BIOLOGICAL OVERVIEW

Crumbs protein is essential for the biogenesis of the adherens junction and the establishment of apical polarity in ectodermally derived epithelial cells. The adherens junction is a multiprotein complex that attaches one cell to another in an epithelial layer. The junction is not spread randomly between the cells, but is found in a belt-like, zonular structure encircling the apical side of the cell.

The apical side of the cell faces the outside of the embryo, in opposition to the inward facing basal side. Placement of the adherens junction is critical because it signals to the cell which side is out and which side is in, preventing the mixing of apical cell membrane tissue with the biochemically distinct basal cell membrane, and thereby assuring cell polarity.

DE-cadherin (shotgun) and Armadillo comprise the two main constituents of the adherens junction. DE-cadherin is a homophilic adhesion transmembrane molecule that links the outside of the cell with the inside. Armadillo, the Drosophila homolog of beta-catenin, is a molecule that links the adherens junction with the cell's cytoskeleton.

crumbs mutants fail to establish adherens junctions and thus fail to establish epithelial cell polarity. Crumbs protein delimits the apical border, thus establishing the proper position for the border's placement . The defect in crumbs minus mutants results in a misdistribution of Armadillo and DE-cadherin, resulting in a disruption of tissue integrity. Curiously, despite the lack of adherens junction formation in such mutants, there is no accompanying loss of membrane polarity. This supports the view that membrane polarity exists prior to the formation of adherens junctions, and establishes the pattern of proper placement of the junction (Grawe, 1996).

Crumbs protein is distributed over the entire apical cell surface of epithelial cells and accumulates at the outer margin of the apical membrane where neighboring cells are in contact. However, no Crumbs protein is detected at the zonula adherens. This suggests that the polarizing activity of Crumbs arises from a direct or indirect binding of the Crumbs protein to adherens junction material at the outer rim of the marginal zone. The retention of adherens junction material in direct contact with the marginal zone would facilitate the formation of the zonula adherens from patches of adherens junction material that assemble through interaction with Crumbs protein (Tepass, 1996).

A conserved motif in Crumbs is required for E-cadherin localisation and zonula adherens formation in Drosophila

A conserved motif in Crumbs is required for E-cadherin localisation and zonula adherens formation in Drosophila. Expression of just the short membrane-bound cytoplasmic domain is sufficient to rescue major defects associated with the loss of crumbs function. The cytoplasmic domain of Crumbs is highly conserved in two putative crumbs homologs in C. elegans. To assess the significance of conserved residues, various point mutations and deletions were introduced into this region. Two functional domains were revealed: an amino-terminal region and the carboxy-terminal amino acids EERLI. Both are necessary for rescue of the crumbs phenotype. The EERLI motif interacts with Discs Lost (now redefined as Drosophila Patj), a cytoplasmic protein containing PDZ domains. Overexpression of the Crumbs cytoplasmic domain induces a transition from the single-layered epithelium to a multilayered tissue. This transition is associated with redistribution of the Drosophila homolog of the cell adhesion molecule E-cadherin, and depends on the presence of the EERLI motif (Klebes, 2000).

Two C. elegans genes that encode transmembrane proteins with multiple EGF-like repeats and short cytoplasmic domains were detected in the database. The cytoplasmic domains of both proteins, called CeCrb1 and CeCrb2, also consist of 37 amino acids, nine of which are conserved in all three proteins. A transgene (CD2-IntraCE), encoding the cytoplasmic domain of CeCrb1 fused to the rat transmembrane protein CD2 (which provides a transmembrane domain and a tag) was expressed in wild-type Drosophila embryos. The phenotypic consequences were compared with those induced by overexpression of a corresponding Drosophila fusion protein (CD2-IntraWT). Both CD2-Intra proteins induce the same phenotype, which is indistinguishable from that caused by the expression of Myc-IntraWT: the epidermis became multilayered and DE-cadherin and phosphotyrosine-containing epitopes are mislocalized. This shows that the functionally important regions responsible for inducing formation of a multilayered epidermis are conserved in the cytoplasmic domain of CeCrb1 (Klebes, 2000).

Data presented here suggest a model in which the Drosophila Crb protein organizes the assembly of an apically localized protein scaffold in epithelial cells that is required for the proper formation and localisation of the ZA. This scaffold includes the protein Dlt and probably other, as yet unidentified, proteins, its assembly depends on the carboxy-terminal segment of Crb. The model further suggests that the Crb-mediated control of DE-cadherin localization depends on interaction between the Crb cytoplasmic domain and the PDZ protein Dlt. Neither DE-cadherin nor Dlt are localized in crb mutant embryos, whereas both proteins are sequestered by mislocalized Crb. However Dlt remains apically localized after overexpression of DE-cadherin. The interaction of Crb with Dlt depends on Crb's carboxy-terminal motif, EERLI. This motif is also necessary for misdistribution of DE-cadherin upon Crb overexpression and for the rescue of crb mutant embryos. The presence of four PDZ domains in Dlt makes it an ideal partner for recruiting other proteins into a hypothetical Crb-dependent, membrane-associated protein network. PDZ domains have been shown to act as versatile organizers of multiprotein complexes. In many cases, the binding site of the interacting protein, often a transmembrane protein, is localized at its carboxyl terminus and ends with a hydrophobic amino-acid residue. Class I PDZ domains bind a conserved S/T-X-V motif (where X is any amino acid), whereas class II domains recognize ligands that carry a hydrophobic amino-acid residue at the -2 position. Since the Dlt-binding site in Crb differs from these motifs, the first PDZ domain of Dlt, which binds to Crb in vitro, may belong to a different class. The presence of the ERLI motif in both C. elegans homologs and the similarities between the phenotypes produced by overexpression of CD2-IntraWT and CD2-IntraCE in the Drosophila embryo suggest that this region might mediate comparable interactions in the nematode. Not surprisingly, a protein similar to Drosophila Dlt has also been detected in the C. elegans database, pointing to the possible conservation of additional components of the postulated protein network (Klebes, 2000).

Data indicate that the EERLI motif is necessary, but not sufficient, to rescue the phenotype of crb mutant embryos. Rescue also requires an intact amino-terminal region of the cytoplasmic domain. It is tempting to speculate that the region containing the mutated amino acids may be involved in additional protein-protein interactions. A comparable situation is provided by a group of transmembrane proteins, including glycophorin C, beta-neurexin and syndecans, that have been identified, respectively, as ligands for the class II PDZ proteins p55, CASK and syntenin. The cytoplasmic tails of these proteins terminate in the tetrapeptides EYFI, EYYV and EFYA, respectively, and show additional conservation in their amino-terminal regions. For glycophorin C it has been demonstrated that the 12-residue sequence immediately adjacent to the membrane binds directly to protein 4.1, a member of the 4.1 superfamily, which includes, among others, the so-called ERM proteins (ezrin, radixin, moesin). The latter proteins provide a linkage between cell-surface receptors and the spectrin/actin cytoskeleton. The 12-residue sequence of glycophorin C that binds protein 4.1 includes a Gly8-Thr9-Tyr10 motif, which is Gly8-Ser9-Tyr10 in beta-neurexin and all syndecans. The corresponding region of Drosophila Crb also contains a Gly8-Thr9-Tyr10 motif at an equivalent position (Gly-His/Lys-Tyr in the C. elegans proteins); mutating Tyr10 to alanine completely abolishes the rescuing function. It is unlikely that the Drosophila protein 4.1 homolog, encoded by coracle, is a partner of Crb in wild-type embryos. Coracle is associated with septate junctions, which are localized basally to the ZA, and colocalizes with Discs Large, a PDZ-domain protein, and beta-neurexin IV. The amino-terminal region conserved between Drosophila and the two C. elegans homologs extends further, to Gly8-X9-Tyr10-X(11-15)-Glu16. The data clearly show that Glu16, which is also a charged amino acid in syndecans, glycophorin C and neurexin, is also of crucial importance for the rescuing function (Klebes, 2000).

There is a further indication of possible involvement of the amino-terminal region of the Crb cytoplasmic domain in interactions with other, as yet unknown, proteins closely associated with the plasma membrane. All CD2 fusion proteins used in this study fail to rescue crb mutant embryos, even those containing the full-length cytoplasmic domain. Whereas the Myc fusion proteins contain the Crb transmembrane domain, immediately followed by the cytoplasmic portion, CD2 fusion proteins contain the CD2 transmembrane domain and provide a spacer of 45 amino acids between the membrane and the cytoplasmic tail of Crb. This spacing could prevent interactions between the cytoplasmic segment of Crb and a hypothetical partner localized at the membrane. At present, however, it cannot be determined whether it is this spacer, the lack of the Crb transmembrane domain, or some other feature of the CD2 fusion protein that is responsible for the lack of rescuing function (Klebes, 2000).

Crb is the earliest zygotically expressed apical transmembrane protein, but nothing is known about the cis-regulatory sequences that target it to the apical face of the cell nor the mechanisms and proteins required for this process. Nothing is known about the function of the large extracellular domain; its overexpression in a secreted or membrane-anchored form (lacking the cytoplasmic domain) does not induce any mutant phenotype. Embryos devoid of maternal Dlt fail to localize Crb. Since the blastoderm epithelium of these embryos itself lacks cell polarity, however, all other defects, including improper Crb localisation, could be regarded as secondary effects. In embryos mutant for stardust, Crb is first expressed apically, but during germ band extension it is no longer detectable, making stardust a likely regulator for the maintenance of apical localization of Crb. In agreement with this, stardust mutant embryos develop a phenotype nearly identical to that of crb mutant embryos. Because the molecular nature of the stardust gene is not yet known, no information can be obtained about its relationship with Crb expression at present (Klebes, 2000).

Anisotropy of Crumbs and aPKC drives myosin cable assembly during tube formation

The formation of tubular structures from epithelial sheets is a key process of organ formation in all animals, but the cytoskeletal rearrangements that cause the cell shape changes that drive tubulogenesis are not well understood. Using live imaging and super-resolution microscopy to analyze the tubulogenesis of the Drosophila salivary glands, this study found that an anisotropic plasma membrane distribution of the protein Crumbs, mediated by its large extracellular domain, determines the subcellular localization of a supracellular actomyosin cable in the cells at the placode border, with myosin II accumulating at edges where Crumbs is lowest. This study shows that Crumbs directs aPKC anisotropy which negatively regulate myosin II, probably through Rok. Laser ablation shows that the cable is under increased tension, implying an active involvement in the invagination process. Crumbs anisotropy leads to anisotropic distribution of aPKC, which in turn can negatively regulate Rok, thus preventing the formation of a cable where Crumbs and aPKC are localized (Roper, 2012).

Myosin II has emerged as a key player in morphogenesis because of its ability to form contractile structures together with F-actin that can directly alter the shapes of cells. Different pools of myosin II within epithelial cells undergoing morphogenesis have been observed, namely apical junctional myosin, apical medial myosin, and in addition myosin organized into supracellular structures termed myosin cables or purse-strings. All three myosin II pools have been shown to be important for epithelial morphogenesis, but how much the activities of the pools depend on each other and how their specific assembly is regulated is much less clear (Roper, 2012).

Using the formation of the invagination of the salivary glands in the fly embryo as a model allowed me to analyze a morphogenetic process in which all three different pools of myosin are present. Upon specification of the gland placode, myosin II levels are drastically upregulated in the secretory cells of the placode, and myosin accumulates at cortical regions and medially within the apical 'dome' of each cell. In addition, a supracellular myosin cable surrounding the placode is formed in a process by which parts of existing structures (remnants of parasegmental cables) are joined together with a newly specified dorsal section of the cable (Roper, 2012).

Compared to mesoderm invagination in the fly, a well-studied process that depends on both apical medial and cortical myosin assemblies, the invagination of the tubes of the salivary gland topologically rather resembles wound healing or dorsal closure processes, as the surrounding epidermis is drawn in from around the placode to cover the patch where cells are invaginating into the embryo (Roper, 2012).

All three processes have in common that the patch of cells 'disappearing' from the plane of the epithelium is surrounded by a contractile actomyosin cable. In contrast to wound healing and dorsal closure, the cable in the case of salivary gland tubulogenesis is assembled within the cells on the inside, whereas it is assembled in the surrounding epithelial cells in the former two instances. Thus, the signal for cable assembly is provided by the 'inside' cells in the salivary gland placode (Roper, 2012).

The laser ablation data presented in this study clearly demonstrate that the cable around the placode is under increased tension, even when compared to other myosin enriched edges. The tension is in magnitude comparable to the tension determined for shorter supracellular myosin cables observed during germband extension in the fly embryo. This increased tension indicates active involvement in the invagination process. Previous modeling studies on sea urchin invagination have shown that a contractile apical ring surrounding a placode could be a driving force for invagination. Interestingly, upon laser ablation the cable around the salivary gland placode was very quickly repaired, suggesting a continuous signal to assemble myosin at the outermost surface of the placode. This fast repair precluded laser ablation as a means of probing function of the cable in the invagination in contrast to medial and junctional myosin (Roper, 2012).

Crumbs, the transmembrane component of the apical protein complex, shows a very striking anisotropic localization at the border of the placode, that is complementary to the accumulation of myosin II forming the cable. The data strongly support a model whereby Crumbs intracellular tails at cell edges facing toward the inside of the placode recruit aPKC, which can act as a negative regulatory factor impinging on Rok, thus preventing cable assembly at edges containing high levels of Crumbs tails. This leaves active Rok at the cell edges forming the placode boundary, where it acts to recruit myosin into the cable.

Interestingly, only the presence or artificial introduction of cortical anisotropy of Crumbs and downstream aPKC has this effect. The central cells of the placode that are not forming the boundary all show strongly upregulated levels of Crumbs, aPKC, Rok, and myosin II, but in these cells a high density of Crumbs tails does not preclude accumulation of junctional membrane-proximal myosin. Thus, the change in density of Crumbs tails, not the overall concentration, is instructive in this system (Roper, 2012).

Crumbs has previously been shown to have an effect on salivary gland morphogenesis through a proposed regulation of the apical membrane domain and has been implicated in tracheal pit invagination through regulation of phospho-Moesin). Also, members of the Crumbs polarity complex have been shown to be able to interact with the Par3 (Bazooka)/Par-6/aPKC complex (e.g., Par-6 can bind to Crumbs^intra; aPKC can phosphorylate Crumbs^intra. Also, an anticorrelation between localization of Crb and aPKC compared to Lgl and myosin has been described in the denticle belts of the fly epidermis (Kaplan, 2010). This work now describes a potential link from Crumbs through aPKC to Rok and myosin II, which would link the interaction of two different polarity factors directly with the coordination of morphogenesis through myosin II at a molecular level (Roper, 2012).

The large extracellular domain of Crumbs has long posed an enigma with regard to its role. Crumbs' function in epithelial polarity can mostly be mediated by its intracellular domain (Klebes, 2000). Only for photoreceptor morphogenesis, the extracellular domain appears required within the fly, though its molecular role is unclear. The protein domains present in the extracellular domain, namely EGF repeats and lamG domains, are both found in many classical and nonclassical cadherins. Data presented in this study suggest that Crumbs could be organized in the plasma membrane through homophilic interactions of the extracellular domains between molecules on neighboring cells: Crumbs shows highly anisotropic localization within the wild-type placode but also within wild-type cells bordering a crumbs mutant clone (Chen, C. L., 2010) or in clusters of Crumbs expressing cells in a null mutant embryo and within cells at the edge of an ectopic step change in Crumbs expression levels. Also in vitro, Crumbs accumulates at contact zones between expressing cells. The extracellular domain appears the ideal candidate to mediate this anisotropy, which is supported by the following findings: (1) the Crb^TMextra-GFP shows anisotropic localization at borders with cells not expressing the construct; (2) endogenous Crumbs in wild-type cells is induced to localize in an anisotropic fashion when neighboring cells are depleted of endogenous Crumbs and only express the intracellular domain; and (3) the Crb^intra-FLAG shows uniform expression in cells. These observations exclude that another transmembrane protein that interacts with the intracellular domain of Crumbs in equal stoichiometry could mediate the anisotropy, though it cannot formally exclude that another extracellular factor might act as an intermediary between two Crumbs extracellular domains. These data are strongly supported by recent evidence from zebrafish, where vertebrate Crumbs isoforms appear to mediate homophilic interactions to promote orderly arrays of photoreceptors. Also, recent data analyzing the establishment of polarity in the Drosophila follicular epithelium suggest a role for cis-interaction of Crumbs molecules within a single cell (Fletcher, 2012). Thus, a clear role for the Crumbs extracellular domain in organizing plasma membrane domains through homophilic interactions in cis and in trans is prominently emerging (Roper, 2012).

Data presented in this study describe a link between the transmembrane protein Crumbs and myosin II structures actively engaged in controlling morphogenesis. Crumbs' ability to interact in trans allows the step change in Crumbs levels between placode and surrounding cells to be translated into a subcellular asymmetry, the anisotropic localization of Crumbs. This mechanism provides the cells at the border of the salivary gland placode with the means of sensing this positional information and allows them to turn the positional information into a morphogenetic readout: myosin cable formation. In the future it will be interesting to determine if the arrangement of Crumbs and myosin II described in this study is conserved during topologically similar processes of tube invagination, such as, for instance, the side budding of branches during lung or mammary gland morphogenesis (Roper, 2012).

Crumbs regulates rhodopsin transport by interacting with and stabilizing myosin V

The evolutionarily conserved Crumbs (Crb) complex is crucial for photoreceptor morphogenesis and homeostasis. Loss of Crb results in light-dependent retinal degeneration, which is prevented by feeding mutant flies carotenoid-deficient medium. This suggests a defect in rhodopsin 1 (Rh1) processing, transport, and/or signaling, causing degeneration; however, the molecular mechanism of this remained elusive. This paper shows that myosin V (MyoV) coimmunoprecipitates with the Crb complex and that loss of crb led to severe reduction in MyoV levels, which could be rescued by proteasomal inhibition. Loss of MyoV in crb mutant photoreceptors was accompanied by defective transport of the MyoV cargo Rh1 to the light-sensing organelle, the rhabdomere. This resulted in an age-dependent accumulation of Rh1 in the photoreceptor cell (PRC) body, a well-documented trigger of degeneration. It is concluded that Crb protects against degeneration by interacting with and stabilizing MyoV, thereby ensuring correct Rh1 trafficking. The data provide, for the first time, a molecular mechanism for the light-dependent degeneration of PRCs observed in crb mutant retinas (Pocha, 2011b).

The role of the Crb complex in polarity is well studied, but the mechanism behind its ability to prevent light-dependent retinal degeneration is poorly understood. Some insight into the latter came from studies reporting that feeding flies a vitamin A (carotenoid)-depleted medium prevented the light-dependent degeneration of crb, sdt, and DLin7 mutant PRCs. These data suggested that degeneration in Crb complex mutants involves Rh1; however, the molecular mechanism behind this remained unknown. This study provides the missing link by showing that the Crb complex interacts with MyoV, an unconventional myosin, which has an established role in the transport of Rh1 to the rhabdomere. MyoV levels are reduced by ~90% in crb mutant retinas, which can be largely rescued by inhibition of the proteasome, and Rh1 transport is defective in crb mutant PRCs. Therefore, it is proposed that the Crb complex protects against light-dependent degeneration by interacting with and maintaining MyoV levels, thereby ensuring proper Rh1 transport to the rhabdomere (Pocha, 2011b).

Blocking proteasome activity also allowed assessment of the localization of MyoV in the absence of Crb. Apical localization of MyoV was observed; however, rather than adopting the WT localization that spans the entire rhabdomere base, the rescued MyoV was seen in large clumps, which only partially covered the base of the rhabdomere. The steady-state WT localization of MyoV reflects its role in transporting Rh1 from the cell body to the rhabdomere base. Therefore, these large accumulations may suggest that some level of MyoV degradation is also important for maintaining efficient transport by the total pool of MyoV. Thus, the levels of MyoV and its ability to transport Rh1 to the rhabdomere base may depend on external cues (e.g., light), which alter the balance between stabilization and degradation (Pocha, 2011b).

IPs of both Sdt and DPatJ contain the respective other members of the Crb complex, and together with these, MyoV is precipitated specifically. The strong reduction in MyoV protein seen in crb mutant photoreceptors raises the question of whether stability of MyoV is dependent on Crb itself or on the integrity of the Crb complex. As loss of Crb results in the loss of Sdt and the delocalization of DPatJ and DLin7, the data obtained using crb^11A22 mutants can be used to analyze the role of the Crb complex. Data obtained from crb^8F105 mutants, however, show that the integrity of the Crb complex is not required for the crb-dependent stabilization of MyoV. This was further supported by experiments in S2R+ cells that showed Crb alone, in the absence of Sdt, can recruit MyoV-GFP to the plasma membrane, suggesting that the interaction observed between Crb and MyoV is not mediated by any of the other core components of the Crb complex. As the interaction was detected by IP, the possibility remains that the interaction between Crb and MyoV is mediated by another still unknown protein (Pocha, 2011b).

Interestingly, loss of MyoV in crb^11A22 mutants cannot be overcome by overexpression of a MyoV transgene expressed under the control of an exogenous system, the UAS/Gal4 system. This demonstrates that Crb is required to maintain MyoV stability posttranscriptionally. This was investigated further by inhibiting proteasomal degradation, and a marked increase of MyoV staining was observed in crb mutant photoreceptors compared with controls. These findings support the conclusion that the interaction between Crb and MyoV is stabilizing the latter by protecting it from degradation by the proteasome (Pocha, 2011b).

crb is known to have two main functions in the eye, one during development of the retina to ensure correct morphogenesis of the PRCs and the other to prevent degeneration of the adult eye in constant light. This study shows that MyoV does not show a polarized distribution at early pupal stages nor is its localization perturbed by loss of Crb in early stages, the time at which morphogenetic defects in crb mutants start, suggesting that the interaction between Crb and MyoV is not required for proper morphogenesis to occur. This is supported by reports that MyoV-null mutant adults display only mild morphological defects, which are distinct from those observed in crb mutants (Pocha, 2011b).

The finding that MyoV fails to start accumulating apically in crb mutant cells during late pupal stages after Rh1 expression starts corroborates the conclusion that the Crb-MyoV interaction is required for the second role of Crb in the retina, preventing light-dependent degeneration. It is also plausible that the steady-state localization of MyoV seen in the adult is largely the result of its role in Rh1 transport to the rhabdomere, as MyoV is seen evenly distributed throughout the cell before Rh1 expression starts. This assumption is supported by published data showing that the localization of MyoV in the adult is light dependent and therefore reflects the status of Rh1 activation and transport. Fittingly, the apical accumulation of MyoV at later pupal stages coincides with increased MyoV staining and increased colocalization of MyoV with Crb (Pocha, 2011b).

The effect that loss of Crb has on Rh1 was tested, and it was demonstrated that in normal 12-h light/12-h dark conditions, defects in Rh1 staining are seen only in old flies. This is suggestive of a subtle defect in Rh1 transport that is only visible at steady state if allowed to accumulate over time or if the system is under stress (i.e., constant light). As it was reported that only minimal MyoV activity is required for proper Rh1 localization, it is probable that the remaining 10% of MyoV seen in crb mutants is sufficient for Rh1 transport in young flies, but, over time, the effect of this deficiency accumulates, resulting in the retention of Rh1-positive punctae in the cell body. Together with the results from the Rh1 pulse-chase assay, it is concluded that in crb mutant tissue, Rh1 transport to the rhabdomere is delayed and that the cumulative effect of this delayed transport leads to the accumulation of Rh1 within the cell body, which is associated with a gradual deterioration of the rhabdomeres (Pocha, 2011b).

Previous findings have shown that Crb-mediated protection against light-dependent retinal degeneration is not solely dependent on the ability of Crb to assemble and integrate into the Crb complex. Photoreceptors of crb^8F105 mutants that express a Crb protein lacking the Sdt-interacting ERLI motif do not undergo light-dependent degeneration. This observation is in agreement with the findings presented in this study that MyoV is retained in Crb^8F105 mutant photoreceptors. In addition, overexpression of a Crb transgene encoding the transmembrane and intracellular domains was not able to rescue the light-dependent degeneration observed in crb^11A22 mutants. Interestingly, this membrane-tethered intracellular domain-encoding transgene does rescue the morphogenetic defects observed in both crb^8F105 and crb^11A22 mutants. Therefore, the two roles of Crb in the retina photoreceptor morphogenesis and maintenance appear to occur through distinct mechanisms. Correct morphogenesis seems to necessitate the assembly of the Crb complex through the Crb ERLI motif. In contrast, Crb-mediated protection against light-dependent degeneration and stabilization of MyoV does not require an intact Crb complex. How do these finding correlate with reports of light-dependent retinal degeneration in other members of the Crb complex? It is proposed that in sdt and DPatJ mutants, it is the concomitant loss of Crb that is responsible for the degeneration phenotype rather than the loss of an intact Crb complex itself (Pocha, 2011b).

The absence of endogenous Crb and Sdt from cultured Schneider S2R+ cells made them particularly useful to identify the regions of Crb required for its interaction with MyoV that were determined to include the membrane-spanning and first 14 amino acids of the cytoplasmic domain. However, the readout for this interaction the recruitment of MyoV-GFP to the plasma membrane may not reflect the purpose of this interaction in vivo, particularly considering the highly polarized and functionally specialized nature of PRC. Indeed, the finding that the majority of the residual MyoV in crb mutant photoreceptors localizes to the rhabdomere base suggests that in photoreceptors, the role of the Crb-MyoV interaction is primarily to stabilize MyoV and not to recruit it to the rhabdomere base. In addition, the ability of Crb lacking the extracellular domain to recruit MyoV to the plasma membrane of S2R+ cells but not to rescue light-dependent degeneration suggests that S2R+ cells lack many qualities (morphology, protein expression, and functionality) of PRCs. Considering the requirement of the extracellular domain, it is possible that in the context of a light-sensing photoreceptor, Crb responds to a stimulus that is transmitted via the extracellular domain, which then initiates the interaction with and/or the stabilization of MyoV. This hypothesis is an intriguing one, as the only function of Crb that requires its extracellular domain is its role in preventing light-dependent degeneration, and, to date, no known partner of the extracellular domain has been identified (Pocha, 2011b).

A model is proposed in which the interaction between Crb and MyoV stabilizes the latter, maintaining a complete Rh1 transport cycle. In crb mutants, this cycle is slowed down at the MyoV-dependent stage of delivery to the rhabdomere. Whereas in normal light/dark conditions the effect of this is minimal, upon exposure to constant light, Rh1 accumulates in the cell body, suggesting that the rate of removal from the rhabdomere (as a result of constant activation) exceeds the rate of delivery to the rhabdomere. As previously discussed, photoreceptors are extremely sensitive to perturbations in the phototransduction cascade, and it has been well documented that mutations that affect the synthesis, delivery, and recycling of Rh1 lead to degeneration. Together with previously published data showing the rescue of Crb-dependent retinal degeneration in the absence of vitamin A, this strongly supports a model that the accumulation of Rh1 in the cell body as a result of a deficiency of Rh1 transport in crb mutants leads to degeneration (Pocha, 2011b).

These data provide a molecular mechanism for the light-dependent degeneration observed in crb mutant animals. Recent findings that myoVIIa mutant mice display light-dependent degeneration as a result of defects in rod protein translocation (Peng, 2011) suggest that the efficient transport of opsins by myosins is crucial to prevent degeneration across species. Therefore, it will be intriguing to see whether the mechanism we identified here is conserved and whether human photoreceptors from patients with CRB1 mutations also display reduced myosin levels and delays in Rh1 transport (Pocha, 2011b).

Crumbs, Moesin and Yurt regulate junctional stability and dynamics for a proper morphogenesis of the Drosophila pupal wing epithelium

The Crumbs (Crb) complex is a key epithelial determinant. To understand its role in morphogenesis, this study examined its function in the Drosophila pupal wing, an epithelium undergoing hexagonal packing and formation of planar-oriented hairs. Crb distribution is dynamic, being stabilized to the subapical region just before hair formation. Lack of crb or stardust, but not DPatj, affects hexagonal packing and delays hair formation, without impairing epithelial polarities but with increased fluctuations in cell junctions and perimeter length, fragmentation of adherens junctions and the actomyosin cytoskeleton. Crb interacts with Moesin and Yurt, FERM proteins regulating the actomyosin network. Moesin and Yurt distribution at the subapical region depends on Crb. In contrast to previous reports, yurt, but not moesin, mutants phenocopy crb junctional defects. Moreover, while unaffected in crb mutants, cell perimeter increases in yurt mutant cells and decreases in the absence of moesin function. These data suggest that Crb coordinates proper hexagonal packing and hair formation, by modulating junction integrity via Yurt and stabilizing cell perimeter via both Yurt and Moesin. The Drosophila pupal wing thus appears as a useful system to investigate the functional diversification of the Crb complex during morphogenesis, independently of its role in polarity (Salis, 2017).

This study aimed at unveiling the function of the Crumbs complex in epithelial morphogenesis. Although Crb was discovered several decades ago in Drosophila, the severe apico-basal polarity defects associated to crb inactivation in embryos have hampered the full exploration of its function during epithelia development. The results indicate that Crb also acts during pupal wing morphogenesis, where the absence of crb function does not impair AP/BL polarity and does not lead to the dramatic tissue alterations often seen in other tissues. The pupal wing thus represents an attractive model system, well suited to dissect additional functions of the Crb complex during epithelial morphogenesis, independently of its role in polarity (Salis, 2017).

The redistribution of Crb at the subapical region (SAR) at the end of hexagonal packing, as well as the defects in cells orientation observed in crb mutants suggest that Crb is required to stabilize the actin cytoskeleton and E-cadherin at the adherens junctions at the end of tissue rearrangement. Alterations in F-actin and Myosin II (Myo) distribution in crb mutant cells strikingly mimic those observed in embryos mutant for the actin-binding protein Canoe/Afadin, which links the actomyosin network to AJs. Canoe loss diminishes this coupling leading to reduced cell shape anisometry and defects in germ band elongation. As for crb, canoe mutant cells still retain some ability to change their shape and germ band elongation is delayed and not completely impaired. The defects observed in crb mutant cells support the hypothesis that Crb is a crucial regulator of the interconnection between the actomyosin cytoskeleton and AJs (Salis, 2017).

The fragmentation of AJs upon Crb depletion has been already described, for example in embryo or during follicular morphogenesis. However, in these two systems the function of Crb has been related to the role of Moe in the regulation of the actomyosin cytoskeleton, while the role of Yurt has never been addressed or has been excluded. The current data support that in pupal wing cells the role of Crb in the stability of the AJs is likely established via Yurt. Crb is shown to modulate Yurt localization at the SAR at the end of hexagonal packing and yurt mutant cells phenocopy crb mutant cortical defects. Nonetheless, previous studies in cultured cells have established that Yurt participates in epithelial polarity and organization of apical membranes by negatively regulating the activity of the Crb complex. On the contrary, this study shows that, whereas Crb modulates Yurt distribution at the SAR at the end of hexagonal packing of wing cells, Yurt depletion does not impact Crb association to the SAR, with the exception of the E-cad- and F-actin-devoid gaps. Yurt and Crb similarly act on actomyosin and E-cad organization at the cell-cell junctions suggesting that the coordinated function of these two proteins is regulated by different mechanisms in different tissues. On the other hand, moe depletion does not specifically modify Crb distribution at the SAR, a finding coherent with the evidence that Moe is not implicated in stability of AJs in this tissue, as opposed to other models (Salis, 2017).

Studies based on in vivo mechanical measurements or mathematical/physical modeling have proposed that epithelial cell packing results from a balance between intrinsic cell tension and extrinsic tissue-wide forces to establish a correct and robust order in the tissue. Hence, the tension generated by the actomyosin cortex and the pressure transmitted through adherens junctions are the two main self-organizing forces driving tissue morphogenesis. Tension shortens cell-cell contacts and pressure of individual cells counteracts tension to maintain cell size. The current data indicate that Crb recruits at SAR Moe and Yurt, which show opposite effects on pupal wing morphogenesis. While Moe promotes cell expansion, Yurt controls cell constriction and the stability of the AJs and of the actomyosin network. In crb mutant cells, the absence of variation in the cell perimeter might be explained by the simultaneous loss of positive and negative regulators. Therefore, Crb acts as a coordinator of the two self-organizing mechanisms implicated in morphogenesis. Additionally, the dynamic redistribution of Crb at the SAR at the end of hexagonal packing, together with the disruption of cell orientation in crb mutants, is consistent with the hypothesis that Crb is required to stabilize cell shape and pattern in order to properly progress throughout tissue development (Salis, 2017).

In conclusion, these functional analyses during pupal wing morphogenesis allowed the unraveling Crb-dependent mechanisms that are integrated to produce shape changes during development independently of epithelial polarity. Furthermore, the results show that the interplay between Crb and FERM proteins is tissue-regulated and that their epistatic interactions differ in a spatio-temporal manner (Salis, 2017).

aPKC is a key polarity molecule coordinating the function of three distinct cell polarities during collective migration

Apical-basal polarity is a hallmark of epithelia and it needs to be remodeled when epithelial cells undergo morphogenetic cell movements. This study used border cells in Drosophila ovary to address how the apical-basal polarity is remodeled and turned into front-back, apical-basal and inside-outside polarities, during collective migration. Crumbs (Crb) complex is required for the generation of the three distinct but inter-connected cell polarities of border cells. Specifically, Crb complex, together with Par complex and the endocytic recycling machinery, ensures a strict distribution control of two distinct populations of aPKC at the inside apical junction and near the outside lateral membrane respectively. Interestingly, aPKC distributed near the outside lateral membrane interacts with Tiam1/Sif and promotes the Rac-induced protrusions, whereas alteration of the aPKC distribution pattern changed protrusion formation pattern, leading to disruption of all three polarities. Therefore, this study demonstrates that aPKC, spatially controlled by Crb complex, is a key polarity molecule coordinating the generation of three distinct but inter-connected cell polarities during collective migration (Wang, 2018).

This study demonstrates that the Crb complex is required for the collective migration of border cells. Loss of function of Crb, Sdt or Patj each delayed border cell migration, which was likely to be a result of the combined effect of disrupting three distinct cell polarities. Most importantly, the front-back polarity of the border cell cluster was disrupted, as demonstrated by the ectopic formation of large actin-rich protrusions in border cells located at the side and back of the cluster. Furthermore, Patj RNAi or sdt RNAi caused border cell clusters to extend major protrusions at random angles relative to the apical-basal axis, unlike the wild-type clusters that restrict protrusion formation to the lateral region, thus extending the protrusions perpendicular to their inherent apical-basal axis. Such restriction of lateral protrusion formation would ensure that protrusions are parallel to the migration direction, resulting in efficient forward movement of the entire cluster. Mutation in crb or the expression of active forms of aPKC expanded the outside membrane area, and overexpressing Crb or reducing aPKC activity suppressed the outside membrane characteristics, causing disruption in inside-outside polarity for each border cell. Interestingly, crb mutant border cells sometimes exhibited ectopic actin patches (containing large aPKC spots) between the adjacent cells, where the inside membrane is normally located. Taken together, these results raise the following question: is there a common mechanism that is affected during the disruption of all three cell polarities? In other words, are these cell polarities interconnected and coordinated by the same mechanism? (Wang, 2018).

A common feature of loss of Crb complex components is that mislocalized aPKC generates ectopic Rac-dependent protrusions in border cells at the side and back of the cluster and at the apical and inside (junctional) region of individual border cells, leading to disruption of all three cell polarities. This indicates that there is a common mechanism involving aPKC that organizes all three polarities. First, the ectopic protrusions and the loss of these three polarities as a result of loss of Patj are likely to be mediated by the ectopically localized aPKC, since reduction of aPKC was able to rescue the ectopic protrusions. Interestingly, loss of other apical polarity proteins (Crb, Sdt, Par6, Cdc42) except for aPKC and Baz also led to similar phenotypes, including disrupted aPKC localization in the apical junctions, ectopic actin patches colocalized with large aPKC spots, and increased F-actin levels and Rac activity at or near the outside membrane. By contrast, loss of aPKC resulted in few protrusions and reduced F-actin levels at the outside membrane, while overactivation of aPKC led to increased F-actin levels and Rac activity, which are mediated by the downstream Sif. These results suggest that an important role of the Crb and Par complexes is to sequester most of the aPKC in the apical junction, leaving only a moderate level near the outside membrane to promote protrusion formation. The major pool of aPKC at the apical junction (together with Crb and Par complex components) is likely to function similarly to its classical role in epithelial cells, which is to promote apical polarity and integrity of apical and subapical junctions. However, the minor aPKC pool near the outside lateral membrane might function differently in that it can activate Sif to increase Rac-mediated actin dynamics. Such a difference might arise if complexes at the apical junction restrict or inhibit the Sif-promoting activity of aPKC. Conceivably, such inhibition would not apply to aPKC near the outside lateral membrane (Wang, 2018).

A crucial function of the Crb complex and Par complex is to produce a high level of membrane-bound aPKC at the inside apical junction and a moderate level of cytoplasmic aPKC near the outside lateral membrane so that the three distinct, but related, cell polarities can be properly established. Furthermore, polarized endocytic recycling of vesicles associated with aPKC and other apical polarity molecules ensures the polarized distribution of two aPKC pools within each border cell. Finally, it is interesting to note that the front-polarized recycling and exocytosis within the wild-type cluster, as mediated by PVF-PVR guidance signaling, could cause aPKC to be much more enriched at the outside membrane of the leading edge (to promote leading protrusion) than at the outside membrane at the side and back (to promote minor side protrusions) of the border cell cluster. When cells migrate collectively under developmental, physiological and pathological contexts, the migrating sheets or clusters of cells often display part-epithelial and part-mesenchymal characteristics. It will be interesting to determine whether aPKC together with Crb and Par complexes and the endocytic recycling machinery also play conserved roles in coordinating these three cell polarities in other types of collective migration (Wang, 2018).

Anisotropic Crb accumulation, modulated by Src42A, is coupled to polarised epithelial tube growth in Drosophila

Tube size control and how tubular anisotropy is translated at the cellular level are still not fully understood. This study investigated these mechanisms using the Drosophila tracheal system. The apical polarity protein Crumbs transiently accumulates anisotropically at longitudinal cell junctions during tube elongation. Evidence is provided indicating that the accumulation of Crumbs in specific apical domains correlates with apical surface expansion, suggesting a link between the anisotropic accumulation of Crumbs at the cellular level and membrane expansion. This study finds that Src42A is required for the anisotropic accumulation of Crumbs, thereby identifying the first polarised cell behaviour downstream of Src42A. The results indicate that Src42A regulates a mechanism that increases the fraction of Crb protein at longitudinal junctions, and genetic interaction experiments are consistent with Crb acting downstream of Src42A in controlling tube size. Collectively, these results suggest a model in which Src42A would sense the inherent anisotropic mechanical tension of the tube and translate it into a polarised Crumbs accumulation, which may promote a bias towards longitudinal membrane expansion, orienting cell elongation and, as a consequence, longitudinal growth at the tissue level. This work provides new insights into the key question of how organ growth is controlled and polarised and unveils the function of two conserved proteins, Crumbs and Src42A, with important roles in development and homeostasis as well as in disease, in this biological process (Olivares-Castineira, 2018).

Since Crb has been proposed to regulate tube length by promoting apical membrane growth, Crb accumulation was examined in the Dorsal Trunk (DT, the main tracheal trunk connecting to the exterior through the spiracles). Crb can localise to different subdomains of the apical membrane during tracheal development: the SubApical Region (SAR) and the Apical Free Region (AFR). The AFR corresponds to the most apical domain, free of contact with other epithelial cells and in direct contact with the lumen in the case of tubular organs like the trachea, while the SAR corresponds to the most apicolateral membrane domain of contact between neighboring epithelial cells. Previous work has shown that during the stages of higher longitudinal DT growth, stage 15 onwards, Crb accumulated strongly in the SAR, displaying a mesh-like pattern that identifies the apical junctional domain. Strikingly, Crb was anisotropically (not uniformly) distributed in the SAR of cell junctions. Cell junctions are classified as longitudinal cell junctions (LCJs), mainly parallel to the longitudinal axis of the tube, and transverse cell junctions (TCJs), perpendicular to the longitudinal axis. Crb accumulation was found to be more visible at LCJs than at TCJs; several examples were observed where accumulation of Crb at TCJs was almost absent. The accumulation of Crb (total fluorescence intensity/junctional length) was quantified at LCJs and TCJs; accumulation was found to be biased to LCJs, where levels were around 30% higher than at TCJs (average % of difference of Crb accumulation at LCJs and TCJs). To compare different embryos the LCJ/TCJ ratio was calculated of Crb accumulation, which showed an average of 1,5 (n = 15 embryos), indicating that Crb is anisotropically distributed, i.e. polarised. In contrast to Crb, DE-Cadherin (DE-cad), a core component of the Adherens Junctions (AJs), was equally distributed among all cell junctions. The ratio of accumulation in LCJ/TCJ was close to 1, indicating that the anisotropic distribution is not a general feature of all junctional proteins. These results indicated that a larger proportion of LCJs accumulate higher levels of Crb than TCJs (Olivares-Castineira, 2018).

To further investigate this observation time-lapse imaging was carried out in embryos carrying the viable and functional CrbGFP allele as the only source of functional Crb protein. Enrichments were observed of Crb protein at LCJs, and less conspicuous accumulations were found at TCJs, from late stage 15 and during stage 16 over a period of 1,30-2 hours. This correlated with an increase in tube length of around a 30% and a moderate increase in tube diameter of an 11% (Olivares-Castineira, 2018).

Altogether these results point to a polarised accumulation of Crb that correlates with an anisotropic growth along the longitudinal axis of the DT during stage 16. It is worth pointing out that anisotropies of Crb, like the one described in this study, or of other apical determinants, have important implications in morphogenesis (Olivares-Castineira, 2018).

Different molecular mechanisms could underlie the preferential accumulation of Crb at LCJs, such as specific Crb degradation at TCJs, specific stabilisation at LCJs, targeted intracellular trafficking, differential protein recycling, among others. To investigate the possible mechanism behind the anisotropic pattern of Crb accumulation FRAP analysis was performed at either LCJs or TCJs of embryos carrying the CrbGFP allele. The amount of fluorescent protein, relative to the pre-bleach value, mobilized during the experimental time (mobile fraction, Mf) was significantly higher at LCJs compared to TCJs, indicating a higher recovery of CrbGFP protein at LCJs. To assess the recovery kinetics the half-time (t1/2, time to reach half of the Mf) was calculated. The half-time was not significantly different at LCJs and TCJs, suggesting that the recovery rate is comparable at the differently oriented junctions. Kymographs of the bleached regions suggested that the recovery was not due to lateral diffusion. Altogether the results indicated a higher mobility of Crb protein at LCJs but a constant rate of incorporation in all junctions (Olivares-Castineira, 2018).

Hence, on the one hand higher levels of Crb accumulate at LCJs, and on the other, FRAP experiments show that Crb protein is more mobile at LCJs. These results could suggest the existence of two molecularly defined different pools of Crb in the junctions with different mobility: a basal level-pool with lower mobility and an enrichment-pool with higher mobility. The basal level-pool would be present in all junctions, while a mechanism acting specifically at LCJs would ensure also the presence of the enrichment-pool there. The increased mobility/instability of the enrichment-pool of Crb at LCJs would contribute to increase the total Crb mobility at LCJs. Further experiments will be required to test this possibility and to understand how the molecular mechanism underlying the increased accumulation of Crb at LCJs relates to the differential mobility of Crb protein that was documented (Olivares-Castineira, 2018).

It was next asked how the anisotropic distribution of Crb is regulated. To investigate this question attention was turned to Src42A, as it triggers one of the mechanisms regulating tube elongation, orienting membrane growth on the longitudinal axis. In conditions of Src42A loss of function, LCJs do not expand and tubes become shorter. Crb accumulation was analyzed in loss of function conditions for Src42A. In one case the Src42AF80 allele was used that lacks the distinct accumulation of phosphorylated Src42A (pSrc42A) at the apical junctional region but does not affect the stability or membrane localisation of the protein. This mutation renders a kinase non-activatable protein that was previously shown to strongly affect tracheal tube elongation. In contrast, a kinase-dead dominant negative form of Src42A (Src42DN) was expressed in the trachea, that was also previously shown to affect tube elongation. In both cases a more uniform distribution was observed of Crb at LCJs and TCJs. Quantification of Crb levels indicated that the differences between the accumulation of Crb at LCJs compared to TCJs were reduced. Analysis of the LCJ/TCJ ratio of Crb clearly showed a significant decrease when compared to the control, indicating a more uniform accumulation in Src42A loss of function conditions. DE-cad LCJ/TCJ ratio in Src42A loss of function conditions remained close to 1, indicating a homogeneous distribution. Altogether these results show that a decrease in Src42A activity leads to a decrease of the anisotropic accumulation of Crb (Olivares-Castineira, 2018).

To investigate whether Src42A promotes an increased accumulation of Crb protein at LCJs or a depletion at TCJs the total levels of protein accumulation were quantified at LCJs and TCJs and control (i.e. heterozygotes) and Src42A mutants (i.e. Src42AF80 homozygotes) were compared from the same experiment. While variability was observed within each genotypic group, different independent experiments indicated that in control embryos there is an increased accumulation of Crb protein at LCJs that is lost in Src42AF80 mutants. In Src42A mutant conditions the levels of Crb accumulation at LCJs and TCJs were similar to those of TCJs of control embryos, indicating that Src42A regulates a mechanism that increases the fraction of Crb protein at LCJs (Olivares-Castineira, 2018).

Consistent with a role for Src42A in regulating directly or indirectly Crb accumulation partial co-localisation was found of Crb and Src42A protein, and with pSrc42A at the SAR. However, no polarised accumulation was detected of the active pSrc42A fraction during tube elongation, as previously documented. While there may be transient anisotropies of pSrc42A accumulation that cannot be detected with the available antibodies, this result suggests that other factors (e.g. mechanical or chemical) modulate the activity of pSrc42A in the different junctions to regulate the anisotropic accumulation of Crb (Olivares-Castineira, 2018).

To further explore Src42A requirement FRAP experiments were performed in CrbGFP embryos in which Src42A was downregulated. Clear differences were found with respect to control: while in the control the Mf and recovery curves of LCJs and TCJs were clearly different, in Src42DN conditions the Mf and recovery curves of LCJs and TCJs were comparable. The Mf at the LCJs of Src42DN was significantly lower than the Mf at the LCJs in control embryos, and was similar to the Mf at TCJs in control and mutant embryos. The halftime recovery, t1/2, was comparable to that of control embryos, indicating a recovery rate similar in all cases (Olivares-Castineira, 2018).

Altogether these results indicate that Src42A contributes to Crb preferential enrichment at LCJs and that it increases Crb mobility there. The fact that Crb levels and Crb recovery are affected particularly at LCJs when Src42A is downregulated strongly suggests that Src42A is (more) active precisely at LCJs. The results are consistent with the proposed model in which a mechanism acting specifically at LCJs, that is now proposed to be mediated by Src42A, would ensure the accumulation of an enrichment-pool of Crb at LCJs with high protein mobility. In the absence of this Src42A-mediated activity, only the basal level-pool of Crb would be present at LCJs and TCJs, leading to comparable levels and mobility of Crb in all junctions. Src42A would not regulate the basal level-pool of Crb, and would instead be required to top up Crb at LCJs with an enrichment-pool of Crb. Future experiments addressing the molecular mechanism by which Src42A regulates Crb accumulation and its mobility will help to fully understand how it regulates the anisotropic accumulation of Crb at LCJs. Src42A-independent accumulation of Crb in tracheal cells together with other Src42A-independent mechanisms of apical membrane growth may be responsible for tube growth in the absence of Src42A (Olivares-Castineira, 2018).

Src42A was found to be required for the anisotropic accumulation of Crb at LCJs. It was then asked whether the overelongation of tubes observed in Src42A overactivation conditions (either overexpression of a wild type form of Src42A or expression of a constitutively active protein) was due to an increased accumulation of Crb at LCJs. The results did not support this expectation. Crb was found to be strongly decreased in the SAR of DT cells both in conditions of overexpression (UASSrc42A) or constitutive activation (UASSrc42ACA). In conditions of mild overexpression of wild type Src42A, rare cases (around 5-8% of embryos) were found where it was possible to detect some levels of Crb in the SAR, which accumulated preferentially at LCJs, as expected. These results suggested that the tube length defects produced by Src42A overexpression/overactivation were caused by a mechanism different than the one operating in physiological conditions. To investigate this possible mechanism the levels and distribution of the total Src42A protein and the pSrc42A active fraction were analyzed in both Src42A overexpression and overactivation conditions. Levels of Src42A protein were found to be increased but still enriched in the membrane region. Interestingly, pSrc42A was not restricted any more to the junctional apical region as in the wild type and instead it was expanded along the whole apicobasal membrane. The increase and expansion of pSrc42A accumulation observed in overexpression and in overactivation conditions indicate that Src42A activity is overactivated in both cases and may explain the similarity of phenotypes. Further analysis also indicated that Src42A overexpression/overactivation leads to a general loss of cell organisation and membrane polarity, as evidenced by the miss-localisation of markers of membrane polarity, like the Septate Junction protein Megatrachea. These results indicate that an unregulated accumulation of active pSrc42A leads to a generalised miss-organisation of the cell and prevents proper Crb accumulation (Olivares-Castineira, 2018).

To investigate the cause of tube overelongation found in Src42A overexpression /overactivation conditions other known tube length regulators were analyzed. One of them is Serp, which regulates the aECM organisation. In Src42A and Src42ACA overexpression conditions Serp was found to be lost from the luminal compartment, although, as in wild type, tracheal cells accumulate Serp at early stages. This result provides explanation for the tube elongation defects observed under these conditions, as Serp absence leads to tube overelongation. Interestingly, defects in Serp accumulation could not be detected in Src42 mutants or in Src42ADN conditions, as previously reported. These results suggested again that Src42A overactivation use a different mechanism than the one used in physiological conditions to drive tube elongation. Hence, the analysis of Src42A overactivation provides new results that allow to revisit and reinterpret previously published work (Olivares-Castineira, 2018).

Altogether the results indicate that an unregulated accumulation of active pSrc42A leads to a generalised miss-organisation of the cell and prevents proper accumulation of Crb and Serp. In addition, it was also observed that DE-cad was not properly localised either. Interestingly, it has been shown that the tracheal accumulation of these proteins depends on their recycling. Thus, the results could suggest a role of Src42A in protein trafficking. In this context, the loss of cell organisation and membrane polarity produced by mislocalisation of pSrc42A could interfere with protein trafficking. Roles for Src42A in protein trafficking have been proposed in different contexts. Src42A could regulate protein trafficking directly, or indirectly through the regulation of the actin cytoskeleton. The actin cytoskeleton plays a capital role in protein trafficking and Src42A acts as a regulator of the actin cytoskeleton. A disruption of actin organisation in Src42A overactivation could lead to defects in the sorting of different cargoes as well as defects in endosomal maturation. Further experiments will be required to investigate a possible involvement of Src42A in protein trafficking during tracheal development (Olivares-Castineira, 2018).

After identifying an anisotropic accumulation of Crb regulated by Src42A, it was asked how this mechanism relates to tube elongation. Crb has been proposed to promote apical membrane growth independently of its role in apicobasal polarity at late stages of epithelial differentiation. In the trachea Crb was proposed to mediate tube elongation by promoting apical membrane growth. Interestingly, the results show an enrichment of Crb in the SAR of LCJ during tube elongation. This observation raises the hypothesis that it is precisely this accumulation of Crb in the SAR of LCJs what favours or facilitates apical membrane expansion (either by membrane growth or membrane transformation leading to cell shape changes), orienting cell elongation and as a consequence the longitudinal growth of the tube. Crb recycling during tracheal development could favour the mobilisation of cellular and/or membrane components facilitating membrane growth or membrane transformation. To investigate this possibility Crb accumulation in the SAR was analyzed in different experimental conditions in which apicobasal polarity was unaffected (Olivares-Castineira, 2018).

Altogether the results confirm a role of EGFR in regulating the accumulation of Crb in the SAR or AFR, at least in tubular organs. EGFR regulates the trafficking of different cargoes, in particular Crb and Serp, raising the possibility that the regulation of the apical surface area depends on targets different than Crb. However, the fact that Serp is not present in the SGs and that Crb has already been proposed to promote apical membrane growth, strongly suggest that Crb is at least one of the targets downstream of EGFR regulating apical expansion. On the other hand, the results correlate apical cell expansion with Crb subcellular localisation in the SAR. It is suggested that Crb accumulation in the SAR of LCJs could promote their expansion facilitating the elongation of the cell along the longitudinal axis, in agreement with the proposed role of Crb promoting apical membrane expansion. Previous observations such as the expansion of the photoreceptor stalk membrane upon Crb overexpression support this hypothesis, indicating that this can be a general mechanism (Olivares-Castineira, 2018).

Crb was proposed to regulate tube size by promoting apical membrane growth. Accordingly, it was found that a weak overexpression of Crb in an otherwise wild type background caused a mild increase in DT dimensions (a significant 12% enlargement of DT and a non-significant 9% diameter expansion) without perturbing the epithelial integrity and polarity. Src42A was shown to control tube elongation through interactions with dDaam and the remodelling of AJs. This study is now showing that Src42A regulates Crb levels, suggesting that Src42A may control tube elongation at least in part through regulation of Crb. Thus, it was asked whether increased levels of Crb can bypass or compensate the requirement of Src42A in tube growth. To evaluate this possibility genetic interaction experiments were performed to test the ability of a weak Crb overexpression in a Src42A loss of function background. Interestingly, Crb overexpression was found to produced a partial but significant rescue of the short-DT phenotype of Src42A loss of function. This result indicates that Crb acts downstream or in parallel of Src42A. Because it was also observed that Src42A is required for Crb preferential enrichment at LCJs, the hypothesis is favored that Crb acts downstream of Src42A contributing to its function in tube elongation (Olivares-Castineira, 2018).

Remarkably, besides a rescue in DT length, an increase was also detected in the diameter of the tube when Crb was overexpressed in a Src42A loss of function background (a 25% expansion with respect to Src42ADN mutants). Under these conditions, the DT diameter was not perfectly smooth and often showed dilations that were not detected in Src42ADN or Crb overexpression conditions on their own. This isometric expansion of the DT along the diametrical and longitudinal directions is interpreted to be the result of an isotropic excess of Crb. Because in the absence of Src42A activity Crb accumulation is not properly polarised, this may promote a non-polarised increase of tube growth. To find support for this interpretation Crb accumulation was analyzed in conditions of weak Crb overexpression. High levels of Crb were detected in the whole apical domain and in vesicles that precluded a proper analysis of Crb localisation and a systematic quantification of Crb accumulation. However, it was possible to observe in examples in which a distinct accumulation of Crb could be detected that the overexpression of Crb in a wild type background leads to high enrichments of the protein particularly at LCJs. This result suggests that the activity of Src42A biases the increased accumulation of Crb to the LCJs, correlating with a preferential growth mainly along the longitudinal axis. In contrast, a more generalised pattern of Crb overexpression could be detected in a Src42A loss of function mutant background, consistent with the isometric tube growth observed. In summary, although it was not possible to directly test whether an anisotropic accumulation of Crb can exclusively compensate tube elongation in Src42A loss of function conditions, the results are consistent with the hypothesis that it is the anisotropic accumulation of Crb, regulated by Src42A, that mediates or promotes oriented tube growth along the longitudinal axis. Future experiments involving the generation of new tools designed to specifically localise Crb protein at desired subcellular domains will be needed to prove the current model and to confirm an instructive and causal role of the anisotropic accumulation of Crb in cell elongation and polarised tracheal tube growth (Olivares-Castineira, 2018).

To summarise, this study finds that Crb is transiently enriched in the SAR of DT cells in a polarised/anisotropic manner. This polarised distribution correlates with different dynamics or turnover of Crb protein, which appears to be more mobile and accumulate more at longitudinal junctions than at transverse ones. This polarised distribution also correlates with the anisotropic expansion of the apical membrane, axially-biased, that drives the longitudinal enlargement of the tracheal tubes. Interestingly it was also found that Src42A is required for this anisotropic accumulation of Crb. Src42A was already known to regulate tube growth along the longitudinal axis, and this study now proposes that it performs this activity at least in part by promoting a Crb anisotropic enrichment. Src42A was also proposed to act as a mechanical sensor, translating the polarised cylindrical mechanical tension (an inherent property of cylindrical structures) into polarised cell behaviour. Hence, it is proposed that Src42A would sense differential longitudinal/transverse tension stimuli and translate them into the cell by polarising Crb accumulation. It is likely that this Crb anisotropic accumulation in the SAR of LCJs mediates apical membrane expansion in the longitudinal direction, which would help to orient cell elongation and as a consequence longitudinal tube growth. A causal role for this Crb anisotropic accumulation in orienting cell elongation awaits definitive confirmation (Olivares-Castineira, 2018).

In light of the current results, the following model is now proposed. Different mechanisms operate to regulate tube growth. On the one hand secretion drives apical membrane growth along the transverse axis independently of Src42A. In addition, a basal level-pool of Crb accumulation independent of Src42A may promote or contribute to isotropic apical expansion. On the other hand the presence of a properly organised luminal aECM also controls tube growth by restricting tube elongation. A Src42A-dependent mechanism acts in coordination with these other mechanisms. Src42A would contribute to tube elongation through interactions with dDaam, the remodelling of AJs and topping up Crb accumulation at LCJs with an enrichment-pool of Crb. This increased accumulation of Crb at LCJs would bias the growth of the tube along the longitudinal axis, counteracting the restrictive activity of the aECM on tube elongation. In the absence of Src42A activity, the Src42A independent mechanism/s of membrane growth would still operate, and would favour a compensatory growth along the transverse axis as observed, as diametrical growth is not restricted by the aECM (Olivares-Castineira, 2018).

The regulation of size and shape of tubular organs is important for organ function, as evidenced by the fact that loss of regulation can lead to pathological conditions such as polycystic kidney disease (PKD), cerebral cavernous malformation (CCM) or hereditary hemorrhagic telangiectasia (HHT). Src proteins have been implicated in malformations like PKD, highlighting the importance of investigating the mechanisms underlying their activities. While Src42A was proposed to regulate polarised cell shape changes during tracheal tube elongation through interactions with dDaam and the remodelling of AJs, no polarised downstream effectors have been identified up to date. Hence, identifying that Crb anisotropy is one of the downstream effects of Src42A activity adds an important piece to the puzzle. Src42A and Crb are conserved proteins with important roles in development and homeostasis and are involved in different pathologies. This work provides an ideal model where to investigate the molecular mechanisms underlying their activities, their interactions, and their roles in morphogenesis (Olivares-Castineira, 2018).

The apical protein Apnoia interacts with Crumbs to regulate tracheal growth and inflation

Most organs of multicellular organisms are built from epithelial tubes. To exert their functions, tubes rely on apico-basal polarity, on junctions, which form a barrier to separate the inside from the outside, and on a proper lumen, required for gas or liquid transport. This study has identified apnoia (apn; CG15887), a novel Drosophila gene required for tracheal tube elongation and lumen stability at larval stages. Larvae lacking Apn show abnormal tracheal inflation and twisted airway tubes, but no obvious defects in early steps of tracheal maturation. apn encodes a transmembrane protein, primarily expressed in the tracheae, which exerts its function by controlling the localization of Crumbs (Crb), an evolutionarily conserved apical determinant. Apn physically interacts with Crb to control its localization and maintenance at the apical membrane of developing airways. In apn mutant tracheal cells, Crb fails to localize apically and is trapped in retromer-positive vesicles. Consistent with the role of Crb in apical membrane growth, RNAi-mediated knockdown of Crb results in decreased apical surface growth of tracheal cells and impaired axial elongation of the dorsal trunk. It is concluded that Apn is a novel regulator of tracheal tube expansion in larval tracheae, the function of which is mediated by Crb (Skouloudaki, 2019).

Animal organs consist of epithelial tissues, which form the boundaries between internal and external environment. During development, epithelia are instrumental to shape the various organs. Many epithelial tissues form tubular organs, such as the gut, the kidney or the respiratory system. A fundamental feature of epithelial tubes and sheets is to keep the balance between the maintenance of structural integrity and tissue rigidity during organ growth and morphogenesis. To understand how this balance is achieved during rapid, temporally regulated developmental transitions from juvenile to adult body shapes, several studies in various animal models have focused on elucidating how cell proliferation, cell polarity, cell shape changes and trafficking contribute to the formation of the tubular lumen length and diameter. The correct coordination of these processes is crucial for normal organ function. This is reflected in the fact that several human diseases are linked to defects in epithelial tube formation and maintenance, such as polycystic kidney disease or cystic fibrosis (Skouloudaki, 2019).

The developing tracheae of Drosophila melanogaster, a network of branched epithelial tubes that ensure oxygen supply to the cells of the body, has emerged as an ideal system to study cell fate determination and morphogenesis of epithelial tubes. The available genetic tools, as well as the ease to image the tracheal system in the fly embryo, has provided detailed insights into the developmental processes required to form tubular structures with defined functional lumens and have contributed to elucidating the interplay between tissue growth, differentiation and cell polarity (Skouloudaki, 2019).

The stereotypically branched tracheal system of Drosophila is set up at mid-embryogenesis. Once a continuous tubular network has formed, the tube expands to accommodate an increased oxygen supply to all tissues during animal growth. Tube expansion occurs by growth along the diameter and along the anterior-posterior axis. Growth is accompanied by the formation of a transient cable, comprised of a chitinous apical extracellular matrix (aECM), which fills the lumen of the tube. The generation of this cable requires the secretion of chitin and chitin-modifying enzymes. Mutations in genes affecting secretion or organization of the chitin cable result in excessively elongated tracheal tubes or tubes with irregular diameter (with constricted and swollen areas along the tube). Axial growth, on the other hand, depends on the proper elongation of tracheal cells along the anterior-posterior axis. At later stages of embryogenesis, the lumen becomes cleared and filled with air (Skouloudaki, 2019).

After hatching, the larvae undergo two molts, a process during which animals rapidly shed and replace their exoskeleton with a new one, bigger in size. For this, a new chitinous aECM is secreted apically, thus surrounding the old tube. Remodeling of this aECM permits tissue growth between larval molts. The molting process is initiated by the separation of the old aECM from the apical surface of the epithelial cells and the secretion of chitinases and proteinases, which partially degrade the old cuticle. The remnants of the old cuticle in each metamer are shed through the spiracular branches. This process, called ecdysis, is followed immediately by clearance of the molting fluid and air filling. Interestingly, while the diameter of the dorsal trunk only increases at each molt, tube length increases continuously throughout larval life, particularly during intermolt periods. Despite the importance of tube expansion and elongation for larval development, the underlying mechanisms that control tracheal growth at this stage remain poorly understood (Skouloudaki, 2019).

A well-established regulator of apical domain size in developing epithelia is Crumbs (Crb). Crb is a type I transmembrane protein, which acts as an apical determinant of epithelial tissues. It has a large extracellular, a single transmembrane and a short cytoplasmic domain. Loss- and gain-of-function experiments have shown that apical levels of Crb are important for proper cell polarity, tissue integrity and growth. For instance, absence of Crb in embryonic epithelia results in loss of apical identity and disruption of epithelial organization. In contrast, overexpression of Crb triggers apical membrane expansion, which leads to a disordered epithelium, abnormal expansion of tracheal tubes and/or tissue overgrowth. These results underscore the importance of Crb levels for epithelial development and homeostasis (Skouloudaki, 2019).

Several mechanisms have been uncovered that ensure proper levels of apical Crb. These include: stabilization of Crb at the membrane, mediated through interactions of its cytoplasmic domain with scaffolding proteins, e.g. Stardust (Sdt), or by homophilic interactions between Crb extracellular domains, regulation of Crb trafficking, including endocytosis by AP-2, Rab5 or Avalanche, membrane delivery by Rab11, recycling by the retromer and endocytic sorting by the ESCRT III component Shrub/Vps32 (Skouloudaki, 2019).

To gain further insight into the molecular mechanisms that regulate Crb and its activity during epithelial growth, this study set out to identify novel interacting partners of Crb using the yeast two-hybrid system. One of the candidates identified, CG15887, encodes a transmembrane protein, which localizes to the apical surface of tracheal tubes. This study found that the CG15887 protein physically interacts with Crb. Based on the phenotype of mutations in CG15887, which is characterized by defects in tracheal growth and inflation during larval stages, this gene was named apnoia (apn). apn mutant animals die as second instar larvae with dorsal trunks displaying reduced axial growth and impaired apical surface area expansion, resulting in shorter tubes. This phenotype is correlated with the absence of Crb from the apical surface. RNAi knock-down of crb phenocopies the apn mutant phenotype of impaired longitudinal growth. These results identify Apn as a novel regulator of tracheal tube growth in the larvae, which acts through Crb to control axial tube expansion (Skouloudaki, 2019).

This work identifies Apn as an essential protein for airway maturation in Drosophila larval stages. Apn is localized apically in tracheal epithelial cells, where it co-localizes and physically interacts with Crb. apn1 mutant larvae exhibit loss of tracheal tissue structure, manifested by tube size defects and impaired gas filling, resulting in body size reduction and lethality at second instar. At the cellular level, exclusion of Crb from the apical membrane in apn1 mutant larval tracheae goes along with apical cell surface reduction and an overall tracheal tube shortening. Absence of apn leads to Crb inhibition and accumulation in enlarged, Vps35/retromer-positive vesicles (Skouloudaki, 2019).

Elongation of the tracheal tube has been extensively studied in embryos where it has been shown to rely on different mechanisms, such as the organization of the aECM and cell shape changes. Anisotropic growth of the apical plasma membrane is an additional mechanism to achieve proper longitudinal tube expansion. However, only few proteins have been described so far to regulate this process. One of these, the protein kinase Src42A, is required for the expansion of the cells in the axial direction, and loss of Src42A function results in tube length shortening, which is associated with an increased tube diameter. Src42A has been suggested to exert its function, at least in part, by controlling DEcad recycling and hence adherens junctions remodeling and/or by its interaction with the Diaphanous-related formin dDAAM (Drosophila Dishevelled-Associated Activator of Morphogenesis), loss of which results in reduced apical levels of activated pSrc42A. More recently, Src42A has been suggested to control axial expansion by inducing anisotropic localization of Crb preferentially along the longitudinal junctions. However, this study never observed any anisotropic distribution of Crb in wild type larval tracheal cells, making it unlikely that, at this developmental stage, axial expansion is regulated by a Src42A-dependent mechanism. This assumption is corroborated by the observation that, unlike in Src42A mutants, the lack of longitudinal expansion in apn¹ mutant larval tubes is not associated with circumferential expansion. Another protein regulating tube elongation in the embryo is the epidermal growth factor receptor, EGFR. Expressing a constitutively active EGFR results in shortened tracheal tubes with smaller apical cell surfaces, but with increased diametrical growth. In this condition, Crb shows altered apical distribution. This phenotype differs from the apn¹ phenotype, where apical localization of Crb is almost completely lost and only longitudinal tube growth is affected. This suggests that Apn executes tube length expansion by a different mechanism (Skouloudaki, 2019).

How does decrease in tubular growth lead to loss of tracheal structure? During development, the larval body, including the tracheal tissue, elongates about 8-fold. Impaired axial tracheal cell growth in apn¹ mutants thus may affect the balance between the forces exerted by apical membrane growth on the one hand and the resistance provided by the luminal aECM on the other, an important mechanism described previously to control tube shape in the embryo. This could lead to physical rupture of tubes mutant for apn¹, allowing fluid entry. The presence of fluid would, in turn, disrupt proper gas filling, resulting in hypoxia and, consequently, in impaired body growth (Skouloudaki, 2019).

Several studies have shown that, in some tissues, Crb accumulation on the apical membrane is mediated by the retromer complex, which controls either the retrograde transport of Crb to the trans-Golgi or the direct trafficking from the endosomes to the plasma membrane. The physical interaction of Apn and Crb, the functional requirement of Apn for Crb apical localization and the fact that in apn¹ mutants Crb is trapped in Vps35-positive/retromer vesicles all suggest that Apn is required for trafficking and/or maintenance of Crb at the apical membrane (Skouloudaki, 2019).

However, the increase in the size of Vps35-positive vesicles in apn¹ mutant cells, which is, to some extent, due to the accumulation of Crb, suggests defects in retromer function, which may prevent Crb lysosomal degradation. Further studies will help to elucidate at which level Apn controls Crb trafficking in larval tracheae (Skouloudaki, 2019).

Regulation of apical constriction via microtubule- and Rab11-dependent apical transport during tissue invagination

The formation of an epithelial tube is a fundamental process for organogenesis. During Drosophila embryonic salivary gland (SG) invagination, Folded gastrulation (Fog)-dependent Rho-associated kinase (Rok) promotes contractile apical myosin formation to drive apical constriction. Microtubules (MTs) are also crucial for this process and are required for forming and maintaining apicomedial myosin. However, the underlying mechanism that coordinates actomyosin and MT networks still remains elusive. This study shows that MT-dependent intracellular trafficking regulates apical constriction during SG invagination. Key components involved in protein trafficking, such as Rab11 and Nuclear fallout (Nuf), are apically enriched near the SG invagination pit in a MT-dependent manner. Disruption of the MT networks or knockdown of Rab11 impairs apicomedial myosin formation and apical constriction. MTs and Rab11 are required for apical enrichment of the Fog ligand and the continuous distribution of the apical determinant protein Crumbs (Crb) and the key adherens junction protein E-Cadherin (E-Cad) along junctions. Targeted knockdown of crb or E-Cad in the SG disrupts apical myosin networks and results in apical constriction defects. These data suggest a role of MT- and Rab11-dependent intracellular trafficking in regulating actomyosin networks and cell junctions to coordinate cell behaviors during tubular organ formation (Le, 2021).

MTs have a crucial role in stabilizing apical myosin during epithelial morphogenesis both in early Drosophila embryos and in the Drosophila SG. In the SG, MTs interact with apicomedial myosin via Short stop, the Drosophila spectraplakin, emphasizing a direct interplay between the MT and the apical myosin networks. The data of this study reveal another key role of MTs in regulating protein trafficking to control the apical myosin networks during tissue invagination. During SG invagination, a network of longitudinal MT bundles is observed near the invagination pit. These data show apical enrichment of Rab11 in the same area is MT dependent and that this enrichment is important for forming the apicomedial myosin networks, suggesting a link between localized intracellular trafficking along MTs to apical myosin regulation (Le, 2021).

The dorsal/posterior region of the SG, where Rab11 is apically enriched in a MT-dependent manner, correlates with localized Fog signaling activity that promotes clustered apical constriction. Disruption of MTs or Rab11 knockdown reduces Fog signals in the apical domain of SG cells and causes dispersed Rok accumulation and defective apicomedial myosin formation. It is consistent with a previous study that the absence of Fog signal results in dispersed apical Rok and defects in apicomedial myosin formation. It is therefore proposed that MT- and Rab11-dependent apical trafficking regulates Fog signaling activity to control apical constriction during epithelial tube formation through transporting the Fog ligand. As recycling of membrane receptors to the cell surface plays an important role in regulating overall signaling activity, it is possible that Rab11 is involved in recycling the as yet unidentified SG receptor(s) of Fog to regulate Fog activity in the SG. Indeed, several GPCRs are recycled via Rab11. During epithelial invagination in early Drosophila embryogenesis, the concentration of the Fog ligand and receptor endocytosis by 'β-arrestin-2 have been shown as coupled processes to set the amplitude of apical Rho1 and myosin activation. It is possible that the movement of Fog receptor(s) that have internalized as a stable complex with 'β-arrestin is recycled back to the cell surface by Rab11. The Fog signaling pathway represents one of the best understood signaling cascades controlling epithelial morphogenesis. Although best studied in Drosophila, the pathway components have also been identified in other insects, suggesting a more widely conserved role for Fog signaling in development. Further work needs to be done to fully understand the regulatory mechanisms underlying the trafficking of Fog and its receptor(s) during epithelial morphogenesis (Le, 2021).

Analysis of apicomedial myosin shows that reduced Rab11 function not only causes a decrease of the myosin intensity but also causes myosin to be dispersed rather than forming proper myosin web structures in the apicomedial domain of SG cells. These data support the idea that Rab11 function is required for both concentration and spatial organization of apicomedial myosin. This can be explained by the combined effect of multiple cargos that are transported by Rab11, including Fog, Crb, and E-Cad. Time-lapse imaging of myosin will help determine how the dynamic behavior of apicomedial myosin is compromised when Rab11 function is disrupted (Le, 2021).

During branching morphogenesis in Drosophila trachea, MTs and dynein motors have a critical role in the proper localization of junctional proteins such as E-Cad. This is consistent with the observations with MT-dependent uniform distribution of E-Cad at adherens junctions in the invaginating SG, suggesting a conserved role of MT-dependent intracellular trafficking in junctional remodeling and stabilization during epithelial tube formation. The data further suggest that the MT networks and Rab11 have key roles in apical distribution of Crb and E-Cad in the SG and that proper levels of apical and junctional proteins are important for apical constriction during SG invagination. Based on these data, it is proposed that MT- and Rab11-dependent apical trafficking of Crb and E-Cad is critical for apical constriction during SG invagination. Alternatively, MTs have an additional role in assembling/anchoring these apical components through the regulation of unidentified molecules. Recent studies in Drosophila mesoderm invagination showed that MTs help establish actomyosin networks linked to cell junction to facilitate efficient force transmission to promote apical constriction. In Ko (2019), however, MT-interfering drugs and RNAi of CAMSAP end-binding protein were used to prevent MT functions and the effect cannot be directly compared with the current data where spastin was used to sever existing MTs. Direct monitoring of MT-dependent transport of Crb and E-Cad during SG invagination will help clarify the mechanism (Le, 2021).

On knockdown of crb or E-Cad, less prominent apicomedial myosin web structures are observed in invaginating SGs, suggesting a requirement of Crb and E-Cad in proper organization of apical actomyosin networks during SG tube formation. Crb acts as a negative regulator of actomyosin dynamics during Drosophila dorsal closure and during SG invagination. It is possible that proper Crb levels are required for modulating myosin activity both in the apicomedial domain and at junctions during SG invagination. Both of which contribute to apical constriction and cell rearrangement, respectively. Anisotropic localization of Crb and myosin was observed at the SG placode boundary, where myosin accumulates at edges where Crb is lowest. Planar polarization of Rok at this boundary is modulated through phosphorylation by Pak1 downstream of Crb. A further test will help understand whether and how Crb might affect junctional myosin dynamics and SG invagination. As contractile actomyosin structures exert forces on adherens junction to drive apical constriction, it is speculated that apical constriction defects on E-Cad RNAi might be due to reduction of cell adhesion and/or of improper force transmission. It will be interesting to determine if the coordination of apical and junctional proteins and apical cytoskeletal networks through intracellular trafficking is conserved during tubular organ formation in general (Le, 2021).

Dhc is also apically enriched in the dorsal/posterior region of the invaginating SG. The data show that knockdown of Dhc64C not only affects Rok accumulation and apicomedial myosin formation but also disrupts MT organization in the SG. These data are consistent with previous findings that cytoplasmic dynein is associated with cellular structures and exerts tension on MTs. For example, dynein tethered at the cell cortex can apply a pulling force on the MT network by walking toward the minus end of a MT. Dynein also scaffolds the apical cell cortex to MTs to generate the forces that shape the tissue into a dome-like structure. In interphase cells, the force generated by dynein also regulates MT turnover and organization (Le, 2021).

In klar mutants, on the other hand, MT organization is not affected in the SG, suggesting that reduction of dynein-dependent trafficking by loss of klar does not cause changes in the MT networks. Notably, although the intensity of apicomedial myosin does not change on Dhc64C knockdown or in the klar mutant background, formation of apicomedial myosin web structures is affected. These data suggest a possible scenario that dynein function is not required for myosin concentration in the apical domain but is only needed for the spatial organization of apicomedial myosin. However, it cannot be ruled out that the zygotic knockdown of Dhc64C by RNAi is not strong enough to affect the intensity of apicomedial myosin. Dhc64C has strong maternal expression and is essential for oogenesis and early embryo development. Embryos with reduced maternal and zygotic pools of Dhc64C showed a range of morphological defects in the entire embryo, some of which were severely distorted. Precise roles for dynein and dynein-dependent trafficking in regulating apicomedial myosin formation remain to be elucidated (Le, 2021).

Apical polarity proteins recruit the RhoGEF Cysts to promote junctional myosin assembly

The spatio-temporal regulation of small Rho GTPases is crucial for the dynamic stability of epithelial tissues. However, how RhoGTPase activity is controlled during development remains largely unknown. To explore the regulation of Rho GTPases in vivo, this study analyzed the Rho GTPase guanine nucleotide exchange factor (RhoGEF) Cysts, the Drosophila orthologue of mammalian p114RhoGEF, GEF-H1, p190RhoGEF, and AKAP-13. Loss of Cysts causes a phenotype that closely resembles the mutant phenotype of the apical polarity regulator Crumbs. This phenotype can be suppressed by the loss of basolateral polarity proteins, suggesting that Cysts is an integral component of the apical polarity protein network. Cysts was demonstrated to be recruited to the apico-lateral membrane through interactions with the Crumbs complex and Bazooka/Par3. Cysts activates Rho1 at adherens junctions and stabilizes junctional myosin. Junctional myosin depletion is similar in Cysts- and Crumbs-compromised embryos. Together, these findings indicate that Cysts is a downstream effector of the Crumbs complex and links apical polarity proteins to Rho1 and myosin activation at adherens junctions, supporting junctional integrity and epithelial polarity (Silver, 2019).

Antagonistic interactions between apical and basolateral polarity regulators position AJs at the apico-lateral membrane to form a junctional complex. In turn, AJs are thought to maintain apical-basal polarity through the segregation of the apical and basolateral membrane domains, organization of the cytoskeleton, and direct polarity by acting as signaling centers for polarity complexes. Although a number of Drosophila RhoGEFs and RhoGAPs have been implicated in epithelial polarity and AJ stability, no single RhoGEF or RhoGAP has been found to phenocopy the polarity or junctional defects that are seen in embryos compromised for factors such as Crb, aPKC, or E-cadherin. The current findings suggest that loss of the RhoGEF Cysts causes a polarity phenotype strikingly similar to the loss of core apical polarity proteins. Moreover, this study found that Cyst is recruited to the apico-lateral cortex by the action of polarity proteins and, by activating Rho1, stabilizes AJ-associated actomyosin, which supports junctional and epithelial integrity (Silver, 2019).

In Cysts-compromised embryos, AJ formation is disrupted in early gastrulation, and AJs do not form a circumferential belt. These defects in AJ assembly or stability correlate with reduced and irregular myosin accumulation at the apico-lateral cortex. Given the molecular function of Cysts as a GEF for Rho1, loss of myosin activity is presumably the immediate cause for the defects in AJ formation and the subsequent loss of apicobasal polarity in many epithelial cells. crb-depleted embryos failed to recruit Cysts to apical junctions and showed a similar decline in junctional myosin. Therefore, a major function of the apical Crb polarity complex appears to be the Cysts-mediated support of junctional actomyosin (Silver, 2019).

While many cells in crb or cyst mutants undergo programmed cell death, others retain or recover polarity and form small epithelial cysts, a process seen from mid-embryogenesis (postgastrulation stages) onward. Several polarity proteins such as Crb, Sdt, and Baz are needed for normal epithelial polarization in early embryos but are not essential for polarization in postgastrulation embryos, which explains the ability of some epithelial cells in these mutants to form epithelial cysts with normal polarization. In fact, when programmed cell death is suppressed, cyst formation is shown by all epithelial cells in crb mutants. Formation of epithelial cysts seen in cysts mutant embryos therefore suggests that Cysts is also not essential for epithelial polarity in late embryos. This view is supported by the decline of Cysts protein accumulation at AJs seen in late embryos (Silver, 2019).

Several observations, including the genetic interaction of cysts with genes encoding basolateral polarity proteins, the dependence of the junctional localization of Cysts on the apical polarity proteins Baz and Crb, the physical interactions between Cysts and apical polarity proteins, and the function of Cysts in stabilizing AJs, indicate that Cysts is an integral part of the apical polarity machinery in early Drosophila embryos. A particularly striking finding was the complete suppression of the cysts phenotype by codepletion of the basolateral polarity proteins Scrib or Lgl, seen in double-mutant embryos that showed phenotypes indistinguishable from single scrib or lgl mutants. This mimics previous observations with double mutants of crb or sdt and scrib, lgl or discs large. Moreover, this study found that a reduction of aPKC enhanced Baz mislocalization in Cysts-compromised embryos, suggesting that aPKC cooperates with Cysts and acts upstream or in parallel to Cysts to organize Baz. These findings emphasize that Cysts, similar to Crb and aPKC, is a component of a negative feedback circuit between apical and basolateral regulatory networks that govern epithelial polarity. The dependence of Cysts localization on Crb and Baz suggests that Cysts acts downstream of these two proteins. Once polarized, Cysts appears to maintain polarity and junctional stability through actomyosin remodeling (Silver, 2019).

In vivo structure-function data indicate that the C-terminal region is essential for Cysts activity. Moreover, it was found that the C-terminal region of Cysts can oligomerize, potentially facilitated by the CC domain. It is speculated that clustering of Cyst could enhance its cortical association. The Crb complex protein Patj represents one possible anchor for Cyst clusters at the cortex. Biochemical data show that the Cysts C-terminal region is sufficient for interactions with Patj. Patj has been implicated as a myosin II activator in the embryo. It is proposed therefore that Crb, Patj, and Cyst form a complex that organizes junctional actomyosin. However, as Patj is not essential for embryonic survival, Cysts may interact with additional binding partners within the Crb complex. Another apical binding partner for Cyst is Baz/Par3, which is required for Cyst cortical recruitment, coprecipitates with the Cyst C-terminal region, and coaggregates with Cyst in HeLa cells (Silver, 2019).

A recent independent study also arrived at the conclusion that Cysts activates Rho1 at AJs during germband extension in the Drosophila embryo (de Las Bayonas, 2019). It is further shown that depletion of Cysts acts downstream of a G protein-coupled receptor (GPCR) and the Gβ13F/Gγ1 heterotrimeric G protein in directing cell rearrangements promoting germband extension, and that germband extension is somewhat reduced when Cysts is depleted. Loss of Gγ1 causes an ~20% reduction in Cyst junctional enrichment (de Las Bayonas et al., 2019). These and the current data suggest that the normal junctional recruitment of Cyst requires at least three distinct inputs: interactions with Baz/Par3 and the Crb complex, and heterotrimeric G protein signaling (Silver, 2019).

This study found that Cysts becomes enriched at the apico-lateral cortex after the mesoderm and endoderm have invaginated and the germband starts to elongate. This localization coincides with the assembly of the apical-cortical actomyosin network. Rho-Rho kinase signaling plays a critical role in the activation of myosin II in this process. Structure-function analysis showed that Cyst contains an essential RhoGEF domain as predicted, and the use of Rho activity probes, genetic interactions, and biochemical assays showed that Cysts preferentially targets Rho1. Although the biochemical assay also revealed stimulation of Rac1 activity by Cysts, all other data point to Rho1 as the primary target of Cyst. It is proposed therefore that Cyst activates Rho1 to organize actomyosin at the cortex at a time when AJs assemble into a circumferential belt (stages 6/7). Consistent with this, it was found that Cysts is important for maintaining normal cortical levels of myosin II. A similar loss in junctional myosin was also observed in Crb-compromised embryos in line with the finding that Crb is required for Cysts junctional recruitment. The cysts mutant phenotype suggests that Cysts is the key RhoGEF that activates Rho1 at ectodermal AJs. In contrast, RhoGEF2 functions in the mesoderm and ectoderm, where it becomes apico-cortically enriched and activates Rho1 to recruit myosin II to the apical-medial cortex. Thus, RhoGEF and Cysts act in parallel on Rho1 to orchestrate the balance of cortical and medial actomyosin dynamics (Silver, 2019).

Cysts is the single orthologue of a group of four mammalian RhoGEFs that target RhoA in cell culture . One of the mammalian orthologues (p114RhoGEF) stabilizes tight junctions and AJs through organization of the actin cytoskeleton associated with cellular junctions (Nakajima and Tanoue, 2011; Terry, 2011; Acharya, 2018). p114RhoGEF is recruited to apical junctions through a mechanism involving CRB3A, Ehm2/Lulu2, Par3, Patj, the heterotrimeric G protein Gα12, and the GPCR Sphingosine-1 phosphate receptor 2 (Acharya, 2018). p114RhoGEF requires the polarity regulator Ehm2/Lulu2 (a homologue of Drosophila Yrt) to activate RhoA. In contrast, this study did not detect genetic or biochemical interactions between Cyst and Yrt in Drosophila. Recently, ARHGEF18, the human orthologue of p114RhoGEF, was identified as a gene associated with retinal degeneration, and a fish orthologue is required to maintain epithelial integrity of the retina. ARHGEF18 mutant retinal defects closely resemble those found in patients carrying mutations in the crb homologue CRB1. It is concluded that the function of Cyst and p114RhoGEF/ARHGEF18 in coupling apical polarity proteins and GPCR signaling to junctional Rho activity and actomyosin function is conserved between flies and vertebrates and likely contributes to retinal health in humans, although some of the molecular interactions may have shifted in relative importance (Silver, 2019).

The other mammalian orthologues of Cyst, p190RhoGEF, AKAP-13, and GEF-H1 have not been implicated as regulators of epithelial polarity. GEF-H1 (also known as ARHGEF2 and Lfc) was shown to be inactive at mature tight junctions. In this case, the tight junction protein Cingulin forms a complex with GEF-H1, preventing it from activating RhoA. Instead, GEF-H1 is thought to promote junction disassembly and cell proliferation, presumably through an association with the mitotic spindle. GEF-H1 was also implicated in the morphogenesis of the vertebrate neural tube, and in the regulation of RhoA activity during cytokinesis. Like GEF-H1, p190RhoGEF has been shown to associate with microtubules. GEF-H1 and AKAP-13 were also found to serve additional functions independent of their RhoGEF activity. Whether and how Cyst might consolidate the functions of its various mammalian orthologues remains to be explored (Silver, 2019 and references therein).

Crumbs complex-directed apical membrane dynamics in epithelial cell ingression

Epithelial cells often leave their tissue context and ingress to form new cell types or acquire migratory ability to move to distant sites during development and tumor progression. Cells lose their apical membrane and epithelial adherens junctions during ingression. However, how factors that organize apical-basal polarity contribute to ingression is unknown. This study shows that the dynamic regulation of the apical Crumbs polarity complex is crucial for normal neural stem cell ingression. Crumbs endocytosis and recycling allow ingression to occur in a normal timeframe. During early ingression, Crumbs and its complex partner the RhoGEF Cysts support myosin and apical constriction to ensure robust ingression dynamics. During late ingression, the E3-ubiquitin ligase Neuralized facilitates the disassembly of the Crumbs complex and the rapid endocytic removal of the apical cell domain. These findings reveal a mechanism integrating cell fate, apical polarity, endocytosis, vesicle trafficking, and actomyosin contractility to promote cell ingression, a fundamental morphogenetic process observed in animal development and cancer (Simoes, 2022).

Drosophila NBs are an outstanding model for scrutinizing the cellular machineries underpinning an EMT-like process with high temporal and spatial resolution. While ingressing, a single NB sequentially loses AJs responding to tensile forces exerted by two pools of actomyosin: a planar polarized pool enriched at anterior-posterior junctions, which disassembles first, and a pulsatile pool at the free apical cortex, which further tugs on shrinking junctions in a ratchet-like manner. However, while actomyosin forces reduce the apical perimeter, it remained unclear how cells lose their apical domain and how polarity regulators contribute to the dynamics of apical domain loss. This study demonstrates that regulation of the Crb complex plays a key role in orchestrating apical domain loss during ingression. During early ingression, cells shrink their apical domain while retaining total levels of Crb, which is crucial for maintaining normal actomyosin in NBs and NICs (neighboring non-ingressing cells) to generate the tension balance required for normal ingression dynamics. During late ingression, Crb is rapidly lost from the apical domain, a process initiated by an interaction between Neur and Sdt, which causes the disassembly of the Crb complex. The loss of the Crb complex then precipitates the concurrent loss of the apical membrane and AJs. A similar regulatory interplay between Crb, Sdt, and Neur was also observed during early neurogenesis in the Drosophila optic lobes. Here, neural stem cells emerge from a wave front in the optic lobes rather than ingress as individual cells. Despite these topological differences, the Crb/Sdt/Neur module appears to be a common cell biological regulator of EMT during early neurogenesis (Simoes, 2022).

Endocytosis and endocytic degradation and recycling are requirements for normal NB ingression dynamics. The apical membrane of neuroepithelial cells is much more active endocytically than the basolateral membrane. Notably, Crb is endocytosed during apical contractions and re-secreted during expansions, suggesting that myosin-driven cell contact contraction promotes endocytosis, consistent with recent data from mammalian cells, whereas expansions allow for enhanced secretion. Blocking endocytosis increases surface levels of Crb and Ecad as expected, and prevents NB ingression, whereas enhancing endocytosis accelerates ingression. Moreover, endocytic trafficking plays a key role in determining ingression speed. Loss of ESCRT complex-mediated degradation appears to enhance apical Crb and slow ingression, whereas loss of Retromer-mediated recycling dramatically reduced surface Crb and accelerated ingression. In fact, Retromer-compromised embryos showed the fastest ingression speed of any condition examined, suggesting that the Retromer not only recycles Crb but also other factors that counteract apical domain loss in NBs. Crb turnover during early ingression maintains a steady Crb surface abundance. During late ingression, Crb endocytosis is enhanced during both apical contraction and expansion as a result of the disruption of the Crb-Sdt interaction by Neur, which likely makes the Crb cytoplasmic tail accessible to the Clathrin adapter AP2. AP2 binds to Crb competitively with Sdt, facilitating the rapid endocytic removal of Crb and the apical membrane (Simoes, 2022).

The relationship between actomyosin contraction, endocytosis, Crb protein levels, and their function in NB ingression illuminates the complexity and robustness of morphogenesis. Disrupting several individual molecular processes, while changing the dynamics of ingression, rarely abrogates ingression entirely. First, although endocytosis is essential for ingression, strong depletion of endocytotic regulator delayed ingression but did not prevent delamination in most cases. Similarly, a dramatic depletion of myosin, which is essential for apical constriction of NBs, extended the ingression period but did not block delamination. Evidence of the mutual dependency of endocytosis and myosin-driven contractions in NBs. Contractions foster endocytosis, whereas endocytosis promotes contractions, a co-dependency that likely finetunes ingression dynamics. Second, maintaining Crb in the apical membrane (through overexpression or expression of a non-endocytosable form of Crb) delayed ingression but did not prevent it in most cases, suggesting that Crb membrane persistence is overcome by other mechanisms such as the Neur-dependent disassembly of the Crb complex. Moreover, failure to resolve the Crb complex in late ingression caused part of the apical membrane to persist as apical plugs. Nevertheless, NB delaminate, detach from apical plugs and animals are viable, suggesting that nervous system development proceeds rather normally. Crb surface abundance is dependent on endocytosis and versicle trafficking, and Crb regulates junctional myosin which contributes to apical contraction. Together, these findings highlight the multilayered regulation of ingression through the co-dependent interactions between apical polarity, vesicle trafficking, and actomyosin contractions (Simoes, 2022).

EMT is thought to be initiated by the expression of EMT transcription factors (EMT-TFs) of the Snail, Zeb, or bHLH families that downregulate key adhesion or polarity proteins such as Ecad and Crb. NBs are specified through the combined action of proneural genes that include bHLH proteins of the Achaete-Scute complex, the Snail family protein Worniu, and the SoxB family protein SoxNeuro. However, although genes that encode Ecad and Crb are transcriptionally downregulated in NBs, this repression does not appear relevant for NB ingression. Replacing endogenous Ecad with a transgene expressing Ecad under the control of a ubiquitous promoter had no impact on NB ingression dynamics. This study shows that surface levels of Crb remained high in NBs during early ingression before Crb is rapidly removed by endocytosis during late ingression. This raises the question of how the upregulation of proneural genes in presumptive NBs elicits enhanced actomyosin contractility and endocytic removal of apical membrane and junctions (Simoes, 2022).

One proneural gene target is neur. Neur is found throughout the neuroepithelium participating in Delta-Notch-mediated lateral inhibition to select the NB from an equivalence group of 5-7 cells. Neur upregulation in ingressing NBs is thought to be part of a positive feedback that stabilizes NB fate through persistent asymmetric Delta-Notch signaling. The increase in Neur may also be important for the effective disruption of the Crb complex to destabilize the apical domain. Neur can disrupt the Crb complex across the epithelium but is normally prevented from doing so by Bearded proteins that act as inhibitors of Neur. Increasing Neur concentration may overcome this inhibition in NBs. This raises the possibility that the proneural gene-dependent upregulation of neur contributes to the timing of ingression, consistent with the observation that in Neur-depleted embryos ingression is prolonged. Furthermore, Neur may enhance actomyosin contractility in NBs seen in late ingression as was reported for Neur in the Drosophila mesoderm. It is hypothesized therefore that Neur could be a central regulator of NB selection and ingression, stabilizing NB fate, driving apical membrane constriction through actomyosin contraction, and disrupting the Crb complex to remove apical membrane and junctions. Interestingly, it was also noted that during ingression, the number of alternative isoforms of Sdt is limited to Sdt3, the isoform susceptible to Neur. Hence, NBs appear to develop the molecular competence for apical membrane removal at least in part through rebalancing Sdt splice forms (Simoes, 2022).

The loss of apical-basal polarity is an early event during EMT marked by the loss of epithelial AJs that can trigger expression of EMT-TFs and the disassembly of cell junctions. However, the findings of this study indicate that the loss of apical-basal polarity in NBs is preceded by a period (~20 min; early ingression) of ratcheted apical contractions that reduce the apical area of delaminating cells. The maintenance of normal Crb levels during early ingression is crucial for normal ingression dynamics. Crb stabilizes junctional myosin through its effector, Cyst, which is recruited to the junctional domain by the Crb complex (Silver, 2019). Although the loss of Crb or loss of Cyst causes similar reductions of junctional myosin in the neuroepithelium (Silver, 2019), NB ingression was consistently faster in Cyst-compromised embryos than in controls. In contrast, NBs in Crb-compromised embryos showed much larger variability of ingression speeds, with a small fraction of NBs ingressing rapidly while the majority was slower than controls. Thus, it is likely that Crb makes other contributions to regulating NB ingression in addition to its Cyst-mediated function in supporting junctional actomyosin. Interestingly, the mouse Crb homolog Crb2 is required for myosin organization and ingression during gastrulation. The predominant defect in Crb2-compromised mice appears to be a failure of ingression, which may be similar to the fraction of NBs showing slower than normal ingression seen with the loss of Drosophila Crb. To what extent the differences in cell behavior caused by the loss of Crb and Crb2 depend on the biomechanical specifics of the tissue context or result from differences in molecular pathways in which Crb and Crb2 operate remains to be explored (Simoes, 2022).

GENE STRUCTURE

There are two RNAs produced by addition of different polyA tracts, but the source of this variation is not known (Tepass, 1990).

cDNA clone length - 7226 bases

Bases in 5' UTR -113 plus

Exons - five

Bases in 3' UTR - 505

PROTEIN STRUCTURE

Amino Acids - 2139

Structural Domains

crumbs encodes a large transmembrane protein with 30 EGF-like repeats and four laminin A G-domain-like repeats in its extracellular domain, suggesting its participation in protein-protein interactions. There is an N-terminal CAX pepeat. The cytoplasmic region consists of 28 amino acids (Tepass, 1990 and Knust, 1993).

Proteins encoded by the tumor suppressor fat gene, the neurogenic slit gene and crumbs gene of Drosophila contain domains homologous with modules identified previously in laminin A. These proteins of Drosophila have a number of features in common: they have large extracellular regions containing laminin A modules linked to epidermal growth factor-like domains, and they are all involved in cell-cell interactions that are crucial for correct morphogenesis of ectodermal tissues (development of midline neuroepithelial, organization of epithelial tissues etc.). Patthy has suggested that the laminin A-type modules of these proteins play important roles in interactions controlling ectodermal differentiation (Patthy, 1992).

date revised: 12 August 2023

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.