test whether they are bona fide TSJs or components in the process of assembly. Alternatively, the old TSJs between dividing cells and neighbors prior to mitosis could undergo a change in distribution from a uniform distribution into clusters, giving rise to the pearl necklace. Redistribution could represent an intermediate step in TSJ disassembly and may explain the fast kinetics of assembly of pearl necklace structure. Future work using, for example, photoconvertible probes would help address this issue (Daniel, 2018).
Finally, loss of Bark resulted in acceleration of the midbody basal displacement. The de novo assembly of TSJs in the pearl necklace structure could be the rate-limiting step that imposes the slower speed of basal displacement. Alternatively, if pearl necklace represents the remodeling of prior TSJ connections with neighboring cells, TSJ disassembly may be the rate-limiting step.
Regardless, the data argue that the TSJ components present in the pearl necklace slow down the midbody basal displacement driven by the BSJ-mediated conveyor belt (Daniel, 2018).
This study reveals differences in midbody maturation and organization of the intercellular bridge between isolated and epithelial cells. In epithelial cells, the densely packed microtubule array that normally sits in the center of the intercellular bridge and the Flemming body were not observed. Instead, an intercellular bridge was found with a uniform density of microtubules that progressively disappear. The electron-dense material detected along the plasma membrane of the intercellular bridge could act as a rigid scaffold preventing membrane constriction. The SJ core components plus MyoII and Pnut at the interface made between the intercellular bridge and the finger-like protrusions might be part of the electron-dense material and therefore contribute to membrane rigidity. Alternatively, this scaffold could prevent tension release within the intercellular bridge, therefore preventing premature abscission (Daniel, 2018).
It was also found that abscission is asymmetric, with the bridge remnant internalized and then degraded in one of the daughter cells in a mode similar to the internalization and autophagy described for isolated cells. However, it cannot be excluded that abscission could also occur on both sides of the bridge, leading to its release followed by its recapture by endocytosis in some epithelial cells (Daniel, 2018).
Finally, whereas epithelial cells seem to be set for abscission about 40 min following the onset of anaphase, cytoplasmic isolation had not occurred 5 hr later. Photoconversion data furthermore suggest that cytoplasmic isolation takes place about one hour prior to one of the sisters entering mitosis, possibly at the mid to late G2 phase as reported for germline stem cells (Daniel, 2018).
Despite the opposite apico-basal positioning of permeability barrier relative to the mechanical barrier, a number of similarities are encountered in vertebrates' and invertebrates' epithelial cells. First, mechanical barrier transmission during epithelial cytokinesis is a multicellular process. Second, the permeability barrier is also maintained throughout epithelial cytokinesis. Third, the recruitment of tricellular junction components in close vicinity to the midbody supports the idea that polarized delivery, membrane composition, and topology at midbody are engineered for tricellular junction remodeling. Fourth, epithelial cytokinesis is polarized along the apico-basal axis, leading to the embedding of midbody en route to abscission within the permeability barrier both in vertebrates and in Drosophila. Based on these findings, it is anticipated that the coordination between permeability barrier transmission and completion of cytokinesis in Drosophila is conserved in vertebrates for maintenance of tissue integrity in proliferative epithelia (Daniel, 2018).
The blood-brain barrier is crucial for nervous system function. It is established early during development and stays intact during growth of the brain. In invertebrates, septate junctions are the occluding junctions of this barrier. This study used Drosophila to address how septate junctions grow during larval stages when brain size increases dramatically. Septate junctions are preassembled as long, highly folded strands during embryonic stages, connecting cell vertices. During subsequent cell growth, these corrugated strands are stretched out and stay intact during larval life with very little protein turnover. The G-protein coupled receptor Moody orchestrates the continuous organization of junctional strands in a process requiring F-actin. Consequently, in moody mutants, septate junction strands cannot properly stretch out during cell growth. To compensate for the loss of blood-brain barrier function, moody mutants form interdigitating cell-cell protrusions, resembling the evolutionary ancient barrier type found in primitive vertebrates or invertebrates such as cuttlefish (Babatz, 2018).
This study has dissected the mechanisms underlying growth of occluding septate junctions in non-dividing Drosophila cells. In primary and secondary epithelia, septate junction strands are mostly generated during embryonic stages. Septate junction protein complexes show a remarkable stability and persist over several days. In primary epithelia, newly synthesized linear and unbranched septate junction strands are added basally to the apically localized adherens junctions. In contrast, in secondary epithelial cells of the Drosophila blood-brain barrier, such newly synthesized septate junction strands are added on both sides of the pre-existing septate junctions. This process depends on the G-protein coupled receptor Moody and F-actin formation to integrate preformed vesicular septate junctions into existing strands. In addition, it was shown that moody mutants activate compensatory mechanisms to cope with the loss of junctional integrity. moody mutant cells activate expression of septate junction proteins and form an excess of membrane cell-cell interdigitations, which establish sufficient barrier function (Babatz, 2018).
Septate junctions are assembled by an astonishing number of proteins in an almost crystalline appearance with considerable stability. This study has demonstrated that septate junctions are indeed stable for several days and that most of the septate junction proteins are generated during early larval development. This is also confirmed by analyzing the developmental expression profile of all septate junction proteins. Moreover, FRAP data are consistent with the notion that septate junctions are arranged in unbranched, long lines connected to tricellular junctions (TCJs). Nevertheless, it is important to note that septate junction strands appear to mature during larval development, and, for example, the loosely associated protein Fas3 is constantly added to the growing septate junctions. Finally, septate junctions of the blood-brain barrier are more stable compared to those found in primary epithelia and use a different mechanism of new strand integration, suggesting alternative characteristics of septate junctions in different tissues (Babatz, 2018).
When epithelial cells grow or divide, the occluding septate junctions have to be adjusted to the changing cell geometry. During growth of the lateral cell membrane, this could imply an opening of existing strands and the subsequent insertion of new septate junction proteins. However, in freeze fracture studies of reorganizing tissues of Rhodnius or hydra, no evidence for such processes was detected. Consequently, it was postulated that existing septa can be redistributed in the plane of the membrane in cell contact areas, meaning septate junction strands stretch across the entire domain upon changes in cell geometry. This study used high-resolution imaging to show that indeed septate junctions are formed in a wavy manner during embryonic stages and unfold during the larval growth period (Babatz, 2018).
The corrugated organization of septate junctions appears to be a more general feature. Salivary gland cells have a conical shape, and the perimeter of the cell increases about two times from apical to basal. Similar to the growing subperineurial glial cells, septate junctions are formed as folded structures at the apical side and stretch out as they move to more basal positions. Likewise, during Drosophila oocyte development, follicle cells initially form corrugated adherens junctions that become straight when cells expand. A similar unfolding of tig (Babatz, 2018).
The unfolding of the bicellular septate junction strands requires force and possibly anchoring of strands to a fixed point. Such a fixed point could be a tricellular junction, which also allows force generation during cytokinesis. Classical freeze fracture studies as well as FRAP data suggest that bicellular occluding junctions are connected to tricellular junctions. This connection might be mediated by the tricellular junction proteins Bark beetle (Anakonda) and Gliotactin, which can also interact with bicellular septate junction proteins (Babatz, 2018).
During epithelial development, tissue convergence and extension correlate with adherens junction dynamics due to local actomyosin-based contractions. However, the initial corrugated-appearing septate junction strands suggest that the initial mechanical forces do not act on the establishment of septate junctions. Only when the cell grows and thus expands its lateral plasma membrane, the pre-established junctions are straightened. When subperineurial glial growth is suppressed, the initial corrugated shape of the septate junctions is not changed. When additional growth of the subperineurial glia is triggered by, for example, excessive neuroblast proliferation as seen in l(2)gl mutant animals, only rare ruptures of septate junction strands were noticed. In contrast, additional septate junction strands appear to be added to compensate for the increased cell growth. This suggests that the growing subperineurial glia senses barrier integrity, and any leakage results in the addition of new septate junction strands. This plasticity appears to depend on gene activity, since upon block of polyploidization in subperineurial glial cells, septate junctions are interrupted. The ability of subperineurial glial cells to react to changing barrier requirements can also be seen in moody mutants, which show an increase of NrxIV expression (Babatz, 2018).
The G-protein coupled receptor Moody is initially found along the entire plasma membrane, but after formation of septate junctions, its localization is restricted to the cell membrane facing the cortex glia with some enrichment at the cell-cell contact sites. Septate junctions form in the absence of Moody, but their arrangement in long, corrugated, and possibly anchored strands fails. Recently, special actin-rich structures have been observed along the lateral borders of the subperineurial glia that are regulated by Moody signaling. Gene dose experiments presented in this study indicate that Moody affects septate junction formation by the local activation of F-actin formation possibly to provide the physical force to integrate septate junction building blocks preformed in vesicular structures (Babatz, 2018).
As the brain grows in size, septate junctions cannot expand in moody mutants. In consequence, the blood-brain barrier established by the subperineurial glial cells becomes leaky, which triggers the expression of additional septate junction proteins, which, however, cannot be properly integrated into the existing strands. Remarkably, moody mutant subperineurial glia also form interdigitating membrane folds, which partially restore barrier integrity. A similar excess of cell interdigitation is induced upon reduction of NrxIV expression, specifically in the subperineurial glia. Interestingly, this type of barrier is also found in several other invertebrates and primitive vertebrates. Thus, moody mutants disclose the principles of an evolutionary ancient form of the blood-brain barrier. The efficient blood-brain barrier based on long, uninterrupted septate junction strands induced by Moody then allowed the evolution of more compact nervous systems, as found in Drosophila (Babatz, 2018).
Smooth septate junctions (sSJs) contribute to the epithelial barrier, which restricts leakage of solutes through the paracellular route of epithelial cells in the Drosophila midgut. Previous work identified three sSJ-associated membrane proteins, Ssk, Mesh, and Tsp2A and showed that these proteins were required for sSJ formation and intestinal barrier function in the larval midgut. This study investigated the roles of sSJs in the Drosophila adult midgut. Depletion of any of the sSJ-proteins from enterocytes resulted in remarkably shortened lifespan and intestinal barrier dysfunction in flies. Interestingly, the sSJ-protein-deficient flies showed intestinal hypertrophy accompanied by accumulation of morphologically abnormal enterocytes. The phenotype was associated with increased stem cell proliferation and activation of the MAP kinase and Jak-Stat pathways in stem cells. Loss of cytokines Unpaired2 and Unpaired3, which are involved in Jak-Stat pathway activation, reduced the intestinal hypertrophy, but not the increased stem cell proliferation, in flies lacking Mesh. The present findings suggest that SJs play a crucial role in maintaining tissue homeostasis through regulation of stem cell proliferation and enterocyte behavior in the Drosophila adult midgut (Izumi, 2019).
In the Drosophila midgut epithelium, the paracellular barrier is mediated by specialized cell-cell junctions known as sSJs. Previous studies revealed that three sSJ-associated membrane proteins, Ssk, Mesh and Tsp2A, are essential for the organization and function of sSJs. In this study, the sSJ proteins were depleted from ECs in the Drosophila adult midgut; they were also required for the barrier function in the adult midgut epithelium. Interestingly, the reduced expression of sSJ proteins in ECs led to remarkably shortened lifespan in adult flies, increased ISC proliferation and intestinal hypertrophy accompanied by accumulation of morphologically aberrant ECs in the midgut. The intestinal hypertrophy caused by mesh depletion was reduced by loss of upd2 and upd3 without profound suppression of ISC proliferation, without recovery of the shortened lifespan, and without recovery of the midgut barrier dysfunction. It also found that Tsp2A mutant clones promoted ISC proliferation in a non-cell-autonomous manner. Taken together, it is propose that sSJs play a crucial role in maintaining tissue homeostasis through regulation of ISC proliferation and EC behavior in the Drosophila adult midgut. The adult Drosophila intestine provides a powerful model to investigate the molecular mechanisms behind the emergence and progression of intestinal metaplasia and dysplasia, which are associated with gastrointestinal carcinogenesis in mammals. Given that Drosophila intestinal dysplasia is associated with over-proliferation of ISCs and their abnormal differentiation, the intestinal hypertrophy observed in the present study should be categorized as a typical dysplasia in the Drosophila intestine. In this study, depletion of sSJ proteins from the ECs was mostly performed through RNAi, which may raise a concern about off-target effects. Nevertheless, it is safe to say that the midgut phenotypes were derived from specific effects of sSJ protein depletion because the essentially same phenotypes were observed in the RNAi lines for different sSJ proteins and additional RNAi lines for mesh and Tsp2A (Izumi, 2019).
Based on these observations, the following scenario is hypothesized for the hypertrophy generation in the sSJ-protein-deficient midgut. First, depletion of sSJ proteins from enterocytes (ECs) leads to disruption of sSJs in the midgut. Second, the impaired midgut barrier function caused by disruption of sSJs results in leakage of harmful substances from the intestinal lumen, thereby inducing the expression of cytokines and growth factors, such as Upd and EGF ligands, in the midgut. Alternatively, disruption of sSJs causes direct activation of a particular signaling pathway that induces expression of cytokines and growth factors by ECs. Third, proliferation of ISCs is promoted by activation of the Jak-Stat and Ras-MAPK pathways. Fourth, EBs produced by the asymmetric division of ISCs differentiate into ECs with impaired sSJs in response to cytokines such as Upd2 and/or Upd3. Consistent with this scenario, increased mRNA expression of upd3 in the ssk- and Tsp2A-deficient midgut has been reported very recently (Salazar, 2018; Xu, 2019). Finally, the ECs fail to integrate into the epithelial layer and thus become stratified in the midgut lumen to generate hypertrophy. Interestingly, loss of upd2 and upd3 reduced the intestinal hypertrophy caused by depletion of mesh, but not the increased ISC proliferation. These findings imply that Upd2 and/or Upd3 preferentially promote enteroblast (EB) differentiation rather than intestinal stem cell (ISC) proliferation. Considering that Upd-Jak-Stat signaling is required for both ISC proliferation and EB differentiation, Upd2 and/or Upd3 may predominantly promote EB differentiation and accumulation of ECs, while other cytokines such as Upd1 and/or EGF ligands may activate ISC proliferation in the sSJ-disrupted midgut. Meanwhile, Upd-Jak-Stat signaling-mediated ISC proliferation also occurs during experimental induction of apoptosis of ECs. Given that apoptosis of ECs is thought to cause the disruption of epithelial integrity followed by midgut barrier dysfunction, it may induce ISC proliferation by the same mechanism as that observed in the sSJ-protein-deficient midgut. Thus, a possibility cannot be excluded that depletion of sSJ proteins initially causes apoptosis of ECs and thereby leads to increased ISC proliferation. This possibility needs to be examined in future studies. Interestingly, the ISC proliferation induced by the loss of sSJ proteins was non-cell-autonomous. Mechanistically, disruption of the intestinal barrier function caused by impaired sSJs may permit the leakage of particular substances from the midgut lumen, which would induce particular cells to secrete cytokines and growth factors, such as Upd and EGF ligands, and stimulate ISC proliferation. Alternatively, sSJs or sSJ-associated proteins may be directly involved in the secretion of cytokines and growth factors through the regulation of intracellular signaling in the ECs (Izumi, 2019).
In this study,abnormal morphology and aberrant F-actin and Dlg distributions were observ ed in ssk-, mesh- and Tsp2A-RNAi ECs. Consistent with these results, Chen (2018) recently reported that loss of mesh and Tsp2A in clones causes defects in polarization and integration of ECs in the adult midgut. In contrast, no remarkable defects in the organization and polarity of ECs were observed in the ssk-, mesh- and Tsp2A-mutant midgut in first-instar larvae, suggesting that sSJ proteins are not required for establishment of the initial epithelial apical-basal polarity. This discrepancy may be explained by the marked difference between the larval and adult midguts: ECs in the larval midgut are postmitotic, while those in the adult midgut are capable of regeneration by the stem cell system. In the sSJ protein-deficient adult midgut, activated proliferation of ISCs generates excessive ECs. These ECs may lack sufficient cell-cell adhesion, because of impaired sSJs, fail to become integrated into the epithelial layer and detach from the basement membrane, leading to loss of normal polarity. Because sSJs seem to be the sole continuous intercellular contacts between adjacent epithelial cells in the midgut, it is reasonable to speculate that sSJ-disrupted ECs have a reduced ability to adhere to other cells (Izumi, 2019).
A recent study revealed that depletion of the tricellular junction protein Gliotactin from ECs leads to epithelial barrier dysfunction, increased ISC proliferation and blockade of differentiation in the midgut of young adult flies (Resnik-Docampo, 2017). In contrast to the findings after depletion of sSJ proteins presented in this study, the gliotactin-deficient flies did not appear to exhibit intestinal hypertrophy accompanied by accumulation of ECs throughout the midgut. Furthermore, the lifespan of gliotactin-deficient flies was found to be longer than that found for sSJ-protein-deficient flies. The difference in phenotypes found between the Resnik-Docampo (2017) study and the present study may reflect the difference in the degrees of sSJ deficiency - disruption of entire bicellular sSJs or tricellular sSJs only. Aging has also been reported to be correlated with barrier dysfunction, increased ISC proliferation and accumulation of aberrant cells in the adult midgut. The hypertrophy formation in the sSJ-disrupted midgut accompanied by increased ISC proliferation and accumulation of aberrant ECs raise the possibility that disruption of sSJs is the primary cause of the alterations in the midgut epithelium with aging (Izumi, 2019).
During the preparation of this manuscript, two groups published interesting phenotypes of the sSJ-protein-deficient adult midgut in Drosophila that are highly related to the present study. Salazar (2018) reported that reduced expression of ssk in ECs leads to gut barrier dysfunction, altered gut morphology, increased stem cell proliferation, dysbiosis and reduced lifespan. That study also showed that upregulation of Ssk in the midgut protects flies against microbial translocation, limits dysbiosis and prolongs lifespan. Meanwhile, Xu (2019) reported that depletion of Tsp2A from ISCs and EBs causes accumulation of ISCs and EBs and a swollen midgut with multilayered epithelium, similar to the current observations. They also showed that knockdown of ssk and mesh in ISCs and EBs results in accumulation of ISCs and EBs. Importantly, that study demonstrated that Tsp2A depletion from ISCs and EBs causes excessive aPKC-Yki-JAK-Stat activity and leads to increased stem cell proliferation in the midgut. That study further showed that Tsp2A is involved in endocytic degradation of aPKC, which antagonizes the Hippo pathway. Those results strongly suggest that sSJs are directly involved in the regulation of intracellular signaling for ISC proliferation. In that study, Tsp2A knockdown in ISCs and EBs caused no defects in the midgut barrier function, in contrast to what is shown in the present study. This discrepancy may be due to differences in the GAL4 drivers used in each study or the conditions for the barrier integrity assay (Smurf assay). In addition, Xu (2019) mentioned that MARCM clones generated from ISCs expressing Tsp2A-RNAi grow much larger than control clones, while this study found no remarkable size difference between Tsp2A-mutant clones and control clones. Such discrepancies need to be reconciled by future investigations. To further clarify the mechanistic details for the role of sSJs in stem cell proliferation, it will be interesting to analyze the effects of sSJ protein depletion on the behavior of adult Malpighian tubules, which also have sSJs and a stem cell system (Izumi, 2019).
This study has demonstrated that sSJs play a crucial role in maintaining tissue homeostasis through regulation of ISC proliferation and EC behavior in the Drosophila adult midgut. The sequential identification of the sSJ proteins Ssk, Mesh and Tsp2A has provided a Drosophila model system that can be used to elucidate the roles of the intestinal barrier function by experimental dysfunction of sSJs in the midgut. However, as described in this study, simple depletion of sSJ proteins throughout the adult midgut causes phenotypes that are too drastic, involving not only disruption of the intestinal barrier function but also intestinal dysplasia and subsequent lethality. To investigate the systemic effects of intestinal barrier impairment throughout the life course of Drosophila, more modest depletions of sSJ proteins are needed for future studies (Izumi, 2019).
During development, many epithelia are formed by a mesenchymal-epithelial transition (MET). This
study examined the major stages and underlying mechanisms of MET during blood-brain barrier (BBB)
formation in Drosophila. Contact with the basal lamina was shown to be essential for the growth of
the barrier-forming subperineurial glia (SPG). Septate junctions (SJs), which provide insulation of the
paracellular space, are not required for MET, but are necessary for the establishment of polarized
SPG membrane compartments. In vivo time-lapse imaging reveals that the Moody GPCR signalling pathway regulates SPG cell growth and shape,
with different levels of signalling causing distinct phenotypes. Timely, well-coordinated SPG growth
is essential for the uniform insertion of SJs and thus the insulating function of the barrier. This
is the first dynamic in vivo analysis of all stages in the formation of a secondary epithelium and
of the key role trimeric G protein signalling plays in this important morphogenetic process (Schwabe, 2017).
This study of Drosophila BBB development represents the first dynamic in vivo study of MET and secondary epithelium formation. The data shed particular light on the roles of the basal lamina and of the insulating SJs, as well as on the function of GPCR signaling in this important morphogenetic process.
Once SPG reach the CNS surface, contact with the basal lamina is essential for the extensive growth of the SPG during epithelium formation. Previous in vitro studies have shown that adhesion to basal lamina components is necessary for cell spreading and proliferation, however this study is the first to demonstrate in vivo that attachment to the basal lamina is essential for non-proliferative cell growth and ensheathment. Attachment to the ECM occurs primarily through focal adhesions and integrins, which in turn can activate MAPK signaling, triggering cell proliferation and growth. In addition, adhesion to the ECM has been shown to provide traction, which facilitates cell spreading. Contact to the ECM may thus provide the SPG with both growth signals and attachment sites. Being highly expressed on the basal lamina facing side of SPG, Dystroglycan (Dg) is an excellent candidate for mediating ECM attachment. However, zygotic mutants of Dg show no BBB defects and germline clones could not be analyzed due to Dg's role in oogenesis (Schwabe, 2017).
Beyond supporting SPG growth, contact with the basal lamina likely provides an important cue for polarizing the cells, as judged by their strong enrichment of Dg at the basal lamina facing (basal) membrane compartment. Previous studies have shown that Dg and its ligand Pcan/Trol are required for the establishment of polarity in follicle cells. However, when the basal lamina and thus its ligand Pcan are depleted, Dg is still expressed and polarized in the SPG, suggesting that glial polarity can be supported by the residual basal lamina or that additional polarizing signals exist (Schwabe, 2017).
Once SJs have formed, the GPCR Moody and the Mdr65 transporter are asymmetrically distributed within the SPG, further demonstrating that these cells possess distinct apical and basal membrane compartments. This polarized distribution is coincident with and dependent on the presence of SJs, demonstrating for the first time that SJs serve a function in cell polarity. By acting as a fence and preventing diffusion of membrane proteins across the lateral compartment, the SJs maintain asymmetric protein distributions, which could result from polarized exocytosis or endocytosis. Intriguingly, a separate study has identified PKA as a crucial antagonistic effector of Moody signaling. PKA has been shown to regulate polarized exocytosis at the trans-Golgi network in different types of epithelia. Apical-basal polarity plays an important morphogenetic role in the continued growth of the SPG epithelium during larval stages and in the function of the BBB (Schwabe, 2017).
Signaling by the GPCR Moody plays a critical role both in regulating the growth of individual SPG and in synchronizing this process across the entire SPG cell population. In Moody pathway mutants, glial growth behavior is more erratic, and more variable between cells. This increased variability of glial cell shape, size, and growth causes a significant delay of epithelial closure of up to 1.5 hrs. This delay is not caused by an earlier delay in glial migration or by a delay in SJ formation (Schwabe, 2017).
The detailed dynamic analysis reveals that, in moody and loco mutants, the spatio-temporal coordination of cell spreading is impaired. Spreading cells, like other motile cells, show fluctuating exploratory motions of the leading edge visible as cycles of protrusion and retraction. This complex process can be broken down into discrete steps: actin protrusion of the leading edge, adhesion to the ECM, and myosin-driven contraction against adhesions. Time-lapse recordings indicate that Moody signaling has its most pronounced effect on the stabilization of protrusions, as evidenced by an increase in the ratio of retractions to extensions, and the marked shift of cell contours over time. The destabilization of protrusions might be due to weaker integrin-mediated interaction of focal adhesions with the ECM, but also due to impaired stress-mediated maturation of focal adhesions. The fact that both under- and overactivity of the Moody pathway impair protrusion stabilization may be due to the feedback between actin-myosin and focal adhesion, which also causes the well-known biphasic response of migration speed to adhesion strength of migrating cells. While the loss of moody has no significant effects on the other parameters that were measured, the loss of loco also reduces the frequency and size of protrusions, suggesting that actin polymerization may be specifically affected by increased GPCR signaling activity. Cumulatively, these impairments in protrusion/retraction behavior lead to retarded, non-isometric growth of SPG and to the irregular cell shapes observed in moody and loco mutants (Schwabe, 2017).
Interestingly, PKA, Rho1 and MLCK have been identified as important downstream effectors of Moody signaling. All three factors are well known to control actin-myosin contraction; Rho1 and MLCK as positive regulators, PKA as a negative regulator. More recently, Rho1 activity has been shown to also drive actin polymerization at the leading edge, and a PKA-RhoGDI-Rho1 regulators feedback loop has been suggested to act as a pacemaker of protrusion- retraction cycles (Schwabe, 2017).
The role of Moody pathway signaling in directed and well-coordinated cell growth is strikingly similar to the function of trimeric G protein signaling in other contexts. In Dictyostelium, G protein signaling is essential for directed cell migration: When all G protein signaling is abolished, cells are still mobile and actively generate protrusions, however these protrusions form in random directions, with the result that the cells lose their directionality. During gastrulation in Drosophila, signaling by the G12 ortholog Concertina and the putative GPCR ligand Folded Gastrulation synchronizes apical actin-myosin constrictions of mesodermal precursor cells, and thereby effects their concerted invagination. Thus, a major role of G protein signaling during development may be to modulate basic cellular behaviors such as cell growth, protrusion, or contraction, and reduce variability within cells and between neighboring cells, with the goal of generating uniform patterns and behaviors (Schwabe, 2017).
Synchronized growth behavior of SPG is not only important for rapid epithelial closure but, ultimately, also for generating an evenly sealed BBB. All the evidence supports the notion that the defects in SJ organization that are responsible for the BBB failure are a secondary consequence of the morphogenetic function of the GPCR pathway. Cell contacts precede and are necessary for SJ formation, and the growth of cell contacts and SJ accumulation are strongly correlated. Delayed and more erratic cell- cell contact formation, as is the case in Moody pathway mutants, is likely to result in uneven circumferential distribution of SJ material later on; conversely, the timing of SJ formation per se is not affected by the pathway, arguing against a direct effect. Since the insulating function of SJs depends on their length, a decrease in the length in some local areas will result in insulation defects. Moreover, since SJs are known to form very static complexes, any irregularity in SJ distribution may be retained for long periods of time (Schwabe, 2017).
Although under- and overactivity of the Moody pathway lead to globally similar outcomes, impaired epithelium formation and failure of BBB insulation, the data point to subtly different subcellular effects of the two types of pathway modulation. During MET, loco mutants (which this study confirms as inducing pathway overactivity) show predominantly retarded growth, presumably as a result of curtailed protrusive activity, while moody mutants show severe fluctuation and variability in growth. It will be very interesting to investigate these distinct outcomes of Moody pathway misregulation in greater detail (Schwabe, 2017).
Stem cells can be controlled by their local microenvironment, known as the stem cell niche. The Drosophila testes contain a morphologically distinct niche called the hub, composed of a cluster of between 8 and 20 cells known as hub cells, which contact and regulate germline stem cells (GSCs) and somatic cyst stem cells (CySCs). Both hub cells and CySCs originate from somatic gonadal precursor cells during embryogenesis, but whereas hub cells, once specified, cease all mitotic activity, CySCs remain mitotic into adulthood. Cyst cells, derived from the CySCs, first encapsulate the germline and then, using occluding junctions, form an isolating permeability barrier. This barrier promotes germline differentiation by excluding niche-derived stem cell maintenance factors. This study shows that the somatic permeability barrier is also required to regulate stem cell niche homeostasis. Loss of occluding junction components in the somatic cells results in hub overgrowth. Enlarged hubs are active and recruit more GSCs and CySCs to the niche. Surprisingly, hub growth results from depletion of occluding junction components in cyst cells, not from depletion in the hub cells themselves. Moreover, hub growth is caused by incorporation of cells that previously express markers for cyst cells and not by hub cell proliferation. Importantly, depletion of occluding junctions disrupts Notch and mitogen-activated protein kinase (MAPK) signaling, and hub overgrowth defects are partially rescued by modulation of either signaling pathway. Overall, these data show that occluding junctions shape the signaling environment between the soma and the germline in order to maintain niche homeostasis (Fairchild, 2016).
The hub regulates stem cell behavior in multiple ways. First, the hub physically anchors the stem cells by forming an adhesive contact with both germline stem cells (GSCs) and cyst stem cells (CySCs). The hub thus provides a physical cue that orients centrosomes such that stem cells predominantly divide asymmetrically, perpendicular to the hub. Following asymmetric stem cell division, one daughter cell remains attached to the hub and retains stem cell identity while the other is displaced from the hub and differentiates. Second, hub cells produce signals, including the STAT ligand Unpaired-1 (Upd), Hedgehog (Hh), and the BMP ligands Decapentaplegic (Dpp) and Glass-bottomed boat (Gbb), that signal to the adjacent stem cells to maintain their identity. As germ cells leave the stem cell niche, two somatic cyst cells surround and encapsulate them to form a spermatocyst. As spermatocysts move from the apical to the basal end of the testis, both somatic cyst cells and germ cells undergo a coordinated program of differentiation. Previous studies have shown that differentiation of encapsulated germ cells requires their isolation behind a somatic occluding junction-based permeability barrier. Specifically, a role was identified for septate junctions, which are functionally equivalent to vertebrate tight junctions, in establishing and maintaining a permeability barrier for each individual spermatocyst (Fairchild, 2016).
During analysis of septate junction protein localization, it was observed that some, notably Coracle, were expressed in both the hub and the differentiating cyst cells. Moreover, knockdown of septate junction components in the somatic cells of the gonad resulted in enlarged hubs. Based on these results, the role of septate junction components in regulating the number of hub cells was explored in detail. To this end, RNAi was used to knock down the expression of the core septate junction components Neurexin-IV (Nrx-IV) and Coracle (Cora) in both the hub and cyst cell populations and the number of hub cells counted in testes from newly eclosed and 7-day-old adults. RNAi was expressed using three tissue-specific drivers: upd-Gal4, expressed in hub cells; tj-Gal4, expressed weakly in hub cells and strongly in both CySCs and early differentiating cyst cells; and eyaA3-Gal4, expressed strongly in all differentiating cyst cells, weakly in CySCs, and at negligible levels in the hub. To visualize hub cells, multiple established hub markers, including upd-Gal4, upd-lacZ, Fasciclin-III (FasIII), and DN-cadherin (DNcad) were used. Surprisingly, it was found that knockdown of Nrx-IV or cora driven by upd-Gal4 gave rise to normal hubs. In comparison, knockdown of Nrx-IV or cora using tj-Gal4 or eyaA3-Gal4 led to large increases in the number of the hub cells. Hub growth was not uniform and varied between testes, but median hub cells numbers in Nrx-IV and cora knockdown testes grew by 30% and 55%, respectively, between 1 and 7 days post-eclosion (DPEs). However, in extreme cases, hubs contained up to five times the number of cells found in age-matched control testes. This result was confirmed using a series of controls that discounted the possibility that hub overgrowth was due to temperature or leaky expression of the RNAi lines. These results suggested that hub growth occurred as a result of knockdown of septate junction proteins in cyst cells rather than the hub. This was further supported using another somatic driver that is not thought to be expressed in the hub, c587-Gal4. However, analysis of c587-Gal4 was complicated by the fact this driver severely impacted fly viability when combined with Nrx-IV or cora RNAi lines (Fairchild, 2016).
Intriguingly, hub growth largely occurred after adult flies eclosed and not in earlier developmental stages. For example, when the driver eyaA3-Gal4 was used to knock down Nrx-IV or cora, hubs from 1-day-old adults were not larger than controls, but hubs from 7-day-old adults were significantly larger. Moreover, overgrowth phenotypes were recapitulated when temperature-sensitive Gal80 was used to delay induction of eyaA3-Gal4-mediated Nrx-IV knockdown until after eclosion. Hub growth manifested both in a higher mean number of hub cells per testis and by a shift in the distribution of hub cells per testis upward, toward larger hubs sizes. This distribution suggested a gradual, stochastic process of hub growth, resulting in a population of testes containing a range of hub sizes. These results reveal progressive hub growth in adults upon knockdown of septate junction components in cyst cells and suggest that this growth is not driven by events occurring in the hub itself but rather by events occurring in cyst cells (Fairchild, 2016).
Niche size has been shown in various tissues, including vertebrate hematopoietic stem cells and somatic stem cells in the fly ovary, to be an important factor in regulating the number of stem cells that the niche can support. In the fly testes, it has been shown that mutants having few hub cells could nonetheless maintain a large population of GSCs. To determine how a larger hub, containing more cells, affects niche function, the number of GSCs and CySCs was monitored after knockdown of septate junction components in cyst cells. Overall, the average number of germ cells contacting the hub grew substantially in Nrx-IV or cora knockdown testes between 1 and 7 DPEs. To confirm that the germ cells contacting the hub were indeed GSCs, spectrosome morphology was studied, and it was found to be to be consistent with that seen in wild-type GSCs. Moreover, in individual testes, there was a positive correlation between the number of hub cells and the number of GSCs. Similar growth was also observed in the number of CySCs, defined as cyst cells expressing Zfh1, but not the hub cell marker DNcad. Control testes (from tj-Gal4 x w1118 progeny) had on average 34.3 CySCs whereas Nrx-IV and cora knockdown testes had 53.4 and 50.2 CySCs, respectively. These results show the importance of maintaining a stable stem cell niche size, as enlarged hubs were active and could support additional stem cells, which may result in the excess production of both germ cells and cyst cells (Fairchild, 2016).
Next, it was of interest to determine the mechanism driving hub growth in adult flies upon knockdown of septate junction components in cyst cells. One possible mechanism that can explain this growth is hub cell proliferation. However, a defining feature of hub cells is that they are not mitotically active. Consistent with this, a large number of testes were stained for the mitotic marker phospho-histone H3 (pH3), and cells were never observed where upd-LacZ and pH3 were detected simultaneously. These results argue that division of hub cells is unlikely to explain hub growth in the adult Nrx-IV and cora knockdown testes. To determine the origin of the extra hub cells, the lineage of eyaA3-expressing cells was traced using G-TRACE (Evans, 2009). eyaA3 was chosed as both the expression pattern of septate junctions, and Nrx-IV or cora knockdown results suggested that hub growth involved differentiating eyaA3-positive cyst cells. The eyaA3-Gal4 driver utilizes a promoter region of the eya gene, which is required for somatic cyst cell differentiation and is expressed at very low levels in CySCs and at high levels in differentiating cyst cells. Using G-TRACE allows identification of both cells that previously expressed eyaA3-Gal4 (marked with GFP) and cells currently expressing eyaA3-Gal4 (marked with a red fluorescent protein [RFP]); additionally, the hub was identified using expression of upd-LacZ and FasIII. In control experiments at both 1 and 7 DPEs, few GFP-positive cells were observed in the hub. Those few GFP-positive cells could be explained by the transient expression of eya in the embryonic somatic gonadal precursor cells that form both hub and cyst cell lineages or extremely low levels of expression in adult hub cells. When G-TRACE was combined with knockdown of Nrx-IV, the results were strikingly different. Initially, 1 DPE, hubs were only slightly larger than controls and few GFP-positive hub cells were observed. In comparison, 7-DPE hubs contained on average more than twice as many cells compared to controls. Importantly, hub growth in Nrx-IV knockdowns was largely attributable to the incorporation of GFP-positive cells. Moreover, a population of upd-LacZ-labeled cells that were also RFP-positive was observed consistent with ongoing or recent expression of eyaA3-Gal4 in hub cells. These results suggest that knockdown of Nrx-IV or cora leads cyst cells to adopt hallmarks of hub cell identity and express hub-cell-specific genes (Fairchild, 2016).
To learn more about the differentiation state of non-endogenous hub cells in Nrx-IV and cora knockdown testes, various markers were used to label the stem cell niche. This analysis showed normal expression of hub cell markers, such as Upd, FasIII, DNcad, as well as Hedgehog (hh-LacZ), Armadillo (Arm), and DE-Cadherin (DEcad). It was asked how cells that were previously, and in some instances were still, eyaA3 positive could express multiple hub-cell fate markers. To answer this question, the signaling mechanisms that determine hub fate were investigated in Nrx-IV and cora knockdown testes. Hub growth phenotypes similar to those produced by Nrx-IV and cora knockdown have been described previously, most notably in agametic testes that lack germ cells, suggesting that the germline regulates the formation of hub cells. One specific germline-derived signal shown to regulate hub fate is the epidermal growth factor (EGF) ligand Spitz. In embryonic testes, somatic cells express the EGF receptor (EGFR), which, when activated, represses hub formation. EGFR-induced mitogen-activated protein kinase (MAPK) signaling, visualized by staining for di-phosphorylated-ERK (dpERK), was active in CySCs and spermatogonial-stage cyst cells. Quantifying dpERK-staining intensity in cyst cell nuclei showed that MAPK activity was lower in CySCs following knockdown of Nrx-IV or cora, suggesting reduced EGFR signaling. Moreover, the effect of Nrx-IV or cora knockdown on MAPK signaling was not restricted to CySCs, as lower dpERK staining was observed at a distance from the hub. To see whether disruption of EGFR signaling could underlie hub defects in Nrx-IV and cora knockdown testes, attempts were made to rescue these phenotypes by increasing EGF signaling. When a constitutively activated EGF receptor (EGFR-CA) was co-expressed in cyst cells along with Nrx-IV RNAi, hub growth was attenuated, resulting in a reduction in the average number of hub cells compared to expressing only Nrx-IV RNAi. Similar results were also observed in the growth of the GSC population, suggesting that reduced EGFR activation in cyst cells contributes to the overall growth of the stem cell niche caused by the knockdown of Nrx-IV or cora. Surprisingly, analysis of testes with loss-of-function mutations in the EGFR/MAPK pathway reveals different phenotypes than those observed: encapsulation is disrupted and CySCs are lost, but hub size is largely unaffected. This result shows that the partial reduction in EGFR/MAPK signaling seen in Nrx-IV and cora knockdown testes results in distinct phenotypes and highlights the complexity of EGFR signaling in the fly testis (Fairchild, 2016).
Another pathway that is documented to regulate hub cell fate is Notch signaling. Notch plays important roles in hub specification in embryos. The Notch ligand Delta is produced by the embryonic endoderm and acts to promote hub cell specification in the anterior-most somatic gonadal precursor cells. Whereas it has been suggested that Notch acts in the adult to regulate hub fate, such a role has not been clearly demonstrated. A reporter for the Notch ligand Delta (Dl-lacZ) was observed in hub cells of both control and Nrx-IV knockdown testes. Intriguingly, reducing Notch signaling efficiently rescued the hub overgrowth seen in adult Nrx-IV knockdown testes. When a dominant-negative Notch (Notch-DN) was co-expressed in the somatic cells, along with Nrx-IV RNAi, the growth of the hub was reduced compared to the expression of Nrx-IV RNAi alone. Growth in the GSC population was not significantly reduced by co-expression of Notch-DN, suggesting that the Notch pathways may modulate hub growth through a different mechanism compared to the EGFR pathway. Because Notch is well established to regulate hub growth in the embryo, temperature-sensitive Gal80 was used to delay expression of Notch-DN and confirm that the reduction in hub cells was due to disruption of post-embryonic Notch signaling. These results suggest that Notch signaling in cyst cells may contribute to the hub overgrowth phenotypes caused by septate junction knockdown in the adult testes (Fairchild, 2016).
In addition to Notch and EGFR, other signaling pathways that regulate hub size may contribute to the hub growth seen upon somatic knockdown of septate junction components. For example, it has been previously shown that the range of BMP signaling is expanded following Nrx-IV or cora knockdown in cyst cells. Constitutive activation of BMP signaling in the germline was shown to increase the size of the hub and the number of GSCs. Additionally, the relative expression levels of the genes drm, lines, and bowl regulate hub size in the adult. In particular, it is known that lines maintains a “steady state” in the testes by repressing expression of a subset of hub genes in the cyst cell population. Unlike lines mutants, Nrx-IV or cora knockdowns generally lack ectopic hubs. This may reflect the more gradual hub growth seen in septate junction knockdowns or, alternatively, highlight key mechanistic differences in how hub growth is achieved in each respective genetic background. The current work is consistent with the model whereby occluding junctions are required for proper soma-germline signaling in the fly testes. This signaling maintains stem cell niche homeostasis by preventing somatic cyst cells from adopting hub cell fate, which would lead to niche overgrowth. It is well established that, in embryonic testes, hub fate is both positively and negatively regulated by signals from the germline and the endoderm.The results, and recent findings about the genes lines and traffic jam, argue that, in the adult testes, hub fate is actively repressed in the cyst cell lineage. Failure to repress hub fate allows cyst cells to exhibit features of hub cells and act as a functional stem cell niche. However, these cyst-cell-derived hub cells are distinct from the true endogenous hub cells in that they show non-hub-cell features, including expression of the differentiating cyst cell markers eyaA3-Gal4 and β3-tubulin. The data suggest that, following disruption of septate junctions proteins, the signaling environment surrounding the somatic cells is altered such that cyst cells gradually begin expressing hub cell markers (Fairchild, 2016).
One major outstanding question is how eyaA3-Gal4-expressing cyst cells become incorporated into the endogenous hub. Previously, it was shown that a septate-junction-mediated permeability barrier forms by the four-cell spermatogonial-stage spermatocyst. The hub growth phenotypes induced by Nrx-IV and cora knockdowns may occur due to defects in cell-cell signaling, possibly involving EGFR and Notch, that manifest in these later spermatocysts. However, this model requires an explanation for how these cyst cells translocate back to and join the hub. Alternatively, signaling defects in these later spermatocysts are somehow instructing earlier cyst cells, such as CySCs, to join the hub. It is easier to envisage the latter model, as early cyst cells are spatially much closer to the hub, but the sequence of signaling events in such a case will be complex and require further elucidation. The ability of CySCs to convert into hub cells in wild-type testes is a controversial subject. However, the incorporation of CySCs into the hub does not necessitate complete conversion into hub cells but could rather involve simple de-repression or activation of genes that confer hub cell function, including regulators of the cell-cycle- and hub-cell-specific signaling ligands. Notably, the transition between CySC and hub cell fate is linked to the cell cycle (Fairchild, 2016).
Why would loss of the septate-junction-mediated somatic permeability barrier result in disruption of signaling between the soma and germline? There are many possible answers, but it is possible to speculate about two such mechanisms that explain hub overgrowth. One possibility is that germline differentiation, which is dependent on the permeability barrier, is required for the release of signals that maintain stem cell niche homeostasis. Another possibility is that the permeability barrier locally concentrates germline-derived signals that repress hub cell fate by trapping them in the luminal space between the encapsulating cyst cells and the germline. The latter scenario could explain the observation that activated EGFR signaling partially rescues hub overgrowth. In this model, septate junctions allow localized buildup of the EGF ligand Spitz, ensuring that sufficient signaling is available to repress hub fate. It is more difficult to draw strong conclusions about how Notch signaling is altered when septate junctions are disrupted, particularly as the Notch ligand Delta appears restricted to the hub. Overall, an unexpected role was found for an occluding-junction-based permeability barrier in mediating stem cell niche homeostasis. This work highlights how the architecture of the stem cell niche system in the fly testes, which is highly regular and contains a reproducible number of stem cells and niche cells, is in fact the result of an active and dynamic signaling environment (Fairchild, 2016).
Cbl-associated protein (CAP) localizes to focal adhesions and associates with numerous cytoskeletal proteins; however, its physiological roles remain unknown. This study demonstrates that Drosophila CAP regulates the organization of two actin-rich structures in Drosophila: muscle attachment sites (MASs), which connect somatic muscles to the body wall; and scolopale cells, which form an integral component of the fly chordotonal organs and mediate mechanosensation. Drosophila CAP mutants exhibit aberrant junctional invaginations and perturbation of the cytoskeletal organization at the MAS. CAP depletion also results in collapse of scolopale cells within chordotonal organs, leading to deficits in larval vibration sensation and adult hearing. This study investigated the roles of different CAP protein domains in its recruitment to, and function at, various muscle subcellular compartments. Depletion of the CAP-interacting protein Vinculin results in a marked reduction in CAP levels at MASs, and vinculin mutants partially phenocopy Drosophila CAP mutants. These results show that CAP regulates junctional membrane and cytoskeletal organization at the membrane-cytoskeletal interface of stretch-sensitive structures, and they implicate integrin signaling through a CAP/Vinculin protein complex in stretch-sensitive organ assembly and function (Bharadwaj, 2013).
Interactions between cells and the extracellular matrix (ECM) are crucial for many biological processes. These include cell migration, directed process outgrowth, basement membrane-mediated support of tissues and maintenance of cell shape. Communication between cells and ECM proteins often occurs through the action of α/β-integrin heterodimers, a receptor complex that forms adhesive contacts, including focal adhesions, hemiadherens junctions, costameres and myotendinous junctions. In response to extracellular forces, focal adhesions undergo structural changes and initiate signaling events that allow adaptation to tensile stress. Vinculin is thought to be the primary force sensor in the integrin complex, mediating homeostatic adaptation to external forces (Bharadwaj, 2013 and references therein).
Vinculin-binding partners include proteins belonging to the CAP (Cbl-associated protein) protein family. However, the physiological significance of this association is unknown. Mammalian CAP proteins are components of focal adhesions in cell culture. In myocytes, CAP localizes to integrin-containing complexes called costameres that anchor sarcomeres to muscle cell membranes. There are three mammalian CAP protein family members: CAP, Vinexin and ArgBP2. CAP associates in vitro with many proteins, including the cytoskeletal regulators Paxillin, Afadin and Filamin, vesicle trafficking regulators such as Dynamin and Cbl, and the lipid raft protein Flotillin. In vitro studies demonstrate that CAP regulates the reassembly of focal adhesions following nocodazole dissolution. However, despite extensive studies on CAP, little is known about its functions in vivo. Cap (Sorbs1) mutant mice are defective in fat metabolism, and targeted deletion of the vinexin gene results in wound-healing defects. Drosophila CAP binds to axin and is implicated in glucose metabolism . Analysis of CAP function in mammals is complicated by potential functional redundancy of the three related CAP proteins. Therefore, the function of Drosophila CAP, the single CAP family member in Drosophila, was examined in vivo (Bharadwaj, 2013 and references therein).
The Drosophila muscle attachment site (MAS) is an excellent system for studying integrin signaling. Somatic muscles in each segment of the fly embryo and larva are connected to the body wall through integrin-mediated hemiadherens junctions. Somatic muscles in flies lacking integrins lose their connection to the body wall. Surprisingly, flies lacking Vinculin, a major component of cytosolic integrin signaling complexes, are viable and show no muscle defects. Thus, unlike its mammalian counterpart, Drosophila Vinculin is apparently dispensable for the initial assembly of integrin-mediated adhesion complexes at somatic MASs (Bharadwaj, 2013).
The fly MAS is structurally analogous to the fly chordotonal organ. These organs transduce sensations from various stimuli, including vibration, sound, gravity, airflow and body wall movements. The chordotonal organ is composed of individual subunits called scolopidia, each containing six cell types: neuron, scolopale, cap, ligament, cap attachment and ligament attachment cells. Chordotonal neurons are monodendritic, and their dendrites are located in the scolopale space, a lymph-filled extracellular space completely enveloped by the scolopale cell. Within the scolopale cell, a cage composed of actin bars, called scolopale rods, facilitates scolopale cell envelopment of the scolopale space. Thus, like the MAS, the actin cytoskeleton plays a specialized role in defining chordotonal organ morphology. Similarities between MASs and chordotonal organs include the requirement during development in both tendon and cap cells for the transcription factor Stripe. Furthermore, both of these cell types maintain structural integrity under force and so are likely to share common molecular components dedicated to this function (Bharadwaj, 2013 and references therein).
This study shows that the Drosophila CAP protein is selectively localized to both muscle attachment sites and chordotonal organs. In Drosophila CAP mutants morphological defects are observed that are indicative of actin disorganization in both larval MASs and the scolopale cells of Johnston's organ in the adult. The morphological defects in scolopale cells result in vibration sensation defects in larvae and hearing deficits in adults. It was also found that, like its mammalian homologues, Drosophila CAP interacts with Vinculin both in vitro and in vivo. These results reveal novel CAP functions required for actin-mediated organization of cellular morphology, lending insight into how CAP mediates muscle and sensory organ development and function (Bharadwaj, 2013).
Integrin-based adhesion complexes are crucial for cell attachment to the extracellular matrix. These complexes change their composition and architecture in response to extracellular forces, initiating downstream signaling events that regulate cytoskeletal organization. This study has investigated the role played by the CAP protein in two stretch-sensitive structures in Drosophila: the MAS and the chordotonal organ. CAP mutants exhibit aberrant junctional invaginations at the MAS and collapse of scolopale cells in chordotonal organs. This study highlights a crucial integrin signaling function during development: the maintenance of membrane morphology in stretch-sensitive structures (Bharadwaj, 2013).
The morphological defects observed in CAP mutants could result from an excessive integrin signaling, or possibly accumulation of additional membranous components related to integrin signaling, in CAP mutants, owing to defects in endocytosis at the MAS. This is consistent with known interactions between CAP family members and vesicle trafficking regulators, including Dynamin and Synaptojanin, which are required for internalization of transmembrane proteins. Alternatively, CAP may be required for proper organization of the actin cytoskeleton at MASs, and the aberrant membrane invaginations that were observe are a secondary consequence of these cytoskeletal defects. This idea garners support from known interactions between CAP and various actin-binding proteins, including Vinculin, Paxillin, Actinin, Filamin and WAVE2. A third possibility is that CAP and Vinculin are regulators of membrane stiffness at the MAS, and aberrant junctional infoldings observed in CAP and vinculin mutants derive from diminished membrane rigidity in the presence of persistent myofilament contractile forces. Biophysical studies demonstrate that Vinculin-deficient mammalian cells in vitro show reduced membrane stiffness. Interestingly, the CAP protein ArgBP2 interacts with Spectrin, a protein important for cell membrane rigidity maintenance. These models for CAP function at MASs, however, are not mutually exclusive. Interestingly, disruption of the ECM protein Tiggrin leads to MAS phenotypes similar to CAP. Future studies on CAP interaction with Tiggrin and other CAP-interacting proteins will shed light on mechanisms underlying CAP function. Nevertheless, this study demonstrates in vivo the importance of CAP in stretch-sensitive organ morphogenesis, and it will be interesting to determine whether this function is phylogenetically conserved (Bharadwaj, 2013).
Apart from the MAS, CAP is also expressed at high levels in chordotonal organ scolopale cells, and this study has found that CAP mutants are defective in vibration sensation, a hallmark of chordotonal organ dysfunction. However, only the initial fast hunching response to vibration is disrupted in CAP mutant larvae. This may result from a partial loss of chordotonal function in these organs in the absence of CAP. A functional defect was also observed in the adult Johnston's organ; CAP mutant flies show diminished sound-evoked potentials. Importantly, the scolopale cells in CAP mutants appear partially collapsed. The extracellular space within the scolopale cell is lined by an actin cage, and CAP may influence the proper assembly of this actin cage or its association with the scolopale cell membrane. Ch organs are mechanosensory detectors and are constantly exposed to tensile forces. Thus, CAP apparently influences cytoskeletal integrity in two actin-rich structures: the MAS and the chordotonal organ, both of which are involved in force transduction (Bharadwaj, 2013).
Mammalian and Drosophila CAP bind to Vinculin. Vinculin is required for the recruitment of the mammalian CAP protein vinexin to focal adhesions in NIH3T3 cells in vitro. Consistent with this observation, a dramatic decrease was seen in CAP levels at MASs in vinculin mutants, but residual levels of CAP protein remain. Furthermore, CAP localization at the muscle fiber Z-lines is completely unaltered in vinculin mutants. These observations indicate that Vinculin is not the sole upstream regulator of CAP localization. vinculin mutants show some of the phenotypic defects observed in CAP mutants; however, these defects are less pronounced. Therefore, the residual CAP pool that is recruited to MASs in a Vinculin-independent manner is apparently sufficient for partial CAP function. Assessment of CAP and Vinculin function at the larval MAS shows that these proteins are required for maintaining the integrity of junctional membranes in the face of tensile forces. CAP proteins may serve as scaffolding proteins at membrane-cytoskeleton interfaces and facilitate the assembly of protein complexes involved in cytoskeletal regulation and membrane turnover (Bharadwaj, 2013).
Mutations in the CAP-binding protein filamin cause myofibrillar myopathy. This, in combination with data showing a crucial role for CAP in regulation of muscle morphology, sets the stage for investigating how loss of CAP protein function might influence the etiology of myopathies (Bharadwaj, 2013).
Tendons are fibrous connective tissue which connect muscles to the skeletal elements thus acting as passive transmitters of force during locomotion and provide appropriate body posture. Tendon-derived cues, albeit poorly understood, are necessary for proper muscle guidance and attachment during development. This study used dorsal longitudinal muscles of Drosophila and their tendon attachment sites to unravel the molecular nature of interactions between muscles and tendons. A genetic screen using RNAi-mediated knockdown in tendon cells was performed to find out molecular players involved in the formation and maintenance of myotendinous junction; 21 candidates were found out of 2507 RNAi lines screened. Of these, 19 were novel molecules in context of myotendinous system. Integrin-βPS and Talin, picked as candidates in this screen, are known to play important role in the cell-cell interaction and myotendinous junction formation validating the screen. Candidates were found with enzymatic function, transcription activity, cell adhesion, protein folding and intracellular transport function. Tango1, an ER exit protein involved in collagen secretion was identified as a candidate molecule involved in the formation of myotendinous junction. Tango1 knockdown was found to affect development of muscle attachment sites and formation of myotendinous junction. Tango1 was also found to be involved in secretion of Viking (Collagen type IV) and BM-40 from hemocytes and fat cells (Tiwari, 2015).
This study used the myotendinous junction of Drosophila flight muscles to explore why many integrin associated proteins (IAPs) are needed and how their function is coordinated. These muscles revealed new functions for IAPs not required for viability: Focal Adhesion Kinase (FAK), RSU1, tensin and vinculin. Genetic interactions demonstrated a balance between positive and negative activities, with vinculin and tensin positively regulating adhesion, while FAK inhibits elevation of integrin activity by tensin, and RSU1 keeps PINCH activity in check. The molecular composition of myofibril termini resolves into 4 distinct layers, one of which is built by a mechanotransduction cascade: vinculin facilitates mechanical opening of filamin, which works with the Arp2/3 activator WASH to build an actin-rich layer positioned between integrins and the first sarcomere. Thus, integration of IAP activity is needed to build the complex architecture of the myotendinous junction, linking the membrane anchor to the sarcomere (Green, 2018).
The adult indirect flight muscles of Drosophila have proved to be an excellent system to identify functions for integrin-associated proteins (IAPs) that are not essential for viability. The mechanical linkage between the last Z-line of each myofibril and the plasma membrane is a well ordered and multi-layered structure, ideal for elucidating the mechanisms by which actin can be organized into different structures at subcellular resolution. In the layer closest to the membrane, the integrin signaling layer, an important counterbalancing is found between IAPs, with FAK inhibiting the activation of integrin by tensin, and RSU1 inhibiting excess PINCH activity. It was discovered that the muscle actin regulatory layer (MARL) has a different composition to the fibroblast ARL, containing a mechanotransduction cascade of vinculin and filamin, which, together with WASH and the Arp2/3 complex, builds an actin-rich zone linking the adhesion machinery at the membrane to the first Z-line (Green, 2018).
The modified terminal Z-lines [MTZ - composed of 4 zones: (1) an integrin signalling layer at the membrane; and then zones containing different actin structures-(2) a force transduction layer (FTL); (3) a muscle actin regulatory layer (MARL); and (4) the first Z-line followed by the first sarcomere] revealed both positive and inhibitory actions of FAK, with the latter consistent with the role of FAK in adhesion disassembly. Both loss of FAK and activated integrin suppressed the phenotypes caused by loss of RSU1 or vinculin, but only activated integrin alleviated the defects caused by the absence of tensin, suggesting that FAK inhibition requires tensin activity, and in turn, tensin elevates integrin activity. This fits with the recent discovery that tensin contributes to the inside-out activation of integrins via talin (Georgiadou, 2017). FAK and tensin thus form a balanced cassette that is thought to respond to upstream signals to regulate integrin activity. Further work is needed to discover how tensin increases integrin activity, how this is inhibited by FAK, and what signals control this regulatory cassette. One model would have tensin activating integrin by direct binding to the β subunit cytoplasmic tail, and FAK inhibition by phosphorylation of tensin, but an alternative is that they have antagonistic roles in integrin recycling (Green, 2018).
RSU1 is part of the complex containing ILK, PINCH and Parvin (IPP complex), and binds the 5th LIM domain of PINCH. Loss of RSU1 causes milder phenotypes than loss of ILK, PINCH or parvin, and these phenotypes have previously been interpreted as a partial loss of IPP activity. The current findings indicate that the phenotypes observed in the absence of RSU1 are due to too much PINCH activity, and therefore the role of RSU1 is to keep PINCH activity in check. This suggests that PINCH is perhaps the key player of the IPP complex, and is recruited to adhesions by integrin via ILK, and kept in check by integrin and RSU1. The importance of regulating active PINCH levels is consistent with the dosage sensitivity of PINCH: reducing PINCH partially rescues the dorsal closure defect in embryos lacking the MAPK Misshapen, and elevating PINCH rescues hypercontraction caused by loss of Myosin II phosphatase. Reducing the interaction of PINCH with ILK had unexpectedly no phenotype, but in combination with the loss of RSU1 becomes lethal; the lethality can now be interpreted as being caused by too much PINCH activity, rather than too little. Excess 'free' PINCH results in elongated membrane interdigitations and elevated paxillin levels. This suggests that PINCH has an important role at the cell cortex, consistent with cortical proteins in the PINCH interactome. Too much parvin activity also causes lethality, which is suppressed by elevating ILK levels. Thus, it is increasingly clear that the functions of IPP components need to be tightly controlled. This study gained some insight into how RSU1 inhibits PINCH activity by demonstrating that ΔLIM4, 5 PINCH still caused longer interdigitations. This rules out RSU1 blocking the binding of another protein from binding LIM5, and suggests instead that RSU1 bound to LIM5 must be inhibiting the activity of LIM1-3 (Green, 2018).
Vinculin has a dual function in the MTZ: its head domain promotes force transduction layer (FTL; containing actin, the C-terminus of talin and vinculin) stability via binding talin, and its tail promotes muscle actin regulatory layer (MARL) formation. This analysis of the vinculin mutant by electron microscopy showed a phenotype within the electron dense layer close to the membrane that is presumed to corresponds to the integrin signalling layer. It suggests that vinculin may mediate interactions between IAPs that aid in keeping this as an even layer. The fact that the disruption to this layer is only evident on the muscle side of the interaction raises the question of how similar the integrin junctions are on the two sides of this cell-cell interaction via an intervening ECM. Many other sites of integrin-mediated adhesion in Drosophila involve integrins on both sides of the interaction and by electron microscopy the electron dense material looks similar on the two sides, and it would be expected that both sides need to resist the same forces. Even with structured illumination microscopy the two sides of the membrane cannot be resolved, but the results show that the C-terminus of talin and vinculin are not pulled away from the membrane in the adult tendon cells. This suggests either that vinculin has a different role in the tendon cell, with a different configuration, as was observed for talin in the pupal wing, or it is absent (Green, 2018).
The vinculin tail function in MARL formation does not require that vinculin is bound to talin, but it is suspected that in the wild type it is talin-binding that converts vinculin into an open conformation, permitting the tail to trigger MARL formation with filamin, as outlined in a working model (see Model of IAP function in the IFM MTZ). A key function of vinculin tail in the MARL is to aid the mechanical opening of the filamin mechanosensitive region. This study presents evidence suggesting this is achieved by the vinculin tail anchoring the C-terminus to actin, but further work is required to determine if there is direct binding between the two proteins. Similarly, the results indicate that the Arp2/3 nucleation promoting factor WASH is part of the same pathway as filamin and acts downstream of it, but the connection between the two has yet to be resolved. This new function for WASH is distinct from its best characterized role regulating actin on intracellular vesicles during endosomal sorting and recycling, but WASH also has additional roles in the nucleus and the oocyte cortex, showing that it is a versatile protein (Green, 2018).
Given the myofibril defects seen with loss of RSU1, tensin, vinculin and filamin it might be expected that mutations in genes encoding these IAPs might be implicated in muscle disease. Indeed, mutations in integrin α7, talin and ILK are associated with muscular myopathies in humans and mice. Mutations in the genes encoding RSU1, tensin and vinculin have not been linked to muscle myopathies, but mutations in filamin are linked to myofibrillar myopathies. However, given the subtlety of these defects in Drosophila, one might predict that mutations in genes encoding these IAPs are associated with subtle defects in humans such as reduced sporting performance or susceptibility to muscle injury. The authors were unaware of any mutations in genes encoding these IAPs being related to athletic performance or injury susceptibility, but these IAPs would be good candidates for further study in this area (Green, 2018).
One way that these IAPs may contribute to athletic performance is by building a muscle shock absorber, the MARL, which protects the myofibrils from contraction-induced damage. The concept of muscle shock absorbers is well established since tendons perform this function. The presence of filamin, Arp3, vinculin and α-actinin in the MARL suggests that the MARL contains branched and bundled actin filaments. Branched actin networks have been shown to be viscoelastic and actin crosslinkers such as filamin have been shown to reduce viscosity and increase elasticity of actin networks. Further study into the functional nature of the MARL should increase understanding of athletic performance and injury susceptibility (Green, 2018).
A key step in tissue repair is to replace lost or damaged cells. This occurs via two strategies: restoring cell number through proliferation or increasing cell size through polyploidization. Studies in Drosophila and vertebrates have demonstrated that polyploid cells arise in adult tissues, at least in part, to promote tissue repair and restore tissue mass. However, the signals that cause polyploid cells to form in response to injury remain poorly understood. In the adult Drosophila epithelium, wound-induced polyploid cells are generated by both cell fusion and endoreplication, resulting in a giant polyploid syncytium. This study identified the integrin focal adhesion complex as an activator of wound-induced polyploidization. Both integrin and focal adhesion kinase are upregulated in the wound-induced polyploid cells and are required for Yorkie induced endoreplication and cell fusion. As a result, wound healing is perturbed when focal adhesion genes are knocked down. These findings show that conserved focal adhesion signaling is required to initiate wound-induced polyploid cell growth (Besen-McNally, 2020).
The spectraplakin family of proteins includes ACF7/MACF1 and BPAG1/dystonin in mammals, VAB-10 in Caenorhabditis elegans, Magellan in zebrafish, and Short stop (Shot), the sole Drosophila member. Spectraplakins are giant cytoskeletal proteins that cross-link actin, microtubules, and intermediate filaments, coordinating the activity of the entire cytoskeleton. This study examined the role of Shot during cell migration using two systems: the in vitro migration of Drosophila tissue culture cells and in vivo through border cell migration. RNA interference (RNAi) depletion of Shot increases the rate of random cell migration in Drosophila tissue culture cells as well as the rate of wound closure during scratch-wound assays. This increase in cell migration prompted an analysis of focal adhesion dynamics. The rates of focal adhesion assembly and disassembly were faster in Shot-depleted cells, leading to faster adhesion turnover that could underlie the increased migration speeds. This regulation of focal adhesion dynamics may be dependent on Shot being in an open confirmation. Using Drosophila border cells as an in vivo model for cell migration, it was found that RNAi depletion led to precocious border cell migration. Collectively, these results suggest that spectraplakins not only function to cross-link the cytoskeleton but may regulate cell-matrix adhesion (Zhao, 2022).
Integrin expression forms focal adhesions, but how this process is physiologically regulated is unclear. Ihog proteins are evolutionarily conserved, playing roles in Hedgehog signaling and serving as trans-homophilic adhesion molecules to mediate cell-cell interactions. Whether these proteins are also engaged in other cell adhesion processes remains unknown. This study reports that Drosophila Ihog proteins function in the integrin-mediated adhesions. Removal of Ihog proteins causes blister and spheroidal muscle in wings and embryos, respectively. Ihog proteins interact with integrin via the extracellular portion and that their removal perturbs integrin distribution. Finally, it was shown that Boc, a mammalian Ihog protein, rescues the embryonic defects caused by removing its Drosophila homologs. It is thus proposed that Ihog proteins contribute to integrin-mediated focal adhesions (Qi, 2023).
Age-related loss of intestinal barrier function has been documented across species, but the causes remain unknown. The intestinal barrier is maintained by tight junctions (TJs) in mammals and septate junctions (SJs) in insects. Specialized TJs/SJs, called tricellular junctions (TCJs), are located at the nexus of three adjacent cells, and this study have shown that aging results in changes to TCJs in intestines of adult Drosophila melanogaster. This study now demonstrates that localization of the TCJ protein bark beetle (Bark) decreases in aged flies. Depletion of bark from enterocytes in young flies led to hallmarks of intestinal aging and shortened lifespan, whereas depletion of bark in progenitor cells reduced Notch activity, biasing differentiation toward the secretory lineage. These data implicate Bark in EC maturation and maintenance of intestinal barrier integrity. Understanding the assembly and maintenance of TCJs to ensure barrier integrity may lead to strategies to improve tissue integrity when function is compromised (Hodge, 2023).
The eggshell that surrounds insect eggs acts as a barrier, protecting against biotic factors and desiccation. The eggshell is a multi-layered structure which is synthesised by the somatic follicle cells that surround the developing oocyte. Although the temporal order of expression of the protein eggshell components goes someway to explaining how the different layers are built up, but how the precise three-dimensional structure is achieved and how lipid components responsible for desiccation resistance are incorporated are poorly understood. This paper demonstrates that wunen, which encodes a lipid phosphate phosphatase, is necessary for fertility in Drosophila females. Compared to sibling controls, females null for wunen lay fewer eggs which subsequently collapse such that no larvae emerge. This is due to a requirement for wunen in the ovarian follicle cells which is needed to produce an ordered and functional eggshell. Knockdown of a septate junction component also results in collapsed eggs, supporting the idea that similar to its role in embryonic tracheal development, Wunen in follicle cells also promotes septate junction function (Mukherjee, 2023).
Although the molecular mechanisms governing abscission of isolated cells have largely been elucidated, those underlying the abscission of epithelial progenitors surrounded by epidermal cells (ECs), connected via cellular junctions, remain largely unexplored. This study investigated the remodeling of the paracellular diffusion barrier ensured by septate junctions (SJs) during cytokinesis of Drosophila sensory organ precursors (SOPs). SOP cytokinesis involves the coordinated, polarized assembly and remodeling of SJs in the dividing cell and its neighbors, which remain connected to the former via membrane protrusions pointing towards the SOP midbody. SJ assembly and midbody basal displacement occur faster in SOPs than in ECs, leading to quicker disentanglement of neighboring cell membrane protrusions prior to midbody release. As reported in isolated cells, the endosomal sorting complex required for the transport-III component Shrub/CHMP4B is recruited at the midbody and cell-autonomously regulates abscission. In addition, Shrub is recruited to membrane protrusions and is required for SJ integrity, and alteration of SJ integrity leads to premature abscission. This study uncovers cell-intrinsic and -extrinsic functions of Shrub in coordinating remodeling of the SJs and SOP abscission (Bruelle, 2023).
Cells in an organism are subjected to numerous sources of external and internal forces, and are able to sense and respond to these forces. Integrin-mediated adhesion links the extracellular matrix outside cells to the cytoskeleton inside, and participates in sensing, transmitting and responding to forces. While integrin adhesion rapidly adapts to changes in forces in isolated migrating cells, it is not known whether similar or more complex responses occur within intact, developing tissues. Changes in integrin adhesion composition were studied upon different contractility conditions in Drosophila embryonic muscles. All integrin adhesion components tested were still present at muscle attachment sites (MASs) when either cytoplasmic or muscle myosin II was genetically removed, suggesting a primary role of a developmental programme in the initial assembly of integrin adhesions. Contractility does, however, increase the levels of integrin adhesion components, suggesting a mechanism to balance the strength of muscle attachment to the force of muscle contraction. Perturbing contractility in distinct ways, by genetic removal of either cytoplasmic or muscle myosin II or eliminating muscle innervation, each caused unique alterations to the stoichiometry at MASs. This suggests that different integrin-associated proteins are added to counteract different kinds of force increase (Bulgakova, 2017).
During Drosophila gastrulation, amnioserosa (AS) cells flatten and spread as an epithelial sheet. This study used AS morphogenesis as a model to investigate how adherens junctions (AJs) distribute along elongating cell-cell contacts in vivo. As the contacts elongated, total AJ protein levels increased along their length. However, genetically blocking this AJ addition indicated that it was not essential for maintaining AJ continuity. Implicating other remodeling mechanisms, AJ photobleaching revealed non-directional lateral mobility of AJs along the elongating contacts, as well as local AJ removal from the membranes. Actin stabilization with jasplakinolide reduced AJ redistribution, and live imaging of myosin II along elongating contacts revealed fragmented, expanding and contracting actomyosin networks, suggesting a mechanism for lateral AJ mobility. Actin stabilization also increased total AJ levels, suggesting an inhibition of AJ removal. Implicating AJ removal by endocytosis, clathrin endocytic machinery accumulated at AJs. However, dynamin disruption had no apparent effect on AJs, suggesting the involvement of redundant or dynamin-independent mechanisms. Overall, it is proposed that new synthesis, lateral diffusion, and endocytosis play overlapping roles to populate elongating cell-cell contacts with evenly distributed AJs in this in vivo system (Goldenberg, 2013).
This study has documented three major behaviours of AJ proteins as AS cell-cell contacts elongate: new addition to the contacts, lateral movement along the contacts, and removal from the contacts. Once delivered to the contacts, AJ proteins appear to be redistributed by the expansion and contraction of fragmented actomyosin networks along the contacts, and by endocytosis from the contacts. However, the even distribution of AJ proteins could not be perturbed by singly disrupting any of these mechanisms, suggesting that they function redundantly for robust localization of AJs during AS morphogenesis (Goldenberg, 2013).
A ~50% increase in total AJ proteins was quantified along elongating contacts from early to mid cell elongation. The exocyst complex closely colocalizes with AJs at all cell-cell contacts in the tissue, suggesting a role in DE-cad exocytosis, as evident in other Drosophila tissues (Langevin, 2005). However, eliminating this net new addition has no noticeable effect on the even distribution of DE-cad along elongating contacts. Notably, within 2h of gastrulation, these mutants begin the lose cell-cell adhesion in the ventral neurectoderm, as holes left from delaminating neuroblasts fail to reform. At this later stage, cell-cell adhesion is still maintained in the AS in the mutants, and the residual maternally supplied DE-cad remains remarkably continuous along the fully elongated contacts. Thus, despite the rapidly expanding circumferences of the cells, net new addition of AJ proteins is not essential, and a stable pool of AJ proteins can apparently be effectively redistributed to maintain AJ continuity and cell-cell adhesion (Goldenberg, 2013).
The fact that total AJ protein levels increase by only ~1.5-fold while the elongating cell-cell contact lengths increase by ~2-fold, suggests that local AJ densities decrease, and thus that AJ proteins are locally lost either though lateral movement or removal from the membrane. Indeed, direct measurements of AJ proteins showed a ~50% decrease in local intensities, and the apparent heights of AJs decreased as well. Furthermore, iFRAP analyses revealed that local AJ patch lengths often increase, or decrease, at rates of 25% per min. Additionally, the local intensities of these patches can drop by up to 30% per min. By quantifying multiple parameters for the same AJ region over time, it was found that decreasing AJ protein levels did not correlate with the lateral spreading of the complexes, suggesting removal from the membrane. Notably, neither the lateral AJ movements nor the AJ losses correlated directly with the lengthening of the overall contacts, suggesting that the redistribution mechanisms are not directly triggered by contact elongation. Instead, it is proposed that local mechanisms result in constitutive expansions and condensations of cadherin-catenin clusters along the contacts, as well as constitutive removal, recycling and re-addition of AJ proteins along the contacts. These mechanisms would result in AJ plasticity that could allow pre-existing AJ proteins to populate elongating cell-cell contacts as they form (Goldenberg, 2013).
A key element regulating AS cell shape change appears to be the loss of actomyosin networks from cell-cell contacts. It is evident that these actomyosin networks restrain protrusive microtubule bundles from initiating elongation of the apical domain and cell rotation [36]. Thus, their weakening appears to permit the cell shape change. This study found that experimentally stabilizing actin networks rigidifies the cell cortex and also inhibits AJ redistribution. Thus, the normal weakening of actomyosin networks may contribute to both AS cell shape change and AJ plasticity along elongating contacts (Goldenberg, 2013).
Weakening actomyosin networks could promote AJ remodeling in two ways: by increasing lateral mobility or by increasing endocytosis. Actin is known to tether and restrain AJs in many systems. For example, in the ectoderm neighbouring the AS, actomyosin levels at AJs are normally maintained at higher levels than in the AS, but disrupting actin or α-catenin leads to the lateral mobility of AJ puncta with residual patches of actin attached. With the normal loss of myosin from AS cells, it was found that its networks fragment, and that these fragments expand and contract. It is proposed that these networks may expand and condense associated cadherin-catenin clusters. Also, the loss of actomyosin networks from the AS may contribute to AJ endocytosis, consistent with the observed increase in AJ protein levels at contacts with actin stabilization (Goldenberg, 2013).
There are clear examples where single AJ remodeling mechanisms play a dominant role in controlling cell-cell adhesion. For example, perturbing dynamin activity has a dramatic effect on AJ localization in the pupal notum, but has no clear effect at elongating AS cell-cell contacts. Such differences may be due to the relative effectiveness of other AJ distribution mechanisms in the tissues. For example, new synthesis of AJ proteins is much lower in the pupal notum versus the early embryo. Tissues with multiple effective AJ distribution mechanisms may maintain adhesion more robustly. Defining such redundancies and their contributions to robust cell-cell adhesion is important for understanding different developmental and homeostatic processes, as well as different disease states, e.g., the propensity of cancerous tissues to metastasize (Goldenberg, 2013).
Morphogenesis is crucial during development to generate organs and tissues of the correct size and shape. During Drosophila late eye development, interommatidial cells (IOCs) rearrange to generate the highly organized pupal lattice, in which hexagonal ommatidial units pack tightly. This process involves the fine regulation of adherens junctions (AJs) and of adhesive E-Cadherin (E-Cad) complexes. Localized accumulation of Bazooka (Baz), the Drosophila PAR3 homolog, has emerged as a critical step to specify where new E-Cad complexes should be deposited during junction remodeling. However, the mechanisms controlling the correct localization of Baz are still only partly understood. This study shows that Drosophila Magi, the sole fly homolog of the mammalian MAGI scaffolds, is an upstream regulator of E-Cad-based AJs during cell rearrangements, and that Magi mutant IOCs fail to reach their correct position. They uncovered a direct physical interaction between Magi and the Ras association domain protein RASSF8 through a WW domain-PPxY motif binding, and showed that apical Magi recruited the RASSF8-ASPP complex during AJ remodeling in IOCs. Further, this Magi complex was required for the cortical recruitment of Baz and of the E-Cad-associated proteins α- and β-catenin. They propose that, by controlling the proper localization of Baz to remodeling junctions, Magi and the RASSF8-ASPP complex promote the recruitment or stabilization of E-Cad
complexes at junction sites (Zaessinger, 2015).
As Magi is the sole Drosophila homolog of the three vertebrate MAGI scaffolds, it offers a powerful system with which to investigate the functions of these important proteins. Using newly generated null alleles, this study has shown that Magi coordinates the number and packing of IOCs in the developing Drosophila pupal eye by regulating AJ dynamics. Magi is necessary in the IOCs to localize the RASSF8-ASPP complex correctly during their junctional remodeling. This ensures the integrity of E-Cad-based junctions and the correct localization of Baz, α- and β-catenin. Based on these observations and on the growing evidence of a role for Baz in AJ remodeling, a model is proposed whereby, during AJ remodeling in IOCs, Magi recruits the RASSF8-ASPP complex, which helps to localize Baz at the membrane and regulates the sites of E-Cad accumulation (Zaessinger, 2015).
Junction remodeling is a key step during morphogenesis, in which cells in a tissue change position and neighbors. For instance, in the developing pupal eye, IOCs found between ommatidia organize as a single row of cells. During this process existing contacts are eliminated and new ones are established by remodeling E-Cad-based junctions. In Magi mutants, rearrangement defects and some incorrect localization of IOCs were observed. At the same time, E-Cad-based AJs were interrupted in Magi mutant cells. It is proposed that this defect in AJ remodeling leads to IOCs remaining at the wrong place in the lattice. The most parsimonious model is that the defects in AJ remodeling trigger the defects in cell numbers seen in Magi mutants by preventing apoptosis, although it was not possible to fully substantiate this as the effect of Magi on apoptosis was not statistically significant. If the model is correct, it still remains unclear how disrupted junctions would lead to a failure in apoptosis. One possibility is that IOCs only receive the correct 'death signal' when they have rearranged to contact the correct cells. Thus, in Magi mutants, the defective AJs would lead to apoptosis failure because the IOCs did not attain their position in the 'death zone' to receive the killing signal (Zaessinger, 2015).
These junctional defects are reminiscent of those seen for magi-1 mutants in the nematode C. elegans, in which magi-1 loss of function enhanced the defects caused by cadherin and catenin mutations and disrupted cell migration during enclosure (Lynch, 2012). MAGI scaffolds are thus implicated in the fine regulation of AJs in both flies and nematodes. A similar role has been suggested for MAGI proteins in mammalian epithelial cells. In overexpression studies, human MAGI1 reduced the Src-induced invasiveness of MDCK cells and stabilized E-Cad-mediated intercellular aggregation (Kotelevets, 2005). By analogy, the overexpression phenotype of Drosophila Magi could thus be due to stronger AJs, although this remains to be experimentally tested. The overexpression effects of MAGI-1b were sensitive to PTEN and AKT activities (Kotelevets, 2005) and mammalian MAGI scaffolds have also been implicated in PTEN activation through their direct binding to PTEN. However, this study did not detect any physical interaction between Drosophila Magi and Pten, and the overexpression phenotype of Magi, at least in the Drosophila eye, appeared insensitive to Pten. Although these are negative observations, they suggest that in Drosophila Magi and Pten do not form a complex to regulate AJs (Zaessinger, 2015).
Despite its effects on eye development, Magi mutants exhibit slightly enlarged wings. Whether this is dependent on E-Cad belt integrity and AJ dynamics remains to be established. The fact that ASPP shows a very similar wing phenotype supports this model (Zaessinger, 2015).
Rather than binding and modulating the activity of Pten, this analysis supports a model whereby Magi, by binding to the RASSF8-ASPP complex, recruits and stabilizes Baz at the membrane. Accumulation of Baz has been shown to specify and initiate the formation of new AJs both in cellularizing embryos and in photoreceptors. It is proposed that Baz recruited at the membrane of IOCs will in turn promote the stabilization or the proper distribution around the cell cortex of AJ material. Since biochemical and genetic experiments suggest that RASSF8 and Magi act together in a complex, it is proposed that the effects of Magi on AJs and on Baz membrane recruitment are mediated by RASSF8, and are thus likely to involve ASPP. Indeed, mammalian ASPP2 binds PAR3 and is required for PAR3 localization at junctions both in cell culture and in the mouse neuroepithelium. This suggests that Magi might control Baz localization through ASPP. However, Baz membrane recruitment is unlikely to be the only step to form correct AJs downstream of Magi/RASSF8/ASPP. Previous studies have implicated C-terminal Src kinase (Csk) and its action on Src kinase, and the relationships between Magi, Baz and Csk should be investigated in the future (Zaessinger, 2015).
During IOC remodeling, Magi therefore appears to be a crucial upstream regulator of AJs. However, the mechanisms governing Magi membrane localization are still unknown. One hypothesis is that the membrane recruitments of different AJ components and regulators are dependent on each other in stabilization loops. However, this is unlikely to be the case for Magi as it is still perfectly localized at the membrane in ASPP, RASSF8 and baz mutants, and in ASPP; RASSF8 double mutants (Zaessinger, 2015).
Another possibility is that Magi would require mature AJs with E-Cad to be at the membrane. No direct correlation was found between E-Cad accumulation around the apical membrane and Magi membrane localization. For instance, in ASPP; RASSF8 double-mutant cells, E-Cad belt interruptions were detected either without or with Magi, indicating that Magi localization does not require E-Cad directly. Furthermore, an extensive domain mapping of Magi failed to identify a single domain (WW or PDZ) that would be required for Magi recruitment, suggesting that it might be independent of these domains or that several redundant mechanisms may be at play. The nature of the signal required for Magi membrane localization thus remains to be uncovered (Zaessinger, 2015).
Even though Magi binds to RASSF8 directly and both proteins function together during Drosophila eye morphogenesis, their mutant phenotypes are not identical. First, RASSF8 mutants have a wing rounding phenotype, which is absent in Magi mutants. Second, whereas RASSF8 has a significant role in the global developmental apoptosis rate in the pupal eye, no significant effect could be detected for Magi and ASPP. Taken together, this suggests that the assembly of a Magi-RASSF8-ASPP complex might be context dependent or that RASSF8 has Magi-independent functions (Zaessinger, 2015).
Although the human N-terminal RASSF (RASSF7-10) proteins lack any PPxY motifs, one is present in ASPP2 and has been shown to bind to MAGI1. It is therefore possible that MAGI-ASPP complexes are formed in all organisms but the precise mode of interaction differs: mediated by RASSF8 in the fly, but direct in humans (Zaessinger, 2015).
MAGI scaffolds have been suggested to play a role in tumorigenesis. First, they are bound and inactivated by several viral oncoproteins. Second, MAGI1 has been shown to exhibit tumor suppressor activity in colorectal cancer cell lines in xenograft model. Finally, mutations in MAGI2 and MAGI3 are reported in colon, prostate and breast cancers. Documented alterations include deletion of the second WW motif of MAGI2 and a MAGI3:AKT3 fusion leading to a disruption of MAGI3. Based on the current work, it is proposed that these are loss-of-function mutations. It would be interesting to investigate whether changes in AJ dynamics are associated with these MAGI mutations in human cancers and whether they contribute to tumorigenesis (Zaessinger, 2015).
The mammalian MAGI proteins play important roles in the maintenance of adherens and tight junctions. The MAGI family of proteins contains modular domains such as WW and PDZ domains necessary for scaffolding of membrane receptors and intracellular signaling components. Loss of MAGI leads to reduced junction stability while overexpression of MAGI can lead to increased adhesion and stabilization of epithelial morphology. However, how Magi regulates junction assembly in epithelia is largely unknown. This study investigated the single Drosophila homologue of Magi to study the in vivo role of Magi in epithelial development. Magi is localized at the adherens junction and forms a complex with the polarity proteins, Par3/Bazooka and aPKC. A Magi null mutant was generated and found to be viable with no detectable morphological defects even though the Magi protein is highly conserved with vertebrate Magi homologues. However, overexpression of Magi results in the displacement of Baz/Par3 and aPKC and leads to an increase in the level of PIP3. Interestingly, it was found that Magi and Baz function in an antagonistic manner to regulate the localization of the apical polarity complex. Maintaining the balance between the level of Magi and Baz is an important determinant of the levels and localization of apical polarity complex (Padash Barmchi, 2016).
A common component of junctional and polarity complexes is modular scaffolding proteins that are capable of binding to each other as well as recruiting other proteins to the complex. Magi proteins are evolutionarily conserved scaffolding proteins and contain multiple domains including a N-terminal catalytically inactive GUK domain, two WW domains and five to six PDZ (PSD95/Dlg/ZO-1) domains (Dobrosotskaya, 1997). There are three MAGI proteins in vertebrates (MAGI-1,2,3) all with multiple splice isoforms. MAGI-1 and MAGI-3 are relatively ubiquitously expressed and localize to a range of junctions including epithelial tight junctions. MAGI-2 (also known as AIP1/S-SCAM/ARIP1) is expressed in the nervous system as a synaptic protein and within glomerular podocytes in the kidney and plays important role in scaffolding synaptic proteins such as NMDA receptors and Neuroligin, the tip-link protocadherin Cadherin23, the Kir4.1 K(+) channel, as well as kidney proteins such as nephrin and JAM4 (Padash Barmchi, 2016).
Within epithelia and endothelia, MAGI-1 and -3 are localized at tight junctions and form a structural scaffold for the assembly of junctional complexes. MAGI-1 also localizes and plays a role in modulating adherens junction adhesion through scaffolding beta-catenin and PTEN. MAGI-1 overexpression stabilizes adherens junctions and epithelial cell morphology through increased E-cadherin and β-catenin recruitment. Silencing of MAGI-1 has the opposite effect with decreased adherens junction adhesion and reduced focal adhesion formation leading to anchorage-independent growth and migration in vitro. MAGI-1 overexpression suppresses the invasiveness of MDCK cells, as well as suppresses tumor growth and spontaneous lung metastasis through the increased recruitment of PTEN or β-catenin and E-cadherin (Padash Barmchi, 2016).
Overall, MAGI proteins play important roles in the stabilization of cell-cell interactions and as such Magi is a key target in polarized epithelia during cell death and viral infection. For instance, MAGI-1 is cleaved by activated caspases during apoptosis, a process thought to mediate the disassembly of cell-cell contacts (Gregorc, 2007). MAGI proteins are also targeted by a number of oncogenic viruses: it is aberrantly sequestered in the cytoplasm by Adenovirus E4orf1, and is targeted for degradation by the E6 oncoprotein of high-risk human papillomavirus. E6-mediated degradation of MAGI-1 in cultured epithelial cells leads to loss of tight-junction integrity (Padash Barmchi, 2016 and references therein).
There is a high degree of conservation of protein structure and function in the invertebrate homologues of Magi in particular with regards to epithelial junction formation and maintenance. In C. elegans, Magi-1 plays a role in the segregation of different cell adhesion complexes into distinct membrane domains along the lateral plasma membrane. In Drosophila, Magi binds Ras association domain protein 8 (RASSF8) and modulates adherens junctions remodeling in late eye development during interommatidial cell (IOC) rearrangements. In this context Magi function is necessary to recruit the polarity protein Par-3 (Drosophila Bazooka, Baz) to the remodeling adherens junction. However, the association of Drosophila Magi or any Magi homologue with any components of the Par polarity complex in stable epithelia has not been determined (Padash Barmchi, 2016).
The Par complex consisting of Par-3/Par-6/aPKC localizes to tight junctions where MAGI is present in vertebrate epithelial cells and is necessary for assembly of this junctional complex as well as for separation of the apical region of the plasma membrane from the basolateral domain. In Drosophila epithelial cells, the Par complex localizes to the apicolateral membrane and demarcates the boundary between the apical and basolateral membrane regions. Mutant embryos for any member of this complex show loss of apicobasal polarity and disruption in the integrity of epithelia. Although the members of the Par complex are important for the establishment of cell polarity, some of the core components of this complex such as Baz are dispensable for the maintenance of cell polarity during later stages of development. Baz localizes to adherens junction and mutant clones of baz in wing imaginal discs are fully viable with no polarity or adherens junction defects. Similarly, Magi function in AJ stability has been determined in many systems, but surprisingly loss of Drosophila Magi has no effect on established, stable AJs (Zaessinger, 2015). Little is known about the convergence of Magi and Par complex function at the adherens junctions and it is possible that Baz and Magi function in established epithelia are redundant. Therefore this study investigated the role of Magi in the established and stable epithelia of the Drosophila wing imaginal disc to test the potential interactions between Magi and members of the Par complex (Padash Barmchi, 2016).
Drosophila Magi was found associated with the PAR polarity complex and is localized at the adherens junction with Baz, Par-6, and aPKC. Overexpression of Magi resulted in the reduction of apical polarity proteins from the membrane and these interactions required the second half of the Magi protein containing the four PDZ domains. Overexpression of Baz resulted in a reduction of Magi from the membrane but an increase in aPKC and Par-6. While Magi mutants were viable with no polarity defects, Magi levels were found to be antagonistic with Baz, and a balance between the two was found to be necessary to regulate the level and localization of Par complex (Padash Barmchi, 2016).
PDZ domain-containing proteins form scaffolding protein complexes with a wide range of roles including cell polarity and signaling. As a MAGUK protein, Magi is part of a scaffold that interacts with members of the polarity complex at the adherens junctions in the epithelia of the imaginal disc. The scaffolding function of Magi has been well established in other systems. In vertebrates epithelial cells MAGI-1 has been shown to act as structural scaffold at tight junctions and adherens junctions. In C. elegans, Magi-1 localizes apical to adherens junction and functions as an organizer to ensure that different cell adhesion complexes are segregated into distinct membrane domains along the lateral plasma membrane. In neuronal cells MAGI-2/S-SCAM was also shown to cluster the cell adhesion molecule Sidekick, and the AMPA and NMDA glutamate receptors at the synapse (Padash Barmchi, 2016).
Given the strong conservation of the Magi protein it is surprising that null mutants of Drosophila Magi exhibit no lasting cellular defects (other than transient defects in the interommatidial cells of the pupal eye and null animals are fully viable. Similarly in C. elegans, magi-1 null worms are healthy with only a few embryos (1.3%) with defects during the ventral enclosure stage. As Magi is highly conserved, it is plausible that Magi may only act in response to cell stress, DNA damage or some other trigger. For example, loss of p53 does not disrupt cellular function under normal conditions and p53 null flies or mice are viable with no cellular defects. However, the role of p53 in response to DNA damage is well established and when these animals are exposed to irradiation apoptosis is not induced. Alternatively, Magi function might be redundant with other components of the apical polarity complex or another protein and that loss of both is necessary for the disruption of cellular function. Core scaffolding components of the apicobasal polarity complex are dispensable for maintaining polarity in the wing imaginal disc epithelia supporting the idea of redundancy in this system. For instance, somatic clones of loss of function mutations in crb, sdt and baz have no effect on the polarity in the wing disc epithelia of the 3rd instar larvae. Baz is a strong candidate for redundancy with Magi given the localization to the adherens junction and function as a PDZ scaffolding protein. As loss of baz in the wing imaginal disc does not disrupt the polarity of wing disc epithelia this leads to the hypothesis that Baz and Magi are redundant. However, somatic clones of a baz null mutant in a Magi mutant background did not lead to a loss of cell polarity or apoptosis. While the two scaffolding proteins do not appear to functionally interact, it was observed that Magi and Baz are in a protein complex and their close proximity within the wing columnar epithelia also suggests a common complex. Overexpression of Magi displaces Baz and aPKC from the apical membrane and, likewise overexpression of Baz displaces Magi from the membrane. The simultaneous over-expression of Magi and Baz suppresses the changes caused by their individual expression, suggesting a balance or competition between the two proteins. The maintenance of a balance between Magi and Baz might be due to a direct physical competition between these two proteins or opposite effects on a common mediator or interactor (Padash Barmchi, 2016).
Baz and vertebrate MAGI proteins bind the lipid phosphatase PTEN and thus the Magi-Baz interaction and balance could be influenced by changes in the level of phosphoinositides such as PtdIns(4,5)P2 (PIP2) or PtdIns(3,4,5)P3 (PIP3). In polarized epithelia, PIP2 is found within the apical domain and PIP3 restricted to the basal-lateral domain. Baz localization in polarized epithelia depends on PIP2 and on the PI4P5 kinase Skittles. Baz in turn can be a positive regulator of PIP2 levels at the plasma membrane by local recruitment of the lipid phosphatase PTEN. This study observed an increase in PIP3 levels with increased expression of Magi, which may reflect the loss of Baz and a loss of PTEN recruitment to the membrane. This study was not able to assess changes in PTEN levels at the membrane with available antibodies. However it was observe that the recruitment of Magi or Baz was not affected in Pten mutant cells. Similarly the changes in PIP3 levels are unlikely to be the cause of Baz loss in the presence of increased Magi as co-expression of PTEN and Magi still resulted in the loss of Baz from the membrane. Prior studies on Magi in Drosophila in the pupal eye did not detect any physical interaction between Drosophila Magi and Pten, and the phenotypes generated by overexpression of Magi in the Drosophila eye were not affected by Pten mutants. Therefore it is likely that loss of Baz in the presence of increased Magi in the wing imaginal disc and vice versa is through competition for a protein component (Padash Barmchi, 2016).
In the developing eye Magi forms a protein complex with RASSF8 (the N-terminal Ras association domain-containing protein) and ASPP (Ankyrin-repeat, SH3-domain, and proline-rich-region containing protein), and this complex plays a role during remodeling of the adherens junctions in the interommatidial cells (IOCs) (Zaessinger, 2015). When IOCs rearrange to create the pupal lattice, this process requires regulation of the E-Cadherin complex where RASSF8 and ASPP regulate adherens junction remodeling and integrity through regulation of Src kinase activity. Magi recruits the RASSF8-ASPP complex in the process of adherens junction remodeling and there are defects in IOC rearrangement in Magi mutants where AJs are frequently interrupted. In the eye the Magi-RASSF8-ASPP complex is necessary for the cortical recruitment of Baz and of the adherens junction proteins α- and β-catenin. A model has been proposed where Magi-RASSF8-ASPP complex functions to localize Baz to remodeling junctions to promote the recruitment or stabilization of E-Cad complexes (Zaessinger, 2015). However, it is not thought that the RASSF8-ASPP complex is the point of competition between Magi and Baz within the wing imaginal disc. In the wing imaginal disc Magi and the RASSF8-ASPP complex are localized to the adherens junction domain independently (Zaessinger, 2015) and while RASSF8 mutants have a wing rounding phenotype, Magi mutants do not. Furthermore no differences were observed in Baz, Ecad or Arm distribution in Magi somatic loss of function clones in the wing imaginal disc. Finally the Magi WW domains are required for the interaction with RASSF8 (Zaessinger, 2015), while the overexpression of the Magi transgene that contains the PDZ domains led to a reduction in Baz suggesting that second half of the Magi protein containing the PDZ domains contains the important sites for this competition (Padash Barmchi, 2016).
Therefore, a strong possibility to explain the reciprocal effects of overexpression is that Baz and Magi compete for a common binding site. Magi was found to interacte with both Baz and aPKC; the latter two are known to interact directly. However, it is unlikely that the shared site is through physical scaffolding of aPKC, as high levels of wild type aPKC had no effect on either Magi or Baz and was not able rescue the changes in Baz levels and localization caused by Magi overexpression. In addition the overexpression of Magi also led to a reduction in aPKC. It is unlikely that the loss of Baz is responsible for this displacement as aPKC is not mislocalized in Baz clones and Baz is not mislocalized in Par-6, aPKC or Cdc42 null clones. Further investigation is required to explore the mechanisms that underlie Magi interactions with components of the apical polarity complex and the adherens junction complex (Padash Barmchi, 2016).
Although Snail is essential for disassembly of adherens junctions during epithelial-mesenchymal transitions (EMTs), loss of adherens junctions in Drosophila melanogaster gastrula is delayed until mesoderm is internalized, despite the early expression of Snail in that primordium. By combining live imaging and quantitative image analysis, the behavior of E-cadherin-rich junction clusters were tracked, demonstrating that in the early stages of gastrulation most subapical clusters in mesoderm not only persist, but move apically and enhance in density and total intensity. All three phenomena depend on myosin II and are temporally correlated with the pulses of actomyosin accumulation that drive initial cell shape changes during gastrulation. When contractile myosin is absent, the normal Snail expression in mesoderm, or ectopic Snail expression in ectoderm, is sufficient to drive early disassembly of junctions. In both cases, junctional disassembly can be blocked by simultaneous induction of myosin contractility. These findings provide in vivo evidence for mechanosensitivity of cell-cell junctions and imply that myosin-mediated tension can prevent Snail-driven EMT (Weng, 2016).
This study shows that during Drosophila gastrulation, subapical junctions are repositioned toward the apical surface and are strengthened as the cortical tension increases. Both these phenomena follow apical myosin activation and thus may reflect a mechanosensitive response of junctional complexes to the tension generated by this activation of myosin. The junctional responses occur on the time scale of individual myosin pulses and are temporally correlated with those pulses. Such junctional changes depend on myosin activity but do not require Sna, given that ectopic myosin activation recapitulates similar junctional responses in Sna-negative tissues. This phenomenon may not be restricted to Drosophila embryos. The increased contractile actomyosin on the apical cortex of human cell lines deficient for the cortex actin regulator Merlin is associated with a condensation of adherens junctions toward the apical surface, suggesting that the response of adherens junctions to cortical tension can be of general significance (Weng, 2016).
The changes in junction mass and density suggest that, rather than being simple passive anchors for contractile actomyosin filaments, adherens junctions respond to the contractile actomyosin by restructuring and repositioning themselves, potentially involving aggregation and rearrangement of E-Cad molecules within the plasma membrane or vesicle-based redistribution of E-Cad. Indeed, actomyosin organization has been shown to be critical in the lateral clustering of E-Cad molecules. The change in E-Cad clustering is considered an active mechanosensitive mechanism to strengthen the adhesion. Alternatively, the adhesion can also be remodeled through the vesicle-based mechanisms, and endocytosis of E-Cad has been shown to be up-regulated when junctions are under actomyosin-generated stress. The repositioning could also arise through restructuring rather than passive dragging, if for example recycling and turnover rates in the basal regions of the junctions differ from apical regions. Overall, regardless of the underlying mechanism, this mechanosensitivity may be advantageous, providing a direct self-corrective mechanism that allows junctions to adjust their localization and intensity to match the mechanical force they experience (Weng, 2016).
Although the molecular mechanism for the junction strengthening requires further investigation, the data suggest that it is resistant to the posttranscriptional disassembly of adherens junctions downstream of Sna. The phenotype of myosin knockdown in this study resembles that previously described for cta; T48 double mutants, in which apical actomyosin cannot be activated and junctions are lost only in the ventral mesodermal cells. In all scenarios in which Sna expression is associated with junction loss (ventral cells in cta; T48 mutants, ventral cells in myosin knockdown mutants, and ectodermal cells with ectopic Sna expression), Sna is expressed in cells in the absence of myosin contractility. Maintenance of adherens junctions ultimately relies on the balance between assembly and disassembly rates of junctional components. Thus mechanical force likely modulates the assembly/disassembly balance and therefore remains in a homeostatic relationship with the junctions bearing the force (Weng, 2016).
In the early stages of embryogenesis analyzed in this study, E-Cad is maternally provided and thus not subject to direct transcriptional repression. The disassembly of junctions in the absence of myosin contraction must therefore reflect a posttranscriptional regulation on junctions, likely performed by one or several of Sna’s transcriptional targets. Much effort has been invested in identifying transcription targets of Sna, but it is not known which, if any, of its known targets might play such a role. One mesodermally expressed gene, Traf4, is required for fine-tuning junction morphology, but its expression appears to depend on the other mesodermal determinant, Twist, rather than Sna. One gene repressed by Sna in Drosophila mesoderm, bearded, is required for the subapical positioning of adherens junctions in cells not expressing Snail. It is not clear, however, whether Bearded plays a direct role in junction disassembly or a more general role in apical polarity or the apical myosin contractility that drives repositioning. The posttranscriptional regulation of adherens junction disassembly may allow more rapid and effective EMT than a disassembly relying on transcriptional down-regulation of junctional components such as E-Cad. Identifying and characterizing the relevant Sna targets in Drosophila may provide insights into the underlying mechanism for this disassembly, especially with respect to its apparent sensitivity to externally exerted tension. The force-dependent resistance to this Sna function may help in dissecting the underlying molecular functions. Further exploration of Sna’s posttranscriptional effect on junctions and how myosin contraction antagonizes Sna will shed light on understanding of EMT processes (Weng, 2016).
In epithelia, specialized tricellular junctions (TCJs) mediate cell
contacts at three-cell vertices. TCJs are fundamental to epithelial
biology and disease, but only a few TCJ components are known, and how they
assemble at tricellular vertices is not understood. This study describes a
transmembrane protein, Anakonda (Aka), which localizes to TCJs and is essential for the formation of tricellular, but not bicellular, junctions in Drosophila. Loss
of Aka caused epithelial barrier defects associated with irregular TCJ
structure and geometry, suggesting that Aka organized cell corners. Aka is necessary and sufficient for accumulation of Gliotactin at TCJs, suggesting that Aka initiated TCJ assembly by recruiting other proteins to tricellular vertices. Aka's extracellular domain had an unusual tripartite repeat
structure that might mediate self-assembly, directed by the geometry of
tricellular vertices. Conversely, Aka's cytoplasmic tail is dispensable
for TCJ localization. Thus, extracellular interactions, rather than
TCJ-directed intracellular transport, appear to mediate TCJ assembly (Byri, 2015).
Epithelial cells are linked via intercellular junctions that provide paracellular diffusion barriers, maintenance of polarity, and cell-to-cell communication. Bicellular junctions (BCJs) make up the most abundant intercellular contacts in epithelia. They connect two neighboring plasma membranes and are organized into distinct complexes along the apical-basal axis, collectively forming the apical junctional belt. However, at certain positions along the cell perimeter, three cell corners meet and the bicellular junctional complex is disjointed. Here, specialized tricellular junctions (TCJs) connect epithelial cells. TCJs play important roles in epithelial barrier functions and cytoskeletal organization, and are preferential sites for trans-endothelial migration of neutrophils and metastatic cancer cells, as well as for the spreading of intracellular pathogens (Byri, 2015).
Due to the geometry of three-cell vertices, the sealing of the epithelium at these sites requires a dedicated junctional organization. Ultrastructural analyses using freeze-fracture electron microscopy (EM) showed that the zonula occludens, tight junctions (TJs) in vertebrates and septate junctions (SJs) in arthropods, changes characteristics when approaching a three-cell vertex. Instead of continuing parallel to the epithelial plane around the cell perimeter, the junctional strands extend basally, forming a bicellular seal along the apical-basal axis. In vertebrates, these structures are termed central sealing elements, and three such parallel TJ extensions enclose a narrow (approximately 10 nm) central canal at each TCJ. Similarly, in invertebrates the SJ strands turn by 90 degrees when approaching a tricellular corner, forming three parallel limiting strands that surround the tricellular juncture space, resembling the central sealing element in vertebrates. Within and perpendicular to the vertical juncture space are a series of diaphragms, which appear linked not only to three limiting septa, but also to the three cell corner membranes, thereby forming true tricellular contacts (Byri, 2015).
Despite the fundamental biological importance of TCJs, only few of their components are known, and the mechanism of their localized assembly at three-cell vertices is not understood. In vertebrates, the Occludin family protein Tricellulin localizes to TCJs and is recruited there by lipolysis-stimulated lipoprotein receptor (LSR) and related proteins (ILDR1 and ILDR2). Additionally, the cytoplasmic PDZ-domain-containing protein Tjp2iso3 associates with Tricellulin in Sertoli cells. The Neuroligin-like transmembrane protein Gliotactin in Drosophila is the only TCJ protein characterized so far in invertebrates. It is not clear what kind of cues direct the accumulation of these proteins at tricellular vertices, and the features of known TCJ proteins do not explain the distinct structure of three-cell contacts observed by EM. This study describes a transmembrane protein, Anakonda (Aka), which accumulates at TCJs in Drosophila epithelia. Aka can initiate TCJ assembly and may do so through its large extracellular domain, which exhibits an unusual triple-repeat structure (Byri, 2015).
Ultrastructural analyses revealed a unique junctional architecture at points in epithelia where three cells meet. Yet, only few TCJ-specific proteins are known, and how they assemble into a tightly localized complex exclusively at tricellular vertices is not understood. This study describes transmembrane protein Anakonda, which is shown to play a critical role in TCJ assembly and epithelial barrier formation. First, Aka was shown to localize to TCJs in ectodermal and endodermal epithelia, suggesting that Aka is a core TCJ component. Second, Aka was demonstrated to be specifically required for the assembly and correct geometry of tricellular, but not of bicellular SJs, and Aka was shown to localize to TCJs independently of bicellular SJs. Third, Aka is required for recruiting or maintaining Gli at tricellular vertices. Conversely, Gli is not required for TCJ localization of Aka. Fourth, Aka mis-expression causes premature accumulation of Gli at TCJs, indicating that Aka acts upstream of Gli in initiating TCJ formation (Byri, 2015).
Aka protein contains a large extracellular domain with a conserved tripartite repeat structure. This unique structure distinguishes Aka from other known TCJ proteins. Tricellulin, a four-pass transmembrane protein in vertebrates, localizes to the central sealing elements, suggesting that it participates in the specialized bicellular contacts that surround each three-cell vertex. Tricellulin is recruited to TCJs by the immunoglobulin domain transmembrane protein LSR, but how LSR localizes to TCJs is still unclear. In Drosophila, Gli, a transmembrane protein with a cholinesterase domain lacking catalytic activity, accumulates at TCJs, but is also involved in bicellular SJ organization. Phosphorylation-dependent endocytic turnover and indirect association with Dlg are required for accumulation of Gli at TCJs. However, the mechanisms underlying TCJ-specific localization are not understood for Gli or any other TCJ protein (Byri, 2015).
The results suggest that Aka acts at an early step during TCJ assembly by recruiting other TCJ components, including Gli, to tricellular vertices. TCJ localization of Aka and Gli does not require Aka’s C terminus, including the PDZ-binding motif. Similarly, Gli was found to localize to TCJs independently of its PDZ-binding motif. These findings suggest that Aka and Gli might be targeted to TCJs through their extracellular domains, rather than through a cytoplasmic localization machinery. Because mislocalized Aka protein is not sufficient to recruit Gli to ectopic locations, Aka might not interact directly with Gli, or only do so in the context of TCJs. Consistent with this notion, we were not able to detect co-immunoprecipitation of Aka and Gli. Interestingly, overexpression of Gli or absence of Aka causes Gli to spread from TCJs to BCJs. Thus, Gli does not have an intrinsic propensity to localize to TCJs, but by default localizes to the apicolateral membrane domain occupied by SJs along the cell perimeter. TCJ accumulation of Gli therefore requires (1) association with the apicolateral membrane and (2) Aka-dependent recruitment of Gli specifically to TCJs. The findings show that unlike Gli, Aka does not depend on bicellular SJs for its localization to TCJs, and suggesting that Aka protein might have intrinsic properties that lead to its accumulation at TCJs (Byri, 2015).
Tricellular vertices display a unique geometry where three plasma membranes are in close proximity and at fixed angles. The exceptional curvature of the plasma membrane at these sites implies distinct physical properties compared to bicellular contacts. Intriguingly, it was found that TCJ assembly depends on Aka expression in all three cells adjoining a vertex, suggesting that extracellular interactions between Aka molecules from different cells are essential for TCJ formation. Considering the size of Aka's extracellular domain (303 kDa), it is likely that a single Aka protein spans the entire TCJ canal (25–30 nm), although shorter Aka isoforms may constitute different structures. Shorter Aka species might carry out functions that may or may not be related to the function of full-length Aka at TCJs. Alternatively, Aka fragments may reflect turnover of TCJ complexes, e.g., during junctional remodeling (Byri, 2015).
It is tempting to speculate that the three repeat regions in full-length Aka protein, with apparent similar domain organization, could make equal contacts with the three cell corners in the plane perpendicular to the central TCJ canal. Possibly, interaction with membrane components in the tricellular region occurs via the three SR domains, since such domains are found in receptors that recognize a wide range of molecular patterns, including surface proteins, carbohydrates, lipids, lipopolysaccharides, and peptidoglycans associated with pathogens or apoptotic cells. Because such three-way contacts may, for steric reasons, only be possible at vertices, they might selectively stabilize Aka complexes at these sites. Computer simulations suggest that the enrichment of Aka at three-cell vertices could theoretically be enhanced by the reduction of dimensionality in the tricellular region, which may promote stacking interactions between Aka molecules within the central TCJ canal. This idea is consistent with the regular structure of the TCJ diaphragms and the equal dihedral angles near TCJs as observed with electron microscopy. Stacking might occur with Aka molecules rotated by 120 or 240 degrees within the stack, perhaps in a helical array, depending on the contributing cell. Notably, such a scenario would explain the finding that TCJ formation requires Aka protein production by all three cells adjoining a vertex. A high priority of future studies will be to investigate the arrangement of Aka proteins at TCJs. Together, the current results on Aka localization, the geometry of three-cell vertices, and the triple-repeat structure of Aka protein suggest a mechanism of TCJ formation, which is promoted by self-assembly of Aka at tricellular contacts. Such self-assembly might additionally involve interactions with other membrane-associated or extracellular components, and could cooperate with bicellular adhesion molecules that zip up bicellular contacts. It will be interesting to test whether the geometry of tricellular vertices and the specific properties of Aka protein are sufficient to direct its accumulation to TCJs. Conversely, perturbed TCJ geometry upon loss of Aka may have long-range effects, such as the loss of cell-cell adhesion that this study observed in late-stage aka embryos. Interestingly, depletion of Tricellulin from mammalian cells affects cell shape and the F-actin network, suggesting that TCJs may in fact have organizing activity on the entire cell (Byri, 2015).
The existence of aka homologs in invertebrates and cephalochordates correlates with the presence of SJs in these groups. However, the proposed self-assembly model for TCJ formation in Drosophila may apply also to vertebrates, although the corresponding proteins remain to be discovered. A better understanding of TCJ assembly will be a key step toward elucidating how these poorly characterized cellular structures provide epithelial barrier function, while at the same time allowing the passage of migrating lymphocytes, metastatic cells, and intracellular pathogens (Byri, 2015).
Remodeling of cell-cell junctions drives cell intercalation that causes tissue movement during morphogenesis through the shortening and growth of bicellular junctions. The growth of new junctions is essential for continuing and then completing cellular dynamics and tissue shape sculpting; however, the mechanism underlying junction growth remains obscure. This study investigated Drosophila genitalia rotation where continuous cell intercalation occurs to show that myosin II accumulating at the vertices of a new junction is required for the junction growth. This myosin II accumulation requires the adhesive transmembrane protein Sidekick (Sdk), which localizes to the adherens junctions (AJs) of tricellular contacts (tAJs). Sdk also localizes to and blocks the accumulation of E-Cadherin at newly formed growing junctions, which maintains the growth rate. It is proposed that Sdk facilitates tAJ movement by mediating myosin II-driven contraction and altering the adhesive properties at the tAJs, leading to cell-cell junction extension during persistent junction remodeling (Uechi, 2019).
To generate tissue shapes, cell collectives show various dynamics, such as cell division and cell deformation. Among them, cell intercalation is a multicellular behavior in which cells change their position through the remodeling of cell-cell contacts, leading to the directional elongation and expansion of tissues across species. Especially in epithelia, this cell-cell junction remodeling involves the shortening and loss of bicellular junctions and the subsequent growth of bicellular junctions in a new direction. Junction shortening initiates tissue dynamics and is driven in a conserved manner by contractile forces generated by actomyosin (actin and non-muscle myosin II complex) associating with the cadherin-catenin core complex, including E-Cadherin, β-Catenin, and other related adherens junction (AJ) components, at the AJs of shortening junctions. Junction growth is also essential for continuing and then completing cellular dynamics and tissue shape sculpting. Several studies using flies have suggested that myosin II has a role in junction growth during developmental events. In the germ band, medial pulses of myosin II in the cells surrounding junctions and toward the posterior ectoderm regulate junction growth during cell intercalation-driven convergent extension [germ band extension (GBE)]. A similar contribution of myosin II pulses in the surrounding cells to junction extension is also observed in the apical cell oscillation of amnioserosa cells during dorsal closure. In developing wing epithelia, a decrease in myosin II levels at newly formed junctions facilitates junction growth to organize the epithelial cellular pattern. However, despite its importance, the mechanisms underlying junction growth remain unclear, in contrast to junction shortening (Uechi, 2019).
Previous studies demonstrated that cell intercalation also contributes to the tissue rotational movement observed for Drosophila male genitalia. The fly genitalia are located at the animal's posterior end, and the male genitalia are surrounded by epithelia known as the A8 segment at the anterior side. At 24 h after puparium formation (APF), the genitalia and the A8 epithelia begin dextral rotation that terminates at around 36 h APF. The rotation consists of an initial 180° movement of the posterior compartment of A8 (A8p) along with the genitalia and a subsequent 180° movement of the anterior component of A8 (A8a), the latter of which starts at around 26 h APF. From 26 h APF in the A8a cells, myosin II accumulates to a greater extent at AJs, forming a right oblique angle with the anterior-posterior (AP) axis than at junctions forming a left oblique angle. This polarized myosin II distribution gives rise to right-biased junction shortening in relation to the AP axis and leads to left-right asymmetric cell intercalation, which is persistently observed during the movement. By combining numerical simulations, it was demonstrated that this repeated junction remodeling in the confined space generates the A8a movement. In this movement, newly formed junctions are sufficiently elongated within a certain time frame to execute the next round of cell intercalation. Incomplete genitalia rotation leads to male sterility (Uechi, 2019).
This study performed time-lapse imaging, developed an optogenetic tool, and analyzed the adhesive protein Sidekick (Sdk), which is known to regulate retinal development in flies and mice and showed that myosin II accumulating at the tricellular contacts (tAJs) of growing junctions is required for bicellular junction growth in A8a cells. Also, Sdk regulates the myosin II and E-Cadherin distributions at the tAJs, thereby maintaining the junction growth rate. These findings suggest that the tAJ is a specialized point promoting cell-cell junction extension (Uechi, 2019).
The process of junction shortening is well characterized and is organized by the contractile forces of actomyosin, which is transmitted to cell-cell contacts via AJ components, such as E-Cadherin. These proteins have important roles in the dynamics of multicellular deformation. Since junction formation and growth are important for the continuation and completion of multicellular dynamics and tissue architecture shaping, it is likely that active mechanisms underlie the extension of cell-cell junctions. Indeed, recent reports suggest that actin and myosin II at the bicellular junctions are involved in the junction extension in cell rearrangement and in cell-shape formation during Drosophila wing and eye development. Polarized medial pulses of myosin II in the cells surrounding junctions regulate junction extension in the Drosophila germ band and amnioserosa. This study used an optogenetic tool that allows for the spatiotemporal inactivation of endogenous myosin II and revealed that myosin II accumulating at the tAJs of newly formed junctions is required for junction growth in the A8a epithelia. This study also demonstrated that the myosin II accumulation and junction growth require the tAJ-localizing protein Sdk. Thus, this report that tAJs are an additional point promoting the extension of bicellular junctions (Uechi, 2019).
Sdk transiently localizes to newly formed junctions as well as tAJs, causing a downregulation of E-Cadherin and a slight increase in intercellular spaces at the AJs of growing junctions, indicative of less tight cell-cell contacts. Recent studies in zebrafish showed that the presence of extracellular spaces and the disassembly of cell-cell contacts contribute to fluidize tissues. During body axis elongation, the extracellular spaces render mesodermal cells fluidized and uncaged and associated with large fluctuations in the lengths of cell-cell contact. Decreases in cell-cell contacts through the destabilization of junctional E-Cadherin, accompanied by an increase in extracellular spaces, induces the fluidization of blastoderm cells and consequently allows blastoderm spreading at the onset of morphogenesis. Analogous to these properties, it is possible that the presence of Sdk at growing junctions confers flexible dynamics to the cell-cell contacts at the level of the AJs of the growing junctions. This study proposes mechanisms of junction growth in which Sdk has dual roles. First, Sdk mediates a driving force of junction growth by anchoring myosin II at tAJs; the contractility of the actomyosin then retracts the membrane of the surrounding cells at tAJs. Second, Sdk assists in the myosin II-driven junction growth by localizing to and decreasing the accumulation of E-Cadherin at the growing junctions and their tAJs; this composition of E-Cadherin and Sdk causes contacts between the vertices of the surrounding cells and the cells forming the growing junction to be less tight. Such adhesion can render the tAJs of growing junctions more sensitive to contractile forces at the vertices of the surrounding cells, supporting the retraction of the membrane of the surrounding cells at tAJs. The latter mechanism is indeed likely to contribute to junction growth since inducing sdk RNAi only in the cell forming the growing junction was sufficient to reduce the junction growth rate, even when the surrounding cells consisted of WT cells (Uechi, 2019).
The precise mechanism by which Sdk blocks the accumulation of E-Cadherin at newly formed junctions is still unclear. While Sdk was already present at growing junctions from the step of four-way vertex resolution, E-Cadherin would be newly recruited to the growing junctions since E-Cadherin is removed from remodeling junctions by endocytosis during junction shortening. A recent study using fluorescence recovery after photobleaching (FRAP) revealed two ways that E-Cadherin is re-distributed to cell-cell junctions, lateral diffusion within the plasma membrane and delivery from the cytoplasm by vesicular trafficking. Since (1) E-Cadherin and Sdk did not interact despite their localization to AJs, (2) showed complementary distributions at newly formed junctions and even at cellular edges in S2 cells where they were ectopically expressed, and (3) changed their distributions when the other protein was depleted, it is possible that there are repelling forces between E-Cadherin and Sdk molecules, which cause them to exclude each other and may delay the diffusion of E-Cadherin from neighboring junctions into newly formed junctions, where Sdk is already enriched. However, this study does not exclude another possibility that Sdk inhibits machineries that deliver E-Cadherin from the cytoplasm, such as blocking their access to growing junctions or biochemically inactivating them (Uechi, 2019).
This study observed the accumulation of myosin II and decreased E-Cadherin levels at the tAJ of growing junctions. These distributions resemble those occurring during new cell-cell contact formation between daughter cells in epithelia. After cytokinesis, myosin II accumulates at the edges of new cell-cell junctions in the neighboring cells of the daughter cells, in response to the local decrease in E-Cadherin levels at these edges, which participates in new cell-cell junction formation. These reports and the current observations suggest a possible common mechanism underlying new cell-cell contact formation among epithelial multicellular behaviors. Although the dynamics and roles of Sdk in cell division are still unclear, an intriguing possibility is that Sdk regulates the dynamics of new cell-cell junctions in concert with myosin II and E-Cadherin not only in the context of cell intercalation but also global epithelial dynamics including cytokinesis (Uechi, 2019).
Tricellular adherens junctions are points of high tension that are central to the rearrangement of epithelial cells. However, the molecular composition of these junctions is unknown, making it difficult to assess their role in morphogenesis. This study shows that Sidekick, an immunoglobulin family cell adhesion protein, is highly enriched at tricellular adherens junctions in Drosophila. This localization is modulated by tension, and Sidekick is itself necessary to maintain normal levels of cell bond tension. Loss of Sidekick causes defects in cell and junctional rearrangements in actively remodeling epithelial tissues like the retina and tracheal system. The adaptor proteins Polychaetoid and Canoe are enriched at tricellular adherens junctions in a Sidekick-dependent manner; Sidekick functionally interacts with both proteins and directly binds to Polychaetoid. It is suggested that Polychaetoid and Canoe link Sidekick to the actin cytoskeleton to enable tricellular adherens junctions to maintain or transmit cell bond tension during epithelial cell rearrangements (Letizia, 2019).
Sdk is the only protein so far shown to be specifically localized to tAJs and almost excluded from bAJs in most epithelia. Although several proteins were known to be enriched at tricellular contacts at the level of AJs, most of them are also present along the whole bicellular junction. This study found find that Sdk is required for the specific enrichment of Cno, Pyd, and actin at tAJs, but not their localization at bAJs. Consistent with this, cno and pyd mutants have stronger phenotypes than sdk mutants, indicating that these proteins have functions independent of Sdk. In contrast, Sdk localizes to tAJs even in the absence of Cno and Pyd. It is proposed that Sdk is the hub that organizes a protein complex specifically at tAJs to modulate the actin cytoskeleton (Letizia, 2019).
This study haa shown that the C-terminal predicted PDZ-binding motif is required for Sdk function, and directly interacts with Pyd. The requirement for Sdk to recruit Cno to tAJs and the ability of misexpressed Cno to expand Sdk suggest that Cno is also in a complex with Sdk. As the vertebrate homologues of Cno and Pyd can bind to each other, all three proteins may be present in the same complex. However, knocking down ZO-1 proteins in MDCK cells increases the recruitment of Afadin to tAJs, so it is also possible that Cno and Pyd compete for the same binding site on Sdk. Although it is not possible to detect a direct interaction between Sdk and Cno, the experiments did not exclude this possibility. Alternatively, Sdk might recruit Cno indirectly, for example by binding to a GEF that increases Rap activity. Cno is known to interact with Ed, which is necessary for Ecad recruitment to bAJs; it is not yet clear whether Ed is in the same complex as Sdk or whether Cno interacts with the two proteins in a mutually exclusive manner. In addition to anchoring the ends of actin filaments, Cno might recruit other regulators of tension such as the LIM domain protein Smallish. pyd mutant embryos show defects in tracheal intercalation similar to sdk mutants, and pyd and cno genetically interact with sdk in this context, supporting a general role for these factors downstream of Sdk (Letizia, 2019).
Although Cno and Pyd are likely to be important mediators of the effects of Sdk on tension and junctional stability, other partners may also contribute to Sdk function. The cytoplasmic domain of Sdk has several regions of strong evolutionary conservation, which could serve as interaction domains for non-PDZ proteins. Elucidating the nature of the complex that has Sdk as its hub will provide important clues to the structure and function of tAJs. The expression of mouse Sdks in tissues other than the nervous system, such as the ureteric bud during branching morphogenesis of the kidney, suggests that the role of Sdk at tAJs may be conserved, although it is not yet known whether Sdks accumulate at tAJs in epithelia in other organisms (Letizia, 2019).
While Sdk can mediate homophilic interactions at bicellular contacts in cultured cells, in epithelial tissues it localizes almost exclusively to vertices. Several mechanisms might contribute to this localization pattern. It is possible that the topology of membranes at vertices imposes a particular structural conformation on Sdk that promotes its clustering there. The FnIII domains of Sdk molecules can interact with lipids and are thought to lie flat along the plasma membrane at bicellular contacts between parallel membranes, while the first four Ig domains mediate homophilic adhesion; the fourth and fifth Ig domains meet at a relatively rigid angle of 126°. This might make Sdk-mediated adhesion at tricellular interfaces (the point at which cells meet at an average angle of 120°) more energetically favorable. Alternatively or additionally, this angle might favor cis interactions between Sdk molecules through their FnIII domains over FnIII-membrane interactions, contributing to Sdk clustering. Such a geometric model could explain why Sdk is still enriched at tAJs when it is present on only two of the three cells that are in contact. However, this cannot be the only mechanism for Sdk localization, as tricellular vertices can form at a range of angles (Letizia, 2019).
In addition, this study found that tension enhances Sdk recruitment to tAJs. Although the Pyd homologue ZO-1 is stretched by tension, which controls its interactions with some of its binding partners, neither Pyd nor Cno is required for the initial localization of Sdk to tAJs. This argues that Sdk can detect tension by a mechanism independent of and prior to its own role in anchoring actin filaments through Cno and Pyd. Sdk might itself be mechanosensitive, as FnIII domains can stretch in response to force. Force exerted on the plasma membrane to pull it away from Sdk-Sdk adhesions could extend the FnIII domains to open up more interaction sites. Alternatively, as Ecad is necessary for Sdk localization, Sdk could detect tension through the cadherin-catenin complex, perhaps by interacting with α-catenin or another protein that is deformed by mechanical force (Letizia, 2019).
Mechanical forces can regulate tissue morphogenesis by promoting cell intercalation, oriented cell division or cell extrusion. AJs are known to transduce mechanical force, with one prominent mechanism being the force-dependent unfolding of α-catenin, which exposes binding sites for vinculin and other proteins. Vinculin is enriched at tAJs, indicating that tension is highest at these positions and suggesting that tAJs might sense and regulate tension. At bAJs, the cadherin-catenin complex resists tension by linking cell adhesions to the actin cytoskeleton through catch bonds. tAJs may require additional mechanisms to resist contractility because they anchor the ends of actin filaments, generating tension in an actomyosin network both along the cell cortex and in radially directed filaments. The requirement for Sdk to maintain normal levels of tension along cell bonds makes it a good candidate to mediate this tension-modulating function of tAJs. The homophilic adhesive properties of the extracellular domain of Sdk and its interaction with the actin cytoskeleton could allow it to transmit tension between cells or organize supracellular contractile networks that facilitate mechanical coordination across a tissue. Sdks mediate strong and compact adhesions with tightly packed molecules, potentially allowing them to resist force. However, the Ecad complex is sufficient to maintain adhesion at tAJs in the absence of Sdk (Letizia, 2019).
Cell shape changes and cell rearrangements often involve oscillations in apical area driven by pulses of actomyosin contraction. Both reduced tension on cell bonds in the embryo and defects in cell intercalation in the retina in sdk mutants could result from failure to connect the oscillatory network to tAJs. A lack of mechanical coupling of tensile actomyosin networks to tAJs would disrupt cell intercalation by reducing the efficiency of T1 transitions mediated by changes in junction length. In the trachea, Sdk accumulates at autocellular-intercellular vertices and is required for the replacement of intercellular junctions by autocellular ones. Tracheal cell intercalation does not require myosin contractility, suggesting that other actin regulators provide local forces to resolve cell rearrangements. Sdk may mediate the replacement of intercellular junctions by homophilic adhesion and/or by modulating the actin cytoskeleton to provide polarized tension or polarized membrane growth that allows vertex displacement during the zipping process. In general, vertex displacements in complex cell rearrangements may rely on the Sdk complex to transmit tension (Letizia, 2019).
As tTJs can also contribute to actin organization and tension, it will be important to investigate the interactions between the two types of tricellular junctions. Such studies may shed light on the role of tricellular junctions in regulating cell proliferation, stem cell homeostasis, and tissue integrity (Letizia, 2019).
Biological systems tailor their properties and behavior to their size throughout development and in numerous aspects of physiology. However, such size scaling remains poorly understood as it applies to cell mechanics and mechanosensing. By examining how the Drosophila pupal dorsal thorax epithelium responds to morphogenetic forces, this study found that the number of apical stress fibers (aSFs) anchored to adherens junctions scales with cell apical area to limit larger cell elongation under mechanical stress. aSFs cluster Hippo pathway components, thereby scaling Hippo signaling and proliferation with area. This scaling is promoted by tricellular junctions mediating an increase in aSF nucleation rate and lifetime in larger cells. Development, homeostasis, and repair entail epithelial cell size changes driven by mechanical forces; this work highlights how, in turn, mechanosensitivity scales with cell size (Lopez-Gay, 2020).
Convergence-extension is a widespread morphogenetic process driven by polarized cell intercalation. In the Drosophila germ band, epithelial intercalation comprises loss of junctions between anterior-posterior neighbours followed by growth of new junctions between dorsal-ventral neighbours. Much is known about how active stresses drive polarized junction shrinkage. However, it is unclear how tissue convergence-extension emerges from local junction remodelling and what the specific role, if any, of junction growth is. This study reports that tissue convergence and extension correlate mostly with new junction growth. Simulations and in vivo mechanical perturbations reveal that junction growth is due to local polarized stresses driven by medial actomyosin contractions. Moreover, it was found that tissue-scale pulling forces at the boundary with the invaginating posterior midgut actively participate in tissue extension by orienting junction growth. Thus, tissue extension is akin to a polarized fluid flow that requires parallel and concerted local and tissue-scale forces to drive junction growth and cell-cell displacement (Collinet, 2015).
To form regulated barriers between body compartments, epithelial cells polarize into apical and basolateral domains and assemble adherens junctions (AJs). Despite close links with polarity networks that generate single polarized domains, AJs distribute isotropically around the cell circumference for adhesion with all neighboring cells. How AJs avoid the influence of polarity networks to maintain their isotropy has been unclear. In established epithelia, trans cadherin interactions could maintain AJ isotropy, but AJs are dynamic during epithelial development and remodeling, and thus specific mechanisms may control their isotropy. In Drosophila, aPKC prevents hyper-polarization of junctions as epithelia develop from cellularization to gastrulation. This study shows that aPKC does so by inhibiting a positive feedback loop between Bazooka (Baz)/Par-3, a junctional organizer, and centrosomes. Without aPKC, Baz and centrosomes lose their isotropic distributions and recruit each other to single plasma membrane (PM) domains. Surprisingly, loss- and gain-of-function analyses show that the Baz-centrosome positive feedback loop is driven by Par-1, a kinase known to phosphorylate Baz and inhibit its basolateral localization. Par-1 was found to promote the positive feedback loop through both centrosome microtubule effects and Baz phosphorylation. Normally, aPKC attenuates the circuit by expelling Par-1 from the apical domain at gastrulation. The combination of local activation and global inhibition is a common polarization strategy. Par-1 seems to couple both effects for a potent Baz polarization mechanism that is regulated for the isotropy of Baz and AJs around the cell circumference (Jiang, 2015).
The identification of Par-1 as an inducer of Baz-centrosome co-recruitment is surprising given its well-established role in inhibiting Baz complex formation in Drosophila, C. elegans, and mammalian systems. It is proposed that Par-1 contributes to both global inhibition and local promotion of Baz complex assembly, providing a simple and potent Baz polarization mechanism (Jiang, 2015).
The Baz-centrosome positive feedback loop is evident from the specific accumulation of Baz next to cortical centrosomes, the MT requirement for Baz accumulation, the Baz requirement for centrosome recruitment, and the dynein role for drawing Baz and centrosomes together. Significantly, Par-1 is also necessary and sufficient for the loop and seems to have two direct roles. One is promotion of astral microtubules around the centrosome, an effect consistent with known effects of Par-1 on MT regulators, but requiring further elucidation in the Drosophila embryo. The other is the phosphorylation of Baz at Ser-151 and Ser-1085. These modifications have well-characterized inhibitory effects on Baz cortical association, but strikingly, they are also enriched where the Baz-centrosome positive feedback loop occurs and appear necessary for Baz entry into the loop. It is speculated that phospho-regulated Baz-14-3-3 protein interactions mediate further protein interactions, or induce conformational changes, important for Baz-MT association. Indeed, 14-3-3 proteins can bridge MT motors, a Par-3 conformational change induces direct MT binding, Par-3 directly binds a dynein subunit, and other links to MTs are known (Jiang, 2015).
Although the Par-1-Par-3-centrosome pathway can be a potent Baz polarization mechanism, it is normally attenuated within a homeostatic system. During early cellularization, Par-1 localizes over the entire PM and presumably phosphorylates Baz and MT regulators. In response, it is proposed that Baz is continually displaced and diffuses over the PM but is additionally primed for MT interactions. Simultaneously, the two centrosomes found atop each nucleus would provide the positional information for localizing Baz around the apical circumference through dynein-mediated MT associations. As Baz accumulates, it recruits aPKC to the apical domain, from where aPKC then displaces Par-1. Normally, this Baz-aPKC-Par-1 negative feedback loop seems to keep the Par-1-Baz-centrosome pathway in check. In the absence of aPKC, the Par-1- Baz-centrosome pathway continues unabated, leading to excessive Baz and centrosome polarization, loss of AJ isotropy, and later epithelial dissociation (Jiang, 2015).
Intriguingly, focused accumulations of Par-3 and AJs colocalize with cortical centrosomes during C. elegans intestinal development and during zebrafish collective cell migration. Moreover, Par-1 induces centrosomal MT interactions with AJs during human liver lumen formation in vitro and is needed for Baz-centrosome associations during the asymmetric division of Drosophila germline stem cells. Thus, induction of the Par- 1-Par-3-centrosome pathway, with regulated shifts to aPKC or Par-1 activities, may be generally relevant to developmental transitions of animal tissues (Jiang, 2015).
Apoptosis is a mechanism of eliminating damaged or unnecessary cells during development and tissue homeostasis. During apoptosis within a tissue, the adhesions between dying and neighboring non-dying cells need to be remodeled so that the apoptotic cell is expelled. In parallel, the contraction of actomyosin cables formed in apoptotic and neighboring cells drive cell extrusion. To date, the coordination between the dynamics of cell adhesion and the progressive changes in tissue tension around an apoptotic cell is not fully understood. Live imaging of histoblast expansion, which is a coordinated tissue replacement process during Drosophila metamorphosis, shows remodeling of adherens junctions (AJs) between apoptotic and non-dying cells, with a reduction in the levels of AJ components, including E-cadherin. Concurrently, surrounding tissue tension is transiently released. Contraction of a supra-cellular actomyosin cable, which forms in neighboring cells, brings neighboring cells together and further reshapes tissue tension toward the completion of extrusion. A model is proposed in which modulation of tissue tension represents a mechanism of apoptotic cell extrusion, and would further influence biochemical signals of neighboring non-apoptotic cells (Teng, 2017).
This study reports the temporal sequence of events during apoptotic cell extrusion, with a focus on the remodeling of AJs, the cytoskeleton, and mechanical tension. After caspase-3 starts to be activated in the polyploid larval epithelial cells (LECs), those undergoing apoptosis initiate apical constriction. It was reasoned that the initiation of this constriction could be due to a combination of actomyosin cable formation in the dying cell and the activity of caspase-3, which assists in the upregulation of actomyosin contractility. Indeed, it has been shown in tissue culture that the cleavage of Rho associated kinase by caspase- 3 is involved in phosphorylation and activation of myosin light chain, which regulates actomyosin contractility. It is proposed that the actomyosin cable that forms in apoptotic LECs is responsible for the early stages of apoptotic cell extrusion. During apical constriction, the level of AJ components including E-cad strongly reduced in a caspase-3-dependent manner. In the neighboring non- dying cells, this reduction is found only at the interface between the apoptotic cell and its neighbors. Since caspase-3 is not activated in the neighboring cells, it is speculated that the reduction of E-cad is a consequence of a loss of trans-interactions between E-cad of the neighboring cell, and E-cad of the apoptotic cell, which undergoes caspase-3-dependent cleavage. This often, but not always, leads to plasma membrane separation, which is suggestive of a loosening of AJ-dependent adhesion. It has been reported that anillin organizes and stabilizes actomyosin contractile rings at AJs and its knock-down is associated with a reduction of E-cad and β-Catenin levels at AJs, leading to AJ disengagement. A gradual decrease in the level of E-cad, and a gradual increase in MyoII accumulation
in apoptotic cells was observed prior to the strong reduction of E-cad levels. This lead to the hypothesis that mechanical tension exerted on the cell interface between apoptotic LECs and neighboring cells by the contraction of the actomyosin cable, which forms in the apoptotic cell, is large enough to rupture the weakened contacts between plasma membranes at AJs upon the strong reduction of E-cad levels (Teng, 2017).
Interestingly, and by contrast, there are cases when AJs are not disengaged even after the level of E-cad is reduced. In these cases the cells exhibit a separation of actomyosin cables from the membrane. It is speculated that the state of cell-cell contacts at AJs, i.e., whether they will disengage or remain engaged during apoptosis, is dependent on which of the following links is weaker: The link between two plasma membranes, or the link between the plasma membrane and the actomyosin cable. Both of these links would be weakened by a strong, albeit incomplete, reduction of E-cad levels. When the former is weaker than the latter, the two plasma membranes could be detached. When the former is stronger than the latter, the two plasma membranes could remain in contact, and the actomyosin cable could be detached from the plasma membrane (Teng, 2017).
In parallel with the reduction of E-cad levels and the associated release of tension, a supra-cellular actomyosin cable begins to form in neighboring cells. These observations prompted a speculation that the release of tissue tension triggers MyoII accumulation in neighboring cells. Subsequent contraction of this outer ring helps to reshape tissue tension, which is transiently released when E-cad is reduced. As a consequence, the neighboring cells are stretched. Upon
completion of apical constriction, neighboring non-apoptotic cells form de novo AJs and the stretched cells undergo cell division and/or cell-cell contact rearrangement. These processes allow a relaxation of the high tension associated with the stretching of cells. Finally, measurements of caspase-3 activity, and the observations from caspase inhibition experiments, lead to a conclusion that the characteristics associated with apoptotic cell extrusion reported in this study are the consequences of the apoptotic process, rather than the cause (Teng, 2017).
In addition to the progressive remodeling of AJs and modulation of tissue tension during apoptosis, the mechanical role was examined of apoptosis 'apoptotic force' in tissue morphogenesis, which has been proposed, demonstrated, and discussed. It was shown that the mechanical force generated by the contraction of actomyosin cables formed when LECs undergo apoptosis, especially boundary LECs, promotes tissue expansion, along with histoblast proliferation and migration. Nonetheless, it cannot be ruled out that this apical contraction is in part driven by a decrease
in cell volume, which can be triggered by caspase activation. Intriguingly, it was found that apoptosis of non-boundary LECs did not affect tissue
expansion. This raised the possibility that the mechanical influence of apoptosis in neighboring tissues is dependent not only on the physical connections between cells, but also on the mechanical properties of cells, including cell compliance. If a tissue is soft, for instance, the tensile forces generated by apoptotic process could be absorbed by nearest-neighbor cells and would not propagate to cells further than a single cell away. It is speculated that the apoptotic process could mechanically contribute to cell death-related morphogenesis, only when apoptosis takes place at optimal mechanical properties of a tissue (Teng, 2017).
This study presents a framework for understanding how cell adhesions and tissue tension are progressively modulated during apoptosis in a developing epithelium. It is concluded that tissue tension reshaping, including the transient release of tension upon a reduction in the levels of AJ components, represents a mechanism of apoptotic cell extrusion. It would be important to explore how this transient modulation in mechanical tension would further influence the biochemical nature of neighboring non-apoptotic cells (Teng, 2017).
During animal development, epithelial cells forming a monolayer sheet move collectively to achieve the morphogenesis of epithelial tissues. One driving mechanism of such collective cell movement is junctional remodeling, which is found in the process of clockwise rotation of Drosophila male terminalia during metamorphosis. However, it still remains unknown how the motions of cells are spatiotemporally organized for collective movement by this mechanism. Since these moving cells undergo elastic deformations, the influence of junctional remodeling may mechanically propagate among them, leading to spatiotemporal pattern formations. Using a numerical cellular vertex model, this study found that the junctional remodeling in collective cell movement exhibits spatiotemporal self-organization without requiring spatial patterns of molecular signaling activity. The junctional remodeling propagates as a wave in a specific direction with a much faster speed than that of cell movement. Such propagation occurs in both the absence and presence of fluctuations in the contraction of cell boundaries (Hiraiwa, 2017).
Distinct patterns of actomyosin contractility are often associated with particular epithelial tissue shape changes during development. For example, a planar polarized pattern of myosin II localization regulated by Rho1 signaling during Drosophila body axis elongation is thought to drive cell behaviors that contribute to convergent extension. This study developed optogenetic tools to activate (optoGEF) or deactivate (optoGAP) Rho1 signaling. These tools were used to manipulate myosin patterns at the apical side of the germband epithelium during Drosophila axis elongation, and the effects on contractile cell behaviors were analyzed. Uniform activation or inactivation of Rho1 signaling across the apical surface of the germband is sufficient to disrupt the planar polarized pattern of myosin at cell junctions on the timescale of 3-5 min, leading to distinct changes in junctional and medial myosin patterns in optoGEF and optoGAP embryos. These two perturbations to Rho1 activity both disrupt axis elongation and cell intercalation These studies demonstrate that acute optogenetic perturbations to Rho1 activity are sufficient to rapidly override the endogenous planar polarized myosin pattern in the germband during axis elongation. Moreover, these results reveal that the levels of Rho1 activity and the balance between medial and junctional myosin play key roles, not only in organizing the cell rearrangements that are known to directly contribute to axis elongation, but also in regulating cell area fluctuations and cell packings, which have been proposed to be important factors influencing the mechanics of tissue deformation and flow (Herrera-Perez, 2021).
In Drosophila epithelial cells, apical exclusion of Bazooka/Par3 defines the position of the Zonula Adherens (ZA), which demarcates the apical and lateral membrane and allows cells to assemble into sheets. This study shows that the small GTPase Rap1, its effector AF6/Canoe (Cno) and the Cdc42-effector Pak4/Mushroom bodies tiny (Mbt), converge in regulating epithelial E-Cadherin, and Bazooka retention at the ZA. Furthermore, the results show that the localization of Rap1, Cno and Mbt at the ZA is interdependent, indicating their functions during ZA morphogenesis are interlinked. In this context, the Rap1-GEF Dizzy was found to be enriched at the ZA and the results suggest it promotes Rap1 activity during ZA morphogenesis. Altogether, it is proposed the Dizzy, Rap1/Cno pathway and Mbt converge in regulating the interface between Bazooka and AJ material to promote ZA morphogenesis (Walther, 2018).
In the pupal photoreceptor, ZA morphogenesis is orchestrated by a conserved protein network that includes Cdc42, Par6, aPKC, Baz, Crb and its binding partner Sdt, and Par1. In turn, AJ material is an essential part of the regulatory network that orchestrates polarity. Previous work has shown that Mbt regulates pupal photoreceptor development by promoting ZA morphogenesis. During this process Mbt contributes in preventing Baz from spreading to the lateral membrane, a regulation that this study found to depend in part on the phosphorylation of Arm by Mbt at S561 and S688. It is proposed that Mbt regulates photoreceptor polarity by promoting the retention of Baz at the developing ZA. Failure in ZA retention leads to Baz spreading to the lateral membrane where it is eliminated through Par1-mediated displacement. In these cells, failure to retain AJ material, including Baz, at the ZA leads to its shortening along the apical basal axis and can impact on the polarization program of the photoreceptor (Walther, 2018).
This study shows that Mbt function is linked to that of Dzy, Rap1 and Cno. First, Cno and Mbt accumulation at the ZA is interdependent, reflecting a tight coupling between the Rap1 and Cno pathway and Mbt. Second, it was found that Cno promotes Baz retention at the ZA, as cnoIR leads to shorter ZAs that can be depleted of Arm and Baz. This phenotype resembles that of mbt mutant cells and is also seen when overexpressing a version of Arm that cannot be phosphorylated by Mbt. These observations prompted a test or the hypothesis that Rap1, Cno and Mbt might function as part of a linear pathway promoting Baz retention at the ZA. In this pathway, it was reasoned that Mbt could mediate Rap1 function through Arm phosphorylation. In testing this hypothesis, it was found that this is not the case. Instead, the observation that expressing a version of Arm that mimics its constitutive phosphorylation by Mbt does not ameliorate the cnoIR phenotype suggests that Rap1, Cno, and Mbt converge in promoting Baz retention at the ZA, and cannot compensate for each other during this process. This conclusion is well supported by the finding that overexpressing cno in mbt mutant cells does not lead to an amelioration of the mbt phenotype. Third, it was found that Mbt influences the distribution of Rap1 along the apical-basal axis of the cell in that Rap1::GFP no longer accumulates preferentially at the ZA. This correlates with a loss of Dzy::GFP at the plasma membrane, raising the possibility that Mbt might regulate Rap1 through Dzy. However, the dzy phenotype is milder than that seen with Rap1 or cno, in that loss of dzy does not lead to cell delamination from the retina. This suggests that, as has been reported in the cellularizing embryo, other GEFs regulate Rap1 during epithelial morphogenesis (Walther, 2018).
An interesting aspect of the cnoIR phenotype is the defects in apical accumulation of aPKC and Crb. These defects are not observed in the dzy mutant or Rap1IR cells, indicating that Cno might function independently of Rap1 during this process. However, it is noted that while Cno was not detected at the ZA of cnoIR cells, it can still be detected in Rap1IR cells. It is therefore hypothesized that residual Cno in Rap1IR cells supports optimum aPKC and Crb accumulation at the apical membrane. In this model, Dzy, Rap1 and Cno function as part of the same pathway, which includes a function in promoting optimum apical accumulation of Crb and aPKC. Baz is required for Par complex assembly and associated aPKC and Crb recruitment at photoreceptor apical membrane. It is hypothesized that the defects in Crb and aPKC that were detect in cnoIR cells are linked to the failure in retaining Baz at the ZA, which leads to its elimination from the lateral membrane by Par1. More work will be required to understand how exactly AJ material and ZA retention of Baz influences apical membrane specification (Walther, 2018).
Rap1 and cno have been shown to regulate apical-basal polarity in the cellularizing embryo. In this model system, Rap1 and Cno regulate the apical localization of Baz and Arm, which precedes the apical recruitment of Crb. In turn, Baz influences the localization of Cno. This work indicates that similar complex regulations are at play in the pupal photoreceptor. However, unlike in the early embryo, AJ material (Arm) is absolutely required for Baz (and Par6-aPKC) accumulation or retention at the cell cortex in the developing pupal photoreceptor. A model is therefore favored whereby Mbt, Rap1 and Cno influence ZA morphogenesis primarily through regulating the interface between E-Cad or Arm, Baz and the F-actin cytoskeleton. In this model, Mbt regulates this interface both through Arm phosphorylation and cofilin-dependent regulation of F-actin, and Cno contributes to this process, at least in part, through its ability to bind to F-actin (Walther, 2018).
To probe Rap1 and Cno function during photoreceptor ZA morphogenesis, the effect of decreasing Rap1 expression on E-Cad stability was assessed. Consistent with the notion that the function of mbt and Rap1 are linked during ZA morphogenesis, it as found that, as it is the case for Mbt , Rap1 is required to stabilize E-Cad::GFP at the photoreceptor ZA. However, the mobile fraction estimated for E-Cad is much higher in Rap1IR cells than in mbtP1 null cells (evaluated at ~70% for Rap1IR and 45% for mbtP1). Together with the finding that Mbt accumulation at the ZA is decreased in Rap1IR cells, FRAP data are therefore compatible with Mbt mediating part of the function of Rap1 in promoting E-Cad stability. However, the much larger mobile fraction were estimated in the Rap1IR genotype when compared to mbtP1 photoreceptors indicates that Rap1 must also regulate E-Cad stability independently of Mbt. The longer time scale for E-Cad::GFP to recover in Rap1IR cells when compared to mbtP1 mutant cells is compatible with Rap1 functioning, in part, through promoting E-Cad delivery (Walther, 2018).
The reorganization of cells in response to mechanical forces converts simple epithelial sheets into complex tissues of various shapes and dimensions. Epithelial integrity is maintained throughout tissue remodeling, but the mechanisms that regulate dynamic changes in cell adhesion under tension are not well understood. In Drosophila melanogaster, planar polarized actomyosin forces direct spatially organized cell rearrangements that elongate the body axis. This study shows that the LIM-domain protein Ajuba is recruited to adherens junctions in a tension-dependent fashion during axis elongation. Ajuba localizes to sites of myosin accumulation at adherens junctions within seconds, and the force-sensitive localization of Ajuba requires its N-terminal domain and two of its three LIM domains. Ajuba stabilizes adherens junctions in regions of high tension during axis elongation, and that Ajuba activity is required to maintain cell adhesion during cell rearrangement and epithelial closure. These results demonstrate that Ajuba plays an essential role in regulating cell adhesion in response to mechanical forces generated by epithelial morphogenesis (Razzell, 2018).
E-cadherin-mediated cell-cell adhesion is fundamental for epithelial tissue morphogenesis, physiology and repair. E-cadherin is a core transmembrane constituent of the zonula adherens (ZA), a belt-like adherens junction located at the apicolateral border in epithelial cells. The anchorage of ZA components to cortical actin filaments strengthens cell-cell cohesion and allows for junction contractility, which shapes epithelial tissues during development. This study reports that the cytoskeletal adaptor protein Girdin physically and functionally interacts with components of the cadherin-catenin complex during Drosophila embryogenesis. Fly Girdin is broadly expressed throughout embryonic development and enriched at the ZA in epithelial tissues. Girdin associates with the cytoskeleton and co-precipitates with the cadherin-catenin complex protein alpha-Catenin (alpha-Cat). Girdin mutations strongly enhance adhesion defects associated with reduced DE-cadherin (DE-Cad) expression. Moreover, the fraction of DE-Cad molecules associated with the cytoskeleton decreases in the absence of Girdin, thereby identifying Girdin as a positive regulator of adherens junction function. Girdin mutant embryos display isolated epithelial cell cysts and rupture of the ventral midline, consistent with defects in cell-cell cohesion. In addition, loss of Girdin impairs the collective migration of epithelial cells, resulting in dorsal closure defects. It is proposed that Girdin stabilizes epithelial cell adhesion and promotes morphogenesis by regulating the linkage of the cadherin-catenin complex to the cytoskeleton (Houssin, 2015).
This study suggests that Girdin is required for epithelial tissue morphogenesis and integrity. Specifically, Girdin coordinates collective cell migration, a function that probably depends on the ability of Girdin to organize the actin cytoskeleton. Moreover, the data indicate that Girdin strengthens cell-cell adhesion by promoting anchorage of the cadherin-catenin complex to the cytoskeleton. Girdin might realize this function by favoring the polymerization and organization of the cortical F-actin ring associated with the ZA. Alternatively, Girdin might be directly involved in the bridging of the cadherin-catenin complex to microfilaments, an intriguing possibility suggested by the association of Girdin with α-Cat. Multiple α-Cat interaction partners have actin-binding activity, and so do Girdin and mammalian Girdin. The data therefore contribute to the understanding of adherens junction regulation, which is crucial for epithelial tissue morphogenesis, physiology and homeostasis. It is likely that the function of Girdin in epithelial tissue cohesion and morphogenesis is evolutionarily conserved, as mammalian Girdin interacts with Par-3 that sustains cell-cell cohesion, and controls epithelial cyst formation in three-dimensional (3D) cell culture. In line with a putative role for mammalian Girdin in cell-cell adhesion, neuroblasts show cohesion defects in Girdin knockout mice. Thus, these data put into perspective the emerging idea that human Girdin is an interesting target to limit cell invasion in cancer. Girdin inhibition might exacerbate loss of cell-cell adhesion and cell dissemination in tumor cells with altered E-cadherin functions, as suggested by the strong enhancement of the shg zygotic mutant phenotype by loss of Girdin. A better understanding of Girdin functions will help to uncover whether this protein is an attractive target for therapeutic intervention (Houssin, 2015).
Intercellular endosomes (IEs) are endocytosed vesicles shuttled through the adherens junctions (AJs) between two neighboring epidermal cells during Drosophila dorsal closure. The cell-to-cell transport of IEs requires DE-cadherin (DE-cad), microtubules (MTs) and kinesin. However, the mechanisms by which IEs can be transported through the AJs are unknown. This study demonstrate the presence of AJ-associated pores with MTs traversing through the pores. Live imaging allows direct visualization of IEs being transported through the AJ-associated pores. By using an optogenetic dimerization system, it was observe that the dimerized IE-kinesin complexes move across AJs into the neighboring cell. The AJ-associated pores also allow intercellular movement of soluble proteins. Importantly, most epidermal cells form dorsoventral-oriented two-cell syncytia. Together, this study presents a model in which an AJ-associated pore mediates the intercellular transport of IEs and proteins between two cells in direct contact (Yang, 2018).
During epithelial contraction, cells generate forces to constrict their surface and, concurrently, fine-tune the length of their adherens junctions to ensure force transmission. While many studies have focused on understanding force generation, little is known on how junctional length is controlled. This study shows that, during amnioserosa contraction in Drosophila dorsal closure, adherens junctions reduce their length in coordination with the shrinkage of apical cell area, maintaining a nearly constant junctional straightness. This study reveals that junctional straightness and integrity depend on the endocytic machinery and on the mechanosensitive activity of the actomyosin cytoskeleton. On one hand, upon junctional stretch and decrease in E-cadherin density, actomyosin relocalizes from the medial area to the junctions, thus maintaining junctional integrity. On the other hand, when junctions have excess material and ruffles, junction removal is enhanced, and high junctional straightness and tension are restored. These two mechanisms control junctional length and integrity during morphogenesis (Sumi, 2018).
This study has shown that adherens junction length is actively controlled during epithelial contraction, leading to a constant junctional straightness upon cell contraction. It is proposed that two different cellular mechanisms are at the origin of this length control. First, a modulation of actomyosin localization from the medial areas toward junctions allows for integrity maintenance by increasing actomyosin contraction along the junctions upon junctional stretch. Possibly, E-cadherin dilution triggered by junctional stretching is responsible for the myosin recruitment, a possibility supported by the anti-correlation between junctional myosin and E-cadherin. Second, the junction removal rate depends on junctional straightness, maintaining a preferred junctional straightness. Presumably, the endocytosis machinery is at the origin of this maintenance. As junctional tension is dependent on junctional straightness, maintaining junctional straightness could also allow it to maintain junctional tension, which remains approximately constant during DC (Sumi, 2018).
These mechanisms rely on a complex interplay between junctional E-cadherin, actomyosin, and tension. Indeed, this study finds that (1) higher junctional E-cadherin levels are associated with lower myosin levels and (2) decreased junctional straightness is associated with lower junctional tension. These observations suggest that E-cadherin is an essential component for the control of adherens junction length and for actomyosin localization in the cell. In cell culture experiments, E-cadherin has already been determined to be a key player in cell-cell mechanical interplay and shown to interact closely with the actomyosin cortex. In the case of the AS tissue, an enrichment of actomyosin was observed at junctions when E-cadherin levels are reduced. A similar interaction has been previously identified in the context of cytokinesis. Ghd results indicate a general interplay between E-cadherin and myosin that could play a role in several morphogenetic rearrangements. Such an interplay is likely to involve actin-associated proteins that could respond to changes in mechanical states of the adherens junction. Consistent with this idea, previous studies identified dynamic enrichment of the formin Diaphanous or of vinculin specifically at adherens junctions (Sumi, 2018).
Interestingly, this study observed that when junctions are stretched and E-cadherin density decreased, the contractile pulses of the AS tissue are arrested, possibly due to the observed myosin recruitment to junctions. Such pulses of contraction have been identified in several tissues during Drosophila, chicken, and mouse embryo development. They have been associated with global tissue contraction and epithelial remodeling. This study has shown that the pulsatile activity can be tuned by junctional stretch and E-cadherin levels. The results therefore uncover a direct coupling between cell-cell adhesion and the contractile activity of the cell (Sumi, 2018).
Finally, the rate of junction removal appears to depend on junctional straightness. How is junction straightness sensed to dictate junction removal? Laser ablation experiments indicate that junctions with lower straightness also have lower mechanical tension. In single cells, it is known that the endocytic rate is modulated by the tension of the cell membrane. In a developmental context, endocytosis could also be tension sensitive, resulting in the control of junctional straightness through junctional tension. Alternatively, E-cadherin density could play a role in the control of the junction turnover rate. Indeed, an increase of E-cadherin density associated to a decrease in junction straightness could trigger an enhanced endocytic rate, therefore controlling junction straightness and tension.
The mechanisms regulating junction length and integrity identified here involve the activity of two essential cellular machineries. They are therefore likely to act during many morphogenetic events that involve tissue contraction, such as neural tube closure, optic cup morphogenesis, or wound healing (Sumi, 2018).
Coordinated rearrangements of cytoskeletal structures are the principal source of forces that govern cell and tissue morphogenesis. However, unlike for actin-based mechanical forces, knowledge about the contribution of forces originating from other cytoskeletal components remains scarce. This study has establish microtubules as central components of cell mechanics during tissue morphogenesis. Individual cells were found to be mechanically autonomous during early Drosophila wing epithelium development. Each cell contains a polarized apical non-centrosomal microtubule cytoskeleton that bears compressive forces, whereby acute elimination of microtubule-based forces leads to cell shortening. It was further established that the Fat planar cell polarity (Ft-PCP) signalling pathway couples microtubules at adherens junctions (AJs) and patterns microtubule-based forces across a tissue via polarized transcellular stability, thus revealing a molecular mechanism bridging single cell and tissue mechanics. Together, these results provide a physical basis to explain how global patterning of microtubules controls cell mechanics to coordinate collective cell behaviour during tissue remodelling. These results also offer alternative paradigms towards the interplay of contractile and protrusive cytoskeletal forces at the single cell and tissue levels (Singh, 2018).
During development individual cells assemble into complex tissues and organs with specialized forms and functions. Tissue morphogenesis is driven by mechanical forces that are generated by the cytoskeleton within cells and transmitted in a coordinated manner through adhesion molecules across neighbouring cells. The best-studied cytoskeletal component is actin, which, together with other proteins, forms protrusive and contractile arrays, a fundamental constituent of rearrangements on the single cell and tissue levels. Recent work has suggested that microtubules, similar to actin, can also generate forces in cells. However, understanding of the contribution of microtubules to cell mechanics, cell shape changes and force coordination during morphogenesis remains poor. This is mainly due to the fact that many current models describing the mechanical state of tissues during shape changes focus on actomyosin dynamics and/or rely on continuum mechanics. These studies, which are based on coarse-grain observations of cell movements or cell shape changes, reveal only part of the physical mechanisms that drive morphogenesis and do not directly investigate the physicomechanical context of tissue remodelling. To understand the relationship between cell mechanics, force patterning and molecular structure, this study investigated the mechanical properties of microtubules at high spatiotemporal resolution using wing development in Drosophila melanogaster as a paradigm (Singh, 2018).
During pupal wing development, non-centrosomal microtubules form an apical array of parallel microtubule bundles that are globally aligned along the proximal-distal (P-D) axis. Patterning of the microtubule cytoskeleton depends on the Ft-PCP signalling pathway and occurs during the early phase of wing reshaping (that is, between 14 and 18 h after puparium formation, or APF). This patterning is associated with extensive changes in cell shape, cell divisions and cell-cell rearrangements. In the Drosophila wing, the Ft-PCP pathway further orients cell elongation and cell divisions along the P-D axis to induce wing tissue elongation. Intriguingly, rescue of the Hippo pathway in Ft-PCP mutant animals, in which microtubule alignment is impaired, aberrant development results in perturbed cell elongation and an abnormal rounded wing shape, suggesting that there is an interdependence between these events. Therefore this study explored the possibility that microtubule-based cell mechanics control cell and tissue shape during early wing development between 16 and 18 h APF (Singh, 2018).
Tissue remodelling is driven by intrinsic and extrinsic mechanisms, and it has previously been shown that extrinsic mechanical forces act during the late phase of wing reshaping (starting 18 h APF). These forces are generated by hinge contraction of the wing that is attached to the cuticle on the distal side. This study evaluated the mechanical autonomy of individual cells before hinge contraction at an earlier developmental stage (that is, between 16 and 18 h APF). This was done by isolating a single cell (or a small patch of cells) using a single-pulse multipoint procedure to cut AJs, thus mechanically uncoupling individual cells from their surrounding. Strikingly, the shape of individual isolated cells did not change significantly upon laser ablation at 17-18 h APF, when cells in the wing are already elongated. The same result was obtained when patches of cells were isolated. Additional analyses of the Feret's diameter before and after ablation showed a small isotropic decrease in cell size, providing evidence that at this early stage, individual cells are not influenced by the neighbouring cells or by tissue-scale forces in a polarized fashion. Consistently, analysis of animals expressing a mutant form of dumpy protein, an extracellular matrix protein associated with tissue remodelling at later developmental stages, showed no substantial differences in wing shape compared to wild-type wings at 18 h APF. Together, these experiments argue that unlike later stages, cell autonomous forces are the major drivers of initial cell shape changes between 16 and 18 h APF (Singh, 2018).
To identify the molecular mechanism underlying cell autonomous shape formation, the distribution and dynamics of two cytoskeletal force-generators were investigated: microtubules and non-muscle myosin II (MyoII) as a component of the actomyosin cytoskeleton. MyoII was detected at the apical cell cortex at the level of AJs. A subsequent analysis of the signal distribution within single cells revealed a planar polarized distribution of MyoII along the P-D axis, which correlated with increased tension along the same junctions. As MyoII provides contractile forces, this should result in P-D junctional shortening upon laser ablation. However, this is inconsistent with the current ablation experiments, suggesting that there is an opposing force present. Interestingly, staining of microtubules showed planar polarized apical microtubules along the P-D axis at the level of AJs. Microtubules are the stiffest cytoskeletal filaments, with a persistence length on the order of millimetres. Microtubules are therefore well suited to balance the tension generated by actomyosin contraction. Consistently, the distribution of microtubules and MyoII in mechanically isolated cells remain polarized. In addition, microtubule and MyoII polarity was preserved in dumpy mutant wings at 18 h APF, indicating that they are polarized in a cell autonomous fashion. The possible role of the atypical myosin Dachs, a downstream component of the Fat signalling pathway, was also analyzed. Dachs mutant wings showed no change in cell elongation or microtubule polarity, which is consistent with recent work reporting that recombinant Dachs does not have ATPase activity and can therefore not function as a molecular motor. Together, these observations argue that planar polarized microtubules may balance actomyosin tensional forces that pull on P-D junctions and stabilize cell shape (Singh, 2018).
To validate this hypothesis, and to elucidate the dynamic and functional role of microtubules in cell mechanics, their properties were investigated during wing development. Live cell imaging of EOS-α-tubulin (EOS-Tub) showed that microtubules were not static but engaged in complex and dynamic rearrangements. An analysis of microtubule straightness showed that in wing cells, virtually all microtubules along the P-D axis were bent, consistently undergoing short wavelength buckling (~3 μm) near the cell cortex. It was further observed that growing microtubules remain straight and only start to buckle after they reach the cell cortex, exhibiting local short wavelength buckling near these sites. This result indicates that microtubule polymerization can generate considerable compressive forces to induce microtubule buckling (Singh, 2018).
Next, whether buckling of microtubules in Drosophila wing epithelium is indeed a result of forces acting on microtubules was also investigated, as suggested by the current experiments and in vitro studies, or whether the cellular environment yields more flexible microtubules. This is important, as buckling of flexible microtubules would rule out a role in balancing actomyosin contractility. To probe the forces of single microtubule filaments in vivo, individual microtubules were cut by laser ablation and the subsequent relaxation was monitored using live imaging. Previously curved microtubules rapidly straighten out, thus verifying that microtubules are indeed loaded with compressive forces. Finally, it was also observed that local ablation of microtubules triggers a rapid translocation of the adjacent junction. This finding supports the idea that non-centrosomal microtubules continuously generate pushing forces via polymerization that may then be stored as compressive forces in a polarized fashion to balance contractile forces generated by junctional actomyosin (Singh, 2018).
How are microtubules polarized along the P-D axis? While the molecular mechanism has remained elusive, previous work has established that the Ft-PCP signalling pathway aligns the apical microtubule network along the P-D axis by regulating association sites of microtubules with AJs. Considering the observed stability of aligned microtubules, whether directional differences in microtubule dynamics could serve as a mechanism for the planar polarization of microtubules was tested. Monitoring of EB1 tagged with green fluorescent protein (EB1-GFP) revealed two populations of microtubule-plus ends: fast growing microtubules with a growth velocity of 24.43 ± 0.43 7mi;m min-1 (mean ± s.e.m.), and slow growing microtubules with a velocity of 17.06 ± 0.26 7mi;m min-1. A further analysis showed that the microtubule growth rates depended on relative localization within cells as well as the growth angle relative to the P-D axis. Microtubule growth rates in the cell interior were higher compared to the cell cortex. Similarly, microtubules along the P-D axis grew faster than microtubules growing perpendicular to the P-D axis along the A-P axis, establishing a spatial gradient in microtubule growth velocity. The lower growth rate along the A-P axis close to the cell periphery suggests that there is more frequent pausing and switching between polymerization and depolymerization of microtubules, thus indicating a decreased stability of A-P oriented microtubules (Singh, 2018).
It was reasoned that over time, such differences in dynamics and stability may result in predominantly P-D aligned microtubules. To test this hypothesis, the cortical residence time was analyzed of microtubules as a function of their angle with respect to the P-D axis. Intriguingly, it found that microtubules that interact with the P-D cell cortex have a longer lifetime than microtubules interacting with the A-P cortex. Upon closer inspection, dynamic cycles of short-lived interactions of microtubules with A-P junctions were noted followed by depolymerization. Importantly, A-P oriented microtubules do not show buckling behaviour, which is in contrast to P-D oriented microtubules, but rather undergo catastrophe soon after interaction with A-P oriented cell junctions. This result suggests that microtubule-plus ends are less stable at these sites and thus cannot sustain long-lasting interactions with the cell cortex, which are required to generate compressive forces. Building on these observations, in silico probing was performed to see whether the angular difference in lifetime may indeed be sufficient for microtubule polarization. Assuming a random orientation for de novo formed microtubules, the lifetime of each microtubule was defined as a function of the angle with a maximal lifetime along the P-D axis. Upon expiration, individual microtubules were re-introduced into the system at random angles, therefore keeping the total number of microtubules constant. Consistent with the in vivo observations, the simulation reached a steady-state at which a constant fraction of microtubules polarized along the P-D axis. Taken together, these observations point to a mechanism whereby microtubule stability regulates the planar alignment of the microtubule cytoskeleton along the P-D axis, which in turn directs cell mechanics along this axis. These data place directional microtubule stability upstream of proposed mechanisms of how cell shape influences microtubule alignment. Furthermore, these results are consistent with previous findings that microtubule association with P-D oriented AJs during the initial stage of wing development depends on Ft-PCP signalling (Singh, 2018).
Having established that planar polarized microtubule-based forces shape single cells, their mechanical coupling and integration into tissue-level mechanics were investigated. In a first round of experiments, transcellular coupling of microtubules were investigated on the ultrastructural level using transmission electron microscopy (TEM). In agreement with previous work, AJs were juxtaposed in neighbouring cells associated with microtubule filaments that span across cells in wild-type wings, forming supracellular cables analogous to myosin cables. Notably, no such association was observed in ftl(2)fdd1 / ftl(2)fd dGC13 (ft d) and ftl2 fd/ftGRV;ActP-Gal4/UAS-FtΔECDΔN-1 (N1) mutant wings, in which microtubules are randomly oriented in wing cells, therefore providing structural support for the Ft-PCP-dependent stabilization of microtubule-based forces at P-D oriented AJs. Consistently, ft mutant clones showed a fragmented microtubule cytoskeleton, arguing that there is Ft-PCP-dependent stabilization of microtubules via coupling at AJs (Singh, 2018).
To further validate the role of polarized transcellular microtubule stability in tissue mechanics and organization, tissue shape changes were observed upon acute perturbation of microtubule-based forces. To control microtubule dynamics in a precise spatial and temporal manner, he recently developed photostatin (PST1)35, a photo-switchable analogue of combretastatin A-4 (CA4)36 was used. The drug was applied to directly test the requirement of microtubules for cell shape maintenance. Notably, it was found that the exposed wing area contracted along the P-D axis upon microtubule inhibition. Quantitative cell shape analysis showed a small but significant reduction in the elongation index (EI) in selective regions where the drug was activated, arguing that polarized tissue stabilization is via microtubule-based forces. Finally, overexpression of the microtubule-severing protein Spastin increased cell shape heterogeneity. These results are consistent with the hypothesis that an intact polarized microtubule cytoskeleton is not only required for the maintenance of anisotropic cell shape but also critically involved in shaping the whole tissue during morphogenesis via polarized transcellular force stability (Singh, 2018).
Understanding the role of microtubules during animal development has so far been limited, especially because of a shortage of methods suitable to demonstrate causality in vivo. Taking advantage of complementary genetic, chemical, numerical and microscopy approaches, these experiments unveil polarized microtubule-based compressive forces as an alternative principle for stabilizing and maintaining cell and tissue shape during morphogenesis. Alignment of microtubules along the P-D axis was found to be based on increased longevity and polymerization of microtubules interacting with P-D oriented AJs compared to non-polarized microtubules. The result of this microtubule patterning along the P-D axis is an asymmetric distribution of protruding forces, which are stored in a polarized fashion via compressive loads on microtubules. Considering that actomyosin and microtubules are both planar polarized, it is plausible to envision that the observed compressive load on microtubules plays an active role in balancing actin-based contractile forces, resulting in the cell mechanical autonomy observed in the laser ablation experiments. Intriguingly, it was recently shown that acetylation of microtubules increases their mechanical resistance and that microtubules undergo self-repair upon damage. These important features support the role of the microtubule cytoskeleton as a site of long-term compressive force storage. Finally, evidence is provided that planar polarized microtubules are coupled at AJs across individual cells, bridging forces on the tissue level via polarized transcellular stability. Although the molecular identity remains elusive, the data suggests an involvement of AJ-associated proteins organized by the Ft-PCP pathway in this process (Singh, 2018).
Collectively, this work provides evidence that PCP-based planar patterning of the microtubule cytoskeleton not only results in polarized cell-autonomous forces but also coordinates global force patterning during tissue morphogenesis. The proposed mechanism establishes the Ft-PCP pathway at the onset of cell and wing elongation, before shape changes, due to extrinsic mechanical forces. Consistently, in a Ft-PCP mutant, in which initial elongation fails, consecutive remodelling by extrinsic tensile forces cannot rescue these length defects, therefore leading to shorter and rounder adult wings. Considering that the Ft-PCP signalling pathway controls a variety of dynamic cell population in vertebrates, the microtubule-based mechanism described in this study is likely to be physiologically relevant beyond wing development (Singh, 2018).
The Hippo signaling network controls organ growth through YAP family transcription factors, including the Drosophila Yorkie protein. YAP activity is responsive to both biochemical and biomechanical cues, with one key input being tension within the F-actin cytoskeleton. Several potential mechanisms for biomechanical regulation of YAP proteins have been described, including tension-dependent recruitment of Ajuba family proteins, which inhibit kinases that inactivate YAP proteins, to adherens junctions (AJ). This study investigated the mechanism by which the Drosophila Ajuba family protein, Ajuba LIM protein (Jub) is recruited to adherens junctions, and the contribution of this recruitment to the regulation of Yorkie. Alpha-catenin is identifed as the mechanotransducer responsible for tension-dependent recruitment of Jub by identifying a region of α-catenin that associates with Jub, and by identifying a region, which when deleted, allows constitutive, tension-independent recruitment of Jub. Increased Jub recruitment to alpha-catenin is associated with increased Yorkie activity and wing growth, even in the absence of increased cytoskeletal tension. These observations establish alpha-catenin as a multi-functional mechanotransducer and confirm Jub recruitment to alpha-catenin as a key contributor to biomechanical regulation of Hippo signaling (Alegot, 2019).
To evaluate the role of α-catenin in the recruitment of Jub to AJ, Jub localization was examined in wing discs isolated from animals expressing truncated forms of α-catenin. This was achieved by using RNAi to knock-down expression of endogenous α-catenin and expressing RNAi-insensitive Venus- or V5-tagged α-catenin transgenes under UAS-Gal4 control. Transgenes were expressed in posterior cells using an en-Gal4 driver, so that anterior cells could serve as a control for Jub localization. Full-length α-catenin expressed under UAS-Gal4 control rescued both the lethality associated with knockdown of α-catenin, and Jub localization.
α-catenin has similarity to Vinculin in N-terminal, middle, and C-terminal regions termed VH1, VH2, and VH3. An initial series of constructs deleted regions of α-catenin centered around these Vinculin homology regions, as well as separate deletions of either the N-terminal or C- terminal half of VH2. Constructs lacking either the N- or C-terminus of α-catenin (ΔVH1 or ΔVH3) could not rescue the lethality associated with α-catenin knockdown. Thus, to enable visualization of the consequences of these deletions on Jub, conditional knockdown and expression of α-catenin was achieved using a temperature sensitive-allele of Gal80 (Gal80ts) that represses Gal4-driven expression at 18°C but not at 29°C. After a 30 hour shift to higher temperature, Jub was mostly lost from apical cell junctions. However, E-cadherin was also mostly lost, indicating that these regions of α-catenin are required to maintain AJ. This is consistent with the roles of F-actin and α-catenin in stabilizing AJ, as the VH1 region is required for association with β-catenin and thus localization to AJ, and the VH3 region is required for association with F-actin. The ΔVH1 and ΔVH3 constructs also did not themselves localize to junctions. The loss of Jub from cell junctions under these conditions is consistent with studies indicating its association with AJ, but does not provide specific information about its interactions with α-catenin (Alegot, 2019).
Conversely, deletions within the central region of α-catenin increased Jub recruitment to AJ. Deletion of the entire VH2 region increased Jub recruitment but was also associated with abnormal cell morphology and E-cadherin localization. However, two smaller deletions comprising the N- and C-terminal halves of the VH2 region (ΔVH2-N and ΔVH2-C) rescued knock-down of α-catenin, resulting in cells that appear to have normal localization of E-cadherin and α-catenin. The ΔVH2-C deletion also appears to have normal Jub localization. In contrast, the ΔVH2-N deletion is associated with increased Jub localization at AJ. Moreover, in cells with the ΔVH2-N deletion, Jub is distributed relatively uniformly around cell junctions, whereas in cells with wild-type α-catenin, Jub localization to junctions is often punctate. The ΔVH2-N deletion was not only associated with increased Jub recruitment when expressed in place of endogenous α-catenin; it could also promote Jub recruitment even when expressed in the presence of endogenous α-catenin. Thus, expression of this isoform dominantly increases Jub recruitment to AJ (Alegot, 2019).
To further investigate the influence of α-catenin on Jub, smaller deletion constructs were created. To aid in the design of these additional constructs, Phyre2 was used to predict the structure of Drosophila α-catenin, based on its sequence similarity to mammalian α-catenin proteins with experimentally-determined structures. Mammalian α-catenin consists largely of a series of α-helical bundles: two N-terminal 4-helix bundles (N1 and N2) that share one long α-helix; three central 4-helix bundles (M1, M2, and M3), and a C-terminal 5-helix bundle. These structural features are also predicted for Drosophila α-catenin (Alegot, 2019).
The ΔVH2-N deletion that increases Jub recruitment to AJ deletes both the M1 and M2 helical bundles. Thus, separate deletions were created of either the M1 or M2 bundles. Deletion of the M2 bundle (ΔM2) slightly increased Jub localization to AJ, but a much stronger increase in Jub binding was detected when the M1 bundle was deleted (ΔM1a or ΔM1b). Recruitment of Jub to AJ is normally promoted by cytoskeletal tension. To examine the possibility that M1 deletions increase Jub recruitment by increasing tension, levels were examined of junctional myosin (using a GFP-tagged myosin light chain) that correlate with junctional tension, but no difference was observed. F-actin levels were similarly unaffected. Staining was carried out specifically for the phosphorylated (activated) form of myosin regulatory light chain (pMLC) in discs expressing Jub:GFP. This revealed similar levels of pMLC in control cells and cells expressing α-catenin ΔM1b, whereas expression of rok RNAi or an activated form of Rok visibly decreased or increased, respectively, junctional levels of both pMLC and Jub. Since Jub associates with α-catenin, the observation that deletion of the M1 bundle increases Jub localization to AJ without increasing tension suggests that deletion of this region alters the structure of α-catenin in a way that makes a Jub-binding region more accessible. The M1 bundle might thus act as an 'inhibitory' region that prevents Jub binding, with this inhibition normally alleviated in wild-type α-catenin by a conformational change induced by high tension. Alternatively, deletion of the M1 bundle might destabilize a non-Jub binding conformation of α-catenin and thereby indirectly increase accessibility of a Jub-binding region (Alegot, 2019).
While both Jub and Vinculin exhibit tension-dependent association with α-catenin, the results suggest that their interactions are distinct. A region initially identified as inhibitory for Vinculin binding, corresponds to the M3 helical bundle and the linker between VH2 and VH3. Moreover, the M1 helical bundle includes the Vinculin binding interface. To directly compare the influence of α-catenin deletions on Jub and Vinculin binding, advantage was taken of a Drosophila genomic GFP-tagged Vinculin (Vinc:GFP). Both of the M1 deletions substantially reduced, but did not eliminate, Vinculin localization to AJ, which fits with observations that Vinculin interacts with sequences in the M1 bundle, but implies that additional mechanisms also contribute to Vinculin localization. Deletion of the M2 bundle increased Vinculin localization to AJ, consistent with observations that M2 participates in interactions that stabilize a conformation of α-catenin that is 'closed' with respect to Vinculin binding (Alegot, 2019).
If M1 deletions mimic the influence of cytoskeletal tension on the ability of α-catenin to bind to Jub, the increased recruitment of Jub in M1 deletion isoforms could occur even in the absence of tension. To test this, Jub localization was examined in flies expressing α-catenin constructs and with cytoskeletal tension decreased by RNAi of Rho-associated protein kinase (Rok), a promoter of myosin activity. In the presence of full-length α-catenin, rok RNAi decreases Jub recruitment to junctions. Conversely, in the presence of deletions that include the M1 bundle, Jub recruitment to junctions remains elevated. Quantitation of Jub at junctions, normalized to E-cadherin, revealed a decrease in Jub recruitment when tension is lowered even in cells expressing M1 deletions, but Jub recruitment nonetheless remains above that in control cells. Thus, these deletions recruit Jub even under low-tension conditions, which implicates α-catenin as the key mechanotransducer responsible for tension-dependent recruitment of Jub (Alegot, 2019).
Cytoskeletal tension promotes Yki activity, and Yki activity is suppressed by knockdown of Jub. However, other mechanisms by which tension might increase Yki activity have also been suggested, such as an influence on spectrins. The observation that deletions of the M1 bundle lead to increased Jub at AJ without increasing myosin identifies a condition under which it was possible to determine whether recruitment of Jub to junctions is sufficient to increase Yki activity, and thus distinguish the contribution of Jub from other potential influences of cytoskeletal tension (Alegot, 2019).
Yki activity was evaluated by examining ex-lacZ expression, which is a reporter for the Yki target gene expanded (ex). Expression of α-catenin with deletion of the M1 bundle increased ex-lacZ, whereas expression of full length α-catenin did not. Increased Yki activity is associated with increased accumulation of Yki in the nucleus, and nuclear levels of Yki were slightly increased in wing disc cells expressing M1 region deletions. To further examine the influence of the M1 deletion, adult wing size was examined, as wing growth is promoted by Yki activity. Expression of M1 deletion isoforms throughout the developing wing under nub-Gal4
control increased wing size as compared to wings expressing wild-type α-catenin. The increased Yki activity and wing growth in M1 deletion mutants are Jub-dependent, because they are reversed by RNAi of jub. As mutation or knock-down of Vinculin has only minor phenotypic consequences in Drosophila, the effects of M1 deletion on Yki activity and wing growth cannot be attributed to loss of Vinculin. Altogether, these observations establish that increased Jub recruitment to junctions can be sufficient to elevate Yki activity. This strongly supports the conclusion that Jub recruitment to AJ is a key component of the biomechanical response linking cytoskeletal tension to Yki activity (Alegot, 2019).
However, analysis of the larger ΔVH2N deletion suggests that the role of α-catenin in promoting Yki activity is more complex. Although expression of ΔVH2N α-catenin increased Jub recruitment at AJ, it did not detectably increase ex-lacZ expression or wing size. Similarly, even though deletion of the M2 helical bundle modestly increased Jub recruitment to AJ, it did not detectably increase ex-lacZ expression or wing size. Thus, it is inferred that additional features of α-catenin may contribute to Yki regulation (Alegot, 2019).
Jub requires α-catenin to localize to junctions. To map regions of
α-catenin that mediate association with Jub, co-immunoprecipitation experiments were performed in Drosophila S2 cells expressing transfected constructs. Initially, co- immunoprecipitation of Jub with α-catenin full length, VH1, VH2, and VH3 region constructs was compared. Significant association of Jub and a VH1 region construct was detected. Conversely, association of Jub with full length, VH2, or VH3 constructs was weaker, and close to the non- specific background. The association with the VH1 region of α-catenin was not as strong as binding of Jub to Warts, which was included as a positive control. Association of Jub with the VH1 region is consistent with reports that the mammalian Ajuba protein can associate with an N- terminal fragment of α-catenin, corresponding roughly to VH1. The stronger association of Jub with a VH1 fragment as compared to full length α-catenin is consistent with normal binding depending upon junctional tension, as S2 cells are not epithelial, so it would be expected that the transfected α-catenin would be in a low-tension conformation. Moreover, co-immunoprecipitation experiments comparing M1 bundle deletion constructs to full length α-catenin revealed that M1 deletions significantly increased association of Jub with α-catenin (Alegot, 2019).
To further refine the Jub-binding region, the predicted Drosophila α-catenin structure was used to design constructs corresponding to the N1 or N2 helical bundles. No significant co-immunoprecipitation with the N1 bundle was detected, whereas co- immunoprecipitation with N2 was comparable to that for the VH1 region construct. Thus, it is inferred that the primary site of association of Jub with α-catenin is within N2. While the simplest model would be that Jub directly binds to this region of α-catenin, it remains possible that their association is mediated through other proteins. The N2 bundle is near the M1 bundle, but they do not directly contact each other in the predicted structure. Thus, the effect of M1 deletion on Jub binding may be an indirect consequence of a conformational change in α-catenin when this region is deleted, or reduced stability of a more closed conformation, rather than the M1 bundle directly obscuring a binding site within N2. Attempts were also mad to examine the consequences of loss of Jub recruitment to junctions by expressing α-catenin with N2 deleted in vivo. However, this protein failed to rescue the viability of wing disc cells expressing α-catenin RNAi, so this could not be examined. Moreover, when expressed in wild-type cells, the ΔN2 construct failed to localize to adherens junctions (Alegot, 2019).
These results establish key features of α-catenin mechanotransduction and tension- dependent regulation of Yki activity. They implicate α-catenin as a multi-functional mechanotransducer that associates with both Vinculin and Jub upon a tension-induced conformational change in α-catenin, but through distinct sites. The complete structure of the open conformation of α-catenin has not been determined, but it is inferred that it makes Jub- associating regions accessible. M1 deletions also make the Jub-associating region more accessible, but it's not clear whether they do so in a manner similar to, or distinct from, the effect of tension. The observation that M1 deletions increase Yki activity also supports the crucial role of Jub recruitment to AJ in promoting Yki activity. This does not exclude the possibility that of other consequences of cytoskeletal tension contribute to Yki regulation, but clearly implicates recruitment of Jub as a key factor (Alegot, 2019).
alpha-Catenin is a key protein of adherens junctions (AJs) with mechanosensory properties. It also acts as a tumor suppressor that limits tissue growth. This study analyzed the function of Drosophila alpha-Catenin (alpha-Cat) in growth regulation of the wing epithelium. Different alpha-Cat levels led to a differential activation of Hippo/Yorkie or JNK signaling causing tissue overgrowth or degeneration, respectively. alpha-Cat can modulate Yorkie-dependent tissue growth through recruitment of Ajuba, a negative regulator of Hippo signaling to AJs but also through a mechanism independent of Ajuba recruitment to AJs. Both mechanosensory regions of alpha-Cat, the M region and the actin-binding domain (ABD), contribute to growth regulation. Whereas M is dispensable for alpha-Cat function in the wing, individual M domains (M1, M2, M3) have opposing effects on growth regulation. In particular, M limits Ajuba recruitment. Loss of M causes Ajuba hyper-recruitment to AJs, promoting tissue-tension independent overgrowth. Although M binds Vinculin, Vinculin is not responsible for this effect. Moreover, disruption of mechanosensing of the alpha-Cat ABD affects tissue growth, with enhanced actin interactions stabilizing junctions and leading to tissue overgrowth. Together, these findings indicate that alpha-Cat acts through multiple mechanisms to control tissue growth, including regulation of AJ stability, mechanosensitive Ajuba recruitment, and dynamic direct F-actin interactions (Sarpal, 2019).
One key factor that determines the impact of α-Cat on tissue growth, leading either to overgrowth or tissue degeneration, is the amount of α-Cat at AJs. Analysis of phenotypic defects resulting from the differential reduction of gene function revealed that Drosophila α-Cat is a negative regulator of tissue growth. Moderate α-Cat overexpression or moderate depletion of α-Cat or DEcad caused Yki activation and overproliferation. Moreover, consistent with these findings, a previous analysis of strong loss-of-function conditions for α-Cat, DEcad, or Arm led to the conclusion that a loss of the cadherin-catenin complex (CCC) reduces Yki activity and causes JNK-meditated tissue degeneration. This conclusion was at odds with some mammalian studies where the loss of E-cadherin or αE-catenin causes YAP or TAZ-dependent overgrowth in a number of different tissues and cell lines. The current data showing that α-Cat and DEcad limit Yki activity and tissue growth in Drosophila similar to mammalian tissues suggest a conserved functional relationship between AJs and the regulation of Yki/YAP/TAZ activity. Only when the Drosophila CCC is strongly depleted does a substantive activation of JNK signaling override Yki activation, causing tissue degeneration. This could potentially involve a direct inhibition of Yki by JNK (Sarpal, 2019).
α-Cat loss-of-function conditions in conjunction with a block of cell death defined three distinct phenotypic classes that broadly align with the progression sequence of epithelial cancer from adenoma (epithelial overgrowth), to adenocarcinoma (overgrowth associated with a partial loss of epithelial integrity), to carcinoma (loss of epithelial integrity with cells showing protrusive activity). Similarly, in mammalian cancer, such as human colon cancer is the down-regulation of αE-catenin associated with an increased propensity for cells to become invasive. On the other hand, loss of αE-catenin was reported to suppress colorectal adenomas induced by APC loss-of-function, an effect that could be mediated by the Rho-Rho kinase signalling-dependent cell death elicited by the loss of the CCC in an APC mutant background. Also in Drosophila, it was found that Rho1 is required for cell death resulting from the loss of α-Cat, likely mediated by the Rho1 signalling dependent activation of the JNK pathway (Sarpal, 2019).
Loss of α-Cat leads to a corresponding decline of DEcad and Arm at AJs. Quantification of CCC proteins in the late stage embryonic epidermis of zygotic α-Cat null mutants suggested that AJs can be retained and support normal tissue architecture when CCC levels are reduced to less than 10% of normal. As heterozygous α-Cat animals are normal, it is anticipated that reduction of α-Cat to somewhere between 50% and 10% of wild-type levels will cross a threshold that will increase Yki activity without activating JNK signaling sufficiently to cause cell death. How strongly α-Cat and DEcad have to be reduced to cause an activation of Yki remains to be explored. For example, reducing α-Cat could compromise the interactions between the AJ protein Echinoid and Salvadore, a Hippo binding partner important for normal Hippo activity, or compromise the interactions between Crb and Ex that could deregulate Hippo signaling. Loss of α-Cat could also affect actin polymerization as mature AJs suppress actin polymerization, whereas enhanced actin polymerization is a known activator of Yki. Reducing α-Cat could therefore directly promote actin polymerization prior to an overt defect in cell adhesion, and consequently stimulate Yki activity and tissue growth. Finally, loss of α-Cat may affect Yki not through the Hippo pathway as was documented for αE-catenin in mammalian keratinocytes (Sarpal, 2019).
Mammalian studies raised the possibility that αE-catenin could act independently of AJs to regulate tissue growth. In contrast, several observations argue that Drosophila α-Cat acts as an AJ component to limit tumorigenesis: (1) The mechanosensory properties of α-Cat regulate the recruitment of Jub to AJs, which stimulates Yki activity and tissue growth and implies that α-Cat is suspended between the E-cadherin/β-catenin complex and actomyosin. In particular, α-Cat mutants that disrupt mechanosensing and cause overgrowth (α-CatR-ΔM1 and α-CatR-H1) are effectively recruited to AJs where they support normal epithelial organization, suggesting that a specific junctional defect disrupts growth regulation. (2) A reduction of DEcad is also associated with hyperplastic tissue overgrowth similar to the partial loss of α-Cat. (3) The expression of a DEcad::αCat fusion protein restores both the epithelial organization and normal growth of α-Cat compromised wing discs, and rescues α-Cat null mutant clones which normally fail to develop in wing discs. Together, these data suggest that α-Cat acts as a component of the CCC at AJs to regulate tissue growth (Sarpal, 2019).
The mechanosensitive interactions between Jub and α-Cat are thought to transmit tissue tension into growth regulatory signals. Cytoskeletal tension enhances recruitment of Jub to junctional α-Cat where Jub forms a complex with Wts, preventing it from phosphorylating and hence deactivating Yki. Supporting this model, Jub and Yki-dependent tissue overgrowth was observed that correlates with an enhanced recruitment of Jub to AJs. In particular, the data suggest that the M1 domain acts as a gatekeeper for Jub recruitment. In the absence of M1, junctional Jub levels become strikingly high, suggesting that M region mechanosensing has become ineffective in limiting Jub recruitment, causing Yki activation and overgrowth. Tension is thought to cause a conformational change in the M region and an unfurling of the M1 domain exposing a Vinc binding site. However, Vinc is not required for limiting Jub recruitment and deletion of M1 causes a reduction of junctional Vinc but not a complete loss. This leaves the mechanism of how M1 moderates Jub recruitment unresolved. A second unknown binding partner of M1 may be involved or an intramolecular interaction between M1 and the Jub binding site in the N domain could control Jub binding (Sarpal, 2019).
This Jub recruitment-dependent model of how mechanotransduction by the α-Cat M region regulates tissue growth does not explain all the observations and therefore needs to be extended to incorporate additional mechanisms of how AJs can control tissue growth. First, this model cannot explain the Yki-dependent overgrowth precipitated by low α-Cat levels, and corresponding low Jub levels at AJs. Second, it was observed that two fusion proteins between DEcad and α-Cat, one containing both full-length proteins (DEcad::αCat) and one lacking the Jub binding domain in α-Cat (DEcadΔβ::αCatΔN), can both support normal growth in α-Cat KD tissue but only DEcad::αCat restored normal junctional Jub levels whereas DEcadΔβ::αCatΔN did not. Third, expression of α-CatR in α-Cat KD tissue restores α-Cat to approximately 63% of normal levels. However, Jub increases to 119% without a noticeable increase in tissue size. One possibility is that mechanical force distributed over fewer α-Cat molecules enhances the mechanosensory response of individual α-Cat molecules in a non-linear manner resulting in higher Jub recruitment. Evidence for such a mechanism was recently reported for the recruitment of Vinc to AJs in the Drosophila germband, and may be similar for Jub recruitment to AJ in that tissue. Fourth, removing the entire M region results in an α-Cat protein that can support normal wing development, implying that M region mechanosensing is not an essential aspect of regulating tissue growth. In light of the results with α-CatR-ΔM1, this can only be explained by assuming that removing M2 and M3 in addition to M1 has a compensatory effect. Whereas α-CatR and α-CatR-ΔM expression in α-Cat depleted PC tissue enhances Jub recruitment above AC control levels, loss of M2 or M3 causes Jub levels to remain below AC levels suggesting that these two domains somehow normally support the ability of α-Cat to recruit Jub, possibly by supporting the stability of the CCC at AJs. Collectively, these findings suggest that M region mechanosensing contributes to Jub recruitment and Hippo/Yki pathway regulation but that the actual mechanisms involved have considerable complexity and require further analysis to be resolved (Sarpal, 2019).
Increased levels of Jub were also observed in response to disrupting the mechanosensory properties of the α-Cat ABD. Disruption of the α1-helix of ABD did not only enhance F-actin binding in vitro but also stabilized the CCC at AJs in the wing epithelium as suggested by the current observations. A corresponding increase in Jub levels at AJs could account for the persistent overgrowth observed in α-Cat KD tissue expressing α1-helix compromised α-Cat (α-CatR-H1). Thus, the mechanosensory properties of the M region and the α-Cat ABD are both important for regulating Jub recruitment to AJs and growth regulation through the Hippo/Yki pathway. As changes in tissue tension are thought to modulate the Hippo/Yki pathway through junctional recruitment of Jub it was asked whether α-Cat mutants that compromise mechanosensing cause changes in tissue tension or viscoelasticity that could indirectly affect Jub recruitment to AJs. We did not observe such changes after laser ablation of cell-cell junction. This is consistent with the finding that replacing α-Cat with α-CatR-ΔM1 did not change junctional myosin II levels, the major tension generator in the wing disc epithelium. Taken together, these data suggest a direct molecular role of α-Cat in Jub recruitment and strongly argue that α-Cat operates as a crucial mechanosensor to regulate tissue growth (Sarpal, 2019).
In summary, it is concluded that α-Cat uses multiple mechanisms to act as an important regulator of tissue growth in the Drosophila wing disc epithelium. It is doing so at least in part by operating as a mechanotransducer, engaging both M region and ABD mechanosensing, to relay cytoskeletal tension into growth regulatory signals. One of these mechanisms involves the recruitment of Jub to the N domain of α-Cat that can be modulated by both mechanosensing mechanisms. However, α-Cat also engages mechanisms that are independent of the junctional recruitment of Jub to control tissue growth, which remain to be explored further (Sarpal, 2019).
Apical-basal polarity is a fundamental property of animal tissues. Drosophila embryos provide an outstanding model for defining mechanisms that initiate and maintain polarity. Polarity is initiated during cellularization, when cell-cell adherens junctions are positioned at the future boundary of apical and basolateral domains. Polarity maintenance then involves complementary and antagonistic interplay between apical and basal polarity complexes. The Scribble/Dlg module is well-known for promoting basolateral identity during polarity maintenance. This study reports a surprising role for Scribble/Dlg in polarity initiation, placing it near the top of the network-positioning adherens junctions. Scribble and Dlg are enriched in nascent adherens junctions, are essential for adherens junction positioning and supermolecular assembly, and also play a role in basal junction assembly. The hypotheses were tested for the underlying mechanisms, exploring potential effects on protein trafficking, cytoskeletal polarity or Par-1 localization/function. The data suggest that the Scribble/Dlg module plays multiple roles in polarity initiation. Different domains of Scribble contribute to these distinct roles. Together, these data reveal novel roles for Scribble/Dlg as master scaffolds regulating assembly of distinct junctional complexes at different times and places (Bonello, 2019).
Identifying the earliest symmetry-breaking events that initially position AJs, thereby setting the boundary between apical and basolateral domains, is a key aspect of understanding how polarity is established. This study reports that Scrib/Dlg, best known for roles as basolateral determinants during polarity maintenance, play a separate and surprising role in organizing AJs during polarity establishment, positioning them at the top of the polarity network (Bonello, 2019).
Scrib and Dlg are multidomain proteins with many partners, allowing them to serve diverse biological functions, from synaptogenesis to oriented cell division. The data reveal they play distinct roles during polarity establishment and polarity maintenance, likely engaging very different sets of binding partners. This is supported by the evolving localization pattern of Scrib/Dlg on the plasma membrane, with sequential co-localization with and roles in positioning AJ versus SJ proteins, suggesting the capacity to engage with and position distinct junctional and polarity proteins. These analyses also begin to dissect the underlying molecular basis. Scrib's PDZ domains are important for the precision of initial polarity establishment but are redundant with other mechanisms for polarity maintenance after gastrulation, though they regulate SJ positioning (Bonello, 2019).
AJs play a key role at the boundary between apical and basolateral domains, and building a functional junction is a multistep process. This includes assembling the core cadherin-catenin complex, positioning it, and supermolecular assembly. Assembly of the core complex appears to occur coincident with synthesis, and thus small puncta are already present before cellularization. As cellularization proceeds, these are captured at the apicolateral interface in a process requiring Baz, Cno, and an intact actin cytoskeleton, where they coalesce into SAJs, with ~1500 AJ complexes and 200 Baz proteins. Cadherin-catenin complexes form independently of either Baz or Cno, but AJ positioning and full supermolecular assembly depend on both. This study found that Scrib/Dlg are also key for AJ apicolateral retention and supermolecular assembly, although Arm and Cno remain associated in misplaced puncta, and thus core AJ complexes remain intact. Further, a second junctional complex that arise during polarity establishment, the BJs, also require Scrib/Dlg for its supermolecular organization. Unlike AJs, BJ organization is not dependent on other polarity determinants including Cno, Rap1 or Par-1. It will be of interest to examine if Scrib/Dlg act via known regulators of cadherin clustering, including intrinsic (e.g., cis- and trans-interactions of cadherins) and extrinsic (e.g., local actin regulation, endocytosis) factors (Bonello, 2019).
The ultimate goal is to define molecular mechanisms underlying polarity establishment. The new data place Scrib/Dlg in a critical position near the top of the network, but also suggest they act via multiple effectors. Perhaps the strongest evidence for multiple roles with distinct effectors comes from analysis of scrib4. Supermolecular organization of both SAJs and BJ must involve interactions with specific partners via the PDZ domains- one speculative possibility is that these include core AJ proteins, as βcatenin can coIP with Scribble and interact with PDZ domains 1 and 4. Testing this idea will be an important future direction. This initial role may also involve modulating Par-1. During cellularization, Scrib/Dlg and Par-1 localize in 'inverse gradients': Scrib and Dlg enriched at the SAJ level, and Par-1 with higher cortical intensity basolaterally. Scrib/Dlg play a role in effective membrane recruitment of Par-1 at this stage, and effects of par-1-RNAi on SAJ protein localization during cellularization are largely similar to those of scrib-RNAi. However, regulating Par-1 is not the only mechanism by which Scrib/Dlg act, as AJs are rescued during gastrulation after par-1-RNAi (Bonello, 2019).
Scrib then plays a second PDZ-independent role as gastrulation begins, ensuring focusing of cadherin-catenin complexes and Baz into apical belt AJs. This requires the N-terminal LRRs but not the PDZs. Positioning Baz at this stage involves at least two inputs which are redundant with one another, one via Par-1 and one via an apical transport mechanism. One speculative possibility is that Scrib/Dlg also regulate protein trafficking, a role they have in other contexts. However, disrupting Scrib/Dlg function has very different consequences than disrupting Rab5-dependent trafficking, suggesting they do not act via Rab5. aPKC also provides important cues at this stage-perhaps Scrib/Dlg regulate aPKC localization or function. It will be important to further explore the nature of this second role (Bonello, 2019).
The initial goal more than a decade ago was to define roles of AJs in polarity establishment. However, it rapidly became apparent AJs are not at the top of the hierarchy. Cno, Rap1 and Baz act upstream of AJ positioning and supermolecular assembly. The new data moves understanding another step upward in the network, revealing a key role for Scrib/Dlg in regulating AJ positioning and assembly. However, they also reveal that the process is not a simple linear pathway, and raise new questions. Loss of Scrib or Dlg almost completely disrupts AJs during cellularization. However, effects on Baz and Cno are less complete-supermolecular assembly is affected, but they are retained in the apical half of the membrane. This suggests other cues are involved. The ultimate polarizing cue during syncytial development is the oocyte membrane, which then directs cytoskeletal polarization. Cytoskeletal cues regulate Cno localization. While the data rule out a role for Scrib/Dlg in establishing basic cytoskeletal polarity, they do not rule out roles, for example, in localizing a special 'type' of actin cytoskeleton in the apical domain. Retention of Cno at the membrane after Scrib/Dlg knockdown suggests that minimally basal Rap1 activity remains intact. Changes to early Par-1, and to a lesser extent Baz, cortical localization with loss of Scrib/Dlg, also raise the possibility that lipid-based regulation is impaired. At this time, it is not known what cues regulate Scrib/Dlg apical enrichment but AJs do not appear to direct this, nor are they essential for polarizing Cno or Baz. Continued characterization of the full protein network and molecular mechanisms governing polarity establishment will keep the field busy for years to come (Bonello, 2019).
Epithelial cells undergo cortical rounding at the onset of mitosis to enable spindle orientation in the plane of the epithelium. In cuboidal epithelia in culture, the adherens junction protein E-cadherin recruits Pins/LGN/GPSM2 and Mud/NuMA to orient the mitotic spindle. In the pseudostratified columnar epithelial cells of Drosophila, septate junctions recruit Mud/NuMA to orient the spindle, while Pins/LGN/GPSM2 is surprisingly dispensable. This study shows that these pseudostratified epithelial cells downregulate E-cadherin as they round up for mitosis. Preventing cortical rounding by inhibiting Rho-kinase-mediated actomyosin contractility blocks downregulation of E-cadherin during mitosis. Mitotic activation of Rho-kinase depends on the RhoGEF ECT2/Pebble and its binding partners RacGAP1/MgcRacGAP/CYK4/Tum and MKLP1/KIF23/ZEN4/Pav. Cell cycle control of these Rho activators is mediated by the Aurora A and B kinases, which act redundantly during mitotic rounding. Thus, in Drosophila pseudostratified epithelia, disruption of adherens junctions during mitosis necessitates planar spindle orientation by septate junctions to maintain epithelial integrity (Aguilar-Aragon, 2020).
Adherens junctions have long been thought to be continuously essential for maintaining epithelial form and function. The current findings demonstrate transient loss of adherens junctions during division of pseudostratified epithelial cells, an event that involves adherens junction remodelling during the extensive rounding up of cell shape in mitosis. This study has furthermore shown that loss of adherens junctions is a direct consequence of the increased Rho activity and actomyosin contractility that drives mitotic rounding, which is both necessary and sufficient to regulate the level of junctional E-cadherin in the pseudostratified wing imaginal disc epithelium of Drosophila. These findings are consistent with previous observations that adherens junctions can be removed via E-cadherin endocytosis upon planar polarised Rho activation and actomyosin-driven junctional shrinkage generated during cell-cell rearrangements in the Drosophila embryo and recent optogenetic experiments in human cells. However, the global loss of E-cadherin observed during mitosis of pseudostratified epithelial cells is unprecedented and may be uniquely required by the rapid transformation of these cells from their highly columnar shape to a rounded sphere at mitosis, which involves a rapid increase in apical area and junctional length. This change in cell chape, driven by global actomyosin contractility during mitotic rounding, may both spread out junctions and disrupt cadherin-cadherin contacts between neighbouring cells to favour endocytosis (Aguilar-Aragon, 2020).
Notably, loss of E-cadherin does not seem to occur during mitosis in cuboidal epithelial cells, which undergo a much milder cell shape change during mitotic rounding, such as the Drosophila follicle cell epithelium or in many human cultured epithelial cell lines. Indeed, E-cadherin was reported to play essential roles in planar spindle orientation in cultured human cells. Thus, the pseudostratified epithelia of Drosophila face a unique challenge of orienting the mitotic spindle in the plane of the epithelium without the use of adherens junctions as a cue, which may explain why these cells instead rely on septate junctions, while planar spindle orientation can occur normally in the cuboidal follicle cell epithelium before septate junctions form. In the absence of septate junctions, spindle-orienting factors such as Pins, Mud, Dlg and Scrib localise to lateral membranes, overlapping with E-cadherin, which can directly interact with Scrib in both Drosophila and human cells (Aguilar-Aragon, 2020).
The findings of this study also shed light on the molecular mechanisms linking the cell cycle with control of Rho activation during mitotic rounding. Downstream of the master mitotic kinase Cdk1, a key role for both Aurora A and B kinases (acting redundantly) was identified in initiating mitosis and maintaining cortical rounding, with Aurora B then acting alone to drive furrow formation during cytokinesis. Aurora kinases are known to activate the key cell cycle kinase Plk1/Polo, which can then activate RacGAP1/MgcRacGAP/CYK4/Tum and ECT2/Pbl, possibly via the kinesin-like protein MKLP1/KIF23/ZEN4/Pav. One report suggested that Aurora B also acts to directly phosphorylate RacGAP1/MgcRacGAP/CYK4/Tum on S387, but this study found that a CRISPR-knockin mutation of this site (tumS387A) is homozygous viable and fertile in Drosophila. In addition, Aurora B can also directly phosphorylate MKLP1/KIF23/ZEN4/Pav to oligomerise and activate RacGAP1/MgcRacGAP/CYK4/Tum and ECT2/Pbl during cytokinesis, via a mechanism involving plasma membrane association of clustered C1 domains from the RacGAP1/MgcRacGAP/CYK4/Tum protein. Accordingly, it was found that a CRISPR-knockin mutation of this site (pavS734A/S735A) is homozygous lethal in Drosophila, suggesting that Aurora A/B could act via this complex during mitotic rounding. Indeed, MKLP1/KIF23/ZEN4/Pav undergoes cell cycle-dependent re-localisation from the nucleus (in interphase) to the cytoplasm (in mitosis), including a clear localisation to the entire plasma membrane during mitosis and then to the cleavage furrow during cytokinesis. The results show that mitotic activation of the MKLP1/KIF23/ZEN4/Pav binding partner RacGAP1/MgcRacGAP/CYK4/Tum also involves translocation from the nucleus to the cytoplasm, similar to ECT2/Pbl, where all three proteins are then available to bind the entire plasma membrane and generate global Rho activity to drive cortical contractility and loss of adherens junctions during mitotic rounding (Aguilar-Aragon, 2020).
The complex of MKLP1/KIF23/ZEN4/Pav and RacGAP1/MgcRacGAP/CYK4/Tum is often referred to as the 'centralspindlin' complex, due to its association with the spindle midzone at anaphase. However, the use of this term was avoided because association with the spindle midzone is not required for the function of this complex in activating ECT2/Pbl and Rho at the plasma membrane during cytokinesis. Furthermore, the results show that the same complex also functions prior to anaphase or cytokinesis in activating ECT2/Pbl and Rho for cortical rounding and downregulation of adherens junctions from the very onset of mitosis (Aguilar-Aragon, 2020).
Importantly, the function of the three cell cycle-regulated Rho activators discussed in this study - MKLP1/KIF23/ZEN4/Pav, RacGAP1/MgcRacGAP/CYK4/Tum and ECT2/Pbl - on the regulation of adherens junctions appears to be dose-dependent and cell shape change-dependent. The sudden re-localisation of these proteins to the cytoplasm at mitosis drives a global increase in Rho activation, cell shape change and loss of junctions. In contrast, the relatively low level of these proteins in the cytoplasm during interphase appears to contribute to the normal maintenance of Rho activity at the junctional actomyosin ring to maintain adherens junctions-at least in mammalian cells. In Drosophila, an interphase role of ECT2/Pbl in contributing to Rho activation and maintenance of adherens junctions is also plausible and should be most easily distinguished from the mitotic role in non-dividing epithelial cells. However, in the ovarian follicle cell epithelium, silencing of ECT2/Pbl by RNAi affected cytokinesis (leading to larger cells) but did not affect overall epithelial architecture after cells arrest their proliferation, suggesting that any interphase function may be obscured by redundancy with other RhoGEFs in this tissue. Note also that, in this cuboidal epithelium, mitotic rounding itself is more subtle and ECT2/Pbl is dispensable for planar spindle orientation and epithelial integrity (Aguilar-Aragon, 2020).
Finally, it is noted that ECT2/Pbl was initially reported to be capable of acting as GEF for another GTPase, Cdc42, in addition to Rho 38. This activity suggested a possible role in regulating the apical Cdc42-Par6-aPKC complex in epithelial polarity and in mitosis. In Drosophila, ECT2/Pbl was found to drive transient apical spreading of the Cdc42-Par6-aPKC complex during mitotic rounding of pupal notum epithelial cells. This study found evidence for a similar function of ECT2/Pbl in activating Cdc42 during interphase in the post-mitotic ovarian follicle cell epithelium, although the loss-of-function phenotype of ECT2/Pbl is obscured due to redundancy with other Cdc42 GEFs such as beta-PIX. Loss of both ECT2/Pbl and beta-PIX reduced the level of the Cdc42-Par6-aPKC complex localising to the apical domain of post-mitotic follicle cells and also reduced apical ZO-1 localisation in human Caco2 intestinal epithelial cells in culture. In contrast, the gain-of-function phenotype caused by ECT2/Pbl overexpression is a neoplastic tumour-like phenotype and clearly involves ectopic spreading of the apical Cdc42-Par6-aPKC complex, in addition to persistent cell rounding in both cuboidal follicle cells and pseudostratified wing epithelial cells (Aguilar-Aragon, 2020).
In conclusion, the findings provide new insights into the cell biology of mitotic rounding, identifying remodelling of adherens junctions as a key event in pseudostratified epithelia, where rounding is extensive, but not cuboidal epithelia, where rounding is more subtle. These results are consistent with the hypothesis that Rho activation and actomyosin contractility can stabilise adherens junctions in the absence of mechanical strain, but that Rho activation can induce E-cadherin endocytosis above a critical strain threshold, be it either junctional shrinkage or expansion, both of which may alter the geometry of the junctional actomyosin ring and disrupt cadherin-cadherin contacts between neighbouring cells to favour endocytic internalisation of E-cadherin. The results also clarify the molecular mechanisms linking cell cycle control machinery, particularly the Aurora A and B kinases, with Rho activation and mitotic rounding. Lastly, this work may have direct relevance to certain human epithelial cancers, such as lung cancer or glioma, where overexpression of ECT2/Pbl has been reported to correlate for poor prognosis, and where the findings suggest it could drive not only disruption of epithelial polarity via activation of Cdc42 or Rac but also loss of adherens junctions via sustained Rho activation to promote tumour progression (Aguilar-Aragon, 2020).
The replacement of cells is a common strategy during animal development. In the Drosophila pupal abdomen, larval epidermal cells (LECs) are replaced by adult progenitor cells (histoblasts). Previous work showed that interactions between histoblasts and LECs result in apoptotic extrusion of LECs during early pupal development. Extrusion of cells is closely preceded by caspase activation and is executed by contraction of a cortical actomyosin cable. This study identified a population of LECs that extrudes independently of the presence of histoblasts during late pupal development. Extrusion of these LECs is not closely preceded by caspase activation, involves a pulsatile medial actomyosin network, and correlates with a developmental time period when mechanical tension and E-cadherin turnover at adherens junctions is particularly high. This work reveals a developmental switch in the cell extrusion mechanism that correlates with changes in tissue mechanical properties (Michel, 2020).
This study has analyzed the replacement of LECs by adult histoblasts in the pupal abdominal epidermis in Drosophila. Previous work has shown that the interaction between the growing nest of histoblasts and LECs is required for LEC extrusion. LEC extrusion is closely preceded by caspase activation and involves contraction of a supracellular actomyosin cable. This study has identified a second hotspot of cell extrusion at the dorsal midline of the pupal abdomen. Cell extrusion at the dorsal midline occurs mainly late during development and is independent of histoblasts, but depends on the presence of neighboring LECs. LEC extrusion is preceded by a uniform rise in caspase activity throughout the larval tissue and involves pulsatile contractions of a medial actomyosin network. Mechanical tension on adherens junctions of LECs increases during pupal development. High mechanical tension during late pupal development depends in part on caspase activity and correlates with a high turnover of E-cadherin. E-cadherin turnover depends on Dynamin-dependent endocytosis and blocking Dynamin severely affects LEC extrusion. This work reveals a novel, histoblast-independent mechanism by which LECs extrude during late pupal development (Michel, 2020).
Extrusion of LECs located at the border of histoblast nests during early pupal development involves the remodeling and weakening of adherens junctions between the extruding LEC and its neighboring cells, and the accumulation of Myosin II at the cortices of both the extruding LEC and its neighboring cells. Cortical Myosin II accumulation in neighboring cells results in the formation of a supracellular actomyosin cable. Contraction of this cable facilitates the extrusion of the LEC (Teng, 2017). The data indicate that extrusion of LECs at the dorsal midline during late pupal development involves a distinct mechanism. In addition to a cortical pool of Myosin II and F-actin, Myosin II and F-actin was found also in an apical medial region of the extruding LECs. Similar accumulations of Myosin II and F-actin have been reported in several other epithelia, including in the Drosophila embryo in ventral furrow cells of the epidermis and in amnioserosa cells. The medial actomyosin network is pulsatile in these cells resulting in concomitant fluctuations in apical cell area. Similarly, it was found that in extruding LECs located in the vicinity of the dorsal midline, apical cell area fluctuates and apical constriction correlates with increased medial Myosin II intensity. Over longer time periods, apical cell area shrinks, indicating a ratchet-like mechanism. A similar ratchet-like contraction mechanism is involved in the Drosophila embryo during neuroblast delamination (Simoes, 2017) and the extrusion of amnioserosa cells. The reason for these different extrusion mechanisms of LECs during early and late pupal development remains unclear. It is noted, however, that mechanical tension on LECs increases approximately 5- to 10-fold between early (20 h APF) and late (27 h APF) pupal development, suggesting that overall tissue mechanical tension influences the mechanism by which cells extrude (Michel, 2020).
How is the extrusion of LECs at the dorsal midline initiated? Several mechanisms have been described that drive cell extrusion, including cell competition and tissue crowding. These two mechanisms, however, appear not to play a major role in the extrusion of LECs located at the dorsal midline of the pupal abdominal epidermis. Tissue crowding induces, for example, caspase-dependent cell extrusion at the dorsal midline in the pupal notum. Experimental induction of clones expressing an activated form of Ras, RasV12, leads to the crowding of neighboring wild-type cells and to their extrusion. In the abdominal epidermis, the growth or migration of histoblasts could result in crowding of the LECs. During the analyzed developmental time period, the average area of non-extruding LECs located at the dorsal midline, however, remains nearly constant, indicating that these LECs do not become crowded. Moreover, LECs located at the dorsal midline still extrude when histoblast division is halted or histoblasts are genetically ablated, providing further evidence that tissue crowding is not involved in the extrusion of these cells. Finally, cell competition refers to the out competition of slow-growing cells by fast-growing cell. Again, the finding that LEC extrusion at the dorsal midline is independent of histoblast division, strongly argues that cell competition does not have a major influence on the extrusion of these LECs (Michel, 2020).
During early pupal development, local interactions between histoblasts and LECs trigger caspase activation in LECs. Caspase activation then closely precedes extrusion of LECs, cell by cell, at the border of histoblast nests, indicating that caspase activation triggers extrusion (Teng, 2017). In LECs at the dorsal midline, by contrast, caspase activity, as measured by Apoliner cleavage, shows a population-wide fluctuation during pupal development. Caspase activity peaks during the observed developmental time period twice, around 20 h APF and around 26-29 h APF. Caspase activation in these cells does not appear to trigger their extrusion, because the first peak of caspase activity at around 20 h APF takes place several hours before LECs at the dorsal midline start to extrude. Interestingly, the second rise of caspase activity between 24 h and 26 h APF precedes the extrusion of LECs at the dorsal midline. Taken together with the observation that LEC extrusion at the dorsal midline requires caspase activity, these results indicate that the second rise of caspase activity is a prerequisite for LECs to extrude at the dorsal midline. How the population-wide fluctuations of caspase activity in LECs at the dorsal midline arise will be interesting to explore (Michel, 2020).
This study found that mechanical tension acting on LEC adherens junctions increases during pupal development. Increased mechanical tension is independent of the proliferation of histoblasts, but requires caspase activity, which in turn is necessary for LEC extrusion. It is speculated that LEC extrusion during late pupal development may, in part, facilitate the increased mechanical tension on adherens junctions of the remaining LECs. Mechanical tension may influence cell extrusion in different ways and by different mechanisms. In the pupal notum, for example, a similar developmental increase in mechanical tension has been observed, but, unlike the situation in the pupal abdomen, increased mechanical tension correlates with fewer cell extrusions. This different behavior may result from the different characteristics of the two epithelia. In the notum, cell extrusion occurs within a diploid, proliferating tissue with columnar cells. On the other hand, LECs of the pupal abdomen are polyploid, non-proliferating and squamous in shape. In these respects, the abdominal LECs resemble more the amnioserosa cells of the embryo (Michel, 2020).
How could an increase in mechanical tension on LEC adherens junctions facilitate extrusion? LEC extrusion involves the remodeling of adherens junctions of the extruding cell (Teng, 2017). Adherens junction remodeling depends on E-cadherin. The turnover of E-cadherin was found to have increased at 27 h APF compared with 20 h APF, thus positively correlating with increased mechanical tension. The turnover of E-cadherin depends on lateral diffusion on the plasma membrane and on trafficking between a cytosolic, presumably vesicular, pool of E-cadherin and a junctional pool of E-cadherin. The exchange between these two pools requires Dynamin-dependent endocytosis. Consistent with this notion, E-cadherin turnover was reduced in LECs of flies mutant for shibire1, which encodes Dynamin. Interestingly, extrusion of LECs in the lateral region of the abdominal segments is nearly blocked in shibire1 mutants. Although the possibility cannot be excluded that Dynamin-dependent endocytosis of proteins other than E-cadherin are important for LEC extrusion, these data lend support for the idea that high E-cadherin turnover is required for extrusion of these LECs. Surprisingly, a subset of LECs, those located in medial regions of the segment, do extrude in shibire1 mutants. These findings reveal an unexpected complexity of LEC extrusion that will require future work to be resolved (Michel, 2020).
The following model is proposed of how LECs in the pupal abdominal epidermis extrude. During early pupal development (20 h APF) mechanical tension on LEC junctions is low and E-cadherin turnover is low. LECs extrude only if they receive a specific 'extrusion signal' from the histoblasts. Extrusion is mediated by cortical actomyosin, which forms a contractile supracellular cable driving apical cell constriction (Teng, 2017). Later during pupal development (27 h APF), mechanical tension on LEC junctions, E-cadherin turnover and caspase activity are high. Stochastic extrusions of LECs commence, which are mediated by a pulsatile medial actomyosin network that leads to ratchet-like apical cell contractions (Michel, 2020).
During embryo development, tissues often undergo multiple concomitant changes in shape. It is unclear which signaling pathways and cellular mechanisms are responsible for multiple simultaneous tissue shape transformations. This study focussed on the process of concomitant tissue folding and extension that is key during gastrulation and neurulation. The Drosophila embryo was used as model system and focus was placed on the process of mesoderm invagination. This study shows that the prospective mesoderm simultaneously folds and extends. Mesoderm cells, under the control of anterior-posterior and dorsal-ventral gene patterning synergy, establish two sets of adherens junctions at different apical-basal positions with specialized functions: while apical junctions drive apical constriction initiating tissue bending, lateral junctions concomitantly drive polarized cell intercalation, resulting in tissue convergence-extension. Thus, epithelial cells devise multiple specialized junctional sets that drive composite morphogenetic processes under the synergistic control of apparently orthogonal signaling sources (John, 2021).
Epithelial structure is generated by the dynamic reorganization of cells in response to mechanical forces. Adherens junctions transmit forces between cells, but how cells sense and respond to these forces in vivo is not well understood. This study identified a mechanotransduction pathway involving the Abl tyrosine kinase and Canoe/Afadin that stabilizes cell adhesion under tension at tricellular junctions in the Drosophila embryo. Canoe is recruited to tricellular junctions in response to actomyosin contractility, and this mechanosensitivity requires Abl-dependent phosphorylation of a conserved tyrosine in the Canoe actin-binding domain. Preventing Canoe tyrosine phosphorylation destabilizes tricellular adhesion, and anchoring Canoe at tricellular junctions independently of mechanical inputs aberrantly stabilizes adhesion, arresting cell rearrangement. These results identify a force-responsive mechanism that stabilizes tricellular adhesion under tension during epithelial remodeling (Yu, 2020).
This study shows that Canoe and Abl function in a mechanotransduction pathway that regulates dynamic changes in cell adhesion at tricellular junctions during epithelial remodeling. Canoe localization to tricellular junctions is acutely disrupted by laser ablation, demonstrating that Canoe localization is rapidly modulated by mechanical perturbation. Canoe mechanosensitivity requires Abl-dependent phosphorylation of a conserved tyrosine (Y1987) in the Canoe actin-binding domain, and Canoe localization to tricellular junctions is required to stabilize tricellular adhesion. Conversely, constitutively anchoring Canoe at tricellular junctions arrests cell rearrangement, indicating that Canoe levels can tune the rate of junctional remodeling. These results demonstrate that the mechanosensitivity of this critical junctional regulator is modulated by phosphorylation of a single tyrosine residue and reveal an essential role of Canoe in coupling tricellular adhesion with mechanical forces during epithelial remodeling (Yu, 2020).
Mechanical forces trigger a cascade of molecular events in cells that translate biophysical signals into altered cellular behaviors. However, the mechanosensors that directly change conformation under tension in vivo are not well defined. Cell surface receptors are well positioned to detect forces generated by neighboring cells, but Canoe localization and function at tricellular junctions do not require the PDZ domain that mediates its interaction with known receptors. One possibility is that the Canoe protein itself could act as a mechanosensor. Canoe is predicted to be anchored to the membrane through interactions with Rap1 and the actin cytoskeleton. Thus, cytoskeletal tension could stretch the Canoe protein and expose tyrosine 1987 to phosphorylation by Abl. Alternatively, Abl or its upstream activators could be regulated by tension during this process, because the activity of Abl and other tyrosine kinases such as Src and FAK has been shown to be regulated by tension in vitro. In a third possibility, the regulation of Canoe by Abl could allow Canoe to detect force-induced changes in other molecules at tricellular junctions. Because tyrosine 1987 is in the Canoe actin-binding domain, phosphorylation at this site could allow Canoe to recognize distinct actin structures at tricellular junctions, force-induced conformational changes in the cadherin-catenin complex, or other specialized features of tricellular junction composition or geometry. Once recruited to tricellular junctions, Canoe could stabilize adhesion at these sites by reinforcing the connection between adherens junctions and the actomyosin cytoskeleton (Yu, 2020).
Abl tyrosine kinases influence many structural changes that are driven by mechanical forces in tissues, including epithelial remodeling, tissue invagination, axon guidance, and cell migration. Abl has been shown to regulate a number of proteins that act at tension-bearing structures in cells in addition to Canoe/Afadin recruitment to tricellular junctions, including vinculin localization and β-catenin recycling at bicellular junctions and regulation of membrane curvature by BAR-domain proteins. Together, these results raise the possibility that Abl could transduce mechanical forces into a wide range of structural changes within cells. For example, during cell rearrangement, Abl could simultaneously stabilize tricellular adhesion by recruiting Canoe and destabilize bicellular adhesion by enhancing β-catenin turnover, which could allow bicellular junctions to complete contraction before tricellular junctions are remodeled. Tricellular junctions serve many important roles in epithelial development and homeostasis, including modulating cell rearrangement, orienting mitotic spindles, balancing stem cell proliferation and differentiation, and maintaining epithelial barrier function. An understanding of how mechanical inputs affect the conformation, localization, activity, and interactions of proteins at tricellular junctions will provide insight into how these structures sense and integrate mechanical forces in epithelial tissues (Yu, 2020).
Src kinases are important regulators of cell adhesion. This study has explored the function of Src42A in junction remodelling during Drosophila gastrulation. Src42A is required for tyrosine phosphorylation at bicellular (bAJ) and tricellular (tAJ) junctions in germband cells, and localizes to hotspots of mechanical tension. The role of Src42A was investigated using maternal RNAi and CRISPR-Cas9-induced germline mosaics. During cell intercalations, Src42A was shown to be required for the contraction of junctions at anterior-posterior cell interfaces. The planar polarity of E-cadherin is compromised and E-cadherin accumulates at tricellular junctions after Src42A knockdown. Furthermore, Src42A was shown to act in concert with Abl kinase, which has also been implicated in cell intercalations. These data suggest that Src42A is involved in two related processes: in addition to establishing tension generated by the planar polarity of MyoII, it may also act as a signalling factor at tAJs to control E-cadherin residence time (Chandran, 2023).
Remodeling of cell-cell junctions drives cell intercalation that causes tissue movement during morphogenesis through the shortening and growth of bicellular junctions. The growth of new junctions is essential for continuing and then completing cellular dynamics and tissue shape sculpting; however, the mechanism underlying junction growth remains obscure. This study investigated Drosophila genitalia rotation where continuous cell intercalation occurs to show that myosin II accumulating at the vertices of a new junction is required for the junction growth. This myosin II accumulation requires the adhesive transmembrane protein Sidekick (Sdk), which localizes to the adherens junctions (AJs) of tricellular contacts (tAJs). Sdk also localizes to and blocks the accumulation of E-Cadherin at newly formed growing junctions, which maintains the growth rate. It is proposed that Sdk facilitates tAJ movement by mediating myosin II-driven contraction and altering the adhesive properties at the tAJs, leading to cell-cell junction extension during persistent junction remodeling (Uechi, 2019).
To generate tissue shapes, cell collectives show various dynamics, such as cell division and cell deformation. Among them, cell intercalation is a multicellular behavior in which cells change their position through the remodeling of cell-cell contacts, leading to the directional elongation and expansion of tissues across species. Especially in epithelia, this cell-cell junction remodeling involves the shortening and loss of bicellular junctions and the subsequent growth of bicellular junctions in a new direction. Junction shortening initiates tissue dynamics and is driven in a conserved manner by contractile forces generated by actomyosin (actin and non-muscle myosin II complex) associating with the cadherin-catenin core complex, including E-Cadherin, β-Catenin, and other related adherens junction (AJ) components, at the AJs of shortening junctions. Junction growth is also essential for continuing and then completing cellular dynamics and tissue shape sculpting. Several studies using flies have suggested that myosin II has a role in junction growth during developmental events. In the germ band, medial pulses of myosin II in the cells surrounding junctions and toward the posterior ectoderm regulate junction growth during cell intercalation-driven convergent extension [germ band extension (GBE)]. A similar contribution of myosin II pulses in the surrounding cells to junction extension is also observed in the apical cell oscillation of amnioserosa cells during dorsal closure. In developing wing epithelia, a decrease in myosin II levels at newly formed junctions facilitates junction growth to organize the epithelial cellular pattern. However, despite its importance, the mechanisms underlying junction growth remain unclear, in contrast to junction shortening (Uechi, 2019).
Previous studies demonstrated that cell intercalation also contributes to the tissue rotational movement observed for Drosophila male genitalia. The fly genitalia are located at the animal's posterior end, and the male genitalia are surrounded by epithelia known as the A8 segment at the anterior side. At 24 h after puparium formation (APF), the genitalia and the A8 epithelia begin dextral rotation that terminates at around 36 h APF. The rotation consists of an initial 180° movement of the posterior compartment of A8 (A8p) along with the genitalia and a subsequent 180° movement of the anterior component of A8 (A8a), the latter of which starts at around 26 h APF. From 26 h APF in the A8a cells, myosin II accumulates to a greater extent at AJs, forming a right oblique angle with the anterior-posterior (AP) axis than at junctions forming a left oblique angle. This polarized myosin II distribution gives rise to right-biased junction shortening in relation to the AP axis and leads to left-right asymmetric cell intercalation, which is persistently observed during the movement. By combining numerical simulations, it was demonstrated that this repeated junction remodeling in the confined space generates the A8a movement. In this movement, newly formed junctions are sufficiently elongated within a certain time frame to execute the next round of cell intercalation. Incomplete genitalia rotation leads to male sterility (Uechi, 2019).
This study performed time-lapse imaging, developed an optogenetic tool, and analyzed the adhesive protein Sidekick (Sdk), which is known to regulate retinal development in flies and mice and showed that myosin II accumulating at the tricellular contacts (tAJs) of growing junctions is required for bicellular junction growth in A8a cells. Also, Sdk regulates the myosin II and E-Cadherin distributions at the tAJs, thereby maintaining the junction growth rate. These findings suggest that the tAJ is a specialized point promoting cell-cell junction extension (Uechi, 2019).
The process of junction shortening is well characterized and is organized by the contractile forces of actomyosin, which is transmitted to cell-cell contacts via AJ components, such as E-Cadherin. These proteins have important roles in the dynamics of multicellular deformation. Since junction formation and growth are important for the continuation and completion of multicellular dynamics and tissue architecture shaping, it is likely that active mechanisms underlie the extension of cell-cell junctions. Indeed, recent reports suggest that actin and myosin II at the bicellular junctions are involved in the junction extension in cell rearrangement and in cell-shape formation during Drosophila wing and eye development. Polarized medial pulses of myosin II in the cells surrounding junctions regulate junction extension in the Drosophila germ band and amnioserosa. This study used an optogenetic tool that allows for the spatiotemporal inactivation of endogenous myosin II and revealed that myosin II accumulating at the tAJs of newly formed junctions is required for junction growth in the A8a epithelia. This study also demonstrated that the myosin II accumulation and junction growth require the tAJ-localizing protein Sdk. Thus, this report that tAJs are an additional point promoting the extension of bicellular junctions (Uechi, 2019).
Sdk transiently localizes to newly formed junctions as well as tAJs, causing a downregulation of E-Cadherin and a slight increase in intercellular spaces at the AJs of growing junctions, indicative of less tight cell-cell contacts. Recent studies in zebrafish showed that the presence of extracellular spaces and the disassembly of cell-cell contacts contribute to fluidize tissues. During body axis elongation, the extracellular spaces render mesodermal cells fluidized and uncaged and associated with large fluctuations in the lengths of cell-cell contact. Decreases in cell-cell contacts through the destabilization of junctional E-Cadherin, accompanied by an increase in extracellular spaces, induces the fluidization of blastoderm cells and consequently allows blastoderm spreading at the onset of morphogenesis. Analogous to these properties, it is possible that the presence of Sdk at growing junctions confers flexible dynamics to the cell-cell contacts at the level of the AJs of the growing junctions. This study proposes mechanisms of junction growth in which Sdk has dual roles. First, Sdk mediates a driving force of junction growth by anchoring myosin II at tAJs; the contractility of the actomyosin then retracts the membrane of the surrounding cells at tAJs. Second, Sdk assists in the myosin II-driven junction growth by localizing to and decreasing the accumulation of E-Cadherin at the growing junctions and their tAJs; this composition of E-Cadherin and Sdk causes contacts between the vertices of the surrounding cells and the cells forming the growing junction to be less tight. Such adhesion can render the tAJs of growing junctions more sensitive to contractile forces at the vertices of the surrounding cells, supporting the retraction of the membrane of the surrounding cells at tAJs. The latter mechanism is indeed likely to contribute to junction growth since inducing sdk RNAi only in the cell forming the growing junction was sufficient to reduce the junction growth rate, even when the surrounding cells consisted of WT cells (Uechi, 2019).
The precise mechanism by which Sdk blocks the accumulation of E-Cadherin at newly formed junctions is still unclear. While Sdk was already present at growing junctions from the step of four-way vertex resolution, E-Cadherin would be newly recruited to the growing junctions since E-Cadherin is removed from remodeling junctions by endocytosis during junction shortening. A recent study using fluorescence recovery after photobleaching (FRAP) revealed two ways that E-Cadherin is re-distributed to cell-cell junctions, lateral diffusion within the plasma membrane and delivery from the cytoplasm by vesicular trafficking. Since (1) E-Cadherin and Sdk did not interact despite their localization to AJs, (2) showed complementary distributions at newly formed junctions and even at cellular edges in S2 cells where they were ectopically expressed, and (3) changed their distributions when the other protein was depleted, it is possible that there are repelling forces between E-Cadherin and Sdk molecules, which cause them to exclude each other and may delay the diffusion of E-Cadherin from neighboring junctions into newly formed junctions, where Sdk is already enriched. However, this study does not exclude another possibility that Sdk inhibits machineries that deliver E-Cadherin from the cytoplasm, such as blocking their access to growing junctions or biochemically inactivating them (Uechi, 2019).
This study observed the accumulation of myosin II and decreased E-Cadherin levels at the tAJ of growing junctions. These distributions resemble those occurring during new cell-cell contact formation between daughter cells in epithelia. After cytokinesis, myosin II accumulates at the edges of new cell-cell junctions in the neighboring cells of the daughter cells, in response to the local decrease in E-Cadherin levels at these edges, which participates in new cell-cell junction formation. These reports and the current observations suggest a possible common mechanism underlying new cell-cell contact formation among epithelial multicellular behaviors. Although the dynamics and roles of Sdk in cell division are still unclear, an intriguing possibility is that Sdk regulates the dynamics of new cell-cell junctions in concert with myosin II and E-Cadherin not only in the context of cell intercalation but also global epithelial dynamics including cytokinesis (Uechi, 2019).
Actomyosin contraction generates mechanical forces that influence cell and tissue structure. During convergent extension in Drosophila, the spatially regulated activity of the myosin activator Rho-kinase promotes actomyosin contraction at specific planar cell boundaries to produce polarized cell rearrangement. The mechanisms that direct localized Rho-kinase activity are not well understood. This study shows that Rho GTPase recruits Rho-kinase to adherens junctions and is required for Rho-kinase planar polarity. Shroom, an asymmetrically localized actin- and Rho-kinase-binding protein, amplifies Rho-kinase and myosin II planar polarity and junctional localization downstream of Rho signaling. In Shroom mutants, Rho-kinase and myosin II achieve reduced levels of planar polarity, resulting in decreased junctional tension, a disruption of multicellular rosette formation, and defective convergent extension. These results indicate that Rho GTPase activity is required to establish a planar polarized actomyosin network, and the Shroom actin-binding protein enhances myosin contractility locally to generate robust mechanical forces during axis elongation (Simoes, 2014).
Rho-kinase is an essential regulator of actomyosin contractility, but the mechanisms that generate Rho-kinase asymmetry to produce spatially regulated forces during development are not well understood. This study shows that Rho GTPase signaling is required for the planar polarized localization of Rho-kinase and myosin II during Drosophila axis elongation. Direct interaction between Rho and Rho-kinase recruits Rho-kinase to adherens junctions but is not sufficient for full Rho-kinase planar polarity, suggesting that other mechanisms amplify the effects of Rho signaling. This study provides evidence that the actin-binding protein Shroom regulates Rho-kinase localization and planar polarized actomyosin contractility to promote sustained cell rearrangements during axis elongation. Shroom is present in a planar polarized distribution at adherens junctions in intercalating cells, consistent with a direct and localized function. Shroom planar polarity requires Rho activity, indicating that Shroom is an effector of Rho signaling. In Shroom mutants, Rho-kinase and myosin II junctional localization and planar polarity initiate normally but fail to be amplified and maintained during axis elongation. Consequently, planar polarized contractile forces and multicellular rosette rearrangements are reduced in Shroom mutants, resulting in decreased convergent extension. These results support a role for Shroom in regulating planar polarized actomyosin contractility and junctional remodeling during convergent extension, expanding the morphogenetic functions of this highly conserved protein beyond its known role in apical constriction (Simoes, 2014).
The data support a model in which Rho GTPase and Shroom have distinct functions in regulating Rho-kinase localization and planar polarized myosin contractility during convergent extension. Rho GTPase recruits Rho-kinase to adherens junctions and initiates planar polarity, and Shroom plays a modulatory role in enhancing and maintaining planar polarized myosin contractility downstream of Rho signaling. Rho GTPase binds to Rho-kinase and could regulate its localization directly. Rho does not bind to Shroom but may regulate Shroom planar polarity indirectly through its effect on the actin cytoskeleton. Rho-kinase, usually viewed as a downstream effector of Shroom, feeds back to maintain Shroom planar polarity and its own planar polarized localization. Rho-kinase could directly phosphorylate Shroom to reinforce planar cell polarity. Alternatively, Rho-kinase could promote Shroom localization through remodeling of the actin cytoskeleton, as the Shroom actin-binding domain is necessary and sufficient for targeting to planar junctions, and Rho-kinase can phosphorylate known regulators of actin (Simoes, 2014).
These findings may be relevant to neural tube development in vertebrates, which involves a combination of apical constriction, polarized junctional remodeling, and cell shape changes. Shroom3 is required for neural tube closure in the mouse, frog, and chick, and disrupting the interaction between Shroom and Rho-kinase reduces the number of rosettes in the chick neural plate. Unlike mutants that have disrupted rosette-based movements caused by defects in cell adhesion, the defects in Shroom mutants are likely a result of reduced myosin II activity. Rosette behaviors in Drosophila predominate midway through elongation at stage 8, coinciding with the stage when myosin becomes mislocalized in Shroom mutants. A failure to reinforce actomyosin contractility during elongation in Shroom mutants could selectively disrupt later-onset, higher-order cell rearrangements, with no effect on local neighbor exchange events that are more frequent at earlier stages. Alternatively, rosette formation may require more force, as rosettes form through the contraction of multicellular actomyosin cables that are under a higher level of tension and accumulate more myosin. In Shroom mutants, defects in myosin junctional localization may prevent contractile forces from reaching the levels necessary to produce rosette-based convergent extension movements. It will be interesting to explore whether planar polarized Shroom activity plays a general role in promoting junctional remodeling and enhancing mechanical force generation in processes that require strong actomyosin contractility during development (Simoes, 2014).
Rho GTPase signaling is an excellent candidate to break planar symmetry, as a small fraction of active Rho protein can trigger rapid and dramatic changes in the actin cytoskeleton. In one model, a subtle increase in Rho activity at AP cell boundaries could provide an instructive cue, guiding planar cell polarity by recruiting Rho-kinase, modifying the actin cytoskeleton, and facilitating the cortical association of the Rho-kinase regulator Shroom. Alternatively, Rho could regulate Rho-kinase planar polarity indirectly through its role in promoting Rho-kinase apical localization. Although it is challenging to visualize a small and highly dynamic population of active Rho protein in vivo, several findings support the idea that localized Rho activity could play an instructive role in planar polarity. First, myosin planar polarity and directional cell rearrangements occur normally at early stages in Shroom mutants, suggesting that other signals are able to generate localized myosin activity. The partial planar asymmetry of a fragment containing the RB domain of Rho-kinase, which is predicted to interact with the active pool of Rho GTPase, suggests that Rho could contribute to this asymmetry. Second, Rho is required for the planar polarized localization of Shroom, raising the possibility that Rho signaling could provide an essential source of Shroom asymmetry. Third, the upstream Rho activator RhoGEF2 in Drosophila and PDZ-RhoGEF in the chick display a subtle planar asymmetry during epithelial bending and elongation. Multiple activators and inhibitors of Rho could act together to generate a spatially localized pattern of Rho activity, as is the case for apical constriction. Notably, although Rho GTPase activity is necessary to establish Rho-kinase and myosin planar polarity, it is not sufficient to maintain their activity at high enough levels to allow sustained force generation and rosette rearrangements in Shroom mutants. It is proposed that Rho promotes the recruitment of Shroom as part of a positive feed-forward mechanism that reinforces planar polarized actomyosin contractility during convergent extension (Simoes, 2014).
Planar polarized cell rearrangements require the active maintenance of cell polarity in large populations of dynamically moving cells. This study shows that Shroom and Rho GTPase signaling play distinct roles in the establishment and maintenance of polarized actomyosin contractility during convergent extension. The upstream spatial cues that localize actomyosin contractility to specific planar cellular domains are not known. An asymmetry in the organization of the actin cytoskeleton is the earliest evidence of planar polarity in the Drosophila embryo. Distinct actin-binding domains in different Shroom isoforms have been proposed to target Shroom protein and its effectors to different regions of the cell. Moreover, the actin-binding domain is critical for Shroom planar polarity. These findings support the idea that an asymmetry in the actin cytoskeleton is an essential spatial input that regulates the localization of Shroom, the contractile machinery, and ultimately the forces that control cell rearrangement and tissue structure. The upstream spatial cues that generate these asymmetries could involve an asymmetry in Rho signaling, perhaps through the local activation of upstream signaling proteins that regulate Rho GTPase activity. Alternatively, the critical event in the establishment of planar cell polarity could be a Rho-independent reorganization of the actin cytoskeleton that biases the activity of Shroom, Rho-kinase, and myosin, which in turn modify the cytoskeleton to allow robust and sustained cell polarization. Elucidation of the upstream spatial cues that regulate actomyosin localization and dynamics will provide insight into the mechanisms that direct polarized cell behavior (Simoes, 2014).
Regulation of cell architecture is critical in the formation of tissues during animal development. The mechanisms that control cell shape must be both dynamic and stable in order to establish and maintain the correct cellular organization. Previous work has identified Shroom family proteins as essential regulators of cell morphology during vertebrate development. Shroom proteins regulate cell architecture by directing the subcellular distribution and activation of Rho-kinase, which results in the localized activation of non-muscle myosin II. Because the Shroom-Rock-myosin II module is conserved in most animal model systems, Drosophila melanogaster was used to further investigate the pathways and components that are required for Shroom to define cell shape and tissue architecture. Using a phenotype-based heterozygous F1 genetic screen for modifiers of Shroom activity, several cytoskeletal and signaling protein were identified that may cooperate with Shroom. Two of these proteins, Enabled and Short stop, are required for ShroomA-induced changes in tissue morphology and are apically enriched in response to Shroom expression. While the recruitment of Ena is necessary, it is not sufficient to redefine cell morphology. Additionally, this requirement for Ena appears to be context dependent, as a variant of Shroom that is apically localized, binds to Rock, but lacks the Ena binding site, is still capable of inducing changes in tissue architecture. These data point to important cellular pathways that may regulate contractility or facilitate Shroom-mediated changes in cell and tissue morphology (Hildebrand, 2021).
Tissue architecture is typically defined during specific stages of embryonic development and errors in these processes can result in human disease. One example is formation of the vertebrate neural tube. The neural tube is formed via the concerted effort of many cellular pathways that functionally convert a plate of neural ectoderm into a closed tube. Errors in this process can result in birth defects such as spina bifida, exencephaly, or craniorachischisis. One cellular pathway that controls this process is regulated by the Shroom3 cytoskeletal adaptor protein. Shroom3 controls neural tube morphogenesis via the formation of apically positioned contractile networks of actomyosin and these networks facilitate neural tube closure by inducing apical constriction and the anisotropic contraction of actin filaments. This is accomplished via the modular nature of Shroom3. Shroom3 localizes to the apical compartment of epithelial adherens junctions via a direct interaction with F-actin. This interaction is mediated by the Shroom Domain (SD) 1, a unique actin-binding motif present in most Shroom proteins characterized to date. Shroom3 function is also dependent on Rho-kinase (Rock), such that Shroom3 directly binds to Rock and regulates both its localization and catalytic activity. The interaction between Shroom and Rock has been elucidated at the molecular level and is mediated by the conserved SD2 region of Shroom and a conserved coiled-coil region of Rock. The interaction between Shroom and Rock results in the localized activation of non-muscle myosin II (myosin II) contractility, which provides the mechanical force needed to facilitate neural tube morphogenesis. The regulation of myosin II activity by Rock and other cellular pathways has been well described. Rock modulates myosin II activity in two ways. First, Rock can directly phosphorylate the associated regulatory light chain (RLC), which modulates the actin-associated ATPase activity and the conformation of myosin II. Secondly, Rock negatively regulates the phosphatase that dephosphorylates the RLC, thus preventing the inactivation of myosin II (Hildebrand, 2021 and references therein).
Shroom proteins are required for numerous biological processes and are associated with several human diseases. In mammals, there are three definitive Shroom proteins, Shroom2, Shroom3, and Shroom4, each of which contains an N-terminal PDZ domain, the centrally located SD1, and the C-terminally located SD2. All three proteins can directly interact with F-actin and regulate cell morphology via Rock. In humans, SHROOM2 has been linked to neural tube morphogenesis, colorectal cancer, and medulloblastoma, while in vitro studies indicate it is important for cell migration, vasculogenesis, metastasis, and melanosome biogenesis. SHROOM3 mutations have been implicated in chronic kidney disease, heart morphogenesis, and neural tube closure in humans. Using model organisms or cell culture, Shroom3 has been shown to control neural tube closure, axon growth, intestine architecture, eye morphogenesis, thyroid budding, and kidney development. Finally, SHROOM4 mutations have been associated with X-linked mental defects (Hildebrand, 2021).
The Shroom gene is conserved in Drosophila and encodes multiple protein isoforms that have different subcellular distributions and activities in vivo. The most highly conserved region of Drosophila Shroom is the SD2, the region that binds to Drosophila Rho-kinase (Rok). Drosophila Shroom also contains a divergent SD1 motif and this appears to mediate localization to adherens junctions in polarized epithelia. Consistent with the known activities of mammalian Shroom3, expression of Drosophila Shroom in epithelial cells induces apical constriction in a Rok and myosin II dependent manner. While Shroom3 is essential for mouse and human development, Shroom is not absolutely essential for Drosophila viability, as Shroom null flies can be recovered, albeit with significantly reduced frequency. In Drosophila embryos, Shroom is planarly distributed and works in a complicated network with RhoA, Rok, and myosin II to control convergent extension movements. These elegant studies showing the role of Shroom in regulating directional contractility are supported by observations that Shroom proteins can be polarly distributed in mammalian tissues and cells (Hildebrand, 2021 and references therein).
To better understand the mechanisms that control Shroom-regulated changes in cell and tissue morphology, this study has established tools to perform genetic screens for modifiers of Shroom activity in Drosophila. Shroom gain-of-function phenotypes in the eye and wing can be suppressed or enhanced by known components of the Shroom pathway. Using a candidate approach, several cytoskeletal regulators were identified, including Short stop and Enabled, as participants in Shroom-mediated changes in cell morphology. Shroom regulates the distribution of Ena and this is likely mediated by conserved proline-rich sequences in Shroom and the EVH1 domain of Ena. This study further shows that while Ena is required for the Shroom gain-of-function phenotypes, apical recruitment of Ena is not sufficient to cause changes in cell morphology. Additionally, by using an isoform of Shroom that does not bind Ena, but still engages Rok, this study showed that apical constriction can be modulated by different cellular pathways depending on the context (Hildebrand, 2021).
This study describes a genetic approach to identify cellular pathways that participate in tissue morphogenesis. This method takes advantage of the observation that ectopic Shroom protein can utilize the endogenous contractile machinery within epithelial cells to induce apical constriction and disrupt normal tissue morphology. While this work focuses on candidate genes that encode known regulators of epithelial and tissue architecture, it is predicted these tools can be used to perform unbiased, genome-wide screens to identify novel participants in Shroom-mediated cellular processes. Two different tissues, eye and wing imaginal discs, were used for these studies, and these screens can identify factors that are used in a wide range of tissues and cells to control cell dynamics. This is based on the observations that ShroomA, the isoform most similar to mammalian Shroom3, induces similar cellular phenotypes in both types of imaginal discs, and the phenotypes can be modified in both tissues. A powerful aspect of this screen is that these processes are functionally conserved in vertebrate cells and tissues. Additionally, the simplified nature of the Drosophila genome makes these screens possible. Due to genetic and functional redundancy, it is predicted that the analysis performed in this study would be more complicated using vertebrate or cell culture model systems. Drosophila have single genes for Shroom, Rok, myosin II, and Ena while mammals possess gene families for these factors. In support of this, previous work has shown that both Rock1 and Rock2 must be inhibited to prevent Shroom3-mediated apical constriction in cell culture. This screening approach should allow for the identification of novel genetic interactions in Drosophila that can be further verified in mammalian model systems to define their potential role in human disease (Hildebrand, 2021).
Most of the modifiers identified in this study participate in defining actin or microtubule architecture. Of these, several regulate actin dynamics at the level of polymerization or stability, including Ena, Diaphanous, Chickadee, and Slingshot. Interestingly, three of these proteins can be linked, directly or indirectly, to neural tube formation in mice. It should be noted that several classes of actin regulators did not appear to modify the Shroom phenotypes, including nucleators, binding proteins, or adaptors, suggesting that specific types of actin organization are required for Shroom-induced perturbation of cell architecture. This is further supported by the observation that Tropomyosin was also identified in the screen. Tropomyosin regulates the structure of actin filaments and the binding of other proteins, including myosin II and cofilin, that in turn modulate cell architecture or behavior. It is particularly intriguing to note that Tropomyosin mutations can suppress phenotypes caused by the loss of Flapwing, presumably caused by increased myosin II activity. In addition to the actin cytoskeleton, these studies also support a role for microtubules in Shroom-induced phenotypes. This is consistent with the role of microtubules in apical constriction in Drosophila. Recent evidence indicates that apical-medial microtubules play an important role in ventral furrow invagination and this is mediated by Patronin, a protein known to interact with Shot. These studies show that microtubules stabilize the connection of contractile networks to cell junctions to facilitate tissue morphogenesis. These studies are consistent with the current results in relation to Shroom function and Shot distribution in the wing epithelium. It will be interesting to determine if the identified proteins act upstream or downstream of Shroom. While the data suggest Ena acts downstream of Shroom, proteins such as Tropomyosin could function upstream by regulating the amount of Shroom that can bind to F-actin or downstream by modulating the amount of myosin II that can be recruited or activated by the Shroom-Rok complex. It was surprising that determinants of cell adhesion or polarity, such as cadherins or Par complex proteins, were not identified in this screen. It is possible that these proteins are present in sufficient quantity and reducing the dosage is unable to modify the Shroom overexpression phenotype and thus other genetic approaches will be needed to assess the role of these pathways (Hildebrand, 2021).
The data show that endogenous Shroom protein is expressed in epithelial cells during wing and eye development, suggesting it functions in these tissues under normal circumstances. Shroom null flies that survive to adults do not exhibit significant defects in the eyes or wings, although null embryos do exhibit defects in convergent extension and perhaps this could contribute to the observed reduction in viability. In embryos, Shroom is important for the polarized distribution of contractile myosin II needed for convergent extension. It is possible that Shroom activity in disc epithelial cells is redundant to other pathways that regulate Rok and myosin II and Shroom normally functions to make these pathways more robust or function with higher fidelity. Uncovering these subtle interactions will require additional genetic approaches. The localization of Shroom in the eye and wing disc appears to be highly regulated and is reminiscent of that exhibited by myosin II and phosphorylated Sqh, particularly in the eye imaginal disc. A dramatic increase was observed in Shroom protein in cells that are exiting the morphogenetic furrow and forming the pre-clusters that will give rise to the ommatidia. As the ommatidia form, Shroom expression becomes restricted to the R3/4 cells and eventually is lost from these cells. This distribution is essentially the inverse to that of E-cadherin, which is highest in the radial junctions and lower in the circumferential junctions. This could reflect differences in adhesive interactions between the ommatidia pre-clusters and the inter-ommatidia cells, which facilitates rotation of the ommatidia. This hypothesis is supported by previous studies demonstrating that differential adhesion generates specific cellular organization and compartmentalization in the developing eye. Interestingly, the PCP protein Flamingo is also expressed in R3 and R4 and previous studies have identified interactions between the Shroom3 and PCP pathways in the neural tube. As eye development continues, this study observed Shroom expression in the pigment cells of the pupal retina. In both the imaginal disc and the retina, Shroom distribution is restricted to specific cell junctions, suggesting there are differential adhesive or contractile forces associated with these membranes (Hildebrand, 2021).
In the wing imaginal disc, expression of Shroom protein was observed in rows of cells that border the anterior half of the wing margin. Consistent with the genetic interactions, a similar expression pattern was observed for both Ena and Shot in these cells. It is currently unclear if the co-expression of Shroom, Ena, and Shot is controlled pre- or post-transcriptionally. It is possible that the expression of Shroom, Ena, and Shot is coordinately regulated in a gene network. Alternatively, the stability or apical localization of these proteins may be interdependent or closely orchestrated. This expression pattern in the anterior wing margin is similar to members of the Irre cell Recognition Module (IRM), including cell surface receptors Roughest, Hibris, and Kirre, which help position the sensory organs. This is particularly interesting in light of the fact that the vertebrate orthologs of these genes, Neph and Nephrin-1, and Shroom3 are all involved in formation of podocytes in the glomerulus of the mammalian kidney. It will be exciting to apply genetic analysis to investigate if these pathways cooperate to regulate tissue morphology (Hildebrand, 2021).
Ena and Shroom show extensive co-expression and colocalization in both the wing and eye imaginal disc, although Ena is more widely expressed than Shroom. In both the wing and eye imaginal disc, Ena is expressed in most cells and is localized primarily in the tricellular junctions with lower expression in the adherens junctions. However, as seen in the wing margin and the morphogenetic furrow, cells that express Shroom protein also exhibit high levels of Ena in the cell junctions. Importantly, reducing the amount of Shroom protein perturbs the localization of Ena in the anterior wing margin. The relationship between Ena, Shroom, Rok, and myosin II in defining cell shape is likely to be complicated. This stems from the observations that these factors could be placed both upstream and downstream of Shroom. For example, it has been previously shown that Shroom distribution to the apical adherens junctions is mediated, at least in part, by direct binding to F-actin. However, it has also been established that RhoA and Rok regulate F-actin architecture to influence Shroom distribution, which then facilitates the polarized distribution of Rok and myosin II. Ena has been shown to have multiple roles in Drosophila development, including axon guidance, collective cell migration, and epithelial morphogenesis. The role Ena plays in Shroom-mediated apical constriction is unclear. The current data suggest that Ena functions downstream of Shroom and is recruited to adherens junctions via an LPPPP-EVH1 interaction. Ena is primarily defined as a modulator of F-actin dynamics that facilitates the formation of long filaments by competing with barbed-end capping and promoting the addition of actin monomers to the barbed end. This activity may be important for providing the substrate for activated myosin II to drive cell contraction. This is consistent with studies in vertebrate cells showing that Diaphanous 1, is also required for contractility in adherens junctions and that this study has also identified Dia as a potential modifier of Shroom activity (Hildebrand, 2021).
Elegant studies from several groups have identified many other signaling pathways that control the distribution of contractile myosin II networks during Drosophila development, including the Fog, PCP, HH, Dpp, EGF, Toll, and integrin signaling pathway. How all these signaling pathways are orchestrated and converge on myosin II at the cellular and tissue level is a fascinating question. It has been shown that the above processes use a variety of methods to regulate the small GTPase RhoA, which activates Rok, including several GTP exchange factors or GTPase Activating Proteins. It should be noted that other GTPases such as Rap1 or CDC42 also regulate apical constriction. This work has shown that Shroom3 may activate Rock independent of RhoA, suggesting that there as mechanisms to bypass small GTPases in the activation of myosin II. It will be informative to utilize this screening approach to further test how these pathways might work with ShroomA to control cell morphology (Hildebrand, 2021).
Adherens junctions are a defining feature of all epithelial cells, providing cell-cell adhesion and contractile ring formation that is essential for cell and tissue morphology. In Drosophila, adherens junctions are concentrated between the apical and basolateral plasma membrane domains, defined by aPKC-Par6-Baz and Lgl/Dlg/Scrib, respectively. Whether adherens junctions contribute to apical-basal polarization itself has been unclear because neuroblasts exhibit apical-basal polarization of aPKC-Par6-Baz and Lgl in the absence of adherens junctions. This study shows that, upon disruption of adherens junctions in epithelial cells, apical polarity determinants such as aPKC can still segregate from basolateral Lgl, but lose their sharp boundaries and also overlap with Dlg and Scrib - similar to neuroblasts. In addition, control of apical versus basolateral domain size is lost, along with control of cell shape, in the absence of adherens junctions. Manipulating the levels of apical Par3/Baz or basolateral Lgl polarity determinants in experiments and in computer simulations confirms that adherens junctions provide a 'picket fence' diffusion barrier that restricts the spread of polarity determinants along the membrane to enable precise domain size control. Movement of adherens junctions in response to mechanical forces during morphogenetic change thus enables spontaneous adjustment of apical versus basolateral domain size as an emergent property of the polarising system (Bonello, 2021).
The Notch pathway mediates cell-to-cell communication in a variety of tissues, developmental stages and organisms. Pathway activation relies on the interaction between transmembrane ligands and receptors on adjacent cells. As such, pathway activity could be influenced by the size, composition or dynamics of contacts between membranes. The initiation of Notch signalling in the Drosophila embryo occurs during cellularization, when lateral cell membranes and adherens junctions are first being deposited, allowing the investigation of the importance of membrane architecture and specific junctional domains for signaling. By measuring Notch dependent transcription in live embryos it was established that Notch initiates while lateral membranes are growing and that signalling onset correlates with a specific phase in their formation. However, the length of the lateral membranes per se was not limiting. Rather, the adherens junction likely play an important role in modulating
Notch activity (Falo-Sanjuan, 2021).
Embryogenesis requires cells to change shape and move without disrupting epithelial integrity. This requires robust, responsive linkage between adherens junctions and the actomyosin cytoskeleton. Using Drosophila morphogenesis, this study defined molecular mechanisms mediating junction-cytoskeletal linkage and explores the role of mechanosensing. Focus was placed on the junction-cytoskeletal linker Canoe, a multidomain protein. The canoe locus was engineered to define how its domains mediate its mechanism of action. Surprising, the PDZ and FAB domains, which were thought connected junctions and F-actin, are not required for viability or mechanosensitive recruitment to junctions under tension. The FAB domain stabilizes junctions experiencing elevated force, but in its absence, most cells recover, suggesting redundant interactions. In contrast, the Rap1-binding RA domains are critical for all Cno functions and enrichment at junctions under tension. This supports a model in which junctional robustness derives from a large protein network assembled via multivalent interactions, with proteins at network nodes and some node connections more critical than others (Perez-Vale, 2021).
During tissue morphogenesis, the changes in cell shape, resulting from cell-generated forces, often require active regulation of intracellular trafficking. How mechanical stimuli influence intracellular trafficking and how such regulation impacts tissue mechanics are not fully understood. This study identified an actomyosin-dependent mechanism involving Rab11-mediated trafficking in regulating apical constriction in the Drosophila embryo. During Drosophila mesoderm invagination, apical actin and Myosin II (actomyosin) contractility induces apical accumulation of Rab11-marked vesicle-like structures ("Rab11 vesicles") by promoting a directional bias in dynein-mediated vesicle transport. At the apical domain, Rab11 vesicles are enriched near the adherens junctions (AJs). The apical accumulation of Rab11 vesicles is essential to prevent fragmented apical AJs, breaks in the supracellular actomyosin network, and a reduction in the apical constriction rate. This Rab11 function is separate from its role in promoting apical Myosin II accumulation. These findings suggest a feedback mechanism between actomyosin activity and Rab11-mediated intracellular trafficking that regulates the force generation machinery during tissue folding (Chen, 2022).
As organs and tissues approach their normal size during development or regeneration, growth slows down, and cell proliferation progressively comes to a halt. Among the various processes suggested to contribute to growth termination, mechanical feedback, perhaps via adherens junctions, has been suggested to play a role. However, since adherens junctions are only present in a narrow plane of the subapical region, other structures are likely needed to sense mechanical stresses along the apical-basal (A-B) axis, especially in a thick pseudostratified epithelium. This could be achieved by nuclei, which have been implicated in mechanotransduction in tissue culture. In addition, mechanical constraints imposed by nuclear crowding and spatial confinement could affect interkinetic nuclear migration (IKNM), which allows G2 nuclei to reach the apical surface, where they normally undergo mitosis. To explore how mechanical constraints affect IKNM, an individual-based model was devised that treats nuclei as deformable objects constrained by the cell cortex and the presence of other nuclei. The model predicts changes in the proportion of cell-cycle phases during growth, which were validated with the cell-cycle phase reporter FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator). However, this model does not preclude indefinite growth, leading to a postulate that nuclei must migrate basally to access a putative basal signal required for S phase entry. With this refinement, the updated model accounts for the observed progressive slowing down of growth and explains how pseudostratified epithelia reach a stereotypical thickness upon completion of growth (Hecht, 2022).
Mechanisms communicating changes in tissue stiffness and size are particularly relevant in the intestine because it is subject to constant mechanical stresses caused by peristalsis of its variable content. Using the Drosophila intestinal epithelium, this study investigated the role of vinculin, one of the best characterised mechanoeffectors, which functions in both cadherin and integrin adhesion complexes. Vinculin was found to regulated by &alpha-catenin at sites of cadherin adhesion, rather than as part of integrin function. Following asymmetric division of the stem cell into a stem cell and an enteroblast (EB), the two cells initially remain connected by adherens junctions, where vinculin is required, only on the EB side, to maintain the EB in a quiescent state and inhibit further divisions of the stem cell. By manipulating cell tension, it was shown that vinculin recruitment to adherens junction regulates EB activation and numbers. Consequently, removing vinculin results in an enlarged gut with improved resistance to starvation. Thus, mechanical regulation at the contact between stem cells and their progeny is used to control tissue cell number (Bohere, 2022).
Intracellular trafficking regulates the distribution of transmembrane proteins including the key determinants of epithelial polarity and adhesion. The Adaptor Protein 1 (AP-1) complex is the key regulator of vesicle sorting, which binds many specific cargos. This study examined roles of the AP-1 complex in epithelial morphogenesis, using the Drosophila wing as a paradigm. AP-1 knockdown leads to ectopic tissue folding, which is consistent with the observed defects in integrin targeting to the basal cell-extracellular matrix adhesion sites. This occurs concurrently with an integrin-independent induction of cell death, which counteracts elevated proliferation and prevents hyperplasia. A distinct pool of AP-1, which localizes at the subapical Adherens Junctions, was identified. Upon AP-1 knockdown, E-cadherin is hyperinternalized from these junctions and becomes enriched at the Golgi and recycling endosomes. Evidence is provided that E-cadherin hyperinternalization acts upstream of cell death in a potential tumour-suppressive mechanism. Simultaneously, cells compensate for elevated internalization of E-cadherin by increasing its expression to maintain cell-cell adhesion (Moreno, 2022).
Expansive Arp2/3 actin networks and contractile actomyosin networks can be spatially and temporally segregated within the cell, but the networks also interact closely at various sites, including adherens junctions. However, molecular mechanisms coordinating these interactions remain unclear. This study found that the SCAR/WAVE complex, an Arp2/3 activator, is enriched at adherens junctions of the leading edge actomyosin cable during Drosophila dorsal closure. Myosin activators were both necessary and sufficient for SCAR/WAVE accumulation at leading edge junctions. The same myosin activators were previously shown to recruit the cytohesin Arf-GEF Steppke to these sites, and mammalian studies have linked Arf small G protein signaling to SCAR/WAVE activation. During dorsal closure, Steppke was found to be required for SCAR/WAVE enrichment at the actomyosin-linked junctions. Arp2/3 also localizes to adherens junctions of the leading edge cable. It is proposed that junctional actomyosin activity acts through Steppke to recruit SCAR/WAVE and Arp2/3 for regulation of the leading edge supracellular actomyosin cable during dorsal closure (Hunt, 2022).
Epithelial cells remodel cell adhesion and change their neighbors to shape a tissue. This cellular rearrangement proceeds in three steps: the shrinkage of a junction, exchange of junctions, and elongation of the newly generated junction. By combining live imaging and physical modeling, this study showed that the formation of myosin-II (myo-II) cables around the cell vertices underlies the exchange of junctions in the Drosophila wing epithelium. The local and transient detachment of myo-II from the cell cortex is regulated by the LIM domain-containing protein Jub and the tricellular septate junction protein M6. Moreover, M6 shifts to the adherens junction plane on jub x and that Jub is persistently retained at reconnecting junctions in m6 RNAi cells. This interplay between Jub and M6 can depend on the junction length and thereby couples the detachment of cortical myo-II cables and the shrinkage/elongation of the junction during cell rearrangement. Furthermore, this study developed a mechanical model based on the wetting theory and clarified how the physical properties of myo-II cables are integrated with the junction geometry to induce the transition between the attached and detached states and support the unidirectionality of cell rearrangement. Collectively, this study elucidates the orchestration of geometry, mechanics, and signaling for exchanging junctions (Ikawa, 2023).
Adherens junctions are tensile structures that couple epithelial cells together. Junctional tension can arise from cell-intrinsic application of contractility or from the cell-extrinsic forces of tissue movement. This study reports a mechanosensitive signaling pathway that activates RhoA at adherens junctions to preserve epithelial integrity in response to acute tensile stress. This study identified Myosin VI/Jaguar as the force sensor, whose association with E-cadherin is enhanced when junctional tension is increased by mechanical monolayer stress. Myosin VI promotes recruitment of the heterotrimeric Galpha12 (Concertina) protein to E-cadherin, where it signals for p114 RhoGEF to activate RhoA. Despite its potential to stimulate junctional actomyosin and further increase contractility, tension-activated RhoA signaling is necessary to preserve epithelial integrity. This is explained by an increase in tensile strength, especially at the multicellular vertices of junctions, that is due to mDia1-mediated actin assembly (Acharya, 2018).
Epithelia are subject to tensile forces that can challenge their cell-cell integrity. . This is exemplified by the observation that monolayers fracture at junctions when monolayer contractility is acutely increased by calyculin. Similarly, overactivation of contractility during Drosophila gastrulation disrupts the actomyosin networks that couple cells together. The current experiments now identify a junctional mechanotransduction pathway that is responsible for sensing, and responding to, such tensile stresses. It is propose that Myosin VI is the key sensor of acute tensile stress applied to AJs. It is stabilized and accumulates at AJs when tensile forces are transmitted to E-cadherin. This promotes the formation of an E-cadherin-Gα12 complex that activates the p114 RhoGEF-RhoA pathway to increase the tensile strength of multicellular junctions via mDia1. Of note, RhoA signaling is active at the ZA, even under resting conditions, but this is mediated by other GEFs such as Ect2. Thus, the Myosin VI-Gα12-p114 RhoGEF pathway that this study has identified can be considered a selective response to superadded tensile stress (Acharya, 2018).
At first sight, it seemed paradoxical that stimulation of RhoA would be used to preserve epithelial integrity. RhoA promotes actomyosin assembly at AJs under resting conditions and also in calyculin-stimulated cells. Both F-actin and NMII (Zipper) increased at bicellular junctions upon treatment with calyculin, and this was abrogated by p114 RhoGEF KD. This p114 RhoGEF-stimulated increase in actomyosin might be expected to promote junctional rupture by increasing the line tension in bicellular junctions and enhancing the forces acting to disrupt epithelial integrity, especially those focused on multicellular junctions. One possibility was that enhanced actomyosin also increased the stiffness of junctions to resist tensile stress. However, simulations in a mechanical model predicted that increasing stiffness alone would accelerate monolayer fracture rather than retarding it (Acharya, 2018).
Instead, it is considered that the protective effect of the p114 RhoGEF pathway is better explained by an increase in the tensile strength of AJs. In simulations of the vertex model, increasing tensile strength protected monolayer integrity against calyculin-induced stresses, even if junctional stiffness was also increased. Experimentally, it is suggested that this protective effect is especially important at the multicellular vertices. Physical considerations identify vertices as the junctional sites where cellular forces will be greatest (Higashi and Miller, 2017
), and, indeed, vertices were the principal sites where cell separation first began in these experiments. The accelerated onset of fracture that was seen in p114 RhoGEF KD cells thus implied that tension-activated p114 RhoGEF-RhoA signaling might reinforce vertices against stress.
RhoA signals to both NMII and F-actin. However, calyculin appeared to maximally stimulate NMII, and this was not reduced by p114 RhoGEF KD. In contrast, p114 RhoGEF signaling was necessary to stimulate actin assembly at vertices in response to calyculin, an effect that was mediated by the RhoA-sensitive formin, mDia1. In turn, mDia1 was required to reinforce E-cadherin at vertices and for monolayers to resist tensile stress. Thus, p114 RhoGEF-RhoA-mediated actin assembly appeared to be key to preserving epithelial integrity in these experiments, although NMII regulation may also be relevant when tension is increased by other means. Without excluding possible roles for other membrane proteins found at vertices, it is therefore proposed that tension-activated RhoA signaling increases the tensile strength of monolayers by stimulating mDia1-dependent actin assembly to reinforce E-cadherin adhesion at vertices (Acharya, 2018).
It was noteworthy that RhoA signaling was selectively increased at cell-cell junctions but not at other adhesive sites, especially cell-substrate interactions. This highlights a key role for mechanisms that can confer spatial specificity on the mechanotransduction response. Two elements appear to be responsible for junctional selectivity in this instance. First, Gα12 can interact directly with E-cadherin, and this is necessary for junctional RhoA to be stimulated by tensile stress. The current working model is that Gα12, preactivated by S1P2 (Sphingosine 1-phosphate), is recruited to E-cadherin upon application of mechanical stress, where it then recruits and activates p114 RhoGEF to drive RhoA signaling (Acharya, 2018).
Second, this study identified Myosin VI as the force sensor that promotes the E-cadherin-Gα12 association. This requires both the ability of Myosin VI to associate with E-cadherin and also its pronounced capacity to anchor to actin filaments in response to load. The findings suggest that Myosin VI interacts transiently with E-cadherin under steady-state conditions. However, it is stabilized by load-sensitive anchorage when acute tensile stresses are transmitted through E-cadherin. In contrast, the functional impact of Myosin VI was abrogated by the L310G mutant, which retains processive motor function but has defective nucleotide gating linked to load-sensitivity. How increased F-actin anchorage promotes association of Myosin VI with E-cadherin remains to be determined. One possibility is that the increased dwell time of Myosin VI facilitates post-translational modifications that stabilize its binding to E-cadherin. This stabilized Myosin VI-cadherin complex may then promote the recruitment of Gα12 through conformational changes or accessory proteins. Irrespective, Myosin VI appears to exert its signaling effects via E-cadherin-Gα12, since tension-activated RhoA was abolished if Gα12 was unable to bind cadherin (Acharya, 2018).
In conclusion, these findings identify a mechanotransduction pathway that is selectively elicited to preserve epithelial integrity in response to tensile stress. The selectivity of this pathway implies that junctions may possess multiple mechanisms to sense mechanical signals that operate under different circumstances. Of note, α-catenin is necessary for the elemental force-sensitive association of cadherins with F-actin and also supports Ect 2-dependent RhoA signaling in steady-state AJs. Therefore, α-catenin may confer mechanosensitivity under baseline conditions, whereas the Myosin VI-dependent pathway that this study has identified is activated in response to superadded mechanical stress. Furthermore, the experiments tested the effects of acute application of tensile stress. Other mechanisms contribute when mechanical stresses are applied more slowly or are sustained longer, such as cellular rearrangements and oriented cell division. That epithelia possess such a diversity of compensatory mechanisms attests to the fundamental challenge of mechanical stress in epithelial biolog (Acharya, 2018).
Tracheal tube maturation starts with an apical secretion pulse that deposits extracellular matrix components to form a chitin-based apical luminal matrix (aECM). This aECM is then cleared and followed by the maturation of taenidial folds. Finally, air fills the tubes. Meanwhile, the cellular junctions are maintained to ensure tube integrity. The Osiris (Osi) gene family is located at the Triplo-lethal (Tpl) locus on chromosome 3R 83D4-E3 and exhibits dosage sensitivity. This study shows that three Osi genes (Osi9, Osi15, Osi19), function redundantly to regulate adherens junction (AJ) maintenance, luminal clearance, taenidial fold formation, tube morphology, and air filling during tube maturation. The localization of Osi proteins in endosomes (Rab7-containing late endosomes, Rab11-containing recycling endosomes, Lamp-containing lysosomes) and the reduction of these endosomes in Osi mutants suggest the possible role of Osi genes in tube maturation through endosome-mediated trafficking. Tube maturation was examined in zygotic rab11 and rab7 mutants, respectively, to determine whether endosome-mediated trafficking is required. Interestingly, similar tube maturation defects were observed in rab11 but not in rab7 mutants, suggesting the involvement of Rab11-mediated trafficking, but not Rab7-mediated trafficking, in this process. To investigate whether Osi genes regulate tube maturation primarily through the maintenance of Rab11-containing endosomes, rab11 was overexpressed in Osi mutant trachea. Surprisingly, no obvious rescue was observed. Thus, increasing endosome numbers is not sufficient to rescue tube maturation defects in Osi mutants. These results suggest that Osi genes regulate other aspects of endosome-mediated trafficking, or regulate an unknown mechanism that converges or acts in parallel with Rab11-mediated trafficking during tube maturation (Scholl, 2023).
Small RhoGTPases direct cell shape changes and movements during tissue morphogenesis. Their activities are tightly regulated in space and time to specify the desired pattern of actomyosin contractility that supports tissue morphogenesis. This is expected to stem from polarized surface stimuli and from polarized signaling processing inside cells. This general problem was examined in the context of cell intercalation that drives extension of the Drosophila ectoderm. In the ectoderm, G protein-coupled receptors (GPCRs) and their downstream heterotrimeric G proteins (Galpha and Gbetagamma) activate Rho1 both medial-apically, where it exhibits pulsed dynamics, and at junctions, where its activity is planar polarized. However, the mechanisms responsible for polarizing Rho1 activity are unclear. This study reports that distinct guanine exchange factors (GEFs) activate Rho1 in these two cellular compartments. RhoGEF2 acts uniquely to activate medial-apical Rho1 but is recruited both medial-apically and at junctions by Galpha(12/13)-GTP, also called Concertina (Cta) in Drosophila. On the other hand, Dp114RhoGEF (Dp114), a newly characterized RhoGEF, is required for cell intercalation in the extending ectoderm, where it activates Rho1 specifically at junctions. Its localization is restricted to adherens junctions and is under Gbeta13F/Ggamma1 control. Furthermore, Gbeta13F/Ggamma1 activates junctional Rho1 and exerts quantitative control over planar polarization of Rho1. Finally, Dp114RhoGEF was absent in the mesoderm, arguing for a tissue-specific control over junctional Rho1 activity. These results clarify the mechanisms of polarization of Rho1 activity in different cellular compartments and reveal that distinct GEFs are sensitive tuning parameters of cell contractility in remodeling epithelia (Garcia De Las Bayonas, 2019).
Critical aspects of cell mechanics are governed by spatial-temporal control over Rho1 activity during Drosophila embryo morphogenesis. This work sheds new light on the mechanisms underlying polarized Rho1 activation during intercalation in the ectoderm. Rho1 activity was found to be driven by two complementary RhoGEFs under spatial control of distinct heterotrimeric G protein subunits. Notably, a regulatory module was uncovered specific for junctional Rho1 activation (Garcia De Las Bayonas, 2019).
Dp114RhoGEF was identified as a novel activator of junctional Rho1 in the extending ectoderm. Hence, two RhoGEFs, Dp114RhoGEF and RhoGEF2, coordinate independently the modular Rho signaling during tissue extension of the ectoderm. This has important implications, as it allows refinement of the nature of the interconnection between the two pools of Myo-II in this tissue. It has been shown previously that medial pulses of Myo-II flow toward and merge with the Myo-II pool at vertical junctions. However, to what extent these 'fusion' events contribute to junctional Myo-II was unclear. This study genetically uncoupled the regulation of both pools of Myo-II and showed that the loss of one pool does not compromise activation of Myo-II in the other. Indeed, junctional Myo-II levels and planar polarity are not affected in RhoGEF2 shRNA embryos or in RhoGEF2 germline clone where medial Myo-II is lost. This rules out the possibility of medial pulses being the main source of junctional Myo-II accumulation. Instead, it is concluded that actomyosin flow toward junctions contributes to junction shrinkage because it serves a distinct and direct mechanical function in junction remodeling rather than working by proxy by fueling junctional Myo-II (Garcia De Las Bayonas, 2019).
The division of labor in the molecular mechanisms of Rho1 activation in distinct cellular compartments lends itself to differential quantitative regulation. The activation kinetics of these different GEFs and nucleotide exchange catalytic efficiencies are likely to differentially impact Rho1 activity and therefore Myo-II activation at the junctional and medial-apical compartments. For example, RhoGEF2 mammalian orthologs, LARG and PDZ-RhoGEF, show a catalytic activity that is two orders of magnitude higher as compared with the Dp114RhoGEF orthologs subfamily. This may help to establish specific contractile regimes of actomyosin in given subcellular compartments. It is therefore important to tightly control RhoGEFs localization and activity to ensure a proper quantitative activation of the downstream GTPase (Garcia De Las Bayonas, 2019).
RhoGEF2 is a major regulator of medial-apical Rho1 activity during Drosophila gastrulation. Originally characterized in the invaginating mesoderm, it was found that RhoGEF2 also activates Rho1 medial-apical activity in the elongating ectoderm. There, RhoGEF2 localizes both medial-apically and at junctions where it is also planar polarized. Although RhoGEF2 and active Rho1 are both planar polarized at junctions, in RhoGEF2 mutants, junctional Rho1-GTP is not affected and ectopic recruitment of RhoGEF2 following expression of Gα12/13Q303L does not cause ectopic junctional Rho1-GTP accumulation. Thus, RhoGEF2 localization at the membrane is not strictly indicative of its activation status. Interestingly, Gα12/13/Cta is necessary for RhoGEF2 to translocate from microtubules plus ends to the plasma membrane where it signals. To date, experimental evidence favor a model whereby the binding of active Gα12/13/Cta to the RhoGEF in the vicinity of the cell membrane triggers its conformational change and stabilizes it in an open conformation able to bind to lipids via its PH domain and signal at the plasma membrane. There is no evidence that Gα12/13/Cta-GTP actively destabilizes RhoGEF2-EB1 interaction, but this is a formal possibility to be tested. Importantly, Gα12/13/Cta alone does not account for the restricted activation of Rho1 medial-apically (Garcia De Las Bayonas, 2019).
It is hypothesized that additional factors must regulate the spatial distribution of RhoGEF2 activity. In principle, RhoGEF2 signaling activity could either be specifically induced medial-apically independent of RhoGEF2 recruitment or RhoGEF2 could be inhibited at junctions and laterally. Sequestration of inactive RhoGEFs at cell junctions has been reported previously in mammalian cell cultures, suggesting that such mechanism could be evolutionary conserved. Phosphorylation can control the activity of the RH-RhoGEFs subfamily. Therefore, phosphorylation could promote activation or inhibition of RhoGEF2 activity in specific subcellular compartments in the ectoderm. RhoGEF2 is reported to be phosphorylated in the gastrulating embryo (Garcia De Las Bayonas, 2019).
Complementary to RhoGEF2, Dp114RhoGEF activates junctional Rho1 in the ectoderm. Dp114RhoGEF strictly localizes at junctions, providing a direct explanation for its junctional-specific effect. Gβ13F/G&gamma1 is also enriched at adherens junctions, where it controls Dp114RhoGEF junctional recruitment together with additional upstream regulators. Therefore, it is suggested that Gβ13F/Gγ1-dependent tuning of junctional Rho1 activation could be achieved through its ability to concentrate the GEF at junctions. Gβ/Gγ-dependent regulation of RhoGEFs has been described in mammals. One study proposes that mammalian p114RhoGEF may bind and be activated by Gβ1/Gγ2. Interestingly, recent work demonstrates that Gα12 can also recruit p114RhoGEF at cell junctions under mechanical stress in mammalian cell cultures where it promotes RhoA signaling. However, the region of mammalian p114RhoGEF that binds to Gα12 is absent in invertebrate RhoGEFs. How Gβ13F/Gγ1 controls Dp114RhoGEF at junctions in the Drosophila embryo remains an open question. A recent study reports that Dp114RhoGEF localizes at adherens junctions in the Drosophila ectoderm through multiple mechanisms, including interactions with Baz/Par3 and the Crumbs complex. Therefore, investigating a possible connection between Gβ13F/Gγ1 signaling and Baz/Crumbs should help decipher the mechanisms of Dp114RhoGEF localization (Garcia De Las Bayonas, 2019).
Importantly, neither Gβ13F/Gγ1 nor Dp114RhoGEF are themselves planar polarized at junctions. Hence, their distribution alone cannot explain polarized Rho1 activity at junctions. Strikingly, an increase in Gβ13F/Gγ1 dimers was found to hyperpolarize Rho1 activity and Myo-II at vertical junctions. Gβ13F/Gγ1 overexpression also leads to an overall increase in Dp114RhoGEF levels at junctions, although Dp114RhoGEF is not planar polarized in this condition. This indicates that recruitment at the plasma membrane and activation of Dp114RhoGEF are independently regulated, similar to RhoGEF2. In contrast, Dp114RhoGEF overexpression increases Myo-II at both transverse and vertical junctions, although a slightly stronger accumulation is observed at vertical junctions. Therefore, although Dp114RhoGEF junctional levels are increased in both experiments, only Gβ13F/Gγ1 overexpression leads to an increased planar polarization of Rho1-GTP and Myo-II at vertical junctions. This points to a key role for Gβ13F/Gγ1 subunits in the planar-polarization process associated with but independent from the sole recruitment of Dp114RhoGEF at junctions. In principle, Gβ13F/Gγ1 could bias junctional Rho1 signaling either by promoting its activation at vertical junctions or by inhibiting it at transverse junctions (e.g., RhoGAP polarized activation). Gβ13F/Gγ1 could also control active Rho1 distribution independent of its activation. For instance, a scaffolding protein binding to Rho1-GTP at junctions could be polarized by Gβ13F/Gγ1 to bias Rho1-GTP distribution downstream of its activation. Anillin, a Rho1-GTP anchor known to stabilize Rho1 signaling at cell junctions is a potential candidate in the ectoderm. Last, Toll receptors control Myo-II planar polarity in the ectoderm. Whether Gβ13F/Gγ1 and Tolls are part of the same signaling pathway is an important point yet to address in the future (Garcia De Las Bayonas, 2019).
Finally, this study sheds light on new regulatory differences underlying tissue invagination and tissue extension. This study found that Dp114RhoGEF localizes at junctions in the ectoderm, where it activates Rho1 and Myo-II. In contrast, maternally and zygotically supplied Dp114RhoGEF::GFP is not detected at junctions in the mesoderm. Little if any cytoplasmic signal is seen in this condition, suggesting that Dp114RhoGEF::GFP could be degraded in these cells. Thus, repression of Dp114RhoGEF protein in the mesoderm could be an important mechanism for cell apical constriction and proper tissue invagination. Of interest, Rho1 signaling is absent at junctions in the mesoderm. Therefore, it is tempting to suggest that the absence of Dp114RhoGEF at junction in the mesoderm accounts for cells' inability to activate Rho1 in this compartment. Importantly, the GPCR Smog and Gβ13F/Gγ1 subunits, found to control junctional Rho1 in the ectoderm, are common to both tissues. Dp114RhoGEF differential expression and/or subcellular localization could be a key element to bias signaling toward junctional compartment in the ectoderm (Garcia De Las Bayonas, 2019).
Cell contractility necessitates activation of the Rho1-Rock-MyoII core pathway. During epithelial morphogenesis, tissue- and cell-specific regulation of Rho1 signaling requires the diversification of Rho1 regulators, in particular RhoGEFs, as shown in this study, and RhoGAPs. Some of them are tissue specific with given subcellular localizations and activation mechanisms. The identification of signaling modules, namely Gα12/13-RhoGEF2 and Gβ13F/Gγ1-Dp114RhoGEF, provides a simple mechanistic framework for explaining how tissue-specific modulators control Rho1 activity in a given subcellular compartment in a given cell type. Therefore, it is suggested that the variation of (1) ligands, GPCRs, and associated heterotrimeric G proteins and (2) types of RhoGEFs and RhoGAPs as well as their combination, activation, and localization by respective co-factors underlies the context-specific control of Rho1 signaling during tissue morphogenesis. How developmental patterning signals ultimately control Rho regulators is an exciting area for future investigations (Garcia De Las Bayonas, 2019).
In animal cells, cell division entails drastic cell shape changes necessary for the faithful segregation of the duplicated genome into the two daughter cells. These morphological changes include cell rounding required for correct spindle formation and orientation, as well as cytokinesis to separate the cytoplasms of the daughter cells. Studies on single cells and tissues have shown that these cell shape changes are powered by small RhoGTPases that remodel the actomyosin cytoskeleton. Moreover, in multicellular contexts, RhoGTPases are also critical to couple cell shape changes and junction dynamics to control tissue polarity, cohesion, and architecture. Notably, cell division is tightly linked to cell fate specification as well as tissue growth, morphogenesis, and mechanics. Therefore, characterizing the regulation of RhoGTPases and its implication in cytoskeleton and junction dynamics during cell division in tissues is central to understand how cell number and genome integrity are controlled and how tissue architecture and function are established and maintained (di Pietro, 2023).
The small RhoGTPases Rho, Rac, and Cdc42 are key pleiotropic regulators of the actomyosin cytoskeleton and cell junction dynamics. They switch between an active GTP-bound state that binds downstream effectors and an inactive GDP-bound state. These states are primarily regulated by Rho guanine exchange factors (RhoGEFs) that activate RhoGTPases by exchanging GDP for GTP and by RhoGTPase-activating factors (RhoGAPs) that promote GTP hydrolysis to GDP, thereby inactivating RhoGTPases. Individual RhoGEF/GAPs associated with the regulation of cell shape changes, cell division, migration, and polarity have been identified in cultured cells and, to a lesser extent, by targeted RNAi or mutant analyses in multicellular contexts. In addition, by ectopically expressing all human RhoGEF/GAPs in cultured cell lines, a recent study has defined their localizations and biochemical interactomes, enabling a better understanding of single-cell migration. Therefore, the mechanisms mediating the spatiotemporal activation of small RhoGTPases are best understood in individual cells in interphase. However, the spatiotemporal regulation of RhoGTPases remains far less explored during cell division or in tissues, impeding understanding of actomyosin and junction dynamics during interphase and cell division in epithelial tissues (di Pietro, 2023).
During animal cell cytokinesis, RhoGTPases control the pronounced cell deformations associated with cytokinetic ring constriction. Numerous studies have converged to show that the assembly and constriction of the actomyosin cytokinetic ring is powered by the membrane redistribution of the RhoGEF ECT2 (Drosophila pebble, Pbl) and RacGAP1 (Drosophila tumbleweed, Tum) within the dividing cell. Despite these fundamental findings, the role of most RhoGEF/GAPs during cell division remains poorly explored. In addition, in epithelial tissues, several studies have shown that the drastic cytokinesis cell shape changes are coupled with E-cadherin (Ecad) adherens junction (AJ) remodeling and de novo AJ formation. In particular, epithelial cytokinesis shares general features in several vertebrate and invertebrate tissues: (1) de novo AJ formation is coordinated with cytokinesis and relies on mechanosensing processes involving the dividing cell and its neighbors, and (2) the arrangement of the newly formed cell junctions is defined in late cytokinesis, and it is proposed to modulate tissue topology and morphogenesis. Some of these epithelial cytokinetic features are known to be regulated by the Rho and Rac GTPases, but the mechanisms controlling their local and temporal activations remain unknown (di Pietro, 2023).
Toward achieving an integrated view of the spatiotemporal regulation of actomyosin and junction dynamics in proliferative epithelia in vivo, a complete library of fluorescently tagged Drosophila RhoGEF/GAPs was assembled. Then RhoGEF/GAP localizations were systematically analyzed from interphase to cell division in two Drosophila epithelial tissues by time-lapse microscopy. By doing so, this study unraveled a series of putative regulators of epithelial tissue organization, polarity, and dynamics. These results led to a focus on the RhoGEF Cysts and RhoGEF4, and to characterize their respective roles in mechanosensation and AJ formation during epithelial cell division. Altogether, this work advances the understanding of cell division in epithelial tissues and highlights that the RhoGEF/GAP library will be a relevant resource to investigate how actomyosin and junction dynamics are controlled during development, repair, and homeostatic processes (di Pietro, 2023).
By modulating the activity of small RhoGTPases, RhoGEFs and RhoGAPs control cytoskeleton organization and dynamics in a broad range of processes such as cell morphogenesis, division, and migration in all eukaryotes. The functions of RhoGTPases have been extensively and systematically investigated in individual cells. However, multicellularity entails a complex interplay between the cytoskeleton and cell-cell junctions to regulate cell and tissue architecture and dynamics, thus highlighting the relevance of characterizing RhoGEF/GAP function in tissues. Cell division has emerged as a multicellular process since it entails the deformation of the neighboring cells, the remodeling of the dividing and neighbor cell junctions, and de novo junction formation. To better understand how RhoGEFs and RhoGAPs control the cytoskeleton and cell junction dynamics in proliferative epithelial tissues, a family-wide Drosophila transgenic library of fluorescently tagged RhoGEF/GAPs was generated and their distributions were systematically determined during interphase and cell division in two epithelial tissues. This screen revealed multiple uncharacterized RhoGEF/GAP interphasic localizations as well as a complex choreography of RhoGEF/GAPs during cell division have been better defined. Building on this screen, the processes of mechanosensing and junction formation by delineating how the activities of specific RhoGTPases are controlled during epithelial cytokinesis and de novo junction formation. This study has therefore uncovered the first mechanisms of RhoGTPase activation underlying the multicellularity of epithelial cell division (di Pietro, 2023).
The Drosophila eye develops from the larval eye disc, a flattened vesicle comprised of continuous retinal and peripodial epithelia (PE). The PE is an epithelium that plays a supporting role in retinal neurogenesis, but gives rise to cuticle in the adult. This study reports that the PE is also necessary to preserve the morphology of the retinal epithelium. Depletion of the adherens junction (AJ) components β-Catenin (β-Cat), DE-Cadherin or α-Catenin from the PE leads to altered disc morphology, characterized by retinal displacement (RDis); so too does loss of the Ajuba protein Jub, an AJ-associated regulator of the transcriptional coactivator Yorkie (Yki). Restoring AJs or overexpressing Yki in β-Cat deficient PE results in suppression of RDis. Additional suppressors of AJ-dependent RDis include knockdown of Rho kinase (Rok) and Dystrophin (Dys). Furthermore, knockdown of βPS integrin (Mys) from the PE results in RDis, while overexpression of Mys can suppress RDis induced by the loss of β-Cat. It is thus proposed that AJ-Jub-Yki signaling in PE cells regulates PE cell contractile properties and/or attachment to the extracellular matrix to promote normal eye disc morphology (DeSantis, 2023).
Collective cell movements contribute to tissue development and repair and spread metastatic disease. In epithelia, cohesive cell movements require reorganization of adherens junctions and the actomyosin cytoskeleton. However, the mechanisms that coordinate cell-cell adhesion and cytoskeletal remodeling during collective cell migration in vivo are unclear. This study investigated the mechanisms of collective cell migration during epidermal wound healing in Drosophila embryos. Upon wounding, the cells adjacent to the wound internalize cell-cell adhesion molecules and polarize actin and the motor protein non-muscle myosin II to form a supracellular cable around the wound that coordinates cell movements. The cable anchors at former tricellular junctions (TCJs) along the wound edge, and TCJs are reinforced during wound closure. The small GTPase Rap1 was necessary and sufficient for rapid wound repair. Rap1 promoted myosin polarization to the wound edge and E-cadherin accumulation at TCJs. Using embryos expressing a mutant form of the Rap1 effector Canoe/Afadin that cannot bind Rap1, it was found that Rap1 signals through Canoe for adherens junction remodeling, but not for actomyosin cable assembly. Instead, Rap1 was necessary and sufficient for RhoA/Rho1 activation at the wound edge. The RhoGEF Ephexin localized to the wound edge in a Rap1-dependent manner, and Ephexin was necessary for myosin polarization and rapid wound repair, but not for E-cadherin redistribution. Together, these data show that Rap1 coordinates the molecular rearrangements that drive embryonic wound healing, promoting actomyosin cable assembly through Ephexin-Rho1, and E-cadherin redistribution through Canoe, thus enabling rapid collective cell migration in vivo (Rothenberg, 2023).
One central question for cell and developmental biologists is defining how epithelial cells can change shape and move during embryonic development without tearing tissues apart. This requires robust yet dynamic connections of cells to one another, via the cell-cell adherens junction, and of junctions to the actin and myosin cytoskeleton, which generates force. The last decade revealed that these connections involve a multivalent network of proteins, rather than a simple linear pathway. This study focused on Drosophila Canoe, homolog of mammalian Afadin, as a model for defining the underlying mechanisms. Canoe and Afadin are complex, multidomain proteins that share multiple domains with defined and undefined binding partners. Both also share a long carboxy-terminal intrinsically disordered region (IDR), whose function is less well defined. IDRs are found in many proteins assembled into large multiprotein complexes. This study has combined bioinformatic analysis and the use of a series of canoe mutants with early stop codons to explore the evolution and function of the IDR. This bioinformatic analysis reveals that the IDRs of Canoe and Afadin differ dramatically in sequence and sequence properties. When shorter evolutionary time scales were examined, multiple conserved motifs were identified. Some of these are predicted by AlphaFold to be alpha-helical, and two correspond to known protein interaction sites for alpha-catenin and F-actin. The lesions were identified in a series of eighteen canoe mutants, which have early stop codons across the entire protein coding sequence. Analysis of their phenotypes are consistent with the idea that the IDR, including the conserved motifs in the IDR, are critical for protein function. These data provide the foundation for further analysis of IDR function (Gurley, 2023).
Drosophila trachea is a classical model for analyzing epithelial, especially tubular epithelial biology. This study identified lateral E-cadherin mediated junctions that encircle the cells just basal to the zonula adherens in the larval trachea. The lateral junction is associated with downstream adapters, including catenins, and has a distinct junctional actin cortex. The lateral cortex contributes to the development of a supracellular actomyosin mesh in the late larvae. Establishment of this cytoskeletal structure depends on lateral junction associated Rho1 and Cdc42 GTPases, and Arp and WASP pathways. The supracellular network takes the character of stress fibers along the AP axis in the early hours of pupation. It contributes to the shortening of the epithelial tube albeit in a manner redundant to ECM-mediated compression mechanism. In conclusion, this study shows the in vivo existence of functional lateral adherens junction and suggest a role for it in mediating dynamic cytoskeletal events during tissue scale morphogenesis (Pradhan, 2023).
During embryonic development dramatic cell shape changes and movements re-shape the embryonic body plan. These require robust but dynamic linkage between the cell-cell adherens junctions and the force-generating actomyosin cytoskeleton. View of this linkage have evolved, and it is now realize that linkage is mediated by a mechanosensitive multiprotein complex assembled via multivalent connections. This study combined genetic, cell biological and modeling approaches to define the mechanism of action and functions of an important player, Drosophila Polychaetoid, homolog of mammalian ZO-1. The data reveal that Pyd reinforces cell junctions under elevated tension, and facilitates cell rearrangements. Pyd is important to maintain junctional contractility and in its absence cell rearrangements stall. Next, structured illumination microscopy was used to define the molecular architecture of cell-cell junctions during these events. The cadherin-catenin complex and Cno both localize to puncta along the junctional membrane, but are differentially enriched in different puncta. Pyd, in contrast, exhibits a distinct localization to strands that extend out from the region occupied by core junction proteins.The implications for the protein network at the junction-cytoskeletal interface is discussed, suggesting different proteins localize and function in distinct ways but combine to produce robust connections (Schmidt, 2023).
Bacillus thuringiensis subsp. kurstaki (Btk) is a strong pathogen toward lepidopteran larvae thanks to specific Cry toxins causing leaky gut phenotypes. Hence, Btk and its toxins are used worldwide as microbial insecticide and in genetically modified crops, respectively, to fight crop pests. However, Btk belongs to the B. cereus group, some strains of which are well known human opportunistic pathogens. Therefore, ingestion of Btk along with food may threaten organisms not susceptible to Btk infection. This study shows that Cry1A toxins induce enterocyte death and intestinal stem cell (ISC) proliferation in the midgut of Drosophila melanogaster, an organism non-susceptible to Btk. Surprisingly, a high proportion of the ISC daughter cells differentiate into enteroendocrine cells instead of their initial enterocyte destiny. Cry1A toxins weaken the E-Cadherin-dependent adherens junction between the ISC and its immediate daughter progenitor, leading the latter to adopt an enteroendocrine fate. Hence, although not lethal to non-susceptible organisms, Cry toxins can interfere with conserved cell adhesion mechanisms, thereby disrupting intestinal homeostasis and endocrine functions (Jneid, 2023).
Biological tissues acquire reproducible shapes during development through dynamic cell behaviors. Most of these behaviors involve the remodeling of cell-cell contacts. During epithelial morphogenesis, contractile actomyosin networks remodel cell-cell contacts by shrinking and extending junctions between lateral cell surfaces. However, actomyosin networks not only generate mechanical stresses but also respond to them, confounding understanding of how mechanical stresses remodel cell-cell contacts. This study developed a two-point optical manipulation method to impose different stress patterns on cell-cell contacts in the early epithelium of the Drosophila embryo. The technique allows production of junction extension and shrinkage through different push and pull manipulations at the edges of junctions. These observations were used to expand classical vertex-based models of tissue mechanics, incorporating negative and positive mechanosensitive feedback depending on the type of remodeling. In particular, this study showed that Myosin-II activity responds to junction strain rate and facilitates full junction shrinkage. Altogether this work provides insight into how stress produces efficient deformation of cell-cell contacts in vivo and identifies unanticipated mechanosensitive features of their remodeling (Nishizawa, 2023).
A collective cell motility event that occurs during Drosophila eye development, ommatidial rotation (OR), serves as a paradigm for signaling-pathway-regulated directed movement of cell clusters. OR is instructed by the EGFR and Notch pathways and Frizzled/planar cell polarity (Fz/PCP) signaling, all of which are associated with photoreceptor R3 and R4 specification. This study shows that Abl kinase negatively regulates OR through its activity in the R3/R4 pair. Abl is localized to apical junctional regions in R4, but not in R3, during OR, and this apical localization requires Notch signaling. Abl and Notch interact genetically during OR, and Abl co-immunoprecipitates in complexes with Notch in eye discs. Perturbations of Abl interfere with adherens junctional organization of ommatidial preclusters, which mediate the OR process. Together, these data suggest that Abl kinase acts directly downstream of Notch in R4 to fine-tune OR via its effect on adherens junctions (Koca, 2023).
This study demonstrates that dAbl regulates cell motility during OR. Although loss of Abl function interferes with multiple aspects of photoreceptor development and morphogenesis, overexpression of dAbl in developing ommatidial clusters in eye discs affects specifically OR, suggesting that dAbl has a defined function in rotation. During OR, dAbl appears to have an inhibitory role, as ommatidial clusters with increased dAbl levels under-rotate, whereas dAbl mutant ommatidia tend to rotate faster (Koca, 2023).
The localization pattern of dAbl posterior to the MF provides further insight about its role in OR. dAbl becomes apically localized in photoreceptors R8, R2/R5, and R4, following a steady phase of rotation, at the time when clusters slow down and refine their motility until the completion of the 90° angle. Prominent Abl localization within the apical plane of specific photoreceptors suggests that Abl is likely to have a local function in the apical junctional domain. Under-rotation features observed upon dAbl overexpression are consistent with the notion that dAbl becomes apically localized in specific R cells, toward the later stages of OR, to slow down the process. Interestingly, there is a differential localization of dAbl between R3 and R4 in the apical junctional domain. Considering the role of the R3/R4 pair and associated signaling pathways in OR, it is tempting to speculate that this differential dAbl localization is comparable to the requirement of the Nmo kinase within R3/R4, with Nmo providing a directional impulse to rotation in R433 and dAbl regulating its slowing down. The data argue that dAbl activity within R3/R4 pairs is indeed important for fine-tuning rotation. Knockdown and overexpression of dAbl in R3/R4 pairs lead to over-rotation and under-rotation, respectively, during the active rotation process in eye discs, suggesting that Abl activity negatively regulates rotation. Specifically, knockdown of Abl in R3/R4 leads to over-rotation of ommatidia, which, taken together with the WT localization of Abl being restricted to the R4 apical junctional domain, suggests that Abl is required in R4 within the apical region to slow down rotation. In the case of under-rotation caused by m Δ0.5>Abl overexpression, apical dAbl was detected in both cells of the R3/R4 pair and, importantly, temporally earlier in this background compared with WT, suggesting that early dAbl expression in both cells causes an under-rotation phenotype by interfering with rotation. Taken together, these observations are consistent with the hypothesis that the timing and specificity of apical localization of dAbl in R4 is critical for its normal function in OR (Koca, 2023).
Notably, Abl overexpression does not appear to affect ommatidial chirality and the localization of PCP factors, as Fmi expression and localization remain intact. Furthermore, Abl overexpression causes a specific and severe under-rotation defect, unlikely resulting from deregulation of core PCP factors, which are commonly associated with random ommatidial chirality and rotation. It is most likely that Abl overexpression, under sev- or m Δ0.5-Gal4 drivers, is temporally too late to interfere with Fz/PCP signaling-mediated R3/R4 cell fate decisions, and thus specifically affects OR (Koca, 2023).
Fz/PCP signaling appears dispensable for the R4-specific apical dAbl localization, as the pattern is maintained in core PCP mutant ommatidia. Yet dAbl does synergize with Fmi, when co-overexpressed in the R3/R4 pair, in a rotation specific manner. This OR-associated functional interaction of Abl with membrane-associated core PCP factors, along with the localization pattern of Abl in the apical domain further suggests that dAbl activity is important in R4 in the apical junctional domain. The results identify Notch and Notch signaling in R4 as critical for apical dAbl localization. Notch over-activation within the R3/R4 pair (via expression of stable isoforms of the receptor) induces apical dAbl localization in both cells of the pair. In contrast, expression in R3/R4 pairs of a version of Notch deficient in Delta binding, the key Notch ligand in the eye, and thus interference with ligand induced Notch activation, leads to a loss of apical dAbl in R4. Similarly, reduction of Notch levels in R3/R4 cells (via RNAi-mediated knockdown) also causes a marked decrease in apical dAbl levels in R4. As Notch-dependent transcription is still active in these backgrounds, the combination of these results suggests that Notch-mediated dAbl apical localization is rather direct, and not via a secondary mechanism through transcriptional regulation. This conclusion is corroborated by the co-immunoprecipitation experiments (Koca, 2023).
Several experimental lines support the hypothesis that the Notch receptor physically recruits dAbl to the membrane. In salivary glands, Notch overexpression augments junctional dAbl localization, leaving total dAbl levels unaffected. dAbl co-immunoprecipitates with Notch in third-instar larval eye disc extracts, supporting a membrane-associated Notch-Abl interaction in vivo, independent of nuclear Notch signaling activity. The sev>Abl GOF rotation phenotype is markedly suppressed upon removal of one copy of Notch, further supporting the idea that a functional N-Abl signaling module in the apical domain of R4 regulates OR (Koca, 2023).
dAbl localization appears to be within the apical region and not restricted to the apical membrane ring. There may be multiple reasons for this. As the Notch receptor is cleaved upon ligand binding and its intracellular domain is released to the cytoplasm, distribution of Abl molecules in the apical region may be broader than restricted to the transmembrane fraction of Notch. Abl-Notch interactions likely last after Notch cleavage, considering efficient Abl co-immunoprecipitation with the Notch ICD. Abl can also interact with actomyosin cytoskeletal elements, which are apically enriched in R cells (Koca, 2023).
As the apical diameter of R cells in this region is less than 2 μm, the imaging resolution does not separate the membrane Abl signal from the juxta-membrane cytoplasmic signal. Notably, in Notch overexpression contexts, Abl signal is often detected as a ring at the apical membrane, likely attributable to the presence of more uncleaved membrane-associated Notch. Furthermore, it is possible to detect and quantify Abl at junctions in salivary glands, and thus document the increased levels of membrane-associated Abl upon higher Notch levels. All these data are consistent with the notion that Abl is specifically recruited to the apical junctional membrane domain by Notch (Koca, 2023).
In Drosophila, dAbl has been suggested to act downstream of Notch during axonal pathfinding in embryos. Compelling evidence suggests that a non-canonical Notch signaling branch, which does not entail nuclear Notch activity, instructs axonal pathfinding and axon-guidance-specific genetic interactions between dAbl and Notch argue that a non-canonical Notch signaling pathway via dAbl may be at work in this context (Koca, 2023).
The results are in accordance with these observations and provide further evidence for a non-canonical Notch-Abl signaling module during morphogenesis. Recently, a non-canonical Notch pathway has been reported in the regulation of adherens junction organization during human vascular barrier formation,
with the transmembrane domain of Notch forming complexes with the tyrosine phosphatase LAR, vascular endothelial cadherin, and Rac1GEF Trio to confer barrier function in human engineered microvessels. The Notch transmembrane domain requires the cleavage of the Notch extracellular and intracellular domains in this context.
The data during OR indicate that apical dAbl recruitment in R4 similarly requires Notch activation by Delta. Whether the transmembrane domain of Notch is an essential component of dAbl recruitment and/or regulation remains to be confirmed. There is a growing body of evidence that Notch uses alternative downstream signaling events to regulate cellular morphogenesis and organization, besides canonical transcriptional target gene regulation. (Koca, 2023).
Abl appears to affect junctional N-cad and Arm levels in the R3/R4 pair. N-cad mutants show OR defects. Although the mechanism of N-cad involvement remains unclear, N-cad and/or Arm at the R3/R4 boundary could mediate the communication between these cells to determine relative force generation or other directional behavior to give the rotation direction or impulse/force. Such mechanisms have been suggested in border cell migration through E-cad (Koca, 2023).
N-cad mutant ommatidia appear to over-rotate
unlike Abl-overexpressing ommatidia (in which N-cad is downregulated at the R3/R4 border). Although this seems like a discrepancy, Abl overexpression by m Δ0.5-Gal4 (unlike N-cad mutations) is spatially and temporally restricted to R3/R4s, possibly accounting for the differences observed in these backgrounds. Furthermore, Abl likely affects OR via regulating several downstream effectors, including cytoskeletal regulators, in parallel to N-cad and thus has a more complex impact on OR than N-cad alone (Koca, 2023).
The observation that the non-phosphorylatable isoform of Arm/β-catenin, ArmY667F, rescues the Abl GOF defects, supports the idea that Arm is a key and direct target of dAbl in the OR context. dAbl is involved in the regulation of multi-cellular reorganization in the context of Drosophila germband elongation through the phosphorylation of Arm/β-catenin on tyrosine 667 (Y667), by which it controls adherens junction turnover to promote convergent extension cell movements (Koca, 2023).
The data argue that dAbl may similarly be involved in regulating Arm/β-catenin dynamics through the same residue during the OR process. The under-rotation phenotype associated with the dAbl GOF (sev>Abl) showed a trend toward rescue by co-(over)expression of Arm-WT and ArmY667E, which is likely due to the fact that exogenously overexpressed Arm isoforms compete with endogenous Arm for dAbl binding. Further experiments will be needed to test these hypotheses (Koca, 2023).
The requirement of Abl in R4 for accurate rotation suggests that it acts antagonistically to Nemo which is enriched at junctions in R4 early via core PCP factors and its function is to promote rotation (Koca, 2023).
There is a temporal sequence of apical plane enrichment of factors in R4 with Nemo first to initiate rotation, and Abl a few hours later to slow it down. It was originally proposed that OR is a two-step process, with an initial fast rotation to 45° and a subsequent slower step to achieve the full 90°. However, this idea goes back to the identification of the original allele of nemo, which is a hypomorph, and only affected the rotation process partially (Koca, 2023).
Recent live imaging studies documenting OR dynamics have established that rotation is continuous with comparable speed throughout. Similarly, there is growing evidence that for rotation to occur correctly, adherens junctions need to be dynamically regulated at the interface between all photoreceptors and the non-rotating inter-ommatidial cells, and possibly between individual inter-ommatidial cells.
It is thus very likely that Abl overexpression with m Δ0.5 and sev drivers interferes with rotation by affecting adherens junction regulation and dynamics in all or multiple R cells, like Nemo (Koca, 2023).
Localization of Abl within the apical plane of R4, as well as R2/R5, is detected at late stages of rotation (from rows 7 and 8 onward), when rotation needs to be slowed down and stopped at 90°, indicating that Abl has a role at the late phases of the process, to terminate rotation. There are additional cues that appear to signal within ommatidia to stop rotation. For example, EGFR signaling via Argos (the original allele of argos being 'roulette/rlt') certainly feeds into slowing down rotation, as without the inhibitory EGFR ligand, argosrlt mutant clusters rotate beyond 90° (as the name 'roulette' indicates). Similarly, Scabrous (Sca), a secreted fibrinogen-like factor, has been suggested to regulate the properties of the extracellular matrix to create a barrier to rotation (Koca, 2023).
Although the mechanism of Sca function remains unknown, a direct involvement of the ECM in rotation has been reported with a specific link of Integrin signaling and ECM in the OR process. A model is thus emerging that suggests the degree of rotation depends on an interplay between multiple signaling pathways, including Notch-Abl signaling, and their regulatory input to cell adhesion and cytoskeletal elements (Koca, 2023).
Notch signaling in R3/R4 pairs is critical to coordinate OR via its feeding into the transcriptional regulation of argos,
with Notch signaling directly promoting the transcription of argos, the inhibitory ligand to EGFR, required to fine-tune EGFR signaling activity during OR.
This study shows that Notch signaling regulates OR via apical junctional recruitment of dAbl in R4, linking Notch activity to non-canonical, Abl-mediated Notch signaling and associated local cellular processes, with Abl modulating cadherin/β-catenin-based junctional complexes. Involvement of Notch signaling in cellular morphogenesis has been suggested in various contexts, including Drosophila oogenesis and neuronal pathfinding, zebrafish sensory organ development and human vascular barrier formation among others (Koca, 2023).
Besides the reported Notch signaling-mediated transcriptional inputs into adhesion and cytoskeletal dynamics a direct link from the Notch receptor to cell adhesion has been revealed (Koca, 2023).
This work also suggests a direct input from Notch signaling to cell adhesion dynamics. Many regulators of OR show conservation across developmental processes in vertebrates. The role of Notch signaling in OR suggests a potential involvement for Notch in PCP-mediated morphogenetic events in vertebrates, which has not been reported thus far. Similarly, Abl kinase may have a role in such processes in its interaction with PCP and Notch signaling pathways. Strikingly, the mouse abl-/- arg-/- double mutants exhibit defects in neurulation and delays in neural tube closure, a process generally requiring PCP-regulated features (Koca, 2023).
The work described here provides insight into Notch-Abl signaling in a tissue remodeling, cell motility process. Although all data are consistent with the proposed model, this model is generated by inference from analyses of static fixed tissue samples, genetics, and biochemical studies. As it involves a cell motility process, it would be desirable to analyze the respective mutant genotypes via live imaging in vivo, including studies applying FRAP and other technologies. This would allow a more complete understanding of how Abl affects junctional dynamics during OR. Future studies will be needed to provide insight into the mechanistic details of how Notch and Abl cooperate in regulating junctional complexes and their dynamics during OR and other morphogenetic developmental and disease processes (Koca, 2023).
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Integrin adhesion, focal adhesion, and myotendenous junctions
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