InteractiveFly: GeneBrief

pickel: Biological Overview | References |

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: Flybase name Pickel) 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 claudin Megatrachea protein complex

Claudins are integral transmembrane components of the tight junctions forming trans-epithelial barriers in many organs, such as the nervous system, lung, and epidermis. In Drosophila three claudins have been identified that are required for forming the tight junctions analogous structure, the septate junctions (SJs). The lack of claudins results in a disruption of SJ integrity leading to a breakdown of the trans-epithelial barrier and to disturbed epithelial morphogenesis. However, little is known about claudin partners for transport mechanisms and membrane organization. This study presents a comprehensive analysis of the claudin proteome in Drosophila by combining biochemical and physiological approaches. Using specific antibodies against the claudin Megatrachea for immunoprecipitation and mass spectrometry, 142 proteins associated with Megatrachea were identified in embryos. The Megatrachea interacting proteins were analyzed in vivo by tissue-specific knockdown of the corresponding genes using RNA interference. Known and novel putative SJ components were identified, such as the gene product of CG3921. Furthermore, the data suggest that the control of secretion processes specific to SJs and dependent on Sec61p may involve Megatrachea interaction with Sec61 subunits. Also, the findings suggest that clathrin-coated vesicles may regulate Megatrachea turnover at the plasma membrane similar to human claudins. As claudins are conserved both in structure and function, these findings offer novel candidate proteins involved in the claudin interactome of vertebrates and invertebrates (Jaspers, 2012).

The Drosophila Claudin Kune-kune is required for septate junction organization and tracheal tube size control

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 kune^C309 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 kune^C309 embryos, demonstrating that lethality was due to loss of Kune. Further, embryos trans-heterozygous for kune^C309 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 kune^C309 homozygotes. These results indicate that kune is an essential gene and that kune^C309 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).

The claudin-like megatrachea is essential in septate junctions for the epithelial barrier function in Drosophila

Vertebrate claudin proteins are integral components of tight junctions, which function as paracellular diffusion barriers in epithelia. This study identified Megatrachea (Mega), a Drosophila transmembrane protein homologous to claudins; it acts in septate junctions, the corresponding structure of invertebrates. This analysis revealed that Mega has transepithelial barrier function similar to the claudins. Also, Mega is necessary for normal tracheal cell morphogenesis but not for apicobasal polarity or epithelial integrity. In addition, evidence is presented that Mega is essential for localization of the septate junction protein complex Coracle/Neurexin. The results indicate that claudin-like proteins are functionally conserved between vertebrates and Drosophila (Behr, 2003).

Search PubMed for articles about Drosophila Megatrachea/pickel

Babatz, F., Naffin, E. and Klambt, C. (2018). The Drosophila blood-brain barrier adapts to cell growth by unfolding of pre-existing septate junctions. Dev Cell 47(6): 697-710 e693. PubMed ID: 30482667

Behr, M., Riedel, D. and Schuh, R. (2003). The claudin-like megatrachea is essential in septate junctions for the epithelial barrier function in Drosophila. Dev. Cell 5: 611-620. 14536062

Daniel, E., Daude, M., Kolotuev, I., Charish, K., Auld, V. and Le Borgne, R. (2018). Coordination of septate junctions assembly and completion of cytokinesis in proliferative epithelial tissues. Curr Biol 28(9): 1380-1391. PubMed ID: 29706514

Jaspers, M. H., Nolde, K., Behr, M., Joo, S. H., Plessmann, U., Nikolov, M., Urlaub, H. and Schuh, R. (2012). The claudin Megatrachea protein complex. J Biol Chem 287(44): 36756-36765. PubMed ID: 22930751

Nelson, K. S., Furuse, M., Beitel, G. J. (2010). The Drosophila Claudin Kune-kune is required for septate junction organization and tracheal tube size control. Genetics 185(3): 831-9. PubMed ID: 20407131

Pannen, H., Rapp, T. and Klein, T. (2020). The ESCRT machinery regulates retromer dependent transcytosis of septate junction components in Drosophila. Elife 9. PubMed ID: 33377869

Yamazaki, Y., Palmer, L., Alexandre, C., Kakugawa, S., Beckett, K., Gaugue, I., Palmer, R. H. and Vincent, J. P. (2016). Godzilla-dependent transcytosis promotes Wingless signalling in Drosophila wing imaginal discs. Nat Cell Biol 18(4): 451-457. PubMed ID: 26974662

date revised: 2 November 2021

Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.