The genetic programs that control patterning along the gut dorsoventral (DV) axis have remained largely elusive. The activation of the Notch receptor occurs in a single row of boundary cells that separates dorsal from ventral cells in the Drosophila hindgut. rhomboid, which encodes a transmembrane protein, and knirps/knirps-related, which encode nuclear steroid receptors, are Notch target genes required for the expression of crumbs, which encodes a transmembrane protein involved in organizing apical-basal polarity. Notch receptor activation depends on the expression of its ligand Delta in ventral cells, and localizing the Notch receptor to the apical domain of the boundary cells may be required for proper signaling. The analysis of gene expression mediated by a Notch response element suggests that boundary cell-specific expression can be obtained by cooperation of Suppressor of Hairless and the transcription factor Grainyhead or a related factor. These results demonstrate that Notch signaling plays a pivotal role in determining cell fates along the DV axis of the Drosophila hindgut. The finding that Notch signaling results in the expression of an apical polarity organizer, one which, in turn, may be required for apical Notch receptor localization, suggests a simple mechanism by which the specification of a single cell row might be controlled (Fusse, 2002).
The regionalization of the hindgut tube involves the formation of three major subregions: the small intestine, which localizes to the anterior end of the hindgut; the large intestine, which represents the middle part and the rectum, the posterior part. The formation of the small intestine and the rectum has been shown to depend on Hedgehog and Wingless activities, which coordinate morphogenesis and cell differentiation in the hindgut. The steroid receptor-encoding genes knirps and knirps-related, which are expressed in banded expression domains in the small intestine and the rectum, are target genes of the Hedgehog and Wingless signaling pathways required for restricting endoreduplication cycles to the middle part of the hindgut, the large intestine (Fusse, 2002).
While studying the role of the kni and knrl genes which act redundantly during hindgut development, it was observed that both genes are also coexpressed in the large intestine, from germ band extension stage onward in two rows of lateral cells (20 ± 1) on each side of the tube. This expression is maintained until late stage 16. In the lateral cell rows, kni and knrl are coexpressed with the rhomboid (rho) gene that encodes a transmembrane protein involved in epidermal growth factor receptor (Egfr) signaling. rho gene expression in the lateral cells appears slightly earlier than kni/knrl gene expression and is also maintained until late stage 16. At the transitions to the small intestine and the large intestine, rho is expressed in two circular expression domains. The transmembrane protein and apical polarity determinant Crumbs (Crb) becomes strongly upregulated in the lateral cell rows after germ band extension stage and displays an unusual cellular distribution. This contrasts with the dorsal and ventral cells of the hindgut, where Crb is located at the apical cell margins -- Crb is localized to the entire apical domain of the lateral cell rows, and expression is maintained until the end of embryogenesis. Similarly, Discs lost (now redefined as Drosophila Patj), another apical polarity organizer, is located to the entire apical cell surface in these cells (Fusse, 2002).
Using various cell shape and cell polarity markers, such as the septate junction markers Fas III, Neurexin IV, and Discs lost, it was determined that the cells of the lateral cell rows show a flat and long-shaped morphology and that these cells separate homogenous cell populations in the dorsal and the ventral halves of the large intestine. The cells of the lateral cell rows can thus be considered boundary cells separating dorsal from ventral cells in the large intestine. The dorsal cells, which are big and columnar, express the homeodomain protein Engrailed (En) from extended germ band stage onward until late embryogenesis. In contrast, the ventral cells, which are small and cuboidal, display expression of Delta from extended germ band stage onward until late embryogenesis. Double immunostainings reveal that En expression in the dorsal half of the large intestine is adjacent and nonoverlapping to the kni/knrl/rho expression domains in the boundary cells. Similarly, the Delta expression domain in the ventral half is adjacent to the boundary cells, although coexpression at a low level in the boundary cells cannot be excluded. In summary, dorsal cells express En; boundary cells kni/knrl, rho, crb, and ventral cells express Delta (Fusse, 2002).
To investigate the role of the genes expressed in the large intestine, lack- and gain-of-function studies were performed. In amorphic Notch and Delta mutant embryos, kni/knrl, rho, and high levels of Crb expression on the apical plate are absent in the large intestine, and the boundary cell fate is not established. In contrast, ventral cell morphologies are normal in Notch or Delta mutant embryos, and En expression and dorsal cell fates are unchanged. This indicates that Notch signaling is required to establish the boundary cells but not for dorsal or ventral cell fates. To further test this, gain-of-function experiments were performed using the UAS/Gal4 system. As driver lines, the G445.2 or the 14-3-fkhGal4 strains were used -- they mediate ubiquitous gene expression in the developing hindgut from the extended germ band stage onward until late stage 16. In order to ectopically activate the Notch signaling pathway, flies carrying the Notch intracellular domain fragment, Nicd, under the control of UAS sequences were used. Expressing Nicd ubiquitously in the hindgut results in an ectopic induction of kni and of rho. In addition, the cellular localization of the Crb protein is affected in these embryos. In dorsal and ventral cells of the large intestine of wild-type embryos, Crb is localized to the apical cell margins, whereas it is localized to the entire apical plates of the boundary cells. In the embryos, in which Nicd is ectopically expressed, Crb protein is found on the apical plates of all the hindgut cells; in addition, it is found in high concentrations in vesicles, especially on the baso/lateral sides of the cells. A similar but less intensive ectopic expression of Crb can also be induced if both Kni and Rho are coexpressed in all the hindgut cells, suggesting that crb may be a downstream effector gene of Kni/Knrl and Rho activities. This is consistent with the analysis of rho7M; Df(3L) riXT1 mutants [Df(3L) riXT1 is a deficieny encompassing the kni and knrl transcription units] in which the expression of crb in the boundary cells is strongly reduced. In summary, these results suggest that rho, kni/knrl, and Crb are target genes which are activated in response to Notch signaling in the boundary cells (Fusse, 2002).
To investigate the relationship between rho and kni/knrl in the boundary cells, the expression of the genes in the respective mutants was examined. rho expression is still present in kni mutants and Df(3L) riXT1 mutants. Similarly, kni and knrl expression are maintained in amorphic rho7M mutants or EGF receptor mutants, such as faint little ball (flb). In flbIK35 mutants, the hindgut tube is much shortened due to a reduction of the cell number. However, banded expression of both genes is found in the small intestine and the rectum along the AP axis of the hindgut; expression is also found in a few cells in the large intestine region. Ectopic expression of rho using the corresponding UAS-effector line combined with a driver line that mediates ubiquitous expression in the hindgut does not result in ectopic kni/knrl gene expression and vice versa. This points toward rho and kni/knrl being regulated independently of one another (Fusse, 2002).
To study whether En, which is expressed in the adjacent dorsal cells, contributes to the boundary cell fate, the expression of kni/knrl, rho, and crb was examined in en mutants and in en; invected double mutants (enE), since en and invected are known to act redundantly. Whereas the expression of the Notch target genes remains unchanged in en mutants, it is absent in the large intestine of en; invected double mutants. Morphological studies indicate that the dorsal and the boundary cell fates are not established in these mutants, and the large intestine seems to consist entirely of the ventral cell fates. To investigate the cause for this effect, the expression of Delta was studied in these mutants and it was found to be expressed ubiquitously in the large intestine. These data indicate that a boundary between Delta expressing and nonexpressing cells is required for Notch receptor activation. Ectopic expression of En in the large intestine using the 14-3 fkh driver and UAS-En effector lines results in a repression of kni/knrl and rho gene expression. This indicates that En bears the potential to act as a negative regulator of Notch target genes. Upon ectopic activation of Notch signaling in the entire hindgut by expressing Nicd, En is repressed on the dorsal side of the large intestine, thus allowing ectopic activation of Notch target genes (Fusse, 2002).
In order to investigate whether Notch signaling in the large intestine of wild-type embryos is activated beyond the boundary cells but actively repressed dorsally and ventrally, flies that carry the chimeric Notch receptor/transcription factor fusion construct N-Gal4/VP16 were used and the range of Notch signaling was determined. Upon heat shock, this fusion protein, which is membrane bound, becomes ubiquitously expressed in the embryo. In cells in which the Notch receptor is activated by ligand binding, the intracellular Gal4-VP16 transcription factor moiety is cleaved off and is able to subsequently activate reporter gene expression in cells that carry a UAS-lacZ construct. The ß-Gal expression pattern of such embryos reflects the range of Notch signaling. When anti-ß-Gal stainings of embryos that were heat shocked and carried the N-Gal4/VP16 and UAS-lacZ constructs was performed, ß-Gal expression was observed exclusively in the lateral boundary cells of the large intestine, demonstrating that Notch signaling is restricted to the boundary cells only. To further test this, flies were used carrying a lacZ-reporter construct in which multiple Su(H) binding sites from the Enhancer of Split m8 gene were combined with binding sites for the transcription factor Grainyhead (Grh). In cells, in which Notch signaling is active and Grh is expressed, Su(H) cooperates with Grh to yield high levels of reporter gene expression, whereas reporter gene expression is repressed in cells in which Notch is inactive. Determining the activity pattern of this construct in the hindgut using anti-ß-Gal antibody stainings demonstrates that activation of the reporter gene occurs exclusively in the boundary cells of the large intestine, consistent with the N-Gal4/VP16 data (Fusse, 2002).
In the Drosophila wing imaginal disc, the Notch receptor is activated along the border between dorsal and ventral cells, leading to the specification of cells that express Wingless and organize wing growth and patterning. The range of Notch signaling is determined by the spatial and temporal expression pattern of its ligands, Delta and the transmembrane protein Serrate (Ser), and by the activity of the glycosyltransferase Fringe (Fng). Fng controls ligand selectivity of Notch and plays a major role in the Notch-dependent positioning of sharp compartment boundaries. Fng has been shown to modify the glycosylation state of the receptor in the Golgi complex, thereby lowering its sensitivity to Ser and raising its sensitivity to Delta. To investigate whether Fng or Ser are also taking part in restricting Notch signaling to the boundary cells in the hindgut, expression studies and lack- and gain-of-function analyses were performed. In situ hybridization using a Ser antisense probe or ß-Gal expression studies of a Ser-lacZ enhancer trap line show that Ser is not expressed in the large intestine of the hindgut, and Ser mutants show a normal hindgut. Furthermore, ectopically expressing Ser in all the hindgut cells has no effect on Notch target gene expression. In contrast, Fng is expressed in boundary and dorsal cells as shown by double immunostainings of Fng and En. However, in amorphic fng80 mutants, misspecification of boundary cells occurs only at a low frequency, and ectopic expression of Fng using the 14-3 fkh driver and UAS-Fng flies does not induce an ectopic activation of the kni/knrl and rho genes in the hindgut. These results indicate that Ser seems not to be required, and fng may play only a minor role in restricting Notch signaling to the boundary cells (Fusse, 2002).
These results suggest that the activation of the Notch receptor in the boundary cells of the hindgut is triggered by the binding of Delta, which is expressed at high levels in adjacent ventral cells. If Delta levels are uniform and this boundary condition is lost, as in enE mutants, Notch signaling fails to occur. To further obtain insight into how the spatial control of Notch receptor activation is mediated, the localization of the receptor was determined using antibody stainings to Notch. In ventral and dorsal cells, Notch is expressed in the apical cell margins, as can be demonstrated using coimmunostainings with Neurexin IV. However, in the boundary cells, the Notch receptor is positioned to the entire apical plate where it is colocalized with Crb or Discs-lost. To test whether the apical localization of the receptor is necessary for its signaling activity, amorphic crb mutants were studied in which the sorting of proteins to the apical domain of the cells is affected. In these mutants, a strong reduction of the number of boundary cells was found, although hindgut morphogenesis is only slightly affected. In addition, the remaining boundary cells are mislocalized, and two rows of cells are often found instead of a single row as is found in wild-type. Anti-Notch/anti-Kni double immunostainings of crb mutants demonstrate the reduction of apical Notch receptor localization in crb mutants. Furthermore, in cells in which the Notch receptor is not localized along the apical plate of the cells, the activation of Notch target genes fails to occur. These results indicate that apical localization of the receptor may be important for boundary cell fate determination (Fusse, 2002).
Thus a single row of boundary cells forms in between En-expressing dorsal cells and Delta-expressing ventral cells and is determined by Notch signaling. Unlike in wing imaginal disc development, Ser seems not involved, and the glycosyltransferase Fringe plays only a minor role for the proper positioning of the DV boundary in the large intestine. These results rather suggest two major determinants that control where Notch signaling can occur in the hindgut: (1) the localization of Delta, which is expressed in ventral cells at high levels and not expressed (or at very low levels) in the adjacent boundary cells in which Notch signaling is eventually activated; (2) the Crb-dependent transport of the Notch receptor to the apical membrane domain of the boundary cells. How the initial En and Dl expression domains are set up in the large intestine is not known. It is noted, however, that the En expression domain in the hindgut primordium is initially broader in early stage 7 embryos and only subsequently refines to the dorsal cells, whereas Dl expression is confined to ventral cells from early stage 7 onward. It is thus possible that Notch signaling in the boundary cells leads to a cell-autonomous repression of en expression, consistent with the repression of en upon Nicd overexpression. Immunhistological studies show that the proper specification of boundary cells in crb mutants correlates with the apical localization of the Notch receptor. The finding that Crb expression in the boundary cells depends on Notch signaling suggests the possibility of a feedback loop that ensures proper receptor localization required for establishing the competence of the boundary cells to receive the Delta signal. It cannot be excluded, however, that the failure of Notch signaling in crb mutants may also be caused by the mislocalization of other localized proteins. It is noteworthy that the apical side of the boundary cells faces the lumen of the hindgut. Activating Notch receptors along the entire apical plate of the boundary cells would therefore require also a secreted form of Delta. The extracellular domain of Delta has been found as a soluble product in the supernatant of Drosophila cultured cells and in embryonic extracts, and has been shown to arise by a proteolytic activity of the ADAM metalloprotease Kuzbanian. Both soluble forms of Delta and Serrate are able to act as antagonists and agonists of the Notch pathway in vivo. It is possible that such a form of Delta and/or additional apically localized factors are involved in binding and activating the Notch receptor locally in the boundary cells of the hindgut (Fusse, 2002).
These results further demonstrate that Notch signaling induces the expression of the rho and kni/knrl genes and that both components are required, in turn, for the expression of Crb. It has been suggested recently that Su(H) functions as a core of a molecular switch by which the transcription of Notch target genes is regulated. In the absence of Notch signaling, Su(H) functions as a repressor, and, in the presence of Notch signaling, Su(H) can cooperate synergistically with other transcriptional activators to induce transcription of target genes. The finding that boundary cell-specific reporter gene expression can be induced in the hindgut by using a model Notch response element [composed of binding sites for Su(H) and the widely expressed activator Grainyhead] suggests the possibility that the localized activation of the rho and kni/knrl genes could rely on the same factors and the same molecular switch mechanism that has recently been proposed for this element and for Notch-dependent atonal and single minded expression. In evolutionary terms, the gut is most likely one of the most ancient organs that evolved in multicellular organisms. Consistently, the morphological processes involved in the development of the gastrointestinal tract of animals are highly similar. It remains to be shown whether or not the evolutionarily conserved regulators of the Notch signaling cascade also determine dorsoventral aspects of gut development in other animals, including vertebrates (Fusse, 2002).
These results provide evidence that Notch signaling in the Drosophila hindgut controls the fate of a single row of boundary cells separating the dorsal and ventral halves of the gut tube. Activation of the Notch receptor in the boundary cells is mediated by its ligand Delta that is expressed in adjacent ventral cells. The induction of Notch target genes activate the expression of the apical polarity organizer Crb, which may be required, in turn, for apical Notch receptor localization. These findings identify a simple mechanism that controls the specification of a single row of DV boundary cells in an animal gut (Fusse, 2002).
Among the diverse cellular processes taking place during oogenesis, the delamination and migration of border cells (BCs), a group of anterior follicle cells, represent a powerful model to study cell invasion in a normal tissue. During stage 9 of oogenesis, BCs detach from the outer epithelium to invade the germline cyst compartment. The BC cluster contains two centrally located polar cells surrounded by approximately six outer border cells and undergoes a nearly 6-hour long posteriorward migration to reach the anterior part of the growing oocyte. Together with centripetal cells, they assemble the micropyle, a specialized structure required for sperm entry. domeless was isolated in a screen to identify genes essential in epithelial morphogenesis during oogenesis. The level of dome activity is critical for proper border cell migration and is controlled in part through a negative feedback loop. In addition to its essential role in border cells, dome is required in the germarium for the polarization of follicle cells during encapsulation of germline cells. In this process, dome controls the expression of the apical determinant Crumbs. In contrast to the ligand Upd, whose expression is limited to a pair of polar cells at both ends of the egg chamber, dome is expressed in all germline and follicle cells. However, Dome protein is specifically localized at apicolateral membranes and undergoes ligand-dependent internalization in the follicle cells. dome mutations interact genetically with JAK/STAT pathway genes in border cell migration and abolish the nuclear translocation of Stat92E in vivo. dome functions downstream of upd and both the extracellular and intracellular domains of Dome are required for JAK/STAT signaling. Altogether, the data indicate that Dome is an essential receptor molecule for Upd and JAK/STAT signaling during oogenesis (Ghiglione, 2002).
The dramatic, early follicle cell phenotype contrasts with the essentially normal phenotype of dome mutant cells observed in later stage egg chambers. In this case, follicle cells are viable and divide normally. A similar, dual phenotype has been reported in crumbs mutant chambers. After initial polarization of the follicle cells in the germarium, Crumbs is no longer required and its loss has no visible effects. Importantly, dome controls Crumbs expression in follicle cells, thus providing a novel link between the JAK/STAT signaling pathway and epithelial polarity (Ghiglione, 2002).
In addition to its early function in the germarium, dome is required for the normal expression of several follicle cell markers, including DE-cadherin and Fas3. It is important to note that despite a clear defect in the expression of these markers, dome mosaic egg chambers are morphologically normal. However, because completely mutant egg chambers cannot be obtained because of the early effect of dome in the germarium, one cannot rule out the possibility that large mutant clones would lead to abnormal development of egg chambers (Ghiglione, 2002).
The pattern of epithelial markers in dome mutant cells indicates that the JAK/STAT pathway is active in all follicle cells, a notion that is reinforced by the wide expression of nuclear Stat92E. How is Dome activated during egg chamber development and does this activation follow the same profile at all stages? Given the restricted pattern of upd expression in the egg chamber and its dramatic effect upon overexpression, it is unlikely that Upd is able to signal long distances in the follicular epithelium of late stage egg chambers. Rather, a model by which the JAK/STAT pathway plays a pre-patterning function is favored, acting early during egg chamber development to activate DE-cadherin and Crumbs expression. This view is consistent both with the expression pattern of upd and the distribution of Dome-containing vesicles described in this study. The formation of endogenous vesicles can be promoted by Upd, and a gradient of such vesicles is present around polar cells. Strikingly, these vesicles, which likely indicate active signaling through Dome, are widespread at early stages and become more restricted later on. It is proposed that during early development, the Upd signal produced by anterior and posterior polar cells contributes to the differentiation of all follicle cells. At this stage, Upd would be more diffusible than later, as suggested by the pattern of Dome intracellular vesicles. The study of the mechanisms controlling Dome activation and Upd activity will require additional tools to directly detect Upd, as, for example, Upd-GFP fusion proteins (Ghiglione, 2002).
This study has revealed several new findings about the function of dome and the JAK/STAT pathway during oogenesis. Future work will help to understand how Upd and Dome initially interact at the cell surface and transduce the signal to downstream JAK/STAT pathway members (Ghiglione, 2002).
Formation of tubes of the correct size and shape is essential for viability of most organisms, yet little is understood of the mechanisms controlling tube morphology. A new allele of hairy has been identified in a mutagenesis screen. hairy mutations cause branching and bulging of the normally unbranched salivary tube, in part through prolonged expression of huckebein (hkb). Hkb controls polarized cell shape change and apical membrane growth during salivary cell invagination via two downstream target genes, crumbs (crb), a determinant of the apical membrane, and klarsicht (klar), which mediates microtubule-dependent organelle transport. In invaginating salivary cells, crb and klar mediate growth and delivery of apical membrane, respectively, thus regulating the size and shape of the salivary tube (Myat, 2002).
Hox genes control animal body plans by directing the morphogenesis of segment-specific structures. As transcription factors, HOX proteins achieve this through the activation of downstream target genes. Much research has been devoted to the search for these targets and the characterization of their roles in organogenesis. This has shown that the direct targets of Hox activation are often transcription factors or signaling molecules, which form hierarchical genetic networks directing the morphogenesis of particular organs. Importantly, very few of the direct Hox targets known are 'realizator' genes involved directly in the cellular processes of organogenesis. This study describes a complete network linking the Hox gene Abdominal-B to the realizator genes it controls during the organogenesis of the external respiratory organ of the larva. In this process, Abdominal-B induces the expression of four intermediate signaling molecules and transcription factors, and this expression results in the mosaic activation of several realizator genes. The ABD-B spiracle realizators include at least five cell-adhesion proteins, cell-polarity proteins, and GAP and GEF cytoskeleton regulators. Simultaneous ectopic expression of the Abd-B downstream targets can induce spiracle-like structure formation in the absence of ABD-B protein. It is concluded that Hox realizators include cytoskeletal regulators and molecules required for the apico-basal cell organization. HOX-coordinated activation of these realizators in mosaic patterns confers to the organ primordium its assembling properties. It is proposed that during animal development, Hox-controlled genetic cascades coordinate the local cell-specific behaviors that result in organogenesis of segment-specific structures (Lovegrove, 2006).
To initiate spiracle organogenesis, ABD-B, in combination with local signaling molecules, activates a set of targets within the dorsal area of A8. This study shows that there may be as few as four direct targets for the posterior spiracle. The expression of the primary targets, with their corresponding cofactors, subdivides the organ into specific regions. After this patterning stage, specific cell behaviors are controlled by another set of transcription factors that include the GATA transcription factor Grn to bring about cell rearrangements, and the JAK/STAT signaling pathway, which induces posterior spiracle-cell elongation. The partially overlapping expression of these transcription factors has the potential to activate in particular subsets of spiracle cells different sets of realizator genes. In the spiracles, these realizators include cell-adhesion molecules, apico-basal polarity proteins, and cytoskeletal regulators. Thus, in this way, ABD-B activates a genetic cascade coordinating the local cell-specific behaviors that result in organogenesis (Lovegrove, 2006).
Two main issues may explain why identification of the realizator genes has been so difficult. Primarily, by nature, many of these molecules are required for general functions in all cells. A screen for Hox realizators based on finding segment-specific defects would miss molecules like E-Cad or the Rho GTPases because of generalized embryonic malformations. Thus, their realizator nature can only be uncovered when, through intermediate regulators, a link to the HOX protein is found. This is demonstrated in the case of crb where a specific spiracle enhancer was found, that directs its increased transcription. In the case of the cytoskeleton, the link is made through the use of specific regulatory GEF and GAP proteins that modulate the activity of the GTPases. A second problem has been that some of the realizator molecules function redundantly and therefore a mutational approach yields no result. This is the case with the nonclassic cad88C and cad96C, which only show a mutant phenotype if E-cad is also mutated. Although cell-adhesion molecules had been originally proposed to be realizators, it is surprising to find that there are four nonclassical cadherins with restricted expression in the spiracle (Lovegrove, 2006).
Another unexpected finding has been the observation that the expression of apical- and basolateral-membrane proteins is modulated in the spiracle during the elongation stages. This study has established a link between ABD-B and the apical determinant crb through the JAK/STAT pathway. During invagination, spiracle cells are going through major membrane reorganization, including apical constriction and basal elongation. Thus, Crb, which is required in many epithelia for maintenance of a proper zonula adherens, may be playing an important role for the polarized remodeling along the apico-basal cell axis. Crb upregulation is functionally important for cell elongation, but it is not the only function controlled by STAT. In this respect, it is important to note that spiracle-cell elongation occurs mainly through the increase of basolateral membranes. It is thus likely that the spiracle-gene network will also be controlling basal polarity determinants (Lovegrove, 2006).
A role for ABD-B in regulation of the cytoskeleton in the posterior spiracles was expected because of the initial observations on cell elongation taking place in the spiracular chamber. The observed effects of the dominant-negative and constitutively active forms of Rho GTPases on spiracle development support this hypothesis. The finding of Gef64C regulation by ABD-B in the spiracle cascade and the finding of spiracle invagination defects in RhoGAP cv-c mutants confirms that specific control of the Rho GTPases is an important feature of spiracle development (Lovegrove, 2006).
Although all the realizators analyzed in this study are activated indirectly by ABD-B, the possibility cannot be excluded that ABD-B can also activate some others directly. Direct regulation of realizator genes by HOX may be important for differentiation of specific cell types (Lovegrove, 2006).
This study has linked the activity of a HOX protein, through the regulation of a small number of intermediate regulators, to a battery of realizator genes. The local-specific modulation of these genes that in other contexts control cell adhesion, polarity, and organization of the cytoskeleton, would be sufficient to confer unique morphogenetic properties to the cells leading to the formation of a segment specific organ. Other examples in Drosophila include salivary-gland organogenesis, where SCR initially activates a cascade of downstream genes, and head formation where DFD activates Dll but similar processes must be occurring in Hox-controlled organogenesis in vertebrates (Lovegrove, 2006).
The Hippo tumor-suppressor pathway controls tissue growth in Drosophila and mammals by regulating cell proliferation and apoptosis. The Hippo pathway includes the Fat cadherin, a transmembrane protein, which acts upstream of several other components that form a kinase cascade that culminates in the regulation of gene expression through the transcriptional coactivator Yorkie (Yki). Work in Drosophila has indicated that Merlin (Mer) and Expanded (Ex) are members of the Hippo pathway and act upstream of the Hippo kinase. In contrast to this model, it was suggested that Mer and Ex primarily regulate membrane dynamics and receptor trafficking, thereby affecting Hippo pathway activity only indirectly. This study examined the effects of Mer, Ex and the Hippo pathway on the size of the apical membrane and on apical-basal polarity complexes. It was found that mer;ex double mutant imaginal disc cells have significantly increased levels of apical membrane determinants, such as Crb, aPKC and Patj. These phenotypes were shared with mutations in other Hippo pathway components and required Yki, indicating that Mer and Ex signal through the Hippo pathway. Interestingly, however, whereas Crb was required for the accumulation of other apical proteins and for the expansion of the apical domain observed in Hippo pathway mutants, its elimination did not significantly reverse the overgrowth phenotype of warts mutant cells. Therefore, Hippo signaling regulates cell polarity complexes in addition to and independently of its growth control function in imaginal disc cells (Hamaratoglu, 2009).
The results show that the Hippo pathway regulates the amount of apical protein complexes and thereby the size of the apical domain and that this effect is independent of its growth control function. Importantly, the regulation of apical complexes is a specific effect of the Hippo pathway, since other growth control pathways do not regulate apical complexes. In addition, this effect of the Hippo pathway is a general effect, since upregulation of apical complexes was observed in multiple tissues and cell types. Although overexpression of Crb and aPKC are sufficient to drive extra growth, the results show that the upregulation of apical complexes is not required for the overgrowth phenotype and for the induction of Hippo target genes in wts mutant cells. It is thus concluded that the Hippo pathway regulates the amount of apical complexes in Drosophila imaginal disc cells in addition to and independently of its growth control function (Hamaratoglu, 2009).
It has been suggested that Mer and Ex regulate the levels of membrane receptors independently of the Hippo pathway. However, the current results show that the upregulation of DER, Ft and apical complexes was similar in hpo and wts mutant cells and mer;ex double mutant cells, and that this effect requires Yki. These results thus indicate that Mer and Ex act through the Hippo pathway to exert their effect and that they are bona fide members of the Hippo pathway. Similar conclusions have been drawn based on observations that overexpression of wts suppresses the lethality and overgrowth phenotypes of ex mutants (Hamaratoglu, 2009 and references therein).
How does the Hippo pathway regulate the size of the apical domain and the amount of the apical complexes? The observation that Yki is required and sufficient for the effect on the apical domain indicates that this effect of the Hippo pathway is mediated by transcriptional regulation. However, although the upregulation of Crb is necessary and sufficient for the expansion of the apical domain and for the accumulation of the other apical polarity complex proteins, it is not required for the upregulation of DER and Ft, which still accumulate in wts,crb double mutant cells. Thus a model is favored in which the Hippo pathway regulates the turnover of several apical membrane components, for example through regulation of endocytosis. Notably mer;ex mutant cells in wing imaginal discs have defects in Notch (N) endocytosis, which leads to accumulation of N. Moreover, the endosomal protein Hrs accumulates in hpo mutant follicle cells in Drosophila ovaries, and this study observed a similar accumulation of Hrs in wts mutant clones in imaginal discs. These observations thus support the hypothesis that Hippo signaling regulates the amount of endocytosis and membrane turnover, thereby affecting the amount of apical membrane proteins. The target of Yki that mediates these effects, however, is currently not known (Hamaratoglu, 2009).
Several other studies also demonstrated roles for the Hippo pathway beyond its function in growth control. For example, the Hippo pathway is required for the proper selection of photoreceptor subtypes in the Drosophila eye, and it is required in follicle cells to generate a signal that polarizes the underlying oocyte. For both of these functions, Hippo signals through Yki, but Yki may regulate different sets of target genes, since the phenotypic effects are different. In addition, the Hippo pathway regulates cellular behavior through pathways that may not require Yki and thus may not involve the regulation of gene expression. For example, Yki-independent functions of the Hippo pathway may regulate dendritic tiling of larval neurons and the death of salivary gland cells during metamorphosis. The finding that Hippo regulates apical polarity complexes in addition to and independently of its growth control function in imaginal discs cells thus further reveals the complex function of this pathway in the regulation of cellular behavior (Hamaratoglu, 2009).
The Hippo signaling pathway restricts organ size by inactivating the Yorkie (Yki)/Yes-associated protein (YAP) family proteins. The oncogenic Yki/YAP transcriptional coactivator family promotes tissue growth by activating target gene transcription, but the regulation of Yki/YAP activation remains elusive. In mammalian cells, Brg1, a major subunit of chromatin-remodeling SWI/SNF family proteins, was identified that interacts with YAP. This finding led the authors to investigate the in vivo functional interaction of Yki and Brahma (Brm), the Drosophila homolog of Brg1. Brm was found to function at the downstream of Hippo pathway and interacts with Yki and Scalloped (Sd) to promotes Yki-dependent transcription and tissue growth. Furthermore, it was demonstrated that Brm is required for the Crumbs (Crb) dysregulation-induced Yki activation. Interestingly, it was also found that crb is a downstream target of Yki-Brm complex. Brm physically binds to the promoter of crb and regulates its transcription through Yki. Together, this study has shown that Brm functions as a critical regulator of Hippo signaling during tissue growth and plays an important role in the feedback loop between Crb and Yki (Zhu, 2014).
The core signaling cascade of Hippo pathway has been extensively studied. However, the regulatory mechanism of Yki/YAP activation remains largely elusive. This study found that Brm, a component of SWI/SNF complex, interacts with Yki and regulates organ growth in Drosophila. The findings indicated that Brm is indispensable for Yki activation to drive the expression of target genes. Interestingly, it was also found that the expression of crb is regulated by Yki-Brm complex. The ChIP assay showed that Brm and Yki physically bound to the promoter region of crb, and knockdown of Yki reduced the binding of Brm to crb promoter. Taken together, this study presents a novel feedback loop between Crb and the Yki-Brm complex. Thus, the mutual regulation between apical polarity and Hippo pathway could be critical for tissue growth and homeostasis (Zhu, 2014).
Considering the transcriptional co-dependence with RNA Polymerase II and broad localization in actively transcribed regions, the Brm complex may be present on multiple promoters and is required by global gene transcription. However, the expression of only 872 genes has been observed significantly altered by Brm knockdown. This suggests that there is a selectivity of Brm-mediated transcriptional regulation. In addition to Hippo pathway, morphogens, such as Wingless (Wg), Hedgehog (Hh) and Decapentaplegic (Dpp), also play fundamental roles in organ patterning and growth. Therefore, the readouts of Wg, Hh and Dpp pathway in wing discs with brm knockdown were examined. Interestingly, only Wg secretion was found to be altered, which argued that there was gene specificity of Brm-mediated transcription. Many other factors including the interaction of transcription factors and co-factors, histone modifications and DNA methylation have been also shown essential in this process. Therefore, how Brm regulates the specific targets transcription needs to be further investigated (Zhu, 2014).
These data demonstrate that the enhancement of Crb levels caused by yki overexpression can be suppressed by brm knockdown. However, the overexpression of Brm alone cannot alter the Crb levels. These results suggest that the regulatory function of Brm complex is indispensable for crb but relies on Yki. Together with the fact that Brm physically associate with Yki, it is argued that Brm complex is recruited to target gene promoters through Brm-Yki interaction. Accordingly, another Drosophila transcription factor Zeste has been reported that recruits the Brm complex to chromatin to initiate transcription. Beside of Brm, Moira (Mor), another subunit of the complex, has also been reported to directly interact with Yki and occupy on the promoters of Hpo target genes, which support the working model that Yki facilitates the recruitment of Brm complex onto crb promoters. Taken together, this study draws a conclusion that the specificity of Brm for regulating Hippo targets is determined by Yki recruitment. Given that both the Brm complex and Hippo pathway are conserved in mammals, these results shed a light on a conserved interaction, which might be critical for mammalian growth and tissue homeostasis (Zhu, 2014).
Shark is a single protein that possesses a tyrosine kinases, ankyrin repeat, and Src homology 2 domains. During Drosophila embryogenesis, shark is expressed exclusively by ectodermally derived epithelia and is localized preferentially to the apical surface of these cells. This apical localization persists, even as tissues undergo complex invaginations, moving from the external surface of embryos to form internal structures, but expression is lost when cells lose their polarity. This pattern closely mimics the expression of Crumbs. Shark's structure and localization pattern suggest that it functions in a signaling pathway for epithelial cell polarity, possibly transducing the Crumbs signal (Ferrante, 1995).
Cell interactions mediated by Notch family receptors have been implicated in the specification of tissue boundaries. Tightly localized activation of Notch is crucial for the formation of sharp boundaries. In the Drosophila wing imaginal disc, the Notch receptor is expressed in all cells. However, Notch activity is limited to a narrow stripe of cells along the dorsal-ventral compartment boundary, where it induces the expression of target genes. How a widely expressed protein becomes tightly regulated at the dorsal-ventral boundary in the Drosophila wing is not completely understood. This study shows that the transmembrane protein Crumbs is involved in a feedback mechanism used by Notch to refine its own activation domain at the Drosophila wing margin. Crumbs reduces the activity of the gamma-Secretase complex, which mediates the proteolytic intracellular processing of Notch. These results indicate a novel molecular mechanism of the regulation of Notch signal, and also that defects in Crumbs might be involved in similar abnormal gamma-Secretase complex activity observed in Alzheimer's disease (Herranz, 2006; full text of article).
Crumbs associates with the Stardust and DPATJ proteins through its short cytoplasmic tail to establish apical-basal cell polarity in the embryo. Expression of this cytoplasmic tail in a mutant background for crb is sufficient to partially rescue the failure in apical-basal cell polarity, indicating that the large extracellular domain of Crb is dispensable for this process. Three different observations indicate that the extracellular domain of Crb is required to attenuate Notch signalling and that the intracellular domain is dispensable. Mutant clones for a null allele of stardust (sdtXP96) are able to cover large areas of the wing without any overt phenotype when abutting the DV boundary or when running along the longitudinal veins. The crb mutant wing phenotype can be rescued when simultaneously expressing either full-length Crb or a truncated form of Crb lacking the whole intracellular tail (Crb-Extra-TM). Overexpression of Crb-Extra-TM leads to a mild downregulation of the Notch signalling pathway. In the adult wing, veins are thicker, resembling a Notch loss-of-function phenotype. In the wing imaginal disc, Crb-Extra-TM overexpression reduces the expression levels of Cut at the DV boundary, a target of Notch that requires high levels of Notch activity. Wg expression is not affected (Herranz, 2006).
Signalling centres along compartment boundaries are required to organize the growth and pattern of the surrounding tissue. However, too much of a signal has deleterious effects. The Notch signalling center organizes the growth and pattern of the developing wing primordium, partially through the secreted protein Wingless. Wingless activity contributes to limit Notch activity to cells immediately adjacent to the DV boundary. This study presents evidence that Notch also contributes to the refinement of its activation domain through its target gene crumbs. Crumbs attenuates Notch signalling by repressing the activity of the gamma-Secretase complex. Many loss-of-function mutations in the human homologue of Crumbs, CRB1, cause recessive retinal dystrophies, including retinitis pigmentosa. Given the fact that the gamma-Secretase complex also mediates the intracellular cleavage of the transmembrane protein APP, leading to accumulation of the Aβ peptide in plaques in AD, it is postulated that Crumbs may also be involved in modulating AD pathogenesis. The analysis indicates a role for the extracellular part of the Crb protein in this process. It is interesting to note that many mutations that give rise to retinal dystrophies are missense mutations that affect different EGF or LG domains of CRB1. Thus, molecular interactions mediated by the extracellular domain of Crb may be crucial in both types of disease (Herranz, 2006).
The polarized architecture of epithelial tissues involves a dynamic balance between apical and basolateral membrane domains. This study shows that epithelial polarity in the Drosophila embryo requires the exocyst complex subunit homolog Exo84. Exo84 activity is essential for the apical localization of the Crumbs transmembrane protein, a key determinant of epithelial apical identity. Adherens junction proteins become mislocalized at the cell surface in Exo84 mutants in a pattern characteristic of defects in apical, but not basolateral, components. Loss of Crumbs from the cell surface precedes the disruption of Bazooka and Armadillo localization in Exo84 mutants. Moreover, Exo84 mutants display defects in apical cuticle secretion that are similar to crumbs mutants and are suppressed by a reduction in the basolateral proteins Dlg and Lgl. In Exo84 mutants at advanced stages of epithelial degeneration, apical and adherens junction proteins accumulate in an expanded recycling endosome compartment. These results suggest that epithelial polarity in the Drosophila embryo is actively maintained by exocyst-dependent apical localization of the Crumbs transmembrane protein (Blankenship, 2007).
Epithelial cells in the Drosophila embryo generate molecularly distinct apical and basolateral surfaces that provide structural integrity to the developing embryo. Specialized cell surface domains are separated by intercellular adherens junctions that initiate as diffuse apicolateral accumulations and subsequently coalesce to form a discrete apical band called the zonula adherens. The spatial organization of mature adherens junctions is actively maintained by input from both apical and basolateral proteins. The Crumbs EGF-repeat transmembrane protein and its cytoplasmic binding partners Stardust and PATJ localize to the apical cell surface and are required for epithelial structure and adherens junction morphology. In addition, overexpression of Crumbs leads to a selective expansion of the apical cell surface, demonstrating that Crumbs is necessary and sufficient for apical identity. The localization of mature adherens junctions also requires the basolateral PDZ-domain proteins Discs large (Dlg) and Scribble (Scrib) and the WD40-domain protein Lethal giant larvae (Lgl) Epithelial defects caused by disruption of apical Crumbs activity can be rescued by a simultaneous reduction in the activity of basolateral proteins, indicating that apical and basolateral domains function in opposition to maintain epithelial polarity (Blankenship, 2007 and references therein).
Misregulation of Crumbs activity can have severe effects on cell and tissue function and is associated with human retinal diseases. Multiple mechanisms contribute to Crumbs localization, stability and activity to precisely control its function. The basolateral proteins Dlg, Lgl and Scrib oppose Crumbs activity and restrict its localization in the Drosophila embryo, and the Yurt FERM-domain protein associates with the Crumbs cytoplasmic domain and negatively regulates Crumbs activity at the apicolateral cell surface. Endocytosis of Crumbs protein is also required for tissue morphology; mutations in the Avalanche syntaxin or the Rab5 GTPase lead to Crumbs accumulation and wing imaginal disc overgrowth. In addition, a complex containing the Rich1 Cdc42 GAP protein and the angiomotin scaffolding protein associates with cytoplasmic binding partners of Crumbs and provides a potential link between the Crumbs complex and the endocytic machinery. However, the mechanisms that govern the delivery of Crumbs protein to the cell surface are not known (Blankenship, 2007).
The targeting of transmembrane proteins to specific destinations at the cell surface is a widely used mechanism for establishing cell polarity. The spatial specificity of vesicle trafficking is thought to occur at a late step in this process through the tethering of exocytic vesicles at defined membrane sites by the eight-subunit exocyst (or Sec6/8) complex. Exocyst components were originally identified based on their role in polarized secretion in Saccharomyces cerevisiae and were subsequently shown to form a complex that is highly conserved from yeast to mammals. In multicellular organisms, exocyst components are required for multiple developmental processes including epithelial polarity, membrane integrity, photoreceptor morphogenesis, cell fate determination and synapse formation. These diverse functions demonstrate that polarized exocytosis is a fundamental mechanism for regulating cell morphology (Blankenship, 2007).
This study provided evidence that the Drosophila homolog of the Exo84 exocyst complex subunit is essential for epithelial polarity and apical protein localization in the Drosophila embryo. In Exo84 mutants, adherens junction proteins become mislocalized along the apical-basal axis in a manner reminiscent of cells lacking the Crumbs apical determinant. Loss of Crumbs from the apical surface is the earliest defect detected in Exo84 mutants. Exo84 mutants at advanced stages of epithelial degeneration display defects in trafficking apical and junctional proteins from the recycling endosome to the cell surface. These results demonstrate that the Drosophila homolog of the exocyst complex subunit Exo84 plays an essential role in epithelial polarity by regulating the localization of the Crumbs apical determinant (Blankenship, 2007).
It is concluded that epithelial polarity in the Drosophila embryo is actively maintained by the Exo84-dependent localization of the Crumbs transmembrane protein to the apical surface. Exo84 mutants display an aberrant distribution of junctional proteins that resembles the phenotype of crumbs mutants, and depletion of Crumbs from the apical surface is the earliest defect detected in Exo84 mutants. In addition, the onset of epithelial disruption at stage 9 in Exo84 mutants is comparable with the timing of the crumbs mutant defects, and the Crumbs protein still aggregates in Exo84 embryos with greatly reduced E-cadherin. Exo84 is likely to function as part of the exocyst complex in the Drosophila embryo, in light of the genetic interactions observed between Exo84 and the Sec5 and Sec6 exocyst subunits and the common defects in recycling endosome morphology caused by exocyst disruption in multiple cellular contexts. These results suggest a role for exocyst-dependent membrane trafficking in the maintenance of apical epithelial identity in the Drosophila embryo (Blankenship, 2007).
In contrast to the relatively specific mislocalization of Crumbs in stage 9 Exo84 mutant embryos, by late stage 10 these embryos display defects in the delivery of multiple proteins to the cell surface. Epithelial polarity and the distribution of apical and junctional proteins are established correctly in Exo84 mutants, either because these processes occur independently of Exo84 or because of residual Exo84 activity in this mutant background. The earliest defect observed in Exo84 mutants is a loss of Crumbs from the apical surface during epithelial maturation. As a likely consequence of the loss of cell-surface Crumbs localization, adherens junction proteins become mislocalized to varying positions along the basolateral cell membrane. Mutant embryos at later stages display a cytoplasmic accumulation of apical and adherens junction proteins in an expanded Rab11 recycling endosome compartment, consistent with a defect in vesicular transport to the cell surface. The failure to deliver junctional proteins to the cell surface is unlikely to result from a defect in Crumbs localization, because the cytoplasmic accumulation of junctional proteins does not occur in crumbs mutants. These results indicate that disruption of exocyst-dependent membrane trafficking ultimately results in the failure to deliver both apical and junctional proteins from the recycling endosome to the cell surface. The mislocalization of apical and junctional proteins in Exo84 mutant embryos is associated with a loss of columnar morphology, demonstrating that Exo84 activity is essential for epithelial organization (Blankenship, 2007).
A precise balance between apical and basolateral determination is essential for epithelial integrity and the placement of the zonula adherens in the Drosophila embryo. This balance is actively maintained by Exo84-dependent localization of the Crumbs transmembrane protein to the apical cell surface. Loss of apical or basolateral identity leads to distinct patterns of junctional protein distribution, suggesting that the apical and basal limits of the zonula adherens are defined by different mechanisms. In crumbs mutants, DE-cadherin and Armadillo are restricted to focused puncta at varying locations at the cell surface. By contrast, in embryos defective for the basolateral proteins Dlg and Lgl, junctional proteins are dispersed along the plasma membrane rather than aggregating at a single site. A basolateral expansion of the apical Crumbs domain has also been reported in dlg and lgl mutants. These results suggest that basolateral proteins create a nonpermissive barrier to adherens junction expansion, whereas apical proteins may play a positive role in recruiting or stabilizing junctions at the apical cell surface. Consistent with this possibility, the apical Crumbs domain is closely apposed to the zonula adherens, and it was found that Bazooka and Armadillo colocalize at the cell surface and in the cytoplasm of Exo84 mutant embryos. Exocyst-dependent trafficking of Crumbs to the apical surface may reinforce the apical epithelial domain and stabilize the apicolateral localization of the zonula adherens (Blankenship, 2007).
The recycling endosome is the primary vesicular compartment affected in embryos mutant for the exocyst subunit homolog Exo84, while Golgi, early endosomal and late endosomal compartments remain largely intact. Exocyst proteins are required for recycling endosome morphology in several epithelial and sensory cell types and the Rab11 recycling endosome protein can associate directly with the exocyst subunits Sec5 and Sec15. It was found that Rab11 vesicles in maturing embryonic epithelia are enriched in the apical cytoplasm, where they preferentially accumulate in the plane of the adherens junctions. Conversely, a basal expansion of recycling endosomes during cellularization correlates with a basal bias in membrane addition. These results suggest that there is a spatial correlation between the sites of recycling endosome accumulation and the surface destinations of proteins trafficked through recycling endosomes. The redistribution of the recycling endosome compartment to the apical cytoplasm accompanies the transition from basolateral to apical membrane insertion and may reflect the onset of a critical requirement for Crumbs activity during epithelial maturation (Blankenship, 2007).
The requirement for Exo84 in apical protein localization in the Drosophila embryo is distinct from exocyst functions in other epithelia, in which exocyst components are required for the localization of basolateral or junctional proteins. The results indicate that Exo84 is also required for delivery of DE-cadherin to the cell surface in the embryo, consistent with the demonstrated roles for Sec5, Sec6 and Sec15 in DE-cadherin trafficking in the pupal epithelium. However, although the mislocalization of the apical Crumbs protein is a primary defect of Exo84 mutant embryos, exocyst mutations do not appreciably affect Crumbs localization in pupal epithelial and photoreceptor cells. These results are consistent with a model in which distinct cargo proteins are trafficked by the exocyst complex in different cellular contexts. Alternatively, DE-cadherin and Crumbs may be delivered to the cell surface in an exocyst-dependent fashion in multiple cell types, but undergo different rates of turnover. For example, Crumbs may be dynamically trafficked in the embryo but stably maintained at the surface of pupal epithelial cells. Differential effects on specific target proteins are not atypical of exocyst function, because loss of Sec6 activity in Drosophila photoreceptor cells disrupts the localization of the rhabdomere proteins Chaoptin and Rhodopsin1, whereas the apical localization of Crumbs and DE-cadherin occurs normally. The Drosophila embryonic epithelium undergoes pronounced changes in structure and organization during development that rely on a balance between apical and basolateral surface domains. A requirement for exocyst-dependent Crumbs trafficking during this process may facilitate the dynamic remodeling of epithelial polarity during morphogenesis (Blankenship, 2007).
Three protein complexes control polarization of epithelial cells: the apicolateral Crumbs and Par-3 complexes and the basolateral Lethal giant larvae complex. Polarization results in the specific localization of proteins and lipids to different membrane domains. The receptors of the Notch, Hedgehog, and WNT pathways are among the proteins that are polarized, with subcellular receptor localization representing an important aspect of signaling regulation. For example, in the WNT pathway, differential DFz2 receptor localization results in activation of either the canonical or the planar polarity pathway. Despite the large body of research on the vertebrate JAK/STAT pathway, there are no reports indicating polarized signaling. By using the conserved Drosophila JAK/STAT pathway as a system, it was found that the receptor and its associated kinase are located in the apical membrane of epithelial cells. Unexpectedly, the transcription factor STAT is enriched in the apicolateral membrane domain of ectoderm epithelial cells in a Par-3-dependent manner. These results indicate that preassembly of STAT and the receptor/JAK complex to specific membrane domains is a key aspect for signaling efficiency. These results also suggest that receptor polarization in the ectoderm cell membrane restricts the cell's response to ligands provided by neighboring cells (Sotillos, 2008).
Besides setting up epithelial polarity, apicobasal complexes also modulate the subcellular compartmentalization or localized activation of various signaling molecules. The JAK/STAT signaling pathway is involved in processes ranging from immune response to organogenesis. In the vertebrate-signaling model, inactive STAT is shuttling from the cytoplasm to the nucleus. Ligand binding to the dimerized receptor results in the activation of JAK bound to the receptor. JAK phosphorylates itself and the receptor, creating docking sites for STAT. Inactive cytoplasmic STAT now binds to the phosphoreceptor/JAK complex, where it is phosphorylated by the kinase. Phosphorylated STAT is imported to the nucleus, where it activates the transcription of target genes. In contrast to vertebrates, in which the JAK/STAT core-signaling elements are highly redundant, the Drosophila pathway is composed of only three ligands, Unpaired (Upd), Unpaired2, and Unpaired3; one receptor, Domeless (Dome); one JAK, Hopscotch (Hop); and one transcription factor, STAT92E. Therefore, Drosophila was used as a model to investigate the polarization of the pathway (Sotillos, 2008).
dome, hop, and stat92E mRNAs are maternally provided and ubiquitously transcribed in the embryo. To analyze their protein subcellular localization, specific antibodies were used or functional tagged proteins were expressed by using UAS-dome, UAS-hop-Myc, and UAS-STAT92E-GFP. These constructs were expressed by using either mesodermal or ectodermal Gal4 drivers, and the subcellular localization of the proteins was analyzed, paying special attention to three organs where the endogenous ligand is expressed and the pathway is active: the posterior spiracles (ectodermal origin), the pharyngeal musculature (mesodermal), and the hindgut (an ectodermal tube surrounded by mesoderm) (Sotillos, 2008).
In the pharynx, as expected for a receptor, Dome localizes to the membrane, and does so in a dotted pattern that could correspond to endocytic vesicles. Hop-myc localizes to the cytoplasm, obscuring any membrane localization. This is due to the high levels of Hop-myc expressed, saturating the receptor binding sites and accumulating in the cytoplasm, as simultaneous coexpression of Hop-myc with the receptor relocates Hop to the membrane. This depends on the cytoplasmic domain of Dome, as it also occurs with a construct missing the extracellular domain but not with constructs missing the intracellular domain. STAT is detected in the cytoplasm and is more concentrated in the nuclei, as expected from the activation of the pathway in the pharynx. All of these observations agree with current knowledge of JAK/STAT activation based on vertebrate studies (Sotillos, 2008).
In contrast to the mesoderm, analysis of ectoderm cells shows a different picture. Both in the hindgut and the posterior spiracles, the Dome receptor localizes on the apical membrane. Hop is again cytoplasmic, but after coexpression with Dome both proteins localize to the apical membrane. Surprisingly, by using a specific antibody it was observed that STAT concentrates on the apical membrane of all embryonic ectodermal cells irrespective of the level of activation of the pathway. And, in cells in which the pathway is active, STAT also localizes to the nucleus. The signal detected by the antibody is specific; the same result by using a STAT-GFP fusion protein. STAT membrane localization is more prominent in cells in which the pathway is inactive; for instance, in the trunk epidermis or the spiracle after stage 15. This suggests that STAT translocates from the subapical membrane to the nucleus after pathway activation, returning to the membrane after inactivation (Sotillos, 2008).
To determine if STAT-GFP membrane localization is due to any other of the pathway's components, STAT-GFP localization was analyzed in upd, dome, or hop null mutants. STAT does not disappear from the membrane in a deficiency that removes all three Upd ligands. STAT membrane localization is not affected in null mutants for either dome or hop, demonstrating that apical STAT localization is independent of the pathway (Sotillos, 2008).
STAT localizes to the membrane domain in which the apical complexes are located. This, and the fact that STAT does not localize to the membrane in the mesoderm where Crb and Par-3 complexes are not formed, suggests the apical complexes could be recruiting STAT. To test this, different apical complex proteins were expressed in the mesoderm, and their capacity to modify STAT subcellular localization was studied. Neither the expression of Crb nor aPKC (another member of Par-3 complex) is able to translocate STAT to the membrane. In contrast, expression of Par-3 results in efficient membrane translocation of STAT and STAT-GFP. Moreover, STAT-GFP and Par-3 coimmunoprecipate from embryo extracts overexpressing STAT-GFP and par-3, pointing to Par-3 as the molecule responsible of STAT apical localization. In accordance, STAT-GFP is lost from the membrane in par-3 zygotic mutants, whereas in crb null mutants, where the polarity is highly compromised and Par-3 localization is severely affected, STAT remains in the membrane of cells only where Par-3 is still present. Similarly, in null aPKC embryos, STAT-GFP exclusively remains apical in cells in which Par-3 still localizes at the membrane. Thus, STAT recruitment is independent of Crb or aPKC and may directly depend on Par-3 (Sotillos, 2008).
To analyze if JAK/STAT polarization is functionally relevant, genetic interactions with polarity mutants were tested. Heterozygous polarity mutants or stat92E embryos are viable and normal. In contrast, embryos simultaneously heterozygous mutant for stat92E and either par-3, aPKC, or crb present phenotypes associated to JAK/STAT loss of function, including malformation of the posterior spiracles and abnormal segmentation. A specific readout of the pathway's activity was studied, analyzing the expression of a crb-spiracle enhancer that is directly activated by JAK/STAT. The expression of this enhancer is severely reduced in zygotic par-3 mutants simultaneously heterozygous for stat92E, compared to its expression in heterozygous stat92E embryos or zygotic par-3 mutants. In contrast, the expression of the JAK/STAT independent ems-spiracle enhancer is not affected in the same genetic backgrounds. The capability of Par-3 to induce STAT membrane localization and the strong genetic interaction between stat92E and cell-polarity mutations indicate that the apical polarization of JAK/STAT components is required for full-signaling efficiency in the ectoderm (Sotillos, 2008).
Next, whether the apical localization of all JAK/STAT transducer components in the ectoderm results in signaling occurring exclusively through this membrane domain was tested. For this purpose the posterior hindgut, where JAK/STAT is required in the ectoderm and in the mesoderm surrounding it, was analyzed. Upd expressed from the most anterior ectodermal cells of the hindgut activates in the ectoderm ventral veinless (vvl) and upregulates in the mesoderm dome through the dome-MESO enhancer. Thus, vvl and the dome-MESO autoregulatory enhancer can be used as readouts for JAK/STAT activation in the different hindgut tissues (Sotillos, 2008).
If signaling in the ectoderm were transduced exclusively through the apical membrane, it would be expected that vvl activation on the hindgut would not be possible if Upd is presented from the basal side. To test this Upd was expressed either in the ectoderm or in the mesoderm, and its effect on vvl activation in the ectoderm was analyzed. As a positive control the expression of dome-MESO was analyzed. When expressed throughout the ectoderm, Upd induces ectopic expression of dome-MESO in the mesoderm and of vvl in the ectoderm, behaving as the endogenous Upd. In contrast, when Upd is expressed throughout the mesoderm, dome-MESO is ectopically activated, whereas vvl is not. The unresponsiveness of the ectoderm cells to Upd from the mesoderm is consistent with the endogenous receptor being apically localized in the hindgut ectoderm and, thus, unable to receive any mesoderm signal (Sotillos, 2008).
Many proteins involved in the establishment and maintenance of cell polarity also modulate signaling pathways by modifying or restricting the localization of their signaling components. Precise subcellular distribution may help the activation of the pathway or restrict its activity by sequestering key elements. This study has shown that in the epithelial cells the localization of JAK/STAT components is highly polarized. The apical restriction of the receptor can influence transduction, since only ligand presented to the apical side of the epithelium would be detected. This may be of relevance after septic injury, when circulating haemocytes secrete the Upd3 cytokine into the haemolymph. In this case, the secreted ligand would activate its targets in the fat body without stimulating the ectoderm epithelial cells, since the cell junctions efficiently block Upd diffusion to the apical side (Sotillos, 2008).
Par-3-dependent STAT apical localization is intriguing. The localization of STAT to the subapical membrane seems important for signal transduction, since mutations reducing the amount of cell polarity proteins enhance stat loss of function phenotypes and reduce the activation of direct pathway targets. It is proposed that in ectodermal cells, where the receptor and the kinase locate apically, the existence of a subapical pool of STAT facilitates its rapid translocation to the activated receptor, increasing signaling efficiency. Future research should resolve whether this is achieved simply by the increased local concentration of apical STAT facilitating receptor binding or if there exists some dedicated machinery to translocate STAT from the subapical region to the active receptor similar to the one involved in nuclear import. It is interesting to note that crb expression is upregulated by JAK/STAT signaling in the follicle cells and in the posterior spiracles. Since Crb helps maintaining Par-3 in the apical membrane, upregulation of crb by STAT might increase apical Par-3, reinforcing signal transduction by increasing the apical concentration of STAT (Sotillos, 2008).
There are few reports of polarized vertebrate JAK/STAT signaling. However, analysis of the subcellular localization of two IL-6 receptors in MDCK epithelial cells has shown that gp130 localizes basolaterally and CNTF-R apically. Also, in the mammary glands, the IL-4Ra receptor is localized apically in luminal cells during gestation and lactation. Recently, activated STAT3 has been transiently detected at the membrane in the nascent cell-cell contacts of squamous cell carcinoma of the head and neck. In vertebrates the Par-3 complex functions as a regulator of junction biogenesis. It will be interesting to investigate whether Par-3 also mediates the localization of STAT3 in the membrane. The results suggest that JAK/STAT polarization in epithelia may be a general feature (Sotillos, 2008).
Sorting of integral membrane proteins plays crucial roles in establishing and maintaining the polarized structures of epithelial cells and neurons. However, little is known about the sorting mechanisms of newly synthesized membrane proteins at the trans-Golgi network (TGN). To identify which genes are essential for these sorting mechanisms, mutants were screened in which the transport of Rhodopsin 1 (Rh1), an apical integral membrane protein in Drosophila photoreceptors, was affected. Deficiencies in glycosylphosphatidylinositol (GPI) synthesis and attachment processes were found to cause loss of the apical transport of Rh1 from the TGN and mis-sorting to the endolysosomal system. Moreover, Na+K+-ATPase, a basolateral membrane protein, and Crumbs (Crb), a stalk membrane protein, were mistransported to the apical rhabdomeric microvilli in GPI-deficient photoreceptors. These results indicate that polarized sorting of integral membrane proteins at the TGN requires the synthesis and anchoring of GPI-anchored proteins. Little is known about the cellular biological consequences of GPI deficiency in animals in vivo. These results provide new insights into the importance of GPI synthesis and aid the understanding of pathologies involving GPI deficiency (Satoh, 2013).
In this study, 546 lethal lines were screened for potential defects in Rh1 by examining the localization of Arr2::GFP in FLP/FRT-mediated mosaic retinas using two-color fluorescence imaging. A mutation was found in the Drosophila homolog of human PIG-U (Drosophila PIG-U), which encodes a subunit of GPI transamidase. Mutations in other genes of the GPI synthesis pathway but not in the GPI modification pathway gave rise to the same phenotype. Furthermore, the GPI-linked protein, Chp accumulates in the ER whereas the stalk membrane Crumbs protein and basolaterally localized Na+K+-ATPase were mis-sorted to the rhabdomere. Rh1 was found to be degraded before entering the post-Golgi vesicles, but Crb and Na+K+-ATPase are misrouted into vesicles destined for the rhabdomere in PIG mutant cells (Satoh, 2013).
There are two previous reports concerning GPI requirements for the transport of transmembrane proteins. In zebrafish, GPI transamidase has been found to be essential for the surface expression of voltage-gated sodium channels. In yeast, GPI synthesis is required for the surface expression of Tat2p tryptophan permease, which is associated with detergent-resistant membrane (DRM) in wild-type cells. In GPI-deficient yeast, Tat2p and Fur4p fail to associate with DRM and are retained in the ER. Although DRM forms in the ER in yeast, in mammalian cells, it is likely that DRM formation occurs only after Golgi entry. The reason for this is thought to be that GPI lipid remodeling occurs in different places: the ER in yeast and the Golgi body in mammalian cells. In mammalian cells, lipid rafts are postulated to concentrate some fractions of apically destined proteins owing to their affinity for the TGN or recycling endosomes (Satoh, 2013).
Along with the raft model, there are two possible explanations for the sorting phenotype of PIG mutant fly photoreceptors: (1) the polarized sorting of Rh1 depends on its affinity for the raft/DRM and the raft/DRM is deficient in PIG mutants; (2) unidentified GPI-anchored protein(s) play crucial roles in the polarized sorting of Rh1 and Na+K+-ATPase, and the raft/DRM provides a platform for the GPI-anchored protein(s). The first model predicts raft/DRM deficiency in PIG mutants, Rh1 association with lipid rafts and a stronger phenotype caused by mutations in the genes involved in raft formation. By contrast, the second model predicts that GPI deficiency produces a stronger phenotype than that caused by raft deficiency (Satoh, 2013).
Analysis of lipid raft deficiency does not support the first model in which the loss of polarized sorting of Rh1/Na+K+-ATPase in PIG mutants is a consequence of raft deficiency; instead, the current results support the second model in which unidentified GPI-anchored protein(s) concentrate Rh1 and exclude Na+K+-ATPase and Crb from post-Golgi vesicles destined for the rhabdomeres. Thus, loss of the GPI-anchored sorting protein(s) might cause most Rh1 to be directed into the endocytotic pathway and degraded by lysosomes while simultaneously allowing Na+K+-ATPase and Crb to be loaded into post-Golgi vesicles destined for the rhabdomeres. Chp is the only GPI-anchored protein known to be expressed in fly photoreceptors in the late-pupal stages. However, chp2 mutants do not exhibit any mislocalization phenotype of Rh1 or Na+K+-ATPase. Identifying the GPI-anchored protein(s) responsible for the sorting in the TGN is an important step for understanding this mechanism of polarized transport (Satoh, 2013).
The biosynthetic pathway of GPI-anchored proteins has been well elucidated, but little was known to date about the phenotypic consequences of the loss of GPI synthesis in vivo. The present study demonstrates that GPI synthesis is essential for the sorting of non-GPI-anchored transmembrane proteins, including Rh1 and Na+K+-ATPase, without obvious defects in adherens junctions. Human PIGM or PIGV deficiency causes seizures or mental retardation. These neurological disorders might be also caused by the mis-sorting of some transmembrane proteins in addition to the defects in the formation of GPI-anchoring proteins. These findings aid the understanding of the pathology of diseases involving deficient GPI-anchoring protein synthesis (Satoh, 2013).
Discs lost (Dlt) is expressed abundantly in ovaries. To assess the consequences of the loss of maternal Dlt in embryos, the dominant female sterile technique was used. However, females that lay eggs could not be recovered, even though more than 50% of the control females produce eggs. Hence, Dlt is essential for oogenesis. To circumvent the inability to produce eggs with reduced maternal Dlt, dsRNA interference was carried out. This process had previously been shown to be effective in C. elegans and Drosophila. The effects of dsRNA treatment soften the effects of mutation in other systems. dlt dsRNA-injected embryos express Dlt at very low levels, or not at all. More importantly, 3- to 4-hr-old injected embryos lack the columnar cell morphology. Instead, the epithelium consists of small cuboidal cells that are about one-fourth the length of the same cells in control embryos. These cells have completed cellularization, since low levels of Dlt and Dlg are present in the basal area. However, unlike control embryos where Dlg is localized laterally, Dlg is localized apically and laterally. These observations, combined with the expression pattern of Dlt, indicate that Dlt is required to establish epithelial polarity during cellular blastoderm formation. Since Crumbs is expressed after cellular blastoderm, at the onset of gastrulation, injected embryos were aged to germband extension (stage 11) and immunostained with anti-Dlt and anti-CRB. dlt dsRNA-injected embryos fail to express Dlt but do express CRB. At higher magnification, control-injected embryos show normal cell morphology and localization of cell junction-specific proteins, but dlt dsRNA-injected embryos display a completely aberrant Crb localization and embryo morphology. Thus, removal of Dlt prior to cellularization causes a complete lack of polarity based on morphological features and Crb localization (Bhat, 1999).
To determine whether ectopic dlt expression affects epithelial polarity, dlt was expressed using a heat shock-GAL4/UAS-dlt bipartite system. Four- to seven-hour-old embryos that received a 1 hr heat shock at 37.5 degrees C do not survive, whereas heat-shocked control embryos showed no lethality. The distribution of Dlt and Crb in ectodermally derived epithelia of control embryos is not altered by heat shock alone, and both proteins are localized apically. However, overexpression of dlt leads to an irregular epithelial cell layer with abnormally shaped cells that cannot maintain an epithelial context. A redistribution of Crb localization to basal and baso-lateral areas was also observed in some cells that overexpress Dlt. These data indicate that epithelial polarity is at least partially lost in the presence of additional Dlt protein, possibly because of the aberrant distribution of Crb (Bhat, 1999).
The apical localization of Dlt and Crb suggests that Dlt may interact with Crumbs. Previous studies have shown that deletion of 23 carboxy-terminal amino acids of the Crb protein leads to a null mutant phenotype (Wodarz, 1993 and Wodarz, 1995); therefore, an investigation was carried out to see whether the two proteins interact using a GST-Crb fusion protein containing the 37 cytoplasmic amino acids of CRB. GST pull down experiments show that full-length T7-tagged Dlt interacts with the cytoplasmic domain of Crb (GST-CRB). However, Dlt that lacks the first PDZ domain fails to bind GST-CRB, suggesting that the first PDZ domain is required for Crb binding. In addition, Dlt from adult extracts also binds to GST-Crb, indicating that both proteins bind to one another in vivo (Bhat, 1999).
To determine whether loss of crb had any effect on the localization of Dlt, crb mutant embryos were double labeled with anti-Crb and anti-Dlt. Dlt fails to localize to the apical side in the absence of Crb protein and is distributed over the entire cell membrane and cytoplasm in embryos that lack Crb. Hence, Dlt depends on Crb for its proper subcellular localization at later developmental stages (Bhat, 1999).
Analysis of the mechanisms that control epithelial polarization has revealed that cues for polarization are mediated by transmembrane proteins that operate at the apical, lateral, or basal surface of epithelial cells. Whereas for any given epithelial cell type only one or two polarization systems have been identified to date, the follicular epithelium in Drosophila ovaries uses three different polarization mechanisms, each operating at one of the three main epithelial surface domains. The follicular epithelium arises through a mesenchymal-epithelial transition. Contact with the basement membrane provides an initial polarization cue that leads to the formation of a basal membrane domain. Moreover, mosaic analysis was used to show that Crumbs (Crb) is required for the formation and maintenance of the follicular epithelium. Crb localizes to the apical membrane of follicle cells that is in contact with germline cells. Contact to the germline is required for the accumulation of Crb in follicle cells. Discs lost (Dlt), a cytoplasmic PDZ domain protein that has been shown to interact with the cytoplasmic tail of Crb, overlaps precisely in its distribution with Crb, as shown by immunoelectron microscopy. Crb localization depends on Dlt, whereas Dlt uses Crb-dependent and -independent mechanisms for apical targeting. The cadherin-catenin complex is not required for the formation of the follicular epithelium, but only for its maintenance. Loss of cadherin-based adherens junctions caused by armadillo mutations results in a disruption of the lateral spectrin and actin cytoskeleton. Also Crb and the apical spectrin cytoskeleton are lost from armadillo mutant follicle cells. Together with previous data showing that Crb is required for the formation of a zonula adherens, these findings indicate a mutual dependency of apical and lateral polarization mechanisms (Tanentzapf, 2000).
Drosophila ovaries are composed of ovarioles, each representing an anterior-posterior series of follicles of increasing age. The assembly of follicles takes place in the germarium that is located at the anterior tip of each ovariole. During follicle formation, ~30 follicle cells form an epithelial monolayer that surrounds a cluster of 16 germline cells. Whereas the germline cells increase in size but not in number, the cells in the follicular epithelium (FE) proliferate so that the FE in a mature follicle contains ~650 cells. A second population of follicle cells that does not establish direct contact with germline cells forms short stacks by cell intercalation, giving rise to the interfollicular stalk (Tanentzapf, 2000).
Crb is initially expressed in all follicle cells at stage 1 of oogenesis, but its expression becomes rapidly restricted to the cells of the FE, and is not maintained in the interfollicular stalk. Crb is found in the apical membrane of the cells of the FE. Dlt shows a temporal and spatial distribution similar to Crb in follicle cells, suggesting that both proteins might interact physically in follicle cells as they do in embryonic epithelia. Dlt also associates in vitro with Neurexin IV, a transmembrane component of the septate junction. The septate junction, which blocks paracellular diffusion similar to the chordate tight junction, is located basally to the ZA. To reconcile these data, it has been proposed that Dlt interacts with Crb apically to the ZA, with the ZA itself, and with Neurexin IV basally to the ZA. To clarify the subcellular localization of Dlt, the distribution of Dlt in the FE and in the embryonic ectoderm and epidermis was determined by interference electron microscopy (IEM). IEM reveals that Dlt, similar to Crb, is confined to the apical membrane. Within the apical membrane, Dlt accumulates at the marginal zone, an area of cell-cell contact apical to the ZA, and shows a lower concentration throughout the apical surface. The signal for Dlt at the ZA or basally to it does not exceed background levels, and no signal is observed at septate junctions. These findings suggest that Dlt does not physically interact with Neurexin IV at the septate junction, and further corroborates the notion that Dlt and Crb form a complex at the apical membrane of epithelial cells (Tanentzapf, 2000).
Crb and Dlt are also expressed in the germline, although the distribution of both proteins overlaps only partially. Dlt and Crb colocalize at the membrane of the nurse cells during early and mid oogenesis. High levels of Crb are seen in the plasma membrane of the oocyte, whereas Dlt is not detectable. Dlt accumulates transiently at the ring canals that connect nurse cells. During late stages of oogenesis, when the content of the nurse cells is rapidly transferred to the oocyte, Dlt is found at the nuclear membrane in close association with actin filaments that connect the nuclei of the nurse cells with the plasma membrane. Previous germline clone experiments did not reveal a function for crb in the germline. In contrast, maternal expression of dlt is essential for egg production, suggesting that dlt plays an important role in germline development. The function of dlt in germline development requires more detailed study (Tanentzapf, 2000).
The follicle cells are generated by two stem cells that are located in the middle of the germarium at the boundary of region 2a and 2b. Offspring of these stem cells establish contact with the basement membrane that surrounds the germarium and the follicles. Analysis of agametic ovaries has shown that the contact between follicle cells and germline cells is required for the formation of the FE. Follicle cells continue to proliferate in agametic ovaries and form a column that is two to three cells wide. To determine whether, and to what extent, contact between follicle cells and the basement membrane contributes to the polarization of the FE, follicle cells were examined in agametic ovaries. In agametic ovaries, ßPS-integrin localizes to the basal membrane of follicle cells as in wild type. Markers that normally localize to the lateral membrane (Fasciclin III and N-cadherin), to the lateral and apical membranes (E-cadherin and Armadillo), or to the apical membrane (ßHeavy-spectrin) are excluded from the basal cell pole. Apical and lateral markers show an overlapping distribution at the non-basal cell surface, but are concentrated at the cell pole that opposes the basal membrane. These findings suggest that contact to the basement membrane causes a partial polarization of follicle cells. The basal membrane is established and an asymmetric distribution of apical and lateral markers is observed, but the apical and lateral membrane domains are not clearly demarcated. As the follicle cells express the cadherin-catenin complex constitutively, it appears that basal polarization cues together with the activity of the cadherin-catenin complex are insufficient to fully polarize follicle cells. Interestingly, the presence of germline cells is required for the accumulation of Crb in follicle cells, since Crb is not detected in follicle cells in agametic ovaries (Tanentzapf, 2000).
To study the role of crb and dlt in the development of the FE, homozygous mutant follicle cell clones were generated. Clones were induced for the two protein-negative crb null alleles crb11A22 and crbB82, for the dlt protein negative null allele dltMY10, and the hypomorphic protein-positive allele dltdre1. Two types of mutant follicle cell clones were examined. Clones were generated either before the formation of the FE by inducing mitotic recombination in the follicle stem cells or their immediate offspring. Alternatively, clones were induced after the formation of the FE by inducing mitotic recombination in region 2b or stage 1 follicles. Early induced clones can be distinguished from late induced clones by their larger size, since all follicle cells derive from only two stem cells, and follicle cells continue to proliferate during early stages of follicle development. Clones are considered as induced early (early clones) if they comprise 15% or more of the cells of the FE of an individual follicle. Early clones can make up the entire FE of a single follicle. In contrast, late-induced clones (late clones) typically comprise less than 10 cells at mid to late stages of oogenesis (Tanentzapf, 2000).
A variable phenotype is observed when crb mutant follicle cells are generated before the FE forms. Many experimental follicles show an incomplete FE with areas in which the germline cells are not covered by follicle cells, a defect that is never seen in wild-type follicles. The area of a follicle not covered by a FE varies greatly in size and in some cases comprises most of the follicle. These gaps in the FE are likely caused by the failure of crb mutant follicle cells to integrate into the FE. The missing follicle cells could not be followed and it is presumed that they degenerate within the germarium. In contrast, some crb mutant follicle cells can form a FE with apparently normal morphology. The distribution of various markers was analyzed in crb mutant follicle cells that were part of the FE. Such crb mutant follicle cells retain apical Dlt, although the level of Dlt associated with the apical membrane is reduced in these cells. The level of ßHeavy-spectrin is also slightly reduced compared with wild-type follicle cells. These findings indicating that the apical localization of Dlt and ßHeavy-spectrin depends on Crb, but also on other mechanisms. No significant alteration was noticed in the level or distribution of Arm (Tanentzapf, 2000).
dltMY10 and dltdre1 mutant follicle cell clones, if generated before the formation of the FE, display gaps of variable size in the FE similar to those caused by crb mutations. Since dltdre1 is a protein-positive allele, it could not be determined whether some dltdre1 mutant follicle cells become part of the FE. In contrast to crb mutant follicle cell clones, no dltMY10 mutant follicle cells were found that participate in the formation of the FE in early clones. dltMY10 is a deletion that removes the genes encoding cdc37 and alpha-spectrin, in addition to dlt. The loss of cdc37 is compensated for by a transgene. Thus, dltMY10 mutant clones lack dlt and alpha-spectrin, raising the possibility that the observed mutant phenotype is the consequence of a synergistic effect between dlt and alpha-spectrin mutations, although it has been shown that alpha-spectrin is not required for the formation of the FE. These findings suggests that Dlt is required, and may be essential for the formation of the FE (Tanentzapf, 2000).
Large crb mutant clones within the FE that survive until mid-oogenesis often form a multilayered epithelium indicating that Crb is important for the maintenance of the FE. The multilayering of follicle cells in crb mutant clones is limited to posterior follicle cells that cover the oocyte. To determine whether removal of Crb and Dlt from cells of the FE disrupts the integrity of the FE, small crb or dlt mutant follicle cell clones were examined that were induced after the FE had been established. Small crb mutant cell clones develop normally until late in oogenesis, although they show a substantial reduction in the level of Dlt. Cells in small dlt mutant clones develop with apparently normal morphology, and show a normal distribution of F-actin and Arm. Crb is lost from most dlt mutant clones, indicating that Dlt is important for maintaining apical Crb. Some dlt mutant clones retain Crb, suggesting that a mechanism other than binding to Dlt can contribute to maintaining Crb at the apical membrane. Crb was also undetectable in many cell clones mutant for dltdre1. Dlt protein in dltdre1 mutant cells forms a 'cap' in the center of the apical membrane rather than being distributed throughout. Together, these findings suggest that the dltdre1 allele may carry a mutation that specifically disrupts the interaction of Dlt and Crb, but not the interaction of Dlt with other apical factors (Tanentzapf, 2000).
The consequences of Crb overexpression in the FE were examined to analyze the response of the FE to mislocalization of Crb, and to further study the interactions between Crb and Dlt. Overexpression of Crb in embryonic epithelia causes a severe disruption of epithelial integrity that includes an extension of the apical cell surface and multilayering of epithelial tissues. A truncated form of Crb that lacks all EGF and laminin G domains (UAS>crbintra), was expressed. In embryos, this construct causes a similar phenotype as overexpression of full-length Crb, but, as it is not recognized by the anti-Crb antibody, the effect of the expression of crbintra on the distribution of endogenous Crb can be examined. Expression of UAS>crbintra causes a strong reduction in the level of endogenous Crb, suggesting that crbintra acts competitively with Crb for interaction to binding partners that are important for maintaining Crb in the plasma membrane. Overexpression of full-length Crb leads to misdistribution of Crb into the lateral membrane, at levels that are similar to the apical membrane. In most of these cell clones, no significant mislocalization of Dlt was seen, suggesting again that the apical localization of Dlt is at least in part Crb independent. A fraction of the Crb overexpressing follicle cells show mislocalization of Dlt, a thinning of the epithelium, and a strong reduction of lateral markers such as Arm and Fasciclin III. These findings indicate that overexpression of Crb disrupts the lateral membrane in follicle cells (Tanentzapf, 2000).
The cadherin-catenin complex plays a major role in epithelial polarization because cadherin-mediated adhesive contacts cause the assembly of the lateral surface domain. Consequently, lack of cadherin activity compromises epithelial integrity in many tissues or epithelial cell culture lines. Removal of E-cadherin (Shotgun) from follicle cells causes only mild defects in the development of the FE. In follicle cells that lack E-cadherin, some Arm is retained at adherens junctions, raising the possibility that the FE coexpresses a second cadherin that interacts with Arm. Indeed, N-cadherin is expressed in follicle cells in a pattern that overlaps with E-cadherin in early- to mid-oogenesis. N-cadherin disappears from the FE at stage 10 of oogenesis, whereas E-cadherin is expressed throughout oogenesis. In contrast to E-cadherin, N-cadherin is not expressed in the cells of the germline. However, Arm appears to be the only ß-catenin homolog in Drosophila, in contrast to vertebrates, where ß-catenin can be functionally replaced by plakoglobin in the cadherin-catenin complex. Thus, to effectively remove the cadherin-catenin complex from follicle cells, clones were generated that lack Arm, which is known to interact with both E- and N-cadherin (Tanentzapf, 2000).
Clones were induced for three different mutant arm alleles that carry premature stop codons in the 6th (armYD35), 7th (armXK22), and 10th (armXP33) arm repeat. armXP33 is an intermediate hypomorph; armXK22 is a strong hypomorph, and armYD35 is believed to be a null allele. Embryos derived from germline clones mutant for armXP33 show a dramatic disruption of epithelial morphology that occurs at the onset of gastrulation and is substantially more severe than the defects in epithelial structure seen in crb null mutant embryos. Moreover Crb is needed for the formation of the ZA in embryonic epithelia. If the failure to form the ZA is the major consequence of compromising Crb or Dlt activity in the FE, the lack of the cadherin-catenin complex would be expected to cause similar defects as seen in crb and dlt mutant follicle cells; that is, a failure to form a FE. Surprisingly, follicle cells mutant for any of the three arm alleles form a FE. No follicles were observed in these experiments that show epithelial discontinuities, as seen in crb and dlt mutant follicles. arm mutant follicle cells often show an irregular morphology at early stages of follicle development. The irregularities in epithelial structure increase in severity until the FE is compromised and the follicle degenerates at mid- to late-oogenesis. To determine whether adherens junctions were effectively disrupted in arm mutant follicle cells, the expression of E- and N-cadherin was examined in those cells. Neither E- nor N-cadherin are detected in follicle cells mutant for any of the three arm alleles studied. Taken together, these findings suggest that cadherin-based adherens junctions are not essential for the formation of the FE, but are important for maintaining its epithelial structure (Tanentzapf, 2000).
Advantage was taken of the fact that arm mutant cells in the FE are maintained for several days and their molecular architecture was examined. armXP33 mutant follicle cells, which in most cases have a normal cuboidal to columnar shape, show a decrease of F-actin and alpha-spectrin at the lateral membrane, and an accumulation of these molecules at the apical cell pole. In contrast, the apical marker ßHeavy-spectrin shows a normal distribution in armXP33 mutant cells, suggesting that the apical spectrin cytoskeleton is intact. Follicle cells mutant for armXK22 or armYD35 often develop a squamous cell morphology or show a multilayered structure. alpha-Spectrin remains associated with the narrow lateral membranes in squamous arm mutant cells. ßHeavy-Spectrin, in contrast, is lost from these follicle cells, suggesting that the apical spectrin cytoskeleton is disrupted. To further examine the apical surface domain of arm mutant follicle cells, the distribution of Crb and Dlt was examined in these cells. armXP33 mutant follicle cells typically show a normal apical localization of Crb and Dlt. In contrast, Crb is lost from the apical membrane of follicle cells mutant for strong arm alleles, whereas apical Dlt is retained in these cells. Similar to dltdre1 mutant cell clones, Dlt forms a cap in the center of the apical membrane of arm mutant follicle cells. These observations suggest that the disruption of adherens junctions leads to a breakdown of the lateral membrane domain, as expected, but that this also compromises the apical surface domain. The differential behavior of Crb and Dlt in strong arm mutant cell clones again emphasizes that Dlt can rely on a Crb-independent apical targeting mechanism, and shows that apical Dlt can be retained in the absence of an apical spectrin cytoskeleton (Tanentzapf, 2000).
stardust (sdt) encodes multiple PDZ domain MAGUK (membrane-associated guanylate kinase) proteins that are expressed in all primary embryonic epithelia from the onset of gastrulation. The polarized architecture of these epithelial cells depends on the highly stereotypic distribution of cellular junctions and other membrane-associated protein complexes. In epithelial cells of the Drosophila embryo, three distinct domains subdivide the lateral plasma membrane. The most apical domain comprises the subapical complex (SAC). It is followed by the zonula adherens (ZA) and, further basally, by the septate junction. A core component of the SAC is the transmembrane protein Crumbs, whose cytoplasmic domain recruits the PDZ-protein Discs Lost into the complex. stardust provides an essential component of the SAC. Cells lacking crumbs or the functionally related gene stardust fail to organize a continuous ZA and to maintain cell polarity. Thus, Stardust and Crumbs are mutually dependent in their stability, localization and function in controlling the apicobasal polarity of epithelial cells. Stardust proteins colocalize with Crumbs and bind to the carboxy-terminal amino acids of the Crumbs cytoplasmic tail. Nevertheless, Stardust and Crumbs expression patterns in sensory organs differ. The polarity of neuroblasts requires the function of Bazooka, but not of Stardust or Crumbs. In conclusion, the Stardust proteins represent versatile candidates as structural and possibly regulatory constituents of the SAC, a crucial element in the control of epithelial cell polarity (Buchmann, 2001; Hong, 2001).
The sdt mutant phenotype is characterized by the loss of epithelial integrity and cell shape. sdt mutant embryos fail to concentrate the scattered spot adherens junctions into a continuous ZA. As a consequence, cells lose contact and the epithelium becomes multilayered. The assembly of the ZA requires a functional SAC. Formation of the SAC depends on an intact cytoplasmic tail of Crumbs, which recruits the PDZ protein Discs Lost (Dlt) into the complex. In sdt mutant embryos, the SAC components Crumbs (Crb) and Dlt fail to localize apically from stage 8 onwards. Because Dlt is correctly associated with the ingrowing plasma membrane during cellularization in sdt embryos, it is suggested that sdt is, similarly to crb, essential for maintenance, but not for initiation of Dlt localization. The transmembrane protein Stranded at Second is lost from the apical pole, whereas the basolateral marker Neurotactin, initially retained at the lateral membranes, is delocalized in later stages. Lack of sdt also affects localization of the PDZ protein Bazooka (Baz) in epithelial cells. Baz forms, together with the PDZ protein DmPar-6 and Atypical protein kinase C (DaPKC), another apical protein complex. This complex is essential for the polarity of epithelial cells and neuroblasts, the progenitors of the central nervous system. In the CNS Baz controls spindle orientation and localization of determinants. Lack of sdt function does not affect Baz localization in neuroblasts, showing that sdt, unlike baz, is not required for the apicobasal polarity of neuroblasts (Buchmann, 2001).
Crumbs, a transmembrane protein with EGF-like repeats, is an apical determinant for establishing apicobasal polarity in embryonic epithelia. The sdt and crb mutants exhibit nearly identical defects in epithelial apicobasal polarity, and crb and sdt function in a common genetic pathway. Precise colocalization of Sdt and Crb throughout embryonic development was found in all cells examined except for those in sensory organs: Sdt but not Crb is at the distal dendrites of sensory neurons. In epithelial cells Crb and Sdt are mutually dependent for their localization and stability. There is a marked reduction of the protein levels of Crb in sdt mutants and of Sdt in crb mutants after gastrulation. In both cases, instead of the normal apicolateral distribution, these proteins are found in sparse and discrete random spots. Dlt, which interacts with the Crb intracellular domain, is distributed similarly to Crb in sdt mutants, although its early expression and localization during cellularization is not affected even in sdt germline clone embryos. Conversely, overexpression of the Crb intracellular domain (Crb-intra) is sufficient to cause a 'dominant' phenotype, disrupting cell polarity, expanding the apical domain, and displacing endogenous Crb into cytoplasm. In such embryos a large fraction of Sdt, together with Crb and Crb-intra, is also displaced away from the membrane into the cytoplasm (Hong, 2001).
To test whether the mutual dependence of Sdt and Crb is due to their physical interaction, a GST::Crb-intra pull-down assay was carried out using in vitro translated 35S-labelled Sdt. Both SdtA and SdtB bind specifically to Crb-intra. Two domains of Crb-intra are known: the C-terminal domain containing the EERLI motif, and the amino-terminal domain containing amino acids, such as Y10 and E16, that are conserved in Crb and its C. elegans homologs. Mutation in either domain abolishes the ability of Crb-intra to rescue the crb phenotype, but only the EERLI motif is required for inducing the dominant overexpression phenotype of Crb-intra. In the in vitro binding assay, binding of Sdt to Crb-intra is nearly abolished by removal of the C-terminal EERLI motif, but is not affected by Y10A and/or E16A mutations, indicating that Crb binds Sdt with its C-terminal EERLI motif (Hong, 2001).
The same C-terminal EERLI is also required for Dlt to bind to Crb. It will be of interest to determine whether Sdt and Dlt bind Crb cooperatively to form a tertiary complex, or whether they compete with each other for binding to the Crb intracellular domain. No direct binding between Dlt and Sdt was detected using the same in vitro GST pull-down assay. However, MAGUK proteins frequently show intramolecular auto-inhibition between different binding motifs so it remains possible that Sdt binds Dlt only after it first binds to Crb. Thus, whereas the Crb-Sdt (and possibly Dlt) pathway controls the epithelial but not neuroblast polarity, the Baz-DmPar-6-aPKC complex affects both epithelial and neuroblast polarity. Proteins in both pathways are expressed in epithelial cells with identical apico-lateral localization patterns but only Baz-DmPar-6-aPKC are expressed in neuroblasts. In epithelia, these two pathways are probably partially redundant in controlling apicobasal polarity. Their role in regulating ZA formation seems to be the key to their epithelial and cuticle phenotypes. Sdt and Crb colocalize in epithelia and together serve as apical determinants, but only partially overlap and are likely to serve different functions in sensory organs. A mutation in a human Crb homolog has been implicated as the cause of one form of retinitis pigmentosa, suggesting a potential role for the Crb family of proteins in the localization of a sensory transduction complex. The localization of Sdt to the dendritic tip and scolopale cells, the probable sites for sensory transduction, raises the possibility that Sdt may have a role in the development or function of a sensory transduction apparatus (Hong, 2001).
The apical transmembrane protein Crumbs is necessary for both cell polarization and the assembly of the zonula adherens (ZA) in Drosophila epithelia. The apical spectrin-based membrane skeleton (SBMS) is a protein network that is essential for epithelial morphogenesis and ZA integrity, and exhibits close colocalization with Crumbs and the ZA in fly epithelia. These observations suggest that Crumbs may stabilize the ZA by recruiting the SBMS to the junctional region. Consistent with this hypothesis, it is reported that Crumbs is necessary for the organization of the apical SBMS in embryos and Schneider 2 cells, whereas the localization of Crumbs is not affected in karst (ßHeavy-spectrin) mutants that eliminate the apical SBMS. The data indicate that specifically the 4.1 protein/ezrin/radixin/moesin (FERM) domain binding consensus, and in particular, an arginine at position 7 in the cytoplasmic tail of Crumbs is essential to efficiently recruit both the apical SBMS and the FERM domain protein, Moesin-like. Crumbs, Discs lost, ßHeavy-spectrin, and Moesin-like are all coimmunoprecipitated from embryos, confirming the existence of a multimolecular complex. It is proposed that Crumbs stabilizes the apical SBMS via Moesin-like and actin, leading to reinforcement of the ZA and effectively coupling epithelial morphogenesis and cell polarity (Médina, 2002).
The Crumbs-Discs lost-Stardust pathway is essential for polarity and has been shown to be a major apical signal for establishing the ZA at the apical-lateral boundary. The observation that mutations affecting ßH and Crumbs both cause a junctional phenotype, along with the close colocalization of both proteins in the marginal zone of epithelial cells, suggested a possible connection between the activities of these two proteins. Crumbs can indeed recruit apical ßH together with the FERM domain protein Moesin-like and actin. The data are in good agreement with the hypothesis that polarity cues are used to organize the SBMS, but this is the first time that this has been shown for an apical determinant (Médina, 2002).
Several lines of evidence indicate that Crumbs can recruit ßH into its complex: (a) ßH is mislocalized in embryos mutant for the truncation allele crumbs 8F105, in which the mutant Crumbs protein itself is mislocalized; (b) ßH mislocalization can be induced by overexpression of the Crumbs transmembrane and cytoplasmic domains in vivo; (c) ßH is recruited to Crumbs protein clusters in an S2 cell cocapping assay; (d) Crumbs can be coimmunoprecipitated with ßH; and (e) the protein-null allele crumbs11A22 acts as a dominant enhancer of hypomorphic karst alleles, strongly indicating that a reduction in the normal amount of Crumbs reduces the level of partially functional ßH at the membrane. Moreover, because the karst mutant alleles all produce COOH-terminally truncated proteins, these results further suggest that the Crumbs-ßH interaction site lies in the NH2-terminal portion of the latter. Finally, loss of Crumbs has been shown (Pellikka, 2002b) to eliminate ßH from the stalk membrane of photoreceptors in the adult eye (Médina, 2002).
Current evidence indicates that ßH can be recruited to the membrane in several additional ways. First, it can associate with the specialized basal adherens junctions during cellularization in a Crumbs-independent manner. Second, it is found in the terminal web subtending brush borders in the midgut epithelium that does not express Crumbs. Finally, it has also been shown that ßH is only partially reduced in crumbs11A22 mutant follicle cell clones, indicating that in this Crumbs-expressing epithelium there are multiple mechanisms to recruit ßH. These data provide a compelling explanation for the modest nature of the karst-crumbs genetic interaction. By reducing Crumbs, only one of these pathways is affected. The observation that the karst1 allele produces readily detectable quantities of truncated product, most of which is not recruited to the membrane in any of these epithelia, suggests that there is a general and essential role of the COOH-terminal half of ßH in its stable membrane localization. Together, the above data are consistent with the multifunctional nature of spectrin membrane skeletons and with the idea that specific pathways recruit the SBMS to establish spatially distinct polarized membrane domains, whereas general COOH-terminal membrane association domains permit tight membrane association and network integration (Médina, 2002).
Partial rescue of crumbs mutants has been attained by the crumbsmyc-intra construct. This suggested that the transmembrane and cytoplasmic domains of Crumbs might be sufficient to concentrate ßH to some areas of the apical membrane. This result has been confirmed and extended, showing that the critical region for recruiting ßH is just 9 amino acids from position 6 through 14 of the cytoplasmic domain in the putative FERM domain binding site. Within this region, a conserved tyrosine residue at cytoplasmic domain position 10 (crucial for Crumbs function in vivo) and an arginine at position 7 are both required for this activity. It is worth noting that all Crumbs genes cloned so far contain a charged amino acid residue at position 7 in the cytoplasmic domain, suggesting that this is an evolutionarily conserved interaction site (Médina, 2002).
FERM domains are found in the protein 4.1 family of proteins that link the SBMS to cell-surface receptors as well as several other proteins which organize the cortical actin (ezrin/radixin/moesin). The founding member of this group, protein 4.1, was originally identified as a major component of the erythrocyte SBMS where it facilitates the interaction of spectrin with actin and the transmembrane protein Glycophorin C. Therefore, the presence of a conserved FERM binding domain in the Crumbs cytoplasmic domain suggests that Crumbs may bind to ßH via a FERM domain protein (Médina, 2002).
In Drosophila, the FERM domain family includes the proteins Coracle, Merlin, Moesin-like, and Expanded. Of these four proteins, Coracle is an unlikely candidate to bind to the Crumbs juxtamembrane domain since it is localized to the septate junctions basal to the ZA. However, the Merlin, Moesin-like, and Expanded proteins are localized in part or in whole at the ZA region in epithelia, and could thus be involved in the interaction between Crumbs and ßH. The fact that none of protein 4.1 family members known in Drosophila contains a spectrin-binding domain as defined by the archetypal protein 4.1 does not necessarily abrogate this hypothesis. ßH-spectrin is clearly recruited to the membrane by different mechanisms than its basolateral counterpart, and this specificity would likely be reflected in divergent interaction domains. In this work, it has been found that ßH and Moesin-like can both coimmunoprecipitate Crumbs. Furthermore, the capping assay and embryo expression evidence provide in vivo support for this result. Not only will Moesin-like cocap with the Crumbs cytoplasmic domain, but it is also dependent on exactly the same sequences that recruit ßH. These results, together with the existence of the consensus binding site for a FERM domain protein in Crumbs, strongly support the hypothesis that Moesin-like forms a bridge between Crumbs and the SBMS. A functional test of this relationship must wait until mutations in the Moesin-like locus become available. Thus, the current data, although highly suggestive, do not formally distinguish between the possibility of a Moesin-like bridge between Crumbs and the SBMS, and the existence of two separate complexes with direct interaction between Crumbs and ßH or Moesin-like in each. Significantly, actin does not cap consistently with Crumbs in S2 cells and is not present in immunoprecipitates. This suggests that other components present in epithelial cells are necessary for stabilization of the actin skeleton around the Crumbs complex. It also indicates that ßH is specifically recruited to the proposed complex and is not merely a passive arrival along with bulk actin (Médina, 2002).
These results indicate that Crumbs interacts with at least two different protein networks, a Moesin-like/Spectrin/actin-based network and a PDZ protein scaffold (Dlt/Sdt). However, it is unclear at present whether Dlt/Sdt and Moesin-like/ßH/actin coexist in the same complex with Crumbs. In the erythrocyte model, glycophorin C is linked to spectrin via a ternary complex containing protein 4.1 and the PDZ domain protein p55 bound to a topologically similar pair of binding sites to the two functional regions identified in the Crumbs cytoplasmic domain. If such a ternary complex forms in association with Crumbs, then the observation that the Sdt-binding domain of Crumbs is not required for the interaction between Crumbs and ßH, would indicate that the latter cannot be dependent on Dlt/Sdt for association with Crumbs in such a complex or that both interactions can coexist (Médina, 2002).
Because both the crumbs and karst phenotypes disrupt the ZA, it is hypothesized that Crumbs promotes the accumulation of ßH to the apicolateral region during gastrulation to orchestrate the fusion of spot adherens junctions and/or to stabilize the ZA. Moreover, the observation that karst mutants exhibit morphogenetic defects without any loss of epithelial polarity, whereas dlt mutants exhibit a strong polarity phenotype, suggests that the polarization and junction building functions of Crumbs are separate and parallel pathways. In support of this hypothesis, the FERM domain binding region of Crumbs is indeed required (Izaddoost, 2002) for correct organization of the ZA (Médina, 2002).
The loss of ßH function causes defects in cell shape change that are associated with apical contraction driven by an apically located actomyosin contractile ring. In this context the discovery that this spectrin isoform is complexed with Moesin-like is particularly provocative, since the activity of the latter is strongly correlated with modulation of cell shape and the actin cytoskeleton. Furthermore, the activity of moesin is modulated by phosphorylation in response to activation of Rho-associated kinase (ROK) in parallel with myosin II. Both Moesin and myosin light chain are activated by ROK phosphorylation and by ROK mediated inhibition of the myosin/moesin phosphatase. Therefore, it is speculated that ßH is part of the cytoskeletal network that facilitates such cell shape changes, and that in organizing spectrin at the membrane, Crumbs would appear to be acting as a molecular coordinator of polarity and morphogenesis. Furthermore, the finding that in human, mutations in CRB1 lead to pathologies such as retinitis pigmentosa (RP12) emphasizes the importance of deciphering the molecular networks associated with Crumbs in Drosophila. The human orthologue of ßH, ßV-spectrin, is strongly expressed in photoreceptor cells. This raises the exciting possibility that a similar interaction between CRB1 and ßV-spectrin exists in these cells. This will be examined in future work (Médina, 2002).
Apicobasal cell polarity is crucial for morphogenesis of photoreceptor rhabdomeres and adherens junctions (AJs) in the Drosophila eye. Crumbs (Crb) is specifically localized to the apical membrane of photoreceptors, providing a positional cue for the organization of rhabdomeres and AJs. The Crb complex consisting of Crb, Stardust (Sdt) and Discs-lost (Dlt) colocalizes with another protein complex containing Par-6 and atypical protein kinase C (aPKC) in the rhabdomere stalk of photoreceptors. Loss of each component of the Crb complex causes age-dependent mislocalization of Par-6 complex proteins, and ectopic expression of Crb intracellular domain is sufficient to recruit the Par-6 complex. The absence of Par-6 complex proteins results in severe mislocalization and loss of Crb complex. Dlt directly binds to Par-6, providing a molecular basis for the mutual dependence of the two complexes. These results suggest that the interaction of Crb and Par-6 complexes is required for the organization and maintenance of apical membranes and AJs of photoreceptors (Nam, 2003).
The strong dependence of Crb localization on Sdt and Dlt suggests that Crb may be destabilized or may not be targeted to the membrane in the absence of Sdt or Dlt. It is intriguing that Sdt and Dlt are lost only partially in the absence of Crb. The findings of a direct interaction between Dlt and Par-6 suggest that Sdt-Dlt can still be targeted to the membrane in the absence of Crb through the binding of Dlt to the Par-6 complex. However, it is important to note that Dlt is essentially lost in sdt mutant clones and vice versa. This raises an intriguing possibility that Dlt or Sdt are dependent on each other in vivo to be targeted to the apical membrane via binding to either Crb or Par-6. This mutual dependency between Dlt and Sdt may explain why Dlt and Sdt are lost in the absence of the other, rather than being associated with the Par-6 complex (Nam, 2003).
The interaction between the Crb and Par-6 complexes is mediated by the PDZ3 region of Dlt and the N-terminal domain of Par-6. The N-terminal domain of Par-6 is also used for binding aPKC. Therefore, a potential function of Dlt is to bind Par-6 in competition with aPKC or to facilitate the interaction of Par-6 with aPKC or other Par-6 binding proteins. Mutant analysis indicates that loss of Dlt and Sdt in sdt- clones causes mislocalization of both Crb and Par-6 complex proteins. This suggests that Sdt-Dlt interaction provides a scaffold to recruit Crb complex to the Par-6 complex and enhance the stability of these two complexes rather than functioning as a competitor for aPKC (Nam, 2003).
Proteins in Crb and Par-6 complexes consist of multiple functional domains which may be involved in diverse protein-protein interactions. A recent study has shown that in mammalian cell culture systems the PDZ domain of Par-6 binds not only Par-3 but also the N terminus of Pals1. These results suggest that the crosstalk between the Crb and Par-6 complexes is mediated by multiple domain-specific interactions. Evidence from genetic analysis using mutants suggests that the crosstalk between the two complexes is mutually required for normal organization of apical membranes and AJs in vivo, and also provides a basis for partial redundancy of these complexes in the organization of photoreceptor cell polarity. Interestingly, when either Crb or Sdt is lost, mislocalization or elimination of other associated components including Par-6 complex proteins becomes more severe in the age-dependent manner. This suggests that the Crb complex may be required for the maintenance rather than the formation of the Par-6 complex. The age-dependent degenerative phenotype may be related to the requirement of extensive apical membrane growth to make rhabdomeres and AJs along the growing axis of photoreceptors during pupal stage. Loss of any one component of the Crb complex is likely to be increasingly more detrimental as the process of membrane reorganization proceeds. In crb- or sdt- mutants, significant fractions of Par-6 complex proteins remain in the membrane despite the age-dependent and progressive mislocalization of apical markers. By contrast, loss of Par-6 or aPKC results in mislocalization of Dlt from the apical membrane. This suggests that the Par-6 complex plays essential functions for membrane localization of Crb complex proteins. Furthermore, both Par-6 and aPKC seem to be important for survival and/or proliferation of retinal cells because mutant clones were very small compared with adjacent twin spots and often completely disrupted, probably due to cell death. This is consistent with the findings of frequent apoptosis in aPKC- or par-6- embryos (Nam, 2003).
An important distinction of Par-6 complex in the photoreceptors from other epithelia is the localization of Baz. Baz localizes with Crb complex in the subapical membrane or both the subapical region and AJ in the Drosophila embryonic epithelia. Vertebrate Par-3 also localizes to the apical tight junction in vertebrate epithelial cells. By contrast, Baz in the photoreceptors is specifically positioned in the AJs basal to the all other proteins in the Crb/Par-6 complexes. Baz and Arm are recruited together to ectopic membrane sites by misexpression of CrbJM, suggesting that Baz is an integral component of AJ. However, Baz is not recruited by CrbPBM, whereas Par-6 and aPKC can be ectopically recruited by CrbPBM rather than CrbJM. Therefore, Baz appears to be recruited to AJ independently of Par-6/aPKC (Nam, 2003).
Intriguingly, despite its specific localization to AJs, loss of Baz results in most severe disruption of AJ as well as the more apical Dlt domain. It has been proposed that the Par-6/aPKC cassette is recruited to the site of cell-cell contact and then moves along the most apical zone of the developing cell-cell contact. In this process, an important step for cell polarity formation is to tether the cytoplasmic Par-6/aPKC complex to the site of cell-cell contact at the membrane, which is mediated by the interaction of Par-3 and a membrane protein JAM. Therefore, the results that baz mutation causes loss of Dlt and AJs support the crucial role of Baz in the initial step of cell polarization. However, the distinct localization of Baz from Par-6 and aPKC in the photoreceptors suggests that the mode of Baz localization varies in different systems. In photoreceptors, Baz may be targeted to the membrane with Par-6 but be sorted out from Par-6 in subsequent steps of polarization to remain in the AJs, whereas Par-6-aPKC-Baz cassette remains together in the complex in other epithelia. In contrast to Baz, aPKC localizes to both rhabdomere stalk and AJ, suggesting that Baz and Par-6 are completely separated during polarization while aPKC is not sorted from both Par-6 and Baz. The critical function of Baz in the localization of Crb complex in the rhabdomere stalk is consistent with the requirement of Baz for Crb localization in embryonic epithelia. However, the requirement of Baz in the embryo appears to be dependent on the stage of development since Crb distribution in the absence of Baz becomes normal in late embryos. On the contrary, such stage-dependent recovery of Crb complex localization has not been observed in baz- photoreceptor cells (Nam, 2003).
Recent studies have shown that mutations in human CRB1 cause RP12 and LCA, severe recessive retinal diseases, emphasizing the importance of Crb family proteins in the eyes of mammals including humans. The Drosophila Crb and human CRB1 are localized in analogous subcellular membrane domains of photoreceptors, the rhabdomere stalk and the inner segment in Drosophila and human photoreceptors, respectively. Besides similar subcellular localization, Crb and human CRB1 are functionally conserved. Age-dependent photoreceptor defects in the crb mutant also provide analogy to age-dependent retinal degeneration in RP12/LCA patients. These studies here imply that hCRB1 may function as a protein complex with homologs of Sdt and Dlt and such a complex may interact with a homologous Par-6 complex. Whether such homologous human genes are the targets of inherited retinal diseases such as RP remains to be studied (Nam, 2003).
Formation of multiprotein complexes is a common theme to pattern a cell, thereby generating spatially and functionally distinct entities at specialised regions. Central components of these complexes are scaffold proteins, which contain several protein-protein interaction domains and provide a platform to recruit a variety of additional components. There is increasing evidence that protein complexes are dynamic structures and that their components can undergo various interactions depending on the cellular context. However, little is known so far about the factors regulating this behaviour. One evolutionarily conserved protein complex, which can be found both in Drosophila and mammalian epithelial cells, is composed of the transmembrane protein Crumbs/Crb3 and the scaffolding proteins Stardust/Pals1 and DPATJ/PATJ, respectively, and localises apically to the zonula adherens. In vitro analysis shows that, similar as in vertebrates, the single PDZ domain of Drosophila Par-6 can bind to the four C-terminal amino acids (ERLI) of the transmembrane protein Crumbs. To further evaluate the binding capability of Crumbs to Par-6 and the MAGUK protein Stardust, analysis of the PDZ structural database and modelling of the interactions between the C-terminus of Crumbs and the PDZ domains of these two proteins were performed. The results suggest that both PDZ domains bind Crumbs with similar affinities. These data are supported by quantitative yeast two-hybrid interactions. In vivo analysis performed in cell cultures and in the Drosophila embryo show that the cytoplasmic domain of Crumbs can recruit Par-6 and DaPKC to the plasma membrane. The data presented here are discussed with respect to possible dynamic interactions between these proteins (Kempkens, 2006).
Homology modelling and energy calculations indicate that the PDZ domains of Sdt and Par-6 both show a high affinity for Crb. According to a classification of PDZ domains, the PDZ domains of Par-6 and Sdt fall into different categories: n (neutral) – h (hydrophic) for Par-6 and Sp (small and polar) – h (hydrophobic) for Sdt. The distinction is based on the nature of the amino acids in two critical positions, those immediately following the second β-strand and at the beginning of the second α-helix. However, this classification, based exclusively on the amino acids at two positions, does not adequately describe the complexity of the PDZ family and is not sufficient to predict the specificity of binding. This is borne out by the results presented in this study (Kempkens, 2006).
The crystal structure used as a template in this study (D. melanogaster Par-6, 1RZX.PDB) includes a canonical type I ligand (ESLV), and this can easily be replaced in the structure by the C-terminal tetrapeptide of Crb (ERLI). Although better binding of the Crb C-terminus to Par-6 than to Sdt is predicted, this conclusion should be treated with caution, since the predicted structure of the Crb-Sdt complex is based solely on homology modelling. The analysis of interactions between Crb and PDZ domains showed minor van der Waals clashes (<0.3 kcal/mol), as well as strong polar and hydrophobic ligand/PDZ-domain interactions. An important component of the interaction is electrostatic. This could favour fast association (Kon) and dissociation rates (Koff), which would facilitate flexible regulation of complex formation. These values, which are slightly better for Par-6, could account for the affinity and specificity observed in the experimental interaction studies. The predicted Kd for interaction between the domains considered in this study fall in the usual range for experimental determinations of PDZ-ligand binding affinities reported by many authors. The theoretical determination of binding interactions by FoldX has been tested for many domain-peptide interactions (i.e. SH3, SH2, PDZ, Ras-Rab, etc.), is validated by available experimental data, and shows a high degree of accuracy. In this sense, the theoretical cut-off for non-binders can be reasonably approximated to that used for experimental procedures, normally 1E−4M. Thus, all putative ligands with interaction energies below 1E−4M are considered to be non-binders (Kempkens, 2006).
It has been shown that the PDZ domain of vertebrate Par-6 can bind not only to the C-terminus of CRB3, but also to the C-terminus of the N-type Ca2+ channel and the C-terminus of neurexin, and that the PDZ domains of vertebrate and Drosophila Par-6 proteins can interact with an internal region in the N-terminal portion of Pals1/Sdt. These data support the view that PDZ domains are promiscuous and can bind different ligands, thanks to the plasticity of the carboxylate-binding loop. This conformational flexibility can be modulated by interaction with other proteins. For example, binding of Cdc42 to Par-6 significantly enhances its affinity for the C-terminal ligand, while it has no effect on the interaction with the internal binding sequence of Pals1. To what extent other components facilitate loop rearrangement to accommodate internal ligands to the Par-6 PDZ binding pocket in vivo remains to be determined. Interestingly, although the PDZ domains of both Par-6 and Sdt bind to the C-terminus of Crb, only the PDZ domain of Par-6 interacts with the internal sequence in Sdt (Kempkens, 2006).
Since the binding affinities of the PDZ domains of Sdt and Par-6 for the C-terminus of Crb are in the same range, and all three proteins are expressed in the same cells, other factors must be considered that might influence their interactions. These could include, for example, (1) temporal differences in expression, (2) protein modifications, and (3) interactions with other factors. Par-6 is present in the embryo before Sdt and Crb appear, since it is expressed maternally. In the genetic hierarchy controlling cell polarity and ZA assembly, components of the Baz/Par-6/DaPKC complex appear to form the top tier, with Baz acting very early during cellularisation to establish the apical domain in an adherens junction-independent manner. At this stage, Par-6 is diffusely distributed in the cytoplasm. It becomes localised apically only after cellularisation is complete. Localisation of Par-6 requires an interaction with the monomeric GTPase Cdc42, which, like its vertebrate homologue, binds Par-6 via a conserved CRIB (Cdc42/Rac interactive binding) domain. Thus, overexpression of a dominant-negative form of Cdc42 prevents apical localisation of Par-6, and a mutant version of Par-6 that cannot interact with Cdc42 (Par-6-ΔP) remains diffusely distributed in the cytoplasm (Kempkens, 2006).
Binding of the PDZ domain of Sdt to the C-terminus of Crb may also depend on additional contacts mediated by the other interaction domains of Sdt. Sdt is a member of the MAGUK protein family, with two L27 domains, one SH3, one PDZ and one guanylate kinase (GUK) domain. The L27 domains of Pals1 and Sdt bind to PATJ/DPATJ and mLin-7/DLin-7. No partner(s) for the SH3 and GUK domains of Sdt have been described so far. Recently, a direct interaction was shown to occur between the SH3/Hook domain of MPP5/Pals1 and the GUK domain of another MAGUK family member, MPP4, in mouse photoreceptor cells. Furthermore, yeast two-hybrid experiments have uncovered an interaction between the SH3 and the Hook/GUK domains of Sdt. In other members of the MAGUK family, such as human Discs large (hDlg), CASK and p55, these two domains participate in intra- as well as intermolecular interactions, with the former being preferred, at least in vitro. Currently, the data do not allow a decision as to whether the observed SH3-Hook/GUK interaction is intra- or intermolecular, nor to what extent this might influence the capacity of the PDZ domain to bind to Crb in vivo. Similarly, at present it is only possible to speculate on the possible influence on the binding specificity/activity of the phosphorylation of the intracellular domain of Crb, which has been demonstrated by in vitro assays, but also occurs under certain in vivo conditions. Future experiments will contribute to the elucidation of the dynamic interactions among these proteins within the cell and help in the understanding of how they establish and maintain the polarised phenotype (Kempkens, 2006).
The Crumbs (Crb) complex is a key regulator of epithelial cell architecture where it promotes apical membrane formation. Binding of the FERM protein Yurt to the cytoplasmic domain of Crb is part of a negative-feedback loop that regulates Crb activity. Yurt is predominantly a basolateral protein but is recruited by Crb to apical membranes late during epithelial development. Loss of Yurt causes an expansion of the apical membrane in embryonic epithelia and photoreceptor cells similar to Crb overexpression and in contrast to loss of Crb. Analysis of yurt crb double mutants suggests that these genes function in one pathway and that yurt negatively regulates crb. The mammalian Yurt orthologs YMO1 and EHM2 bind to mammalian Crb proteins. It is proposed that Yurt is part of an evolutionary conserved negative-feedback mechanism that restricts Crb complex activity in promoting apical membrane formation (Laprise, 2006).
The formation and maintenance of cell polarity is essential for epithelial morphogenesis. Patj (the Drosophila homolog of mammalian Patj) is multi-PDZ domain protein that localizes to the apical cell membrane and form a protein complex with cell polarity proteins, Crumbs (Crb) and Stardust (St). Whereas Crb and Sdt are known to be required for the organization of adherens junctions (AJs) and rhabdomeres in differentiating photoreceptors, the in vivo function of Dpatj as a member of the Crb complex in developing eye has been unclear due to the lack of loss-of-function mutations specifically affecting the patj gene. Genetic analysis of hypomorph, null, and RNA inerference reveals distinct dual functions of Patj in developing and mature photoreceptors. The C-terminal region (PDZ domains 2-4) of Patj is not essential for development of the animal but is required to prevent late-onset photoreceptor degeneration. In contrast, the N-terminal region of Patj is essential for animal viability and photoreceptor morphgenesis during development. The localization and maintenance of Crb and Sdt in the apical photoreceptor membrane are strongly affected by reduced level of Patj. Patj is necessary for proper positioning of AJs and the integrity of photoreceptors in the developing retina as well as for the maintenance of adult photoreceptors. This study provides evidence that Drosophila Patj has domain-specific early and late functions in regulating the localization and stability of the Crb-Sdt complex in photoreceptor cells (Nam, 2006).
Dynamic changes take place in developing photoreceptors to reorganize the apical cell membrane during pupal stage. Crb and Sdt are required for growth and maintenance of rhabdomeres and AJs during this time of photoreceptor morphogenesis. Patj binds Sdt to form a conserved heterotrimeric Crb complex (Roh, 2002), but its function in vivo has been unclear. In this study, a key question whether Patj is an essential member of the Crb complex was addressed in vivo (Nam, 2006).
This study made three new observations that demonstrate important functions of Patj: (1) hypomorphic patj shows severe late-onset degeneration of photoreceptor cells in adult eye although the mutant eyes develop relatively normally; (2) analysis of patj null mutant patj RNAi demonstrate that Patj is essential for early development of the animal and for morphogenesis of AJ and apical membrane domains of photoreceptor cells during pupal development (Nam, 2006).
Consistent with these results on the late-onset degeneration of photoreceptor cells in hypomorphic adult eyes, (3) degeneration of adult photoreceptor cells was found. However, despite the findings on the phenotypes of the hypomorphic mutant, it is worth noting that there are important differences between these two studies. First, a second study on patj function (Richard, 2005) is limited to the analysis of a viable hypomorphic condition patj that shows no obvious defects in the pattern of AJs and rhabdomeres in the eyes until approximately 70% late pupal development. In contrast, the current study with the newly generated null mutant and RNAi reveals important developmental functions of Patj in the eye as well as animal viability. Second, the hypomorpic mutant is not only deleted in the PDZ2-4 domain portion of the dpatj gene but also deficient for JTBR and partially CG32327 that are adjacent to the 3' end of patj. In this study, it was shown that patj RNAi causes similar phenotypes of patj null mutant in the eye, suggesting that JTBR and CG32327 do not affect eye development and, thus, have no detectable influence on analysis of hypomorphic and null mutants (Nam, 2006).
The complete loss of patj causes several significant defects in early pupal eyes at approximately 45% pupal development. (1) Both Crb and Sdt are strongly reduced or mislocalized in the absence of Patj, although the loss of patj shows slightly weaker phenotypes than those of crb and sdt mutants. Furthermore, and in contrast to the hypomorphic mutant, (2) null clones or the eyes expressing patj RNAi reveal striking basolateral displacement or expansion of AJ markers such as Arm and Baz. These results suggest that the N- and C-terminal region of Patj protein may have distinct functions in the photoreceptor cells. Because the hypomorphic mutant shows relatively normal development of photoreceptors until later pupal stage or eclosion, the PDZ2-4 domains are not required during early photoreceptor development but are necessary for the maintenance of fully differentiated photoreceptors during very late pupal and adult stage. Of interest, a significant level of Crb and Sdt are apically localized in 45% pupal photoreceptors in hypomorphic mutant eyes (Richard, 2005) but are lost later in adult eyes (Richard, 2005), suggesting that PDZ2-4 domains are required to maintain the Crb complex in late pupal and adult stages. In contrast, the N-terminal region of Patj containing MRE and PDZ1 is crucial for morphogenesis of AJ and rhabdomere during the first half of pupal stage. The MRE motif of Patj interacts with the L27 domain of Sdt. Thus, it is likely that the N-terminal region expressed in hypomorphic mutants might be sufficient for interaction with Sdt, allowing normal development. Conversely, Sdt cannot bind Patj in the null mutant, resulting in the failure of normal development as in crb or sdt mutants (Nam, 2006).
Recently, it has been shown that reduction of Patj by RNAi in MDCK epithelial cell culture leads to delayed tight junction formation and defects in cell polarization. Similarly, mammalian Patj is required for localizing tight junction proteins and stabilizing the Crb3 complex in human intestinal cells. Thus, the findings on the important function of Drosophila Patj in developing photoreceptors are consistent with the results from these mammalian studies, and further provide evidence for the developmental function of Patj in the organization of apical cell membrane in the in vivo animal model. Mouse Patj is closely related to Drosophila Patj, but it has 10 PDZ domains in contrast to the presence of 4 PDZ domains in Drosophila Patj. It will be interesting to see whether the mammalian Patj also show distinct functions of the N-terminal MRE and the multiple PDZ domains in organizing cell polarity and maintaining the stability of the Crb complex, respectively (Nam, 2006).
Mutations in the human CRB1 gene cause retinal diseases such as retinitis pigmentosa 12 (RP12) and Leber congenital amaurosis. The current results that Patj has dual functions in photoreceptor morphogenesis and maintenance in developing and adult animals, respectively, suggest that like CRB1, human Patj may be a target for early- and late-onset retinal diseases. It has been shown that the extracellular domain of Crb is required for preventing photoreceptor degeneration in ageing adult fly eyes. Results from this study and Richard (2005) indicate that PDZ2-4 domains of Drosophila Patj are required for blocking late-onset photoreceptor degeneration. It is currently unknown whether the requirement of Crb extracellular domain and the intracellular function of Drosophila Patj PDZ2-4 domains are related events in the maintenance of adult photoreceptors (Nam, 2006).
The establishment of apicobasal polarity in epithelial cells is a prerequisite for their function. Drosophila photoreceptor cells derive from epithelial cells, and their apical membranes undergo elaborate differentiation during pupal development, forming photosensitive rhabdomeres and associated stalk membranes. Crumbs (Crb), a transmembrane protein involved in the maintenance of epithelial polarity in the embryo, defines the stalk as a subdomain of the apical membrane. Crb organizes a complex composed of several PDZ domain-containing proteins, including Patj (formerly known as Discs lost). Taking advantage of a Patj mutant line in which only a truncated form of the protein is synthesized, Patj was demonstrated to be necessary for the stability of the Crb complex at the stalk membrane and is crucial for stalk membrane development and rhabdomere maintenance during late pupal stages. Moreover, Patj protects against light-induced photoreceptor degeneration (Richard, 2006).
This study presents evidence to show that Patj plays an important role in stabilizing the Crb complex in photoreceptor cells (PRCs). A truncated form of Patj, consisting of the N-terminal L27 and the first PDZ domain, is produced in Patj mutant eyes. It is further shown that the truncated Patj protein fails to stabilize Crb and Sdt at the stalk membrane during late pupal development and in adult eyes. It has been demonstrated that Crb is required for proper localization of Sdt and Patj at the stalk membrane of PRCs and that, in sdtXP96, the maintenance of Crb and Patj is compromised. These data together with the results presented in this study support the view that lack of any component of the Crb complex leads to mislocalization and/or dysfunction of the whole complex in the Drosophila eye (Richard, 2006).
To understand how the Crb complex may ensure a proper morphogenesis of photoreceptor cells, two major events during photoreceptor development have to be considered. In the first half of pupal development, stabilization of the ZAs is essential to maintain adhesion between PRCs during the tremendous cell shape changes that take place later when the cells undergo elongation. Furthermore, from 37% pupal development onward, the apical membrane differentiates into the rhabdomere and the stalk (Richard, 2006).
In crb and sdt mutants, the rhabdomeres are shorter and thicker, suggesting a failure to stabilize adhesion in early stages of pupal development, which in turn prevents proper elongation. This interpretation is consistent with the observation that, in eyes lacking crb function, the continuity of the ZA is interrupted at early stages of pupal development. Patj mutants do not exhibit any obvious defects in ZA development or PRC elongation, which can be explained by the fact that components of the Crb complex are still correctly localized until 70% pupal development. This timing contrasts with crb or sdt mutant PRCs, in which the integrity of the Crb complex is lost at an early stage of pupal development. Several explanations may account for this different behavior of Patj mutant PRCs. (1) The N-terminal portion of Patj may still retain some function during early pupal development, which stabilizes the Crb complex and, hence, the ZA. (2) Alternatively, Patj does not play a major function for the stability of the complex at early stages. (3) Finally, additional factors may interact with Patj and stabilize the Crb complex at early stages of development, and these interactions still occur with a truncated Patj. In fact, recent in vitro studies have suggested direct interactions between Patj and either Drosophila Par-6 or Drosophila PKC, two members of the other apically localized protein complex, which is essential for epithelial cell polarity in the embryo. However, it can be excluded that the suggested interaction between the third PDZ domain of Patj and the N-terminal domain of DmPar-6 plays any role in the stabilization of the complex during the first half of pupal development. The truncated Patj protein studied here lacks the third PDZ domain, yet it remains localized at the apical membrane at this stage (Richard, 2006).
The other major aspect of PRC maturation -- the differentiation of the apical membrane into rhabdomere and stalk -- is affected in crb, sdtXP96, and Patj mutant eyes, suggesting that all three components are necessary for this process. These mutations result in a shortening of the stalk membrane. The weaker phenotype of the Patj mutant relative to that of crb mutants is probably due to the hypomorphic nature of the former. Separation of the apical membrane of PRCs into two distinct domains, the rhabdomere and the stalk, becomes manifest at approximately 55% pupal development and coincides with the restriction of Crb and its associated proteins to this region. No other mutant affecting the length of stalk membrane has been described to date, although some mutants affect individual aspects of the crb or Patj morphogenetic phenotype, displaying thicker (bifocal; DSec61), malformed (Glued; WASp) or missing (overexpression of amphiphysin) rhabdomeres. Thus, the regulation of stalk membrane development seems to be a unique function for members of the Crb complex (Richard, 2006).
One phenotype of Patj mutants observed in this study, the progressive resorption of rhabdomeric microvilli, has not been described to date for any other mutant of the Crb complex. This raises the question whether Patj is involved in other processes in addition to those that are controlled by crb and sdt. The rhabdomere is composed of microvilli, each of which is supported by actin filaments. Rhabdomere morphogenesis and integrity depend on constant renewal of the membrane and on a highly organized actin cytoskeleton. Thus, it is not surprising that mutations in proteins involved in endo- or exocytosis, such as dynamin, Rab1, Rab6, Rab11, Sec6, Sec61, or Sunglasses, affect the integrity of the rhabdomere. It has been suggested that the addition of new membrane occurs at the base of the rhabdomere in Drosophila, while shedding occurs at the distal tip in tipulids. The further analysis of the function of these genes, the subcellular distribution of the respective proteins and their possible interactions with members of the Crb complex will be required to determine any involvement of the Crb complex in these processes. Rhabdomere integrity is also affected in eyes lacking proteins involved in actin structure and remodeling, such as NinaC, Chaoptin, Glued, Moesin or Rac1 but also in mutants for rhodopsin itself, which plays a structural role in addition to its function in signal transduction. In this scenario, Patj could help to stabilize the cytoskeleton and thereby maintain the integrity of the rhabdomere. Alternatively, the four PDZ domains in Patj may mediate the assembly of additional proteins. The identification and functional characterization of these proteins will shed light on the process by which Patj controls the stability of PRCs (Richard, 2006).
At present, the possibility that the defects observed in pigment cells in Patj mutant eyes contribute to the mutant phenotype observed in PRCs cannot be excluded. In vertebrate eyes, the pigment epithelium plays an active role in the renewal of rhodopsin, and defects in the pigment epithelium can lead to degeneration of PRCs. It is not yet known whether pigment cells in the Drosophila eye have a comparable function, although they certainly serve to insulate the PRCs of individual ommatidia from the light impinging on their neighbors. The accumulation and fusion of pigment granules in Patj mutant eyes may point to a defect in vesicular biogenesis and/or secretion. Whether this defect also affects interactions with the PRCs, in other words, whether pigment cells play an active role in the maintenance of the rhabdomeres or PRC function, and if so, whether Patj is involved in this process, is not known (Richard, 2006).
Finally, the results of this study demonstrate that Patj, like Crb, protects PRCs from the deleterious effects of excess light. The degeneration of PRCs observed in Patj mutant eyes may be a direct consequence of the failure to stabilize the components of the Crb complex at the stalk membrane. Previously published data have shown that the absence of crb in Drosophila eyes leads to retinal degeneration under similar lighting conditions. Similarly, mice deficient for CRB1 display signs of retinal degeneration upon exposure to light, which are reminiscent of defects seen in patients bearing mutations in the CRB1 gene. However, the penetrance of degeneration observed in crb eyes is much higher than that observed in Patj eyes, although the cellular features of degeneration observed in both mutants are similar. Taking into account that the crb clones were produced in a white background, whereas Patj eyes are red (due the transgenes that were introduced), it is not unlikely that the pigments could play a protective role in the latter case, as also shown previously for white mutants. Preliminary experiments suggest that the presence of pigments in crb mutant ommatidia indeed slows down the light-dependent degeneration (Richard, 2006).
Taken together, these results extend the knowledge of the genes involved in controlling retinal morphogenesis and preventing light-dependent PRC degeneration in the fly. Mutations in human CRB1 lead to RP12 and LCA, two severe forms of retinal dystrophy, raising the question whether mutations in the homologues of the other members of the complex might result in similar phenotypes. Understanding the molecular mechanisms leading to the mutant phenotype in the fly will certainly contribute to unraveling the pathogenesis of these retinal dystrophies in humans (Richard, 2006).
The integrity of polarized epithelia is critical for development and human health. Many questions remain concerning the full complement and the function of the proteins that regulate cell polarity. This study reports that the Drosophila FERM proteins Yurt (Yrt) and Coracle (Cora) and the membrane proteins Neurexin IV (Nrx-IV) and Na+,K+-ATPase are a new group of functionally cooperating epithelial polarity proteins. This 'Yrt/Cora group' promotes basolateral membrane stability and shows negative regulatory interactions with the apical determinant Crumbs (Crb). Genetic analyses indicate that Nrx-IV and Na+,K+-ATPase act together with Cora in one pathway, whereas Yrt acts in a second redundant pathway. Moreover, it was shown that the Yrt/Cora group is essential for epithelial polarity during organogenesis but not when epithelial polarity is first established or during terminal differentiation. This property of Yrt/Cora group proteins explains the recovery of polarity in embryos lacking the function of the Lethal giant larvae (Lgl) group of basolateral polarity proteins. It was also found that the mammalian Yrt orthologue EPB41L5 (also known as YMO1 and Limulus) is required for lateral membrane formation, indicating a conserved function of Yrt proteins in epithelial polarity (Laprise, 2009).
To clarify the mechanisms of epithelial polarization, the function was examined of the FERM-domain protein Yrt, which was shown to act as a negative regulatory component of the Crb complex in both Drosophila and vertebrates. Crb regulates epithelial apical basal polarity by promoting apical membrane and apical junctional complex formation. Crb also controls the growth of the apical membrane at late stages of epithelial differentiation. Yrt binds to the cytoplasmic tail of Crb and restricts Crb activity in apical membrane growth. However, Yrt is predominantly a basolateral protein that is recruited into the Crb complex only at late stages of epithelial differentiation. Embryos completely lacking maternal and zygotic Yrt (yrtM/Z) displayed polarity defects before Yrt is recruited into the Crb complex at late organogenesis. This raises the question whether Yrt has a function in epithelial organization as a basolateral protein (Laprise, 2009).
yrtM/Z mutants and double-mutant combinations of yrt and genes encoding basolateral proteins were examined for synergistic genetic interactions that could indicate functional cooperation. yrtM/Z embryos showed clear polarity defects at post-gastrula stages of development, as indicated by the basolateral mislocalization of Crb. In contrast, zygotic yrt mutants demonstrated only minor polarity defects in the trunk ectoderm. This indicates that yrt is required for polarity, and that zygotic yrt mutants could provide a sensitized background to reveal genetic interactions. Within the basolateral membrane, Yrt is enriched at the septate junction together with the Na+,K+-ATPase and other septate junction components from stage 14 onwards. Marked genetic interactions were found between yrt and Atpα (which encodes the α-subunit of Na+,K+-ATPase) and between yrt and Nrx-IV, but not between yrt and six other genes that encode septate junction transmembrane proteins. In contrast to wild type and single mutants, apical markers were mislocalized in yrt Atpα and yrt Nrx-IV double mutants similar to yrtM/Z embryos. Enhancement of the Nrx-IV null phenotype by yrt indicates that Nrx-IV and yrt have overlapping functions and do not operate in a linear pathway. The pathway relationship between yrt and Atpα remains uncertain as embryos completely devoid of Na+,K+-ATPase function cannot be analysed. These findings reveal previously unrecognized functions of the Na+,K+-ATPase and Nrx-IV in epithelial polarity (Laprise, 2009).
The developmental timing of the yrtM/Z and the yrt Atpα and yrt Nrx-IV polarity phenotypes is notable. These mutants show no polarity defects during cellularization when epithelial cells first form or during gastrulation when the apical junctional belt is assembled. Polarity defects are first seen at stage 11 or 12, and are most prominent at embryonic stage 13. In contrast to wild type, Crb and other apical markers are found throughout the plasma membrane and colocalize with basolateral markers such as Discs large (Dlg, also known as Dlg1) and Fasciclin 3 in yrtM/Z, yrt Atpα and yrt Nrx-IV mutants. During late embryogenesis, apical markers become restricted again to the apical membrane, which, however, remains abnormally extended and dome-shaped. Thus, Yrt, Na+,K+-ATPase and Nrx-IV cooperate and have critical functions in maintaining epithelial polarity during early organogenesis (stages 11-13), well before septate junction assembly is observed, and Yrt is recruited to the apical membrane at stage 14 (Laprise, 2009).
Na+,K+-ATPase and Nrx-IV are required for septate junction assembly. Because Yrt also accumulates at septate junctions, it was asked whether Yrt has a role in septate junction formation or function. Septate junctions are basal to the adherens junction and, like vertebrate tight junctions, prevent diffusion of solutes between cells. Dye injection assays show that paracellular barriers in epithelia of yrtM/Z embryos are compromised. However, yrtM/Z embryos showed a normal complement of septa when examined ultrastructurally whereas Atpα and Nrx-IV mutants lack septa. Immunoprecipitation experiments demonstrated FERM-domain-dependent interactions between Yrt and the septate junction transmembrane protein Neuroglian (Nrg), and the enrichment of Yrt at septate junctions was less pronounced in Nrg mutants. However, Nrg or Nrg yrt mutant embryos did not display overt defects in epithelial polarity. These findings indicate that Yrt is a bona fide septate junction component that has a function distinct from Nrx-IV and Na+,K+-ATPase because it is not required for septa formation but essential for barrier function (Laprise, 2009).
Next, the analysis extended to cytoplasmic adaptor proteins associated with septate junctions. yrt does not show genetic interactions with dlg or lgl, genes encoding conserved basolateral polarity proteins required for septate junction formation and apical/basal polarity. varicose (vari), which encodes a membrane-associated guanylate kinase, and cora, which encodes a FERM protein that is the Drosophila orthologue of mammalian erythrocyte protein band 4.1 and its paralogues, were examined. vari and cora null mutant embryos did not show defects in apical basal polarity. No functional interactions were seen between yrt and vari. In contrast, yrt cora double-mutant embryos displayed marked apicalization defects, with strongly expanded apical membranes and reduced basolateral membranes in all ectodermal epithelia including the epidermis. This phenotype is significantly stronger than the yrtM/Z or cora null mutant phenotypes indicating that Yrt and Cora have redundant functions in promoting epithelial polarity (Laprise, 2009).
Similar to yrtM/Z mutants, yrt cora double mutants did not show polarity defects during gastrulation. By stage 11, polarity defects were more prominent in yrt cora mutants than in yrtM/Z embryos, which correlates with Cora being expressed from stage 11 onwards. In yrt cora mutants, the most severe polarity defects characterized by overlapping distributions of apical and basolateral markers occurred during stage 13. However, by late embryogenesis, apical and basolateral markers were segregated, although expanded apical membranes and severe defects in tissue organization persisted. Interestingly, the yrt cora apicalization phenotype is very similar to the phenotype that results from high-level overexpression of Crb in both timeline and severity. The segregation of apical and basolateral markers in late-stage yrt cora mutants is also observed in late-stage yrtM/Z, yrt Atpα and yrt Nrx-IV embryos. Thus, polarization mechanisms are at play in late embryos that are not dependent on Yrt and Cora. To address the possibility that this repolarization is the result of redundancy between Yrt/Cora and Lgl-group proteins, which include Scribble (Scrib) as well as Lgl and Dlg, embryos lacking Yrt and Scrib (yrtM/Z scribM/Z) were examined. Apical and basolateral markers segregated in yrtM/Z scribM/Z embryos indicating that a basolateral polarity mechanism exists in late embryos that is independent of Yrt/Cora and Lgl-group proteins (Laprise, 2009).
Together, these findings suggest that Yrt, Cora, Nrx-IV and the Na+,K+-ATPase are a new functionally cooperating group of basolateral epithelial polarity proteins, which is refered to as the 'Yrt/Cora group'. In contrast to the enhancement of the cora and Nrx-IV null phenotypes by yrt mutations, no phenotypic enhancement was seen in cora Nrx-IV or cora Atpα double mutants, indicating that the Yrt/Cora group is composed of two functionally overlapping pathways. Cora, Nrx-IV and the Na+,K+-ATPase belong to one pathway, which is consistent with previously documented biochemical and genetic interactions between these proteins, whereas Yrt defines a second redundant pathway (Laprise, 2009).
One critical aspect of polarity regulation is that apical and basolateral proteins act antagonistically to set up mutually exclusive membrane domains. In gastrulating Drosophila embryos, this interaction occurs between apical factors and basolateral Lgl-group proteins. Similar to lgl, dlg and scrib mutations, yrt mutations partially suppress the crb mutant phenotype. Because Yrt can bind Crb directly, it is suggested that Yrt negatively regulates Crb function as a component of the apical Crb complex in both apical basal polarity and the control of apical membrane size. The data reported in this study argue that Yrt opposes Crb function also as a basolateral polarity protein. To test this hypothesis further it was asked whether the loss of other Yrt/Cora-group genes could suppress the crb mutant phenotype. It was found that mutations in Atpα, Nrx-IV and cora ameliorate the epithelial defects of crb mutants to an extent similar to that of yrt mutations. Remarkably, yrt cora crb triple-mutant embryos not only showed a suppression of the crb mutant phenotype, which was much stronger than the one observed in yrt crb or cora crb double mutants, but also caused a strong suppression of the apicalization effect observed in yrt cora mutants. These findings further support the conclusion that Yrt and Cora act redundantly, and indicate that mutual competition of basolateral Yrt/Cora-group proteins and apical Crb organizes epithelial membrane domains (Laprise, 2009).
Epithelial differentiation during Drosophila embryogenesis can be subdivided into four phases that are characterized by distinct mechanisms governing epithelial polarity. (1) Initial cues for polarity are given before or during cellularization. (2) Fully polarized epithelial cells are established during gastrulation through the interplay of apical Par and Crb complexes and basolateral Lgl-group proteins. (3) The Yrt/Cora group acts during organogenesis to promote basolateral polarity and counteracts apical determinants, thereby functionally replacing the Lgl group. (4) The function of Yrt/Cora and Lgl groups does not account for the polarization of epithelial cells in late embryos, because normalization of polarity is observed in the absence of these factors. This implies the existence of a yet unknown mechanism that stabilizes basolateral polarity. Septate junctions form well after Lgl-group and Yrt/Cora-group proteins have contributed to epithelial polarity, indicating that polarity and septate junction formation are independent functions of these proteins. The identification of a novel group of polarity factors that acts in a discrete time window during development highlights the temporal complexity of the regulation of the epithelial phenotype and further explains why the loss of individual polarity proteins does not completely compromise polarity (Laprise, 2009).
The vertebrate Yrt orthologues Mosaic eyes (Moe, also known as Epb41l5) in zebrafish and EPB41L5 (also known as YMO1 and Limulus) and EPB41L4B (also known as EHM2) in mammals bind to Crb proteins and contribute to epithelial organization. However, as in Drosophila, vertebrate Yrt homologues are predominantly associated with the basolateral membrane and seem to be recruited to the apical membrane only at later stages of epithelial development. To test whether EPB41L5 is required for basolateral differentiation, RNA interference was used in MDCK cells. Transient depletion of EPB41L5 using shRNA resulted in a notable cell flattening and an expansion of the cell perimeter, indicating that lateral membranes were strongly reduced and apical and basal membranes were enlarged. Consistently, it was found that the lateral markers Na+,K+-ATPase, Scrib and E-cadherin were reduced or lost from the plasma membrane. The same effects were observed using siRNA oligonucleotides targeted to a distinct region of EPB41L5. MDCK cells were established that were stably transfected with EPB41L5 short hairpin RNA (shRNA), and lateral membrane formation was examined after Ca2+-switch. After re-addition of Ca2+, EPB41L5 shRNA cells displayed significant delays in lateral membrane formation and recruitment of E-cadherin to cell-cell contacts. At 8 h after Ca2+-switch, E-cadherin levels appeared similar in control and knockdown cells although experimental cells remained slightly flatter. Interestingly, by 24 h after Ca2+-switch, E-cadherin levels at the plasma membrane had decreased again and appeared lower than in control cells but similar to EPB41L5 shRNA cells before Ca2+-switch. EPB41L5 knockdown cells ultimately established normal cell shape. Whether the formation of a normal lateral membrane in EPB41L5 shRNA cells is due to residual EPB41L5 expression, or reflects a transient requirement of EPB41L5 for cell polarization as in Drosophila epithelia, remains unclear. It is concluded that the function of Yrt as a basolateral polarity protein is conserved in mammalian epithelial cells (Laprise, 2009).
The loss of basolateral membrane in Drosophila embryos that lack Yrt/Cora-group function and in MDCK cells depleted of EPB41L5 is reminiscent of the loss of basolateral membrane in MDCK cells depleted of the phosphoinositide PtdIns(3,4,5)P3 and of human bronchial epithelial cells depleted of β2-spectrin or ankyrin-G. Cora/band 4.1 and the Na+,K+-ATPase can associate with either spectrin or the spectrin adaptor ankyrin, and loss or ectopic expression of EPB41L5 can cause defects in basolateral actin. This raises the possibility that Yrt/Cora-group proteins act by stabilizing the lateral actin/spectrin membrane cytoskeleton. Analysis of vertebrate development in the absence of the function of the Yrt orthologues Moe in zebrafish and EPB41L5 in mice revealed defects in epithelial organization that reflect Crb-dependent and probably also Crb-independent functions of Yrt proteins. For example, the abnormal cell shape and multi-layering defects seen in the developing neuroepithelium of mutant mouse embryos could result from defects in basolateral polarity. Moreover, mouse embryos lacking EPB41L5 show defects in epithelial-mesenchymal transition of mesodermal cells during gastrulation. These surprising findings indicate that Yrt proteins are core regulators of animal tissue organization that can enhance epithelial or mesenchymal cell differentiation (Laprise, 2009).
Regulation of epithelial tube size is critical for organ function. However, the mechanisms of tube size control remain poorly understood. In the Drosophila trachea, tube dimensions are regulated by a luminal extracellular matrix (ECM). ECM organization requires apical (luminal) secretion of the protein Vermiform (Verm), which depends on the basolateral septate junction (SJ). This study shows that apical and basolateral epithelial polarity proteins interact to control tracheal tube size independently of the Verm pathway. Mutations in yurt (yrt) and scribble (scrib), which encode SJ-associated polarity proteins, cause an expansion of tracheal tubes but do not disrupt Verm secretion. Reducing activity of the apical polarity protein Crumbs (Crb) suppresses the length defects in yrt but not scrib mutants, suggesting that Yrt acts by negatively regulating Crb. Conversely, Crb overexpression increases tracheal tube dimensions. Reducing crb dosage also rescues tracheal size defects caused by mutations in coracle (cora), which encodes an SJ-associated polarity protein. In addition, crb mutations suppress cora length defects without restoring Verm secretion. Together, these data indicate that Yrt, Cora, Crb, and Scrib operate independently of the Verm pathway. These data support a model in which Cora and Yrt act through Crb to regulate epithelial tube size (Laprise, 2010).
The transmembrane protein Crumbs (Crb) acts as an apical determinant during establishment of epithelial apical-basal polarity. During later stages of epithelial differentiation, Crb promotes apical membrane growth independently of its role in apical-basal polarity. Crb activity is counteracted by different groups of basolateral polarity proteins including the Yrt/Cora group, which is composed of Yurt (Yrt), Coracle (Cora), Na+/K+-ATPase, and Neurexin IV (Nrx-IV). Loss of Yrt results in Crb-dependent apical membrane growth during late stages of epithelial cell maturation in Drosophila. Thus, the equilibrium between the activities of these polarity proteins is important to define the size of the apical domain. Precise control of the apical surface of tracheal cells is crucial to define epithelial tube size in the Drosophila respiratory system, suggesting a potential role for polarity proteins in tube morphogenesis. However, the contribution of polarity regulators to the regulation of epithelial tube shape and size is poorly understood. Controlling length and diameter of the lumen is important for organ function, as illustrated by the deleterious tubule enlargements that occur in polycystic kidney disease (Laprise, 2010).
To better understand the role of polarity regulators and apical membrane growth in epithelial tube morphogenesis, the role of Yrt was investigated in the formation of the Drosophila respiratory system, a network of interconnected tubules that delivers oxygen throughout the body. Yrt is mainly associated with the lateral membrane in tracheal cells and is enriched at septate junctions (SJs), as shown by its colocalization with the SJ marker Nrx-IV. Tracheal development was characterized in zygotic yrt mutants or yrt null embryos devoid of both maternal and zygotic yrt (yrtM/Z). Segmental tracheal placodes invaginated and established a normal branching pattern of tracheal tubes with intersegmental connections in both yrt and yrtM/Z mutants. yrtM/Z embryos display apical-basal polarity defects and irregularities in the tracheal epithelium at midembryogenesis. However, apical-basal polarity normalizes during terminal differentiation. In contrast, zygotic yrt mutants have only minor, if any, polarity defects in tracheal cells. The most apparent defect in yrt mutant trachea was the presence of excessively long and convoluted dorsal trunks compared to the straight dorsal trunks seen wild-type. The average dorsal trunk length was 417 ± 12 μm in wild-type embryos, whereas it was 476 ± 22 μm in yrt and 470 ± 23 μm in yrtM/Z mutant embryos. Similar but milder tube length defects were observed in other tracheal branches. In addition, the diameter of dorsal trunks in yrt and yrtM/Z mutants was uniform, but wider than in wild-type embryos. The average dorsal trunk diameter was 9.1 ± 0.6 μm in tracheal segment seven of wild-type embryos compared to 12.2 ± 0.9 μm in yrt and 12.7 ± 0.6 μm in yrtM/Z mutant embryos. In smaller branches, some diameter expansions were also apparent. In addition, the smaller tracheal branches in yrtM/Z embryos showed frequent interruptions indicating either breaks or a failure in the luminal accumulation of the 2A12 antigen. These findings indicate that Yrt regulates the size of tracheal tubes and supports the integrity of segmental tracheal branches. Remarkably, despite the prominent differences in the apical-basal polarity defects between yrt and yrtM/Z mutant embryos, both mutants exhibit similar dorsal trunk elongation and diameter defects. This finding suggests that the transient loss of apical-basal polarity in yrtM/Z embryos is not the cause of dorsal trunk size defects and that Yrt therefore has distinct functions during early and late stages of tracheal morphogenesis (Laprise, 2010).
The enlargement of the tracheal tube lumen observed in yrt mutants could be caused by an increase in cell number or an increase in the dimension of the apical surface of tracheal cells that surround the lumen. To address this question, the number of dorsal trunk cells in yrt mutants and wild-type embryos was counted. No significant differences in cell numbers between wild-type and yrt mutant embryos were found, indicating that the enlargement of tracheal tubes observed in yrt and yrtM/Z mutants must be accompanied by an increase in the dimension of the apical surface of tracheal cells (Laprise, 2010).
Several other mutants display enlarged dorsal trunks similar to yrt. One group of genes required for limiting tube length encodes components of the SJ, including the Na+/K+-ATPase (α and β subunits), Cora, Nrx-IV, Scribble (Scrib), Lachesin (Lac), Sinuous (Sinu), Megatrachea (Mega), and Varicose (Vari). Among these SJ proteins, Na+/K+-ATPase, Cora, Nrx-IV, and Scrib also play a role as basolateral polarity proteins. In Drosophila and other invertebrates, SJs appear as a ladder-like group of septa basal to the cadherin-based adherens junctions. SJs have functions analogous to vertebrate tight junctions, because they provide a transepithelial diffusion barrier. Yrt is not required for normal septa formation or localization of SJ components such as Cora but is essential for the barrier function of SJs. Zygotic yrt mutants show only minor defects in paracellular barrier function, whereas barrier function is fully compromised in yrtM/Z embryos. This observation supports the notion that transepithelial barrier function and the regulation of tube dimension are independent functions of Yrt because yrt and yrtM/Z mutant embryos show similar tube size defects. This conclusion is consistent with previous findings suggesting that the regulation of tracheal tube elongation and the regulation of the paracellular diffusion barrier are distinct roles of SJ proteins (Laprise, 2010).
A second class of mutants exhibiting abnormally long tracheal tubes are defective in the genesvermiform (verm) and serpentine (serp), which encode enzymes predicted to modify the chitin-based luminal extracellular matrix (ECM), and mutants of which show structural defects in the luminal ECM. The chitin matrix filling the tracheal lumen is transiently present during lumen morphogenesis and is critical for determining lumen diameter and length. Interestingly, all of the mutations affecting SJ components tested so far are associated with a failure to secrete Verm into the tracheal lumen. This suggests that SJ proteins control tube size by regulating apical secretion and remodeling of the apical chitin matrix. Although Yrt is required for the barrier function of SJs, it was found that luminal secretion of Verm and Serp was normal in yrt and yrtM/Z mutants. In contrast, low but detectable levels of Verm were observed in cora mutants. This finding suggests that Yrt regulates tracheal tube length through a pathway that is independent of Verm and Serp (Laprise, 2010).
During late stages of epithelia maturation, Yrt is known to restrict apical membrane growth in epidermal and photoreceptor cells by limiting Crb activity. This interplay between Yrt and Crb governs apical membrane size in stage 14 and later embryos when tracheal tube size is defined. This raises the possibility that Crb-dependent apical membrane growth is responsible for dorsal trunk expansion in yrt mutant embryos. To test this hypothesis, crb dosage was reduced in yrt mutant embryos by introducing one copy of a crb null allele into a yrt mutant background. Loss of one copy of crb suppressed the dorsal trunk elongation defects seen in yrt mutants, because the dorsal trunks appeared similar to wild-type in yrt crb/yrt + mutants. In addition, moderate Crb overexpression increased dorsal trunk length and diameter without interfering with the integrity of the tracheal epithelium or the secretion of Verm or Serp. These results show that Crb is required for promoting the expansion of tracheal tubes at late stages of embryogenesis. It was previously suggested that Crb also acts during early tracheal branch outgrowth in addition to its role in apical-basal polarity. Therefore, Crb plays a critical role at several steps of tracheal development. Together, these findings indicate that the antagonistic interactions between Yrt and Crb determine tracheal tube size (Laprise, 2010).
Verm levels in yrt crb/yrt + and in yrt/yrt mutants were indistinguishable, indicating that the minor reduction in Verm levels sometimes seen in yrt mutants are not the cause of the tracheal elongation defects. Accordingly, reduction of crb dosage in a verm or serp mutant background did not suppress tube size defects. Similarly, loss of one copy of crb in sinu mutant embryos, which fail to secrete Verm, had no impact on the length of dorsal trunks that remained enlarged as in sinu single mutants. These findings suggest that the apical secretion of matrix-modifying enzymes such as Verm and the control of Crb activity by Yrt are two independent and nonredundant modes of tracheal tube size regulation. The data also establish that epithelial tube size control by SJ-associated proteins involves Verm-dependent and Verm-independent mechanisms (Laprise, 2010).
To further characterize the function of SJ-associated polarity proteins in the regulation of tracheal tube size, tube elongation, the integrity of SJs, and the secretion of Verm in scrib, lethal giant larvae (lgl), and discs large (dlg) mutant embryos were examined. Zygotic loss of scrib, lgl, or dlg resulted in excessively long dorsal trunks, indicating that these genes are critical for tube size control. Zygotic loss of lgl expression caused fully penetrant defects in SJ paracellular barrier function, whereas zygotic scrib or dlg mutants did not have compromised transepithelial barriers. Luminal Verm deposition was not detected in lgl mutants but appeared near normal in dlg and in scrib mutants. Thus, Scrib and Dlg act like Yrt by controlling tracheal tube size through mechanisms distinct from Verm secretion (Laprise, 2010).
Further analysis concentrated on Scrib and it was asked whether this protein, like Yrt, controls tracheal tube size by negatively regulating Crb activity. Scrib together with Lgl and Dlg shows antagonistic interactions with Crb to regulate apical-basal epithelial polarity in early Drosophila embryos. However, the tracheal tube defects were not ameliorated in scrib crb/scrib + embryos compared to scrib single mutants. Thus, in contrast to Yrt, Scrib does not seem to limit tube length by restricting Crb activity. Because Yrt and Scrib appear to control tracheal tube size through different mechanisms, tests were performed to see whether yrt scrib double homozygous mutant embryos had a more severe phenotype. The double mutants had Verm levels that were lower compared to yrtM/Z and scrib mutants. Moreover, the tracheal defects also appeared more severe in yrt scrib mutants than in yrt null mutant embryos, particularly in the smaller-diameter branches. The defects in small-diameter branches of the yrt scrib double mutants are not likely caused by the reduction in Verm secretion, because the complete loss of Verm has only a mild effect on smaller branches. The enhanced severity of the yrt scrib double-mutant tracheal defects compared to the defects seen in yrt null embryos and the differences in the genetic interactions of scrib and yrt with crb suggest that Scrib and Yrt act in separate pathways to regulate the size of tracheal tubes and that Scrib does not act by modulating Crb activity. It is possible that Scrib acts through other proteins, such as proteins of the Par complex, that promote apical domain formation. Therefore, SJ-associated polarity proteins use at least two Verm-independent mechanisms to restrict the dimension of tracheal tubes (Laprise, 2010).
Cora is an SJ-associated protein required for optimal secretion of Verm. This suggests that Cora may control tracheal tube length through a Verm-luminal matrix pathway. However, Cora is also a basolateral polarity protein restricting the activity of Crb, which promotes Verm-independent expansion of the dorsal trunk. This led to an investigation of the functional relationship between Cora and Crb in tracheal morphogenesis. In the epidermis, a striking redundancy between yrt and cora is observed in the regulation of apical-basal polarity. Similarly, it was found that tracheal cells in yrt cora double mutants show severe apicalization defects characterized by a broad expansion of the surface distribution of Crb. The antigen recognized by the monoclonal antibody 2A12 was found surrounding tracheal cells and was not confined to the luminal cavity. In addition, the 2A12 antigen was associated not only with tracheal cells but also with epidermal cells. These tracheal defects seen in cora yrt mutant embryos mimic defects that result from high levels of Crb overexpression. This observation argues that the tracheal defects observed in cora yrt double-mutant embryos result from strong Crb overactivation, which is associated with a loss of basolateral polarity and an expansion of apical membrane character. Because epidermal cells did not acquire expression of the tracheal cell marker Tango, it is unlikely that epidermal cells adopt a tracheal cell fate in cora yrt mutants. The association of 2A12 with epidermal cells is therefore presumably due to the apicalization of tracheal cells, which would consequently secrete the 2A12 antigen not only on the luminal side but all around their cell surface, allowing the 2A12 antigen to diffuse and bind to surrounding cells. Accordingly, cuticle deposition, taking place at the apical membrane, was seen at both luminal and abluminal sides of tracheal cells overexpressing Crb (Laprise, 2010).
The data indicate that Yrt and Cora cooperate to control apical-basal polarity of tracheal cells by limiting Crb to the apical cell pole, but they do not reveal whether Cora and Crb interact to control the length of tracheal tubes. To address this question, cora crb/cora + embryos were examined for a suppression of the tracheal size defects seen in cora single mutants. Reduction of crb dosage suppresses tube overelongation defects resulting from the loss of Cora. This restriction of dorsal trunk elongation does not result from the restoration of Verm secretion, because the level of Verm present in the dorsal trunk lumen was as low in cora crb/cora + embryos as in single cora mutants. Together, these data suggest that Crb overactivation is the primary cause of epithelial tube length defects observed in the absence of Cora. Thus, Cora and Yrt act independently from each other to counteract Crb activity and maintain the appropriate size of epithelial tubes. Because the reduction of crb dosage does not rescue the verm mutant phenotype, it is concluded that the residual amount of Verm found in cora mutants is sufficient to maintain Verm pathway activity (Laprise, 2010).
This analysis suggests that basolateral proteins that are enriched at SJs have several critical functions in determining the size of epithelial tubes in the Drosophila tracheal system. This study shows that the increase in tube size is not caused by an increase in cell number and therefore must be accompanied by an increase in the apical surface area of individual tracheal cells. Given that Crb is a well-known regulator of apical membrane size, these findings suggest that the interplay between Yrt, Cora, and Crb modulates the dimensions of the apical surface of tracheal cells to control tracheal tube size. Moreover, this mechanism acts independently of and in parallel to a previously proposed pathway depending on the apical secretion of the matrix-modifying enzymes Verm and Serp, which requires several SJ-associated proteins. Yet another mechanism is revealed by results demonstrating that scrib mutants also have long trachea with normal Verm levels but that, in contrast to cora and yrt, tracheal defects in scrib mutants are not suppressed by loss of one copy of crb. Together, these findings suggest that basolateral proteins utilize at least three distinct mechanisms to regulate tube size in the Drosophila tracheal system. Unexpectedly, these mechanisms involve functional interactions between polarity proteins that appear to be different from those involved in establishing apical-basal polarity at earlier stages of development. For example, in promoting apical-basal polarity, Yrt and Cora act redundantly so that cora mutants show polarity defects only in a yrt mutant background, and polarity defects in yrt mutants are strongly enhanced by removal of Cora. In contrast, both cora and yrt single mutants show similar strong tracheal size defects. Furthermore, Scrib and Crb display antagonistic functional interactions during establishment of apical-basal polarity, but not during tracheal elongation. An important challenge for future investigations will be to uncover the adaptations in the molecular pathways that allow polarity proteins to contribute to different aspects of epithelial development (Laprise, 2010).
Lack of abstrakt function does not compromise the ability of cells to undergo their differentiation program or cellular behaviour typical of their differentiated state. Thus, neurite outgrowth still occurs, and pole cells are able to migrate over large distances, but both types of cells appear to fail to recognize or react to the cues that direct their morphogenetic behavior in the spatially appropriate manner. One reason for this defect might be a loss of subcellular order or polarity. Various aspects of cell polarity were indeed affected in abs mutants. The first abnormality detected was in the localization of mRNAs in the blastoderm. In the wild-type blastoderm, the mRNAs of several genes are distributed unevenly in the cell. For example, the transcripts of short gastrulation (sog) and crumbs (crb) are tightly apposed to the apical cell surface. In abs14B mutants, CRB mRNA is not seen apically but predominantly at the level of the nuclei, while SOG mRNA forms a gradient from the apical towards the basal side of the cell. In the case of crb, the mislocalization of RNA is also seen at later stages and in other tissues. For example, the tight apical localization in the hindgut epithelium is disturbed in abs mutants. Remarkably, the Crumbs protein is synthesized properly and targeted to the apical cell surface, showing that apical-basal cell polarity in the epithelial cells is correctly established and maintained in abs mutants (Irion, 1999).
Drosophila Stardust (Sdt), a member of the MAGUK family of scaffolding proteins, is a constituent of the evolutionarily conserved Crumbs-Stardust (Crb-Sdt) complex that controls epithelial cell polarity in the embryo and morphogenesis of photoreceptor cells (PRCs). Although apical localisation is a hallmark of the complex in all cell types and in all organisms analysed, only little is known about how individual components are targeted to the apical membrane. A structure-function analysis of Sdt was performed by constructing transgenic flies that express altered forms of Sdt to determine the roles of individual domains for localisation and function in photoreceptor cells. The results corroborate the observation that the organisation of the Crb-Sdt complex is differentially regulated in pupal and adult photoreceptors. In pupal photoreceptors, only the PDZ domain of Sdt - the binding site of Crb - is required for apical targeting. In adult photoreceptors, by contrast, targeting of Sdt to the stalk membrane, a distinct compartment of the apical membrane between the rhabdomere and the zonula adherens, depends on several domains, and seems to be a two-step process. The N-terminus, including the two ECR domains and a divergent N-terminal L27 domain that binds the multi-PDZ domain protein PATJ in vitro, is necessary for targeting the protein to the apical pole of the cell. The PDZ-, the SH3- and the GUK-domains are required to restrict the protein to the stalk membrane. Drosophila PATJ or Drosophila Lin-7 are stabilised whenever a Sdt variant that contains the respective binding site is present, independently of where the variant is localised. By contrast, only full-length Sdt, confined to the stalk membrane, stabilises and localises Crb, although only in reduced amounts. The amount of Crumbs recruited to the stalk membrane correlates with its length. These results highlight the importance of the different Sdt domains and point to a more intricate regulation of the Crb-Sdt complex in adult photoreceptor cells (Bulgakova, 2008).
Data presented in this study corroborate the view that distinct mechanisms control localisation of the Crb-Sdt complex in PRCs at different developmental stages. This conclusion is further supported by the observation that a truncated PATJ protein, consisting of only L27 and the first PDZ domain, is localised correctly during the first half of pupal development, but is delocalised in adult PRCs. The stability of the complex at pupal stages seems to depend only on Crb. In pupae, all core components of the complex are mislocalised in crb-mutant PRCs, whereas the absence of sdt, PATJ or Lin-7 does not affect apical localisation of the others. Accordingly, Sdt localisation at this stage only depends on its PDZ domain that binds the cytoplasmic tail of Crb. Neither the non-canonical L27N domain of Sdt, which is responsible for binding PATJ, nor the other protein-protein interaction domains are required for Sdt localisation in pupal PRCs (Bulgakova, 2008).
In the adult Drosophila eye, localisation of Crb-Sdt-complex core proteins to the stalk membrane is mutually dependent, with the exception of Lin-7, which is not required to localise other components. Similarly, in zebrafish the levels of the Crb orthologous proteins require the function of the Sdt orthologue Nagie oko. In the fly eye, changes observed at different developmental stages point to a transition in the mechanisms regulating the building and stability of the complex. This transition occurs gradually in the second half of pupal development. At the same time, Bazooka, which is associated with the adherens junctions in the first half of pupal development, accumulates in the cytoplasm. The transition also correlates with the formation of stalk membrane, which initiates around 55% pupal development and ultimately separates the apical plasma domain into two distinct compartments. This process seems to require additional, more complex control mechanisms, as reflected by the fact that several Sdt domains are required for its proper localisation at later stages. It is very possible that other, yet unknown components contribute to the stability and/or restriction of Sdt at the stalk membrane (Bulgakova, 2008).
Results presented in this study also suggest that in the adult Drosophila eye, localisation of Sdt occurs in several steps that rely on different domains. In the first step, Sdt is brought close to the apical membrane. This function is mediated by the N-terminus, including the two ECR domains and the N-terminal L27 domain. Since Par-6, a known binding partner of the ECR motifs, is localised basolaterally in adult PRCs, PATJ binding is more likely to be crucial for apical recruitment of Sdt. In fact, no localised Sdt is detected in PATJ-mutant adult PRCs. In the absence of all other domains besides the N-terminus (with the exception of L27C), Sdt proteins accumulate at the rhabdomere base, a specialised region that seems to have an important role in PRCs. Many proteins involved in morphogenesis, phototransduction or endocytosis, such as Drosophila moesin, TRPL (transient receptor potential-like) and Rab11, to mention just a few, are enriched there. The final step, recruitment of Sdt to the stalk membrane, requires the PDZ-, the SH3- and the GUK-domain. Whereas the PDZ-domain binds Crb, no binding partners for the SH3- and the GUK-domain are known. It was shown that these two domains can bind each other in vitro. Similar interactions between corresponding domains of the human MAGUK CASK were reported to occur either intramolecularly or intermolecularly between the GUK domain of human CASK and the SH3 domain of hDLG. In the MAGUK PSD-93, binding of a ligand to the PDZ domain releases intramolecular inhibition of the GUK domain by the SH3 domain. This possible complexity currently does not distinguish whether the failure to recruit Sdt to the stalk membrane upon removal of one of these domains is due to either the lack of binding additional partner(s) or the lack of intramolecular interactions, or both (Bulgakova, 2008).
Whereas Sdt is not required to restrict components of the Crb-Sdt complex to the apical membrane in pupal PRCs, the apical localisation of Par-6, a member of the Par-protein network, depends on Sdt at this developmental stage. Recently, several studies suggested a direct interaction between the Crb-Sdt and the PAR complex, but the proposed interactions differ with respect to the partners mediating the link. Results obtained from in vitro analysis have suggested a number of interactions: aPKC with both PATJ and the intracellular domain of Crb; the PDZ domain of Par-6 with either the N-terminus of Sdt and/or Pals1 or the C-terminus of CRB1 or CRB3; and the N-terminus of Par-6 with the third PDZ domain of PATJ. The observations that neither Crb nor PATJ localisation is affected in sdt-mutant pupal PRCs and that expression of Sdt-B2 in sdt-mutant PRCs completely restores Par-6 apical localisation, strongly suggests that in pupal PRCs the interaction between the Crb complex and Par-6 is mediated by the ECR motifs of Sdt. Sdt-A, which carries an additional 433 amino-acid-long stretch between ECR1 and ECR2, only partially restored apical recruitment of Par-6, suggesting that separation of ECR1 from ECR2 interferes with efficient interactions between the two proteins (Bulgakova, 2008).
The results show that in adult PRCs, sdt controls localisation and stability of Crb, PATJ and Lin-7 but the mechanisms differ. Whenever a Sdt protein is expressed that contains binding domains for PATJ or Lin-7, the amount of the latter is, independently of localisation, restored to wild-type levels. By contrast, Crb protein is stabilised only when Sdt is associated with the stalk membrane (expression of Sdt-A, Sdt-B2, Sdt-βL27C and Sdt-βN). Interestingly, none of the constructs used, including the two full-length variants, rescued Crb protein to wild-type levels. One possible explanation is that other, yet uncharacterised Sdt isoforms are expressed in the eye, which, together with Sdt-B2 and/or unknown interaction partners of the Crb-Sdt complex, regulate the amount of Crb at the stalk membrane. Additional Sdt isoforms are predicted by Flybase to exist. They mainly differ from the known forms in their N-termini, which suggests alternative interaction partners (Bulgakova, 2008).
One striking phenotype observed in PRCs mutant for crb, sdt or PATJ is the reduction of stalk-membrane length. This raises questions about how the Crb-Sdt complex regulates the size of this distinct apical membrane compartment. The results provide evidence that the amount of Crb protein is a crucial determinant of stalk-membrane length. This agrees with the observation that Crb overexpression increases stalk-membrane length. Interestingly, overexpression of a Crb protein that lacks the cytoplasmic domain and, hence, the binding site for Sdt, is sufficient to cause this increase. This suggests that either the transmembrane and/or extracellular domain of Crb regulates stalk-membrane growth. Sdt contributes to the stabilisation of Crb at the stalk and, hence, is indirectly involved in the control of stalk-membrane length. It will be interesting to explore the mechanism by which Crb regulates stalk-membrane length (Bulgakova, 2008).
The evolutionarily conserved apical determinant Crumbs (Crb) is essential for maintaining apicobasal polarity and integrity of many epithelial tissues. Crb levels are crucial for cell polarity and homeostasis, yet strikingly little is known about its trafficking or the mechanism of its apical localization. Using a newly established, liposome-based system described in this study, Crb was determined to be an interaction partner and cargo of the retromer complex (See Retromer-mediated sorting). Retromer is essential for the retrograde transport of numerous transmembrane proteins from endosomes to the trans-Golgi network (TGN) and is conserved between plants, fungi, and animals. Loss of retromer function results in a substantial reduction of Crb in Drosophila larvae, wing discs, and the follicle epithelium. Moreover, loss of retromer phenocopies loss of crb by preventing apical localization of key polarity molecules, such as atypical protein kinase C (aPKC) and Par6 in the follicular epithelium, an effect that can be rescued by overexpression of Crb. Additionally, loss of retromer results in multilayering of the follicular epithelium, indicating that epithelial integrity is severely compromised. These data reveal a mechanism for Crb trafficking by retromer that is vital for maintaining Crb levels and localization. A novel function is also shown for retromer in maintaining epithelial cell polarity (Pocha, 2011).
This study aimed to identify factors that interact with the cytoplasmic domain of the type I transmembrane protein Crumbs (Crb) and are involved in its trafficking. A strategy was devised to present the Crb cytoplasmic tail on liposomes, a method uniquely suited to recruit and identify coats, because it mimics the native configuration of a receptor tail at the membrane/cytosol interface (Pocha, 2011).
Proteoliposomes have been used successfully to identify coat complexes and their accessory proteins; however, these studies were restricted to short, chemically synthesized peptides, which severely limited the length of the cytoplasmic tail. To overcome this, this study redesigned the recruitment assay enabling the use of tails expressed and purified from E. coli. A bacterial expression plasmid was designed containing an N-terminal tandem affinity tag followed by a tobacco etch virus (TEV) protease cleavage site and a single cysteine for the chemical coupling to liposomes, to which the cytoplasmic tail of mouse Crb2 (amino acids R1246 to I1282) was fused (Pocha, 2011).
Because the levels of many transmembrane proteins are regulated by sorting decisions in the early (sorting) endosome, phosphatidylinositol 3-phosphate, the predominant inositol phospholipid of early endosomes, was incorporated into proteoliposomes to selectively enrich endosomal trafficking proteins. These proteoliposomes were used for recruiting cytosolic coat components and other interactors from brain extract], followed by protein identification by tandem mass spectrometry (MS/MS). Crb2 was chosen, because it is the predominant Crb gene expressed in the vertebrate brain. Importantly, the tails of all Crb proteins are highly conserved, suggesting that their trafficking mechanisms may also be conserved. Mass spectroscopic analysis confirmed that large amounts of Crb2 (∼600 MS2 spectra) were coupled onto the liposomes. The most abundant protein isolated (as determined by MS2 spectra) with an established role in the recognition and trafficking of transmembrane cargoes was the retromer subunit Vps35. In addition, Vps26B was identified. Western blotting confirmed the presence of Vps35 in our Crb2 recruitment reactions and showed it to be highly enriched relative to two independent controls (Pocha, 2011).
The mammalian retromer is composed of a cargo recognition subcomplex containing Vps35, Vps26, and Vps29 and a membrane interacting subcomplex consisting of SNX1/SNX2 and SNX5/SNX6 heterodimers. Because both Vps35 and Vps26 are crucial for cargo recognition and binding, the recruitment data suggest that Crb2 is a retromer cargo (Pocha, 2011).
To probe the hypothesis that Crb is a retromer cargo, internalization assays were performed by overexpressing Flag-hCrb2 in HeLa cells and analyzing the uptake of anti-Flag antibodies, visualizing compartments through which Crb2 traffics. Previous studies using the classical retromer cargo, the cation-independent mannose-6-phosphate receptor (ciMPR), have shown that retromer subunits and cargo decorate tubules that emanate from endosomes and travel toward the trans-Golgi network (TGN). This study observed colocalization of Crb2 with Vps35 on intracellular vesicles and tubules as well as an overlap with ciMPR- and galactosyltransferase (GalT) label. These data suggest that in HeLa cells, Crb2 travels in retromer-decorated tubules and can traffic via the TGN. However, it should be noted that it does not accumulate there like other retromer cargoes (e.g., ciMPR). Instead, Crb2 appears to undergo rapid transport back to the plasma membrane. RNA interference (RNAi) suppression of Vps35 in HeLa cells displays enhanced localization of Crb2 in lysosomal structures positive for Lamp-I, a phenotype described previously for other retromer cargoes. These data are all in line with Crb being a potential retromer cargo (Pocha, 2011).
To study the functional interaction between Crb and retromer in Drosophila, a previously generated null allele of Vps35, Vps35MH20 was used. As a result of strong maternal contribution, animals homozygous for this allele reach the third larval instar, allowing analysis of Crb in homozygous mutants. Because retromer is required for the retrieval of receptors from endosomes and thus the prevention of their lysosomal degradation, total Crb levels were analyzed and found to be reduced in Vps35MH20 heterozygote third-instar larvae compared to stage-matched wild-type (WT) larvae and dramatically reduced in Vps35MH20 homozygotes. Analysis of the mRNA levels of Crb showed that loss of Vps35 has very little effect on crb transcripts, suggesting that the dramatic reduction in Crb protein that was seem is due to posttranscriptional regulation of Crb by Vps35 (Pocha, 2011).
This led to an investigation of Crb at a cellular level. For this, two different epithelia, wing discs of third-instar larvae and the follicle epithelium, were chosen. Clones of Vps35MH20 mutant cells in wing disc epithelia, labeled with GFP using the mosaic analysis with a repressible cell marker (MARCM) system, were induced by heat shock-Flp at early larval stages. Crb localizes to the subapical region of wing disc epithelial cells. In agreement with results from western blot analysis, Crb staining is decreased in Vps35MH20 clones. Quantification of the fluorescence intensity in the clone and in surrounding tissues revealed that there is an ∼50% reduction in Crb signal within Vps35MH20 clones. The wing discs of Vps35MH20 homozygous animals are small and show variable morphological defects, presumably as a result of defective Wingless secretion. Analysis of Crb localization (by immunofluorescence) and protein levels (by western blotting) in Vps35MH20 hetero- and homozygous wing discs corroborated the data that were obtained using Vps35MH20 clones and larval lysate, respectively (Pocha, 2011).
The stability of the cargo-selective retromer subcomplex is dependent on the presence of all of its components [8 and 16]. To show that the loss of Crb is due to loss of retromer function rather than just the loss of Vps35, the effect was compared of Vps26 and Vps35 knockdown in the posterior compartment of the wing disc using engrailed-Gal4 to drive UAS-Vps26RNAi and UAS-Vps35RNAi. Hedgehog expression, which is unperturbed by loss of retromer, served to label the posterior compartment. Expression of either RNAi construct resulted in a clear reduction of Crb staining in the posterior compartment (∼50% reduction in fluorescence). Expression of engrailed-Gal4 alone had no effect on Crb. From these data, it is concluded that the retromer cargo recognition subcomplex is required for the maintenance of Crb levels (Pocha, 2011).
To further analyze the relation between Crb and retromer, the follicular epithelium, which surrounds the germline cysts of the Drosophila ovary, was examined. Previous work has identified key roles for Crb in polarization of the follicular epithelium. Crb localizes to the entire apical membrane of the follicle epithelial cells, with very little detectable in the cytoplasm. Vps35MH20 clones show strong reduction in Crb staining and protein loss from the apical membrane. Interestingly, although Crb staining at the apical membrane is strongly reduced, it is not detected at increased levels within the cytoplasm, suggesting that Crb is not merely mislocalized but reduced at the protein level, as shown in larvae. The cytoplasmic domain of Crb organizes an apical, membrane-associated protein complex by recruiting the scaffolding proteins Stardust (Sdt), DPATJ, and DLin-7. Therefore, the apical localization of Sdt in the follicular epithelium was assessed, and at was found to be heavily reduced in Vps35MH20 clones. Probing whole larval lysates from third-instar WT and Vps35MH20 hetero- and homozygotes for Sdt confirmed that at the protein level, like Crb, Sdt shows a dose dependence on Vps35. Thus, retromer function in maintaining Crb levels and function is conserved between wing and follicle epithelia (Pocha, 2011).
Interestingly, in some Vps35MH20 clones, the strict monolayer structure of the epithelium is disrupted and the tissue appears multilayered, an indication of polarity defects and characteristic of loss of Crb at early stages of follicle development, whereas loss at later stages results only in the mislocalization of other polarity proteins, without affecting tissue integrity. Multilayering was observed in 19% of Vps35MH20 clones in follicles between stages 7 and 10 and did not appear to be dependent on clone size or position. Given that various links between Crb and Notch have been reported, tests were performed to see whether the multilayering phenotype observed in the follicle epithelium upon loss of Vps35MH20 could be the result of defective Notch signaling. The expression of Notch and Hindsight, a transcription factor downstream of Notch signaling that represses proliferation in the follicle epithelium, were examined in Vps35MH20 mutant clones. Both showed wild-type expression, suggesting that Notch signaling is not affected by loss of retromer, similar to previous findings in the wing disc (Pocha, 2011).
To test whether the loss of Crb in retromer mutants is due to missorting of Crb to the lysosome, follicles harboring Vps35MH20 clones were incubated in leupeptin, a potent inhibitor of lysosomal proteases. After a 3 hr incubation, a dramatic accumulation of Crb was observed in punctae within the cytoplasm of Vps35MH20 cells, a phenomenon that was not seen in WT tissue or in follicles containing Vps35MH20 clones that were incubated in control medium lacking leupeptin. Additionally, colocalization of these intracellular Crb punctae with LysoTracker was observed. Together with the reduction of Crb protein levels and constant crb mRNA levels in Vps35MH20 larvae and tissue, these data strongly suggest that retromer ablation leads to lysosomal degradation of Crb, as observed for other retromer cargoes (Pocha, 2011).
To test whether retromer functions after endocytosis of Crb, internalization of Crb from the plasma membrane was blocked by expression of a dominant-negative construct of shibire (dynamin) or by incubating follicles in dynasore, a dynamin inhibitor. In Vps35MH20 clones, this resulted in the accumulation of Crb at the plasma membrane, confirming that retromer is indeed transporting Crb after internalization from the plasma membrane (Pocha, 2011).
Crb is required, together with atypical protein kinase C (aPKC), to restrict Bazooka/Par3 to the zonula adherens, an adhesion belt at the apex of epithelial cells, in the follicle epithelium, and in photoreceptor cells, thus excluding it from the apical membrane and specifying the border between apical and lateral domains. In previous studies, it was shown that the localization of aPKC and Par6 was dependent on Crb. To test whether loss of retromer phenocopies the loss of Crb, aPKC and Par6 localization were examined in follicles containing Vps35MH20 clones. Indeed, the level of both proteins is reduced at the apical surface in Vps35MH20 clones. Interestingly, unlike Crb and the Crb complex member Sdt, Par6 and aPKC protein levels are not reduced in Vps35MH20 mutant larvae. Therefore, it is likely that the loss of Par6 and aPKC from the apical membrane of Vps35MH20 clones in the follicle epithelium is due to loss of cell polarity in the absence of Crb rather than loss of the proteins themselves (Pocha, 2011).
To test this, Crb was overexpressed in Vps35MH20 clones. Because overexpression of Crb causes defects in epithelial cell polarity, Crb overexpression was induced using GABFc204 Gal4, a follicle epithelium-specific driver that starts expression late in follicle development (stage 8). Thereby, it was possible to rescue the apical localization of Par6 and Sdt. This rescue did not appear to be dependent on clone size or location. From these data, it is concluded that the loss of polarity observed in retromer mutant clones is the direct result of loss of Crb (Pocha, 2011).
The identification of Crb as a retromer cargo confirms the hypothesis that one crucial step in the regulation of Crb occurs at the early (sorting) endosome and, importantly, fills a gap in the current understanding of Crb trafficking. Previous reports showed that transport of Crb to the plasma membrane is reliant on Rab11, the exocyst and Cdc42 in Drosophila embryonic epithelia. Internalization of Crb from the plasma membrane into endosomes is mediated by the syntaxin Avalanche and Rab5. This study has shown that retromer is responsible for sorting Crb away from the degradative pathway and into a recycling one, thus allowing a high level of control over the amount of cellular Crb, previously shown to be vital for maintaining epithelial polarity and integrity, as demonstrated by numerous loss- and gain-of-function studies. Interestingly, retromer was previously shown to play a role in the apical delivery of the polymeric immunoglobulin receptor (pIgR) in Madin-Darby canine kidney cells. However, as for Crb, it remains unclear whether this transport occurs via the TGN, via recycling endosomes, or through alternative pathways. The exact trafficking itinerary of Crb following recycling by retromer remains unclear and may depend upon the purpose of Crb recycling (Pocha, 2011).
Which function of Crb is the prime target of retromer-driven retrieval? Is this a Crb level-sensing mechanism, in which retromer regulates the amount of protein at the plasma membrane, which is crucial for cell homeostasis? To date, all known functions of Crb require an intact Crb complex. By controlling the recycling of Crb and thereby its level at the plasma membrane, retromer could define the amount of Crb available for complex formation. Alternatively, it is tempting to speculate that Crb, much like Wntless (Wls), acts as a transport receptor and that apical delivery of its (yet to be identified) ligand or many ligands is the main purpose of its recycling to the TGN. These are fascinating hypotheses that will be the focus of future research (Pocha, 2011).
Crumbs (Crb), a cell polarity gene, has been shown to provide a positional cue for the apical membrane domain and adherens junction during Drosophila photoreceptor morphogenesis. It has recently been found that stable microtubules in developing Drosophila photoreceptors were linked to Crb localization. Coordinated interactions between microtubule and actin cytoskeletons are involved in many polarized cellular processes. Since Spectraplakin is able to bind both microtubule and actin cytoskeletons, the role of Spectraplakin was analyzed in the regulations of apical Crb domain in developing Drosophila photoreceptors. The localization pattern of Spectraplakin, Drosophila Short stop, in developing pupal photoreceptors showed a unique intracellular distribution. Spectraplakin localized at rhabdomere terminal web which is at the basal side of the apical Crb or rhabdomere, and in between the adherens junctions. The spectraplakin mutant photoreceptors showed dramatic mislocalizations of Crb, adherens junctions, and the stable microtubules. This role of Spectraplakin in Crb and adherens junction regulation was further supported by spectraplakin's gain-of-function phenotype. Spectraplakin overexpression in photoreceptors caused a cell polarity defect including dramatic mislocalization of Crb, adherens junctions and the stable microtubules in the developing photoreceptors. Furthermore, a strong genetic interaction between spectraplakin and crb was found using a genetic modifier test. In summary, a unique localization of Spectraplakin was found in photoreceptors, and the role of spectraplakin in the regulation of the apical Crb domain and adherens junctions was identified through genetic mutational analysis. These data suggest that Spectraplakin, an actin-microtubule cross-linker, is essential in the apical and adherens junction controls during the photoreceptors morphogenesis (Mui, 2012).
This study investigated where Shot localizes compared to apical membrane domain, adherens junction, stable microtubule, and rhabdomere, in mid-stage pupal photoreceptors. The localization results of Shot in pupal eyes strongly indicate that Shot localizes in between the adherens junctions, at the basal side of the apical domain, and at the apical side of the stable microtubules. The rhabdomere terminal web (RTW), where the Shot localizes, may be the interface where the stable microtubules and F-actins of rhabdomere meet together. Since Shot has an actin-microtubule cross-linking activity, Shot might cross-link the two cytoskeletons of actin and microtubules at the RTW (Mui, 2012).
This genetic interaction data of shot and crb strongly suggests that Shot may provide an additional cue for Crb in photoreceptor development because the rough-eye phenotype caused by Crb overexpression was further enhanced by reduced shot gene dosage (shot/+). The relationship between crb and shot might be one of the following possibilities; (1) shot acts at the upstream of crb, (2) shot acts at the downstream of crb, or (3) shot and crb control the parallel pathway in photoreceptor development. From comparative genetic analysis, Crb and Shot require each other reciprocally to localize at their target sites of rhabdomere stalk (apical domain) and RTW (Mui, 2012).
Genetic analysis of the shot mutation strongly indicates that Shot modulates the apical Crb membrane domain during rhabdomere elongation. The apical membrane modulation activity of spectraplakin was further confirmed by spectraplakin overexpression which caused a dramatic misplacement of the apical membrane domain. It is postulated that the spectraplakin might affect the actin (rhabdomere) and/or microtubules based on its dual binding capacity for actin/microtubule, and its activity as an actin/microtubule cross-linker. Therefore, its role in photoreceptor morphogenesis might require its dual actin/microtubule binding, which will be supported by the absence of Crb-mislocalization activity of ShotC, which lacks the actin-binding domain. The Crb-mislocalization activity of ShotA might be based on its dual actin/microtubule binding ability (Mui, 2012).
One important point of the potential cross-talk between the crb and shot is their different spatial localizations in photoreceptors. How does one protein affect the other that is at a different localization in the cell? There might be at least several following possibilities; (1) a potential interaction when they co-localize during the trafficking before their final targeting, (2) a potential interaction in previous developmental time, or (3) a potential interaction at the interface where the two subcellular compartments meet. The 'RTW' is the place where the microtubules and F-actins (rhabdomere) meet each other. Therefore, the Shot might have a potential role in the regulation of stable microtubules and rhabdomere, and thereby the localizations of Crb and adherens junctions might be affected in photoreceptor cells (Mui, 2012).
Shot has a microtubule organizing activity. Therefore, the expected result in the loss of shot is the defects of stable microtubules which were observed in the loss-of-function study of shot mutation. Furthermore, the stable microtubules were mostly defected in the gain-of-function of ShotA-GFP overexpression. Therefore, the primary target of Shot seems to be the stable microtubules. Then, the defected microtubules may further affect the Crb and E-cad through the microtubule-based trafficking and/or other microtubule-based cell polarity. But the other possibilities of direct targeting of Shot toward Crb/E-cad or actin-based cell polarity cannot be excluded. ShotA-GFP overexpression results in ectopic localization of acetylated-tubulin (Acetub; stable microtubulin) around the cells which could be caused by the direct binding of ShotA-GFP to the Acetub. However, another possibility of the indirect mislocalization of Acetub caused by the mislocalized 'RTW' by the ShotA-GFP cannot be excluded (Mui, 2012).
Another potential possibility of Spectraplakin's function in the regulation of apical membrane domain might be through microtubule plus-end-tracking proteins (+TIPS). The +TIPS belong to the class of microtubule-associated proteins, and link microtubule ends with apical actin cytoskeleton. In Drosophila muscle-tendon junctions, Shot regulates microtubule cytoskeleton by forming a complex with the EB1 and APC of +TIPS. Therefore, there is a potential possibility of Shot and +TIPS interaction in apical domain control during photoreceptor morphogenesis (Mui, 2012).
This study has shown that Spectraplakin/Shot is required for correct localization of Crb, adherens junctions, and stable microtubules in the photoreceptors and disruption of Shot function affects photoreceptor morphogenesis. The data strongly suggests that Spectraplakin/Shot plays important functions in the modulation of cell membrane domains including the apical Crb domains of photoreceptors during pupal eye development. Evolutionary conservation in the structure and function of eye morphogenesis genes makes the Drosophila eye an excellent model for studying the genetic and molecular basis of retinal cell organization. Future work will help to uncover other genes that might affect the Crb positioning during the extensive morphological growth phase of the Drosophila pupal eye. Given the high degree of evolutionary conservation of Crb and Spectraplakin genes from Drosophila to higher mammals including humans, similar cooperative mechanism between Crb and Spectraplkain could have a role in the development and degeneration of human photoreceptor (Mui, 2012).
Epithelial cell polarity is essential for animal development. The scaffold protein Bazooka (Baz/PAR-3) forms apical polarity landmarks to organize epithelial cells. However, it is unclear how Baz is recruited to the plasma membrane and how this is coupled with downstream effects. Baz contains an oligomerization domain, three PDZ domains, and binding regions for the protein kinase aPKC and phosphoinositide lipids. With a structure-function approach, this study dissected the roles of these domains in the localization and function of Baz in the Drosophila embryonic ectoderm. A multifaceted membrane association mechanism localizes Baz to the apical circumference. Although none of the Baz protein domains are essential for cortical localization, it was determined that each contributes to cortical anchorage in a specific manner. It is proposed that the redundancies involved might provide plasticity and robustness to Baz polarity landmarks. Specific downstream effects were identified, including the promotion of epithelial structure, a positive-feedback loop that recruits aPKC, PAR-6 and Crumbs, and a negative-feedback loop that regulates Baz (McKinley, 2012).
The PDZ domains of Baz are dispensable for its localization. The current results show that this is due to redundant mechanisms in other parts of the protein. In fact, each PDZ domain plays a unique role in Baz positioning and activity in the Drosophila embryonic ectoderm. The following main roles were identified for the PDZ domains: PDZ1 and PDZ3 recruit Baz to the apical domain, PDZ2 mediates downstream effects on epithelial structure and PDZ1 promotes the turnover of Baz. Each domain also has minor effects that might result from distinct activities or secondary effects of their main activities: PDZ1 and PDZ3 have non-essential but detectable effects on epithelial structure and PDZ2 promotes weak membrane binding (McKinley, 2012).
PDZ1 and PDZ3 activities involve at least two sub-regions of the domains. PDZ domains typically use their peptide-binding pocket to bind the C-termini of their protein partners, but regions outside of these pockets can also mediate interactions. PDZ1 promotes apical surface and circumferential localization independently of its peptide-binding pocket, and its peptide-binding pocket plays a distinct role in promoting Baz turnover. These opposing activities might form a negative-feedback loop that regulates localization of Baz. By contrast, PDZ3 appears to solely promote Baz localization. It can promote apical surface and circumferential localization independently of its peptide-binding pocket, whereas its peptide-binding pocket specifically promotes circumferential anchorage. The binding partners that engage these sites are unknown. However, in vitro studies have shown that the C-termini of Arm and Ed can bind Baz PDZ1-3 in tandem, and that the C-terminus of PTEN can bind Baz PDZ2-3. Binding partners for regions outside the peptide-binding pockets have not been identified for Baz PDZ domains, but the binding of rat PAR-3 PDZ2 to PIPs involves outside regions, as does the binding of C. elegans PAR-3 PDZ1 to PAR-6 (McKinley, 2012).
A major function of Baz PDZ domains is to maintain the protein around the apical circumference. PDZ1 and PDZ3 use peptide-binding-pocket-independent mechanisms to generally localize Baz to the apical domain, but Baz is focused around the apical circumference through mechanisms involving the peptide-binding pockets of these domains. Without these pockets, Baz can saturate its remaining apical anchors and mislocalizes in puncta over the apical surface. PDZ1 appears to prevent this mislocalization by reducing protein levels below saturation, but PDZ2 and PDZ3 might directly bind circumferential proteins or promote an active redistribution of Baz. This activity is weaker for PDZ2 versus PDZ3 (with the former requiring oligomerization and the latter not) and it is possible that the localization activity of PDZ2 is a by-product of its binding to downstream effectors localized to the apical circumference. Thus, it is proposed that PDZ3 has the most direct role in anchoring Baz around the apical circumference (McKinley, 2012).
Because Baz can localize to the apical membrane without its PDZ domains, other localization mechanisms are also involved. The results clarify the importance of two additional mechanisms. The first involves dynamic interactions with apical polarity proteins. Baz has been shown to recruit aPKC to the apical domain as epithelial polarity is first established and to maintain aPKC during later stages. However, aPKC normally localizes at the apical surface with PAR-6 and the Crb complex above Baz and adherens junctions. When BazδPDZ1-3 forms puncta over the apical surface domain it recruits aPKC, PAR-6 and Crb, but the proteins then segregate locally, mimicking their associations around the apical circumference. Indeed, BazδPDZ1-3 might separate from the apical polarity proteins by two known mechanisms: the release of aPKC after it phosphorylates its binding site on Baz, and the loss of PAR-6 binding as a result of competition with Crb. The segregation of aPKC, PAR-6 and Crb from BazδPDZ1-3 suggests that they would not form a stable anchorage site for Baz. However, removal of the aPKC binding region from BazδPDZ1-3 (but not full-length Baz) severely weakens its cortical localization. This suggests that a positive feedback loop exists between Baz and aPKC to maintain localization of each protein. It is proposed that the proteins undergo continuous cycles of attraction and local repulsion to maintain their close but non-overlapping positioning around the apical domain (McKinley, 2012).
An additional Baz localization mechanism involves PIPs. A conserved region of the C-terminal tail of Baz has been shown to bind PIPs (Krahn, 2010), and it was found that the apical surface puncta of BazδPDZ1-3 colocalize with plasma membrane domains enriched with PIPs. Although deletion of the PIP binding region had no effect on full-length Baz (Krahn, 2010), deleting it and the PDZ domains together strongly disrupts plasma membrane binding. Thus, the aPKC binding region and the PIP binding region might both mediate apical localization of Baz in the absence of the PDZ domains. These anchorage mechanisms are also dependent on the oligomerization of Baz, because BazδOD+δPDZ1-3 shows minimal cortical localization. Moreover, the mechanisms appear to support each other because the aPKC binding region cannot compensate for the loss of the PIP binding region from BazδPDZ1-3 and vice versa. Also, membrane binding is abrogated with deletion of the Baz C-terminus, including the aPKC and PIP binding regions (Krahn, 2010). Perhaps the continuous cycles of attraction and local repulsion between Baz, aPKC, PAR-6 and Crb are partly staged on a platform of PIPs (McKinley, 2012).
Interactions with these proteins and lipids might also explain the ability of Baz to partially maintain epithelial structure without its PDZ domains. Indeed, deletion of the aPKC binding region from full-length Baz abrogates its rescue activity (Krahn, 2010), as does deletion of the OD and the PIP binding region, when expressed with a weaker driver, but not a stronger driver (McKinley, 2012).
The results indicate that there are at least five sites in Baz, in addition to its OD, that are involved in membrane localization. No single site is essential, and different combinations of interactions are sufficient for anchorage. This suggests that the individual anchorage mechanisms are relatively weak, as has been shown for the PIP binding region (Krahn, 2010). Cortical localization through multiple weak interactions might provide plasticity and robustness for the role of Baz/PAR-3 as a multifunctional polarity landmark (McKinley, 2012).
The membrane-association mechanism of Baz would allow fine regulation of protein positioning. For example, Baz becomes planar polarized around the apical domain to regulate germband extension in the Drosophila embryo. Rho kinase has been shown to reduce Baz at anterior and posterior cell edges by phosphorylating the Baz C-terminus and inhibiting PIP binding. However, Baz is not fully lost from these edges. Thus, planar polarity might arise from a partial set of membrane-association mechanisms acting along anterior-posterior edges and a more complete set acting at dorsal-ventral edges. Apical localization of Baz is also altered in amnioserosa cells to regulate apical constriction during dorsal closure. Here, Baz forms apical surface puncta in addition to its circumferential localization. Although the mechanisms for this redistribution are unclear, the work suggests that it might involve weakening of PDZ domain activities. Intriguingly, Ed, an in vitro binding partner of the PDZ domains, is specifically absent in the amnioserosa. However, this might not fully explain the redistribution because Baz appears to be localized normally in most ectodermal cells of ed mutants. A more dramatic cellular reorganization occurs as neuroblasts delaminate from the epithelium. As this occurs, adherens junctions and Crb are lost from the cells, but Baz is retained apically and engages with new partners to direct asymmetric cell division after delamination. The mechanisms regulating Baz during this transition are unknown, but its multifaceted membrane-association mechanism might ensure robust apical localization as Baz exchanges molecular interaction networks (McKinley, 2012).
Redundancies in Baz/PAR-3 scaffold activity might also have permitted co-evolution with polarity networks to organize eggs, single-cell embryos, epithelial cells, neurons and stem cells. Indeed, roles for Baz/PAR-3 PDZ domains appear to have diverged. In C. elegans, PDZ2, but not PDZ1 or PDZ3, is essential for embryogenesis, as in Drosophila, but PDZ2 of C. elegans PAR-3 was also shown to be required for proper localization, in contrast to Baz PDZ2. Also, mammalian PDZ2 has also been shown to mediate membrane binding through PIPs, but key residues involved in the interaction are not conserved in Drosophila or C. elegans (McKinley, 2012).
The Baz localization mechanism appears to be unique among characterized polarity scaffold proteins. Other scaffolds also involve multiple mechanisms, but typically there is a primary mechanism that localizes the scaffold to the membrane and secondary mechanisms that focus localization to a particular site. For example, the leucine-rich repeats of Scribble are crucial for its cortical localization in Drosophila epithelia, whereas its second PDZ domain promotes septate junction localization. In Drosophila, the Hook domain of Discs Large is crucial for plasma membrane targeting, whereas particular PDZ domains promote septate junction localization in epithelia and synapse localization in neurons. Similar 'twostep' localization mechanisms have been described for C. elegans and mammalian Discs large, mammalian PSD-95, Drosophila Inscuteable and Pins and Drosophila Stardust. Contrasting these mechanisms, no single site in Baz is essential for membrane recruitment in ectodermal cells. However, any of these mechanisms could be context dependent. Scaffolds shown to localize through a two-step mechanism in one context might use a multifaceted membrane-association mechanism in another, and Baz could localize by one-step or two-step mechanisms in other cell types or developmental stages (McKinley, 2012).
This study has identified a multifaceted membrane-association mechanism that localizes Baz to the apical circumference in epithelial cells. This mechanism integrates with downstream pathways, involving both negative- and positive-feedback loops, which regulate Baz and epithelial polarity. It is important to define the partners for the interaction sites involved, and to dissect how these interactions are controlled (McKinley, 2012).
Epithelial tissues are composed of polarized cells with distinct apical and basolateral membrane domains. In the Drosophila ovarian follicle cell epithelium, apical membranes are specified by Crumbs (Crb), Stardust (Sdt), and the aPKC-Par6-cdc42 complex. Basolateral membranes are specified by Lethal giant larvae (Lgl), Discs large (Dlg), and Scribble (Scrib). Apical and basolateral determinants are known to act in a mutually antagonistic fashion, but it remains unclear how this interaction generates polarity. A computer model of apicobasal polarity was build that suggests that the combination of positive feedback among apical determinants plus mutual antagonism between apical and basal determinants is essential for polarization. In agreement with this model, in vivo experiments define a positive feedback loop in which Crb self-recruits via Crb-Crb extracellular domain interactions, recruitment of Sdt-aPKC-Par6-cdc42, aPKC phosphorylation of Crb, and recruitment of Expanded (Ex) and Kibra (Kib) to prevent endocytic removal of Crb from the plasma membrane. Lgl antagonizes the operation of this feedback loop, explaining why apical determinants do not normally spread into the basolateral domain. Once Crb is removed from the plasma membrane, it undergoes recycling via Rab11 endosomes. The results provide a dynamic model for understanding how epithelial polarity is maintained in Drosophila follicle cells (Fletcher, 2012).
These above results define an apical positive feedback loop that centers on endocytic regulation of Crb. If such a positive feedback loop exists, it must be antagonized by the basolateral determinants to prevent spreading of apical determinants into the basolateral domain. In the computer model, ectopic spreading of apical determinants caused by simulated inhibition of endocytosis (strongly reducing the rate at which apical determinants are removed from the plasma membrane) can be counteracted simply raising the number of basolateral determinants by 5-fold. In follicle cells, inhibiting endocytosis with RNAi against the AP2/clathrin component AP50 leads to ectopic spreading of apical determinants into the basolateral domain, as in the model. Overexpression of Lgl-GFP was sufficient to restore normal polarity even in the presence of AP50 RNAi, again similar to the simulations. Furthermore, expression of Lgl-GFP also rescued the spreading of apical determinants caused by Rab5 RNAi or overexpression of Crb. These results suggest that Lgl may be a rate-limiting basolateral determinant and that it acts to inhibit positive feedback among apical determinants and thereby promote endocytic removal of Crb from the basolateral membrane (Fletcher, 2012).
Once Crb has been endocytosed by the AP2/clathrin machinery, it could be either degraded in the lysosome or recycled. Recent evidence indicates that Crb avoids the lysosome due to the action of the retromer machinery. The recycling endosome protein Rab11 is essential for Crb to remain at the plasma membrane in embryos. By costaining for Crb and Rab11 in follicle cells, it was possible to detect many endosomes that are positive for both proteins. Furthermore, when Rab11 is knocked down by RNAi in follicle cells, a loss of Crb from the plasma membrane was detected and an accumulation in enlarged endosomes. In contrast, RNAi of Rab5 causes accumulation of Crb at the plasma membrane. Accordingly, the Rab11 RNAi phenotype -- unlike that of Rab5 -- cannot be suppressed by coexpression of Lgl-GFP. These results confirm that Crb undergoes Rab11-mediated recycling to maintain its polarized plasma membrane localization (Fletcher, 2012).
One difference between the computer model and in vivo data is that inactivation of apical determinants in the model leads to complete loss of apical determinants from the membrane. However, in follicle cells, mutation of crb does not cause complete loss of apical aPKC from the plasma membrane. This residual aPKC is due to the Bazooka protein (Baz/Par3), which-like Crb-is able to bind to aPKC-Par6 and normally localizes to adherens junctions but can also occupy the apical membrane in the absence of Crb. Whether the Baz system operates by the same positive feedback principle as the Crb system remains to be explored (Fletcher, 2012).
These findings indicate that polarization of Crb in the follicle cell epithelium depends on the combination of two principles: positive feedback and mutual antagonism. The apical domain forms where Crb can recruit additional Crb molecules via Crb-Crb interactions, recruitment of Sdt and aPKC-Par6-cdc42, aPKC phosphorylation of the Crb FERM-binding domain, and recruitment of the FERM-domain protein Ex and its binding partner Kib. Although direct binding between these factors in follicle cells was not shown, work in other model systems indicates that they do bind directly. Disruption of any element of this feedback loop results in endocytosis of Crb from the plasma membrane. In contrast, ectopic activation of various components of this feedback loop-by overexpression of Crb, cdc42V12, or aPKCdeltaN-stabilizes Crb and the other apical determinants at the plasma membrane. The basolateral domain forms where Crb is endocytosed from the plasma membrane because Lgl (which can bind to aPKC-Par6 and inhibit aPKC kinase activity), presumably prevents Crb from engaging in a productive interaction with the other apical determinants, thereby disrupting Crb self-recruitment (Fletcher, 2012).
In conclusion, the model explains how epithelial polarity is a property of a complex system that can emerge spontaneously from the nature of the interactions between apical and basolateral polarity determinants. The principle of combined positive feedback and mutual antagonism outlined in this study in Drosophila follicle cells may prove to be widely used in the generation of polarity in many different cellular contexts (Fletcher, 2012).
In epithelial tissues, growth control depends on the maintenance of proper architecture through apicobasal polarity and cell-cell contacts. The Hippo signaling pathway has been proposed to sense tissue architecture and cell density via an intimate coupling with the polarity and cell contact machineries. The apical polarity protein Crumbs (Crb) controls the activity of Yorkie (Yki)/Yes-activated protein, the progrowth target of the Hippo pathway core kinase cassette, both in flies and mammals. The apically localized Four-point-one, Ezrin, Radixin, Moesin domain protein Expanded (Ex) regulates Yki by promoting activation of the kinase cascade and by directly tethering Yki to the plasma membrane. Crb interacts with Ex and promotes its apical localization, thereby linking cell polarity with Hippo signaling. This study shows that, as well as repressing Yki by recruiting Ex to the apical membrane, Crb promotes phosphorylation-dependent ubiquitin-mediated degradation of Ex. Skp/Cullin/F-boxSlimb/beta-transducin repeats-containing protein (SCFSlimb/beta-TrCP) was identifed as the E3 ubiquitin ligase complex responsible for Ex degradation. Thus, Crb is part of a homeostatic mechanism that promotes Ex inhibition of Yki, but also limits Ex activity by inducing its degradation, allowing precise tuning of Yki function (Ribeiro, 2014).
Recent work in flies and mammals has implicated the apical polarity determinant Crb as a transmembrane receptor for Hpo signaling. Accordingly, clonal loss of crb function leads to Yki derepression and increased growth. However, the observation that Crb is required for Ex membrane localization apparently conflicts with the finding that Crbintra overexpression reduces Ex levels and, like crb loss of function, leads to increased Yki activity. This study reconciles these findings by showing that Crb is not only required for Ex tethering at the apical membrane but also for promoting its degradation via the SCFSlimb/β-TrCP E3 ubiquitin ligase. Indeed, immediately downstream of its FERM domain, Ex contains a sequence that conforms to the D/S/TSGφXS consensus sequence for canonical Slmb targets, which is conserved in Ex orthologs from arthropod species but absent from related FERM domain proteins such as Moe and Mer. In addition, loss of Slmb increases Ex levels in vivo, whereas Slmb depletion prevents Crbintra-induced Ex degradation in cell culture. Thus, in crb mutants, Ex no longer reaches the apical membrane and is protected from degradation in the cytoplasm, where it accumulates but is presumably unable to repress Yki. When Crbintra is overexpressed, Ex turnover at the membrane (or in an endocytic compartment if Ex degradation occurs after Crb internalization) is accelerated, leading to its depletion and consequent Yki activation. Therefore, in both cases, the outcome is Yki derepression, albeit for different reasons (Ribeiro, 2014).
How is the Slmb:Ex association regulated by Crb? Previous observations point to the involvement of a phosphorylation-dependent degradation mechanism, because Crb induces Ex phosphorylation. Accordingly, mutation of the phosphodegron (Ser453) or the putative priming site (Ser462) leads to loss of Slmb:Ex association and Ex stabilization. The most obvious candidate Ex kinase in this context is aPKC, which is known to interact with the Crb polarity complex and to phosphorylate Crb at its FBM. However, aPKC is thought to be recruited to the Crb complex via Par-6, which interacts with Crb through the PBM at the C terminus of the Crb intracellular domain. This is inconsistent with the fact that the PBM is dispensable for Crb to promote both Ex phosphorylation and degradation. Moreover, a dominant-negative version of aPKC is unable to rescue the overgrowth phenotype of Crbintra overexpression in the wing. The Hpo downstream kinase Wts is another attractive candidate, because mammalian LATS1/2 promotes YAP phosphorylation on Ser381, providing priming for phosphorylation by CK1δ/ε and triggering degradation by SCFβ-TrCP. However, neither Ser462 nor Ser453 conform to the Wts/LATS consensus. The identity of the kinases therefore remains open (Ribeiro, 2014).
Regulation of tissue growth during development and adult life depends on the maintenance of tissue architecture, which, in turn, relies on cell-cell and cell-matrix interactions. Due to its intimate coupling to polarity and cytoskeletal regulators, the Hpo signaling pathway is thought to sense epithelial integrity and couple tissue architecture to growth control. In particular, YAP has been shown to sense contact inhibition in cell culture, such that its progrowth activity is silenced as cultured epithelial cells reach confluence. This is thought to depend on the assembly of tight junctions, leading to repression of YAP by the mammalian Crb complex. Recent work in flies and zebrafish has suggested that Crb can form homodimers in trans. This suggests that Crb mediates local cell-cell communication in epithelial tissues. Indeed, clonal loss of Crb leads to Crb depletion in the junctions of wild-type cells abutting the crb mutant tissue. This loss of Crb at the clone boundary is mirrored by loss of Ex, which is dependent on Crb for its apical localization. Thus, loss of Crb caused by loss of polarity or cell death can be transmitted to neighboring cells. This has been proposed as a means of cell- cell communication to induce regenerative proliferation through Yki activation. Indeed, genetic induction of epithelial wounds in imaginal discs has been shown to up-regulate Yki activity in cells neighboring the wound. In addition, Yki/ YAP is activated and promotes tissue regeneration upon injury in the vertebrate and fly intestine, as well as in the mouse liver (Ribeiro, 2014).
These findings suggest that Crb (and perhaps other polarity proteins) functions as a sensor of cell density and tissue integrity during development. In this model, disruption of Crb function would lead to Yki/YAP derepression, which, upon tissue injury, would allow regenerative growth to ensue. Another interesting question is whether liganded Crb behaves differently to unliganded Crb with respect to regulation of Ex stability. For example, it is possible that unliganded Crb promotes Ex turnover faster than its liganded counterpart, which might provide a sensitive means of responding to the status of neighboring cells. Further work will be needed to resolve this issue. The present work indicates that Crb fulfills a dual function in Hpo signaling, both recruiting Ex apically to repress Yki activity and promoting Ex turnover through phosphorylation and Slmb-dependent degradation. This mechanism could ensure constant turnover of Ex at the apical membrane, allowing Yki activity to rapidly respond to changing environmental conditions. This dynamic equilibrium could be particularly important to promote fast tissue regeneration upon injury (Ribeiro, 2014).
Crumbs (Crb) family proteins are crucial for cell polarity. Recent studies indicate that they are also involved in growth regulation and cancer. However, it is not well-understood how Crb participates in mitotic processes. This study reports that Drosophila Crb is critically involved in nuclear division by interacting with Xeroderma pigmentosum D (XPD). A novel gene named galla-1 (CG30152) was identified from a genetic screen for crb modifiers. Galla-1 protein shows homology to MIP18, a subunit of the mitotic spindle-associated MMS19-XPD complex. Loss-of-function galla-1 mutants show abnormal chromosome segregation, defective centrosome positions and branched spindles during nuclear division in early embryos. Embryos with loss-of-function or overexpression of crb show similar mitotic defects and genetic interaction with galla-1. Both Galla-1 and Crb proteins show overlapping localization with spindle microtubules during nuclear division. Galla-1 physically interacts with the intracellular domain of Crb. Interestingly, Galla-1 shows little binding to the Drosophila homolog of XPD, but a related protein Galla-2 (CG7949) binds both Crb and Xpd. Loss-of-function galla-2 mutants show similar mitotic defects as galla-1 and strong genetic interaction with crb. Xpd can form a physical complex with Crb. In imaginal disc, Crb overexpression causes tissue overgrowth as well as DNA damages marked by H2Av phosphorylation. These phenotypes are suppressed by reduction of Xpd. Taken together, this study identifies a novel Crb-Galla-Xpd complex and its function for proper chromosome segregation during nuclear division, implicating a potential link between Crb and Xpd-related genome instability (Yeom, 2014).
The protein O-glucosyltransferase Rumi/POGLUT1 regulates Drosophila Notch signaling by adding O-glucose residues to the Notch extracellular domain. Rumi has other predicted targets including Crumbs (Crb) and Eyes shut (Eys), both of which are involved in photoreceptor development. However, whether Rumi is required for the function of Crb and Eys remains unknown. This study reports that in the absence of Rumi or its enzymatic activity, several rhabdomeres in each ommatidium fail to separate from one another in a Notch-independent manner. Mass spectral analysis indicates the presence of O-glucose on Crb and Eys. However, mutating all O-glucosylation sites in a crb knock-in allele does not cause rhabdomere attachment, ruling out Crb as a biologically-relevant Rumi target in this process. In contrast, eys and rumi exhibit a dosage-sensitive genetic interaction. In addition, although in wild-type ommatidia most of the Eys protein is found in the inter-rhabdomeral space (IRS), in rumi mutants a significant fraction of Eys remains in the photoreceptor cells. The intracellular accumulation of Eys and the IRS defect worsen in rumi mutants raised at a higher temperature, and are accompanied by a approximately 50% decrease in the total level of Eys. Moreover, removing one copy of an endoplasmic reticulum chaperone enhances the rhabdomere attachment in rumi mutant animals. Altogether, these data suggest that O-glucosylation of Eys by Rumi ensures rhabdomere separation by promoting proper Eys folding and stability in a critical time window during the mid-pupal stage. Human EYS, which is mutated in patients with autosomal recessive retinitis pigmentosa, also harbors multiple Rumi target sites. Therefore, the role of O-glucose in regulating Eys may be conserved (Haltom, 2014 PubMed).
The transmembrane protein Crumbs (Crb) functions in apical polarity and epithelial integrity. To better understand its role in epithelial morphogenesis, this study examined Crb localization and dynamics in the late follicular epithelium of Drosophila. Crb was unexpectedly dynamic during middle-to-late stages of egg chamber development, being lost from the marginal zone (MZ) in stage 9 before abruptly returning at the end of stage 10b, then undergoing a pulse of endocytosis in stage 12. The reappearance of MZ Crb was necessary to maintain an intact adherens junction and MZ. Although Crb has been proposed to interact through its juxtamembrane domain with Moesin (Moe), a FERM domain protein that regulates the cortical actin cytoskeleton, the functional significance of this interaction is poorly understood. This study found that whereas the Crb juxtamembrane domain was not required for adherens junction integrity, it was necessary for MZ localization of Moe, aPKC and F-actin. Furthermore, Moe and aPKC functioned antagonistically, suggesting that Moe limits Crb levels by reducing its interactions with the apical Par network. Additionally, Moe mutant cells lost Crb from the apical membrane and accumulated excess Crb at the MZ, suggesting that Moe regulates Crb distribution at the membrane. Together, these studies reveal reciprocal interactions between Crb, Moe and aPKC during cellular morphogenesis (Sherrard, 2015).
These results reveal novel functional interactions between Crumbs, Moesin and aPKC in the follicular epithelium during late stages of Drosophila oogenesis. Loss of Crb, or mutation of its JM domain, results in reduction of both aPKC and active Moe at the MZ. Surprisingly, it was also found that whereas aPKC promotes MZ accumulation of Crb, Moe does the opposite - loss of Moe results in increased MZ accumulation of Crb in the follicular epithelium. These opposing phenotypes suggest that Moe and aPKC function antagonistically to regulate accumulation of Crb in the MZ at these stages (Sherrard, 2015).
How do Moe and aPKC regulate levels of Crb in the MZ? First, although aPKC is generally thought to interact with the Crb complex through binding to Patj and Par-6 via its C-terminal PDZ binding motif, it has been recently suggested that aPKC also interacts with the Crb JM domain. Therefore, aPKC might promote junctional localization of Crb through interaction with two Crb domains, consistent with the observation that MZ localization of Crb is partially lost in crbJMM cells. In contrast to loss of aPKC, excessive accumulation of Crb+ (and aPKC), but not CrbJMM, was observed in the absence of Moe. A previous study has proposed that Moe also interacts with the JM domain, suggesting that Moe and aPKC could compete for Crb binding. Overall, it is hypothesized that aPKC promotes Crb stability through interactions with the Crb JM domain, whereas Moe reduces these stabilizing interactions at the MZ through competition with aPKC (Sherrard, 2015).
A second possible contributing factor in MZ accumulation of Crb is competition between the apical and junctional pools of Crb, with Moe functioning to stabilize apical Crb. This study observed that whereas loss of aPKC does not appear to affect Crb localization at the apical membrane, reduction in Moe function caused decreased Crb on the apical membrane and increased junctional Crb. Moe might stabilize Crb at the apical membrane by linking Crb to cortical actin or through more general effects on the actin cytoskeleton. Destabilized Crb on the apical membrane could then move within the plasma membrane and/or be recycled to the MZ, where it is stabilized by interactions through its PDZ-binding domain (Sherrard, 2015).
The results suggest that, in addition to the previously proposed interactions between Moe and the Crb JM domain, the more general effects of Moe on cortical actin also affect Crb localization and dynamics. In support of this idea, loss of Moe affected the localization of CrbJMM, which should not be able to interact directly with Moe, and this study observed pleiotropic effects on F-actin staining in late follicular cells depleted for Moe protein. Studies in yeast have demonstrated a crucial role for cortical actin in regulating early endocytic events. Actin dynamics are necessary for apical endocytosis in mammalian cells Furthermore, the mammalian ERM protein Ezrin provides a necessary linkage to actin in the recycling of a β-adrenergic receptor. Further disentangling the details of the interactions between Crb, Moe and F-actin will require more specific reagents to identify and manipulate the Crb/Moe binding sites to disrupt specific functions (Sherrard, 2015).
Strikingly, interactions among Crb, pMoe and aPKC were evident after stage 10, coincident with an increase in Crb trafficking and MZ accumulation. Cells undergoing shape changes, such as rapid squamous expansion, must remodel their junctions and membrane domains. Similar to the expansion of amnioserosal cells, but unlike the follicle stretch cells of stage 9, the main-body FCs maintained continuous AJs throughout their expansion. After stage 10, MZ localization of aPKC, F-actin and pMoe, and continuity of Baz, an AJ component, required Crb. This latter result is consistent with a previous study, in which Crb was required for the addition of AJ material to the expanding rhabdomere of the pupal eye. However, another rhabodmere study using transgenic overexpression implicated the Crb JM domain in AJ continuity, in contrast to the current finding that AJs were intact with the crbJMM allele (Sherrard, 2015).
An intriguing possibility is that the putative competition between aPKC and Moe contributes to the role of Crb in morphogenesis, the mechanisms of which are not fully known. In salivary placode invagination, the Crb JM domain recruits Moe and actin to a supercellular, contractile purse string, which is specified by the anisotropic distribution of Crb and aPKC away from the site of the purse string Additional evidence that the JM domain can regulate actin cable formation comes from the defective dorsal closure of crbJMmutants (Sherrard, 2015).
The results suggest a mechanism whereby competition between Moe and aPKC for Crb JM binding could help specify an actin cable. The apical Crb-Par complex might favor aPKC binding in regions of high Crb density, but Moe binding in regions of low Crb density (such as the apical surface, or the site of the purse string in the salivary placode). Thus, competition for binding Crb could amplify the anisotropy of aPKC and Crb, and Moe could recruit the actin for the supercellular cable. The upregulation of Crb endocytosis observed after stage 10 probably plays a role in regulating Crb dynamics during stages 9-11. In other contexts, the retromer regulates Crb membrane levels by influencing its recycling versus degradation. Intercellular homophilic binding of Crb might favor its MZ localization in most tissues (Sherrard, 2015).
However, in egg chambers, the germline cells, which are tightly apposed to the apical surfaces of FCs, also express Crb, which could stabilize Crb apically. Thus, the outgrowth and subsequent regression of microvilli in stages 9-10 could drive the movement of Crb apically and back to the MZ, but this hypothesis remains untested (Sherrard, 2015).
A striking aspect of the effect of Crb loss on Moesin localization was that pMoe and F-actin staining appeared to be noticeably excluded from the MZ in Crb-deficient or CrbJMM-expressing cells. In addition, the width of the zone of pMoe and F-actin loss appeared greater than the width of the MZ Crb staining. It is somewhat puzzling how pMoe and actin could be either excluded from, or fail to be recruited, to this rather broad region in the absence of the relatively narrow JM domain. One possibility is that the zone of pMoe absence represents newly added membrane material, which cannot recruit pMoe or F-actin in the absence of Crb or the Crb JM domain. Alternately, Crb might 'nucleate' addition of F-actin and/or pMoe at the MZ, and it could spread outwards from there (Sherrard, 2015).
The late-stage follicular epithelium displays an unexpectedly dynamic deployment of MZ components, revealing functional relationships not readily apparent in other developmental contexts. Whereas the interactions between Moe, aPKC and Crb uncover a novel connection between the apical PAR network and the apical actin cortex, much remains to be learned about how multifaceted interactions among these proteins and other junctional components direct tissue homeostasis and morphogenesis (Sherrard, 2015).
Maintenance of epithelial polarity depends on the correct localization and levels of polarity determinants. The evolutionarily conserved transmembrane protein Crumbs is crucial for the size and identity of the apical membrane, yet little is known about the molecular mechanisms controlling the amount of Crumbs at the surface. This study shows that Crumbs levels on the apical membrane depend on a well-balanced state of endocytosis and stabilization. The Adaptor Protein 2 (AP-2) complex binds to a motif in the cytoplasmic tail of Crumbs that overlaps with the binding site of Stardust, a protein known to stabilize Crumbs on the surface. Preventing endocytosis by mutations in AP-2 causes expansion of the Crumbs-positive plasma membrane and polarity defects, which can be partially rescued by removing one copy of crumbs. Strikingly, knocking-down both AP-2 and Stardust retains Crumbs on the membrane. This study provides evidence for a molecular mechanism, based on stabilization and endocytosis, to adjust surface levels of Crumbs, which are essential for maintaining epithelial polarity (Lin, 2015).
Data presented in this study, obtained using genetic and biochemical assays, has led to the conclusion that a delicate balance of stabilization and internalization controls proper Crb levels at the surface. This balance is mediated by binding of a conserved amino acid sequence of Crb to either Sdt or AP-2, suggesting a competitive binding between Sdt and AP-2. Such a mechanism, based on stabilization by PDZ-ligand interaction and AP-2-mediated endocytosis, has so far only be described for a few proteins, such as the NR2B subunit of the neuronal NMDA receptor and the glutamate transporter excitatory amino acid carrier 1 (EAAC1) in MDCK cells. In the latter case, AP-2 and the PDZ-domain of PDZK1 antagonistically regulate surface levels of EEAC1 by binding to two closely adjacent binding sites in EEAC1 (Lin, 2015).
Currently, it is only possible to speculate about the mechanisms that may determine which of the binding partners AP-2 or Sdt - binds to Crb. One possibility is that different binding affinities between Crb and its partners modulate the binding preference. Several arguments suggest that binding of AP-2 to Crb seems to be weaker than the Sdt-Crb interaction. First, AP-2 - Crb interactions could only be shown by liposome recruitment assays, but not by standard pull-down experiments, suggesting that the presence of membranes strengthen the interaction between the adaptor and its cargo. In fact, interaction of AP-2 with PtdIns4,5P2-containing membranes induces a conformational change of the AP-2 complex, thus facilitating binding to its cargo (Lin, 2015).
Second, binding of Sdt only depends on the C-terminal leucine and isoleucine residues of Crb. This is in agreement with recently published structural data using crystallography and fluorescence polarization, which show that the four C-terminal amino acids of human Crb1 interact with the PDZ domain of Pals1 by van der Waals contacts and charged interactions (Ivanova, 2015). In contrast, AP-2 binding to Crb requires the glutamine and arginine residues of Crb besides the leucine and isoleucine residues, at least under the in vitro conditions used in this study. The importance of more than one motif for strong binding of AP-2 is not unprecedented. Binding of the human immunodeficiency (HIV)-1 protein Nef to the α-σ2 -hemicomplex of AP-2 requires both a di-leucine- and a di-acidic-motif. Posttranslational modification of the ligand Crb itself could also contribute to a preferred binding to either Sdt/Pals1 or AP-2. It was recently documented that aPKC-mediated phosphorylation of threonine residues near the FBM of Crb abolishes the Crb- Moesin interaction, but not the Crb/Pals1 interaction (Wei, 2015; Lin, 2015 and references therein).
In the case of Drosophila Gliotactin, a transmembrane protein at the tricellular junction, phosphorylation of tyrosine residues is necessary for its endocytosis and ultimately lysosomal degradation, thus preventing overexpression, which would result in delamination, migration and finally apoptosis of cells (Lin, 2015).
Alternatively, the accessibility of the PDZ domain of Sdt/Pals1 to Crb could be modulated. In fact, a ~100-fold stronger binding of the PDZ domain of Pals1 to the Crb tail is achieved upon intra- or intermolecular interactions between the Src homology 3 (SH3)- and the guanylate kinase (GUK)-domain of Pals1. Similarly, the affinity of the PDZ domain of the postsynaptic density protein PSD-95 to its ligand is reduced upon phosphorylation of a tyrosine residue in a linker region between the third PDZ-domain and the subsequent SH3-domain, thereby weakening the intramolecular interaction between the PDZ- and the SH3 domain (Lin, 2015).
A striking observation was the high degree of phenotypic variability upon knock-down/knock -out of AP-2 in different epithelial tissues, and sometimes even in the same epithelium. This prevented study of the different aspects of the AP-2/Crb relation in just one epithelium. In the follicle epithelium, the phenotypes ranged from minor expansion of the apical surface to complete loss of polarity and overgrowth. Complete loss of AP-2α in wing discs leads to cell lethality, whereas mutant clones survived in eye discs. This variability might be due to the time point of induction of mitotic recombination, which could occur before or after establishment of epithelial polarity, or at time points of high or low Crb expression. Alternatively, additional AP-2-independent polarity regulators could act redundantly and in a tissue- and/or time-specific manner, thus modulating the severity of the phenotype, e.g. in the developing eye (Lin, 2015).
The results described in this study are compatible with the assumption that several phenotypes obtained by loss/reduction of AP-2 are a consequence of increased Crb levels on the plasma membrane, since similar phenotypes can be obtained upon overexpression of Crb. (1) The Crb-positive plasma membrane in AP-2α mutants cells is expanded both in the imaginal discs and the follicle epithelium, often at the expense of the lateral membrane. (2) Without functional AP-2α, the monolayered epithelium is often disrupted and becomes multilayered. (3) Surviving AP-2α3 clones in eye imaginal discs show strong Crb enrichment, similar as HEK293 cells, in which AP-2α is knocked-down by RNAi. (4) Assuming that a similar increase in Crb also occurs in AP-2α3 mutant cells induced in wing discs, their elimination could be a consequence of cell competition when next to wild-type cells (Lin, 2015).
Finally, the multi-layering phenotype of follicle epithelia lacking AP-2α can be partially suppressed by removing one copy of crb. A more direct insight into the relationship between Crb and AP-2 comes from an analysis of the garland cells, the functional equivalent of vertebrate podocytes. Podocytes are highly specialized epithelial cells in the kidney of vertebrates, which form long 'foot-processes' connected by slit diaphragms to form a filtration barrier in the renal glomerula. Interestingly, Crb2 is expressed in the kidney of both rats and zebrafish, where it localizes at the slit diaphragm of podocytes. crb2b mutant zebrafish show defects in the formation of the slit diaphragm as well as in arborization of the foot-processes. Furthermore, mutations in human Crb2 are linked to Steroid-resistant nephrotic syndrome (Ebarasi, 2015), a disease causing kidney failure due to defects in differentiation and function of podocytes. This study shows that AP-2α3 mutant garland cells are clearly impaired in Crb endocytosis. Whether loss of crb in garland cells affects their excretory function has to be determined (Lin, 2015).
A complex machinery is required to ensure proper Crb surface levels, which is key for the maintenance of apico-basal epithelial cell polarity. Crb levels can be regulated at multiple levels, including stabilization at the membrane via homophilic interactions of the extracellular domains in cis or trans, interactions of the cytoplasmic tail with scaffolding proteins, endocytosis, degradation and recycling by the retromer. The trafficking pathway offers multiple steps for regulation, and results presented here provide further mechanistic insight how binding of two counteracting PDZ motif-binding proteins, Sdt and AP-2, regulate proper Crb levels (Lin, 2015).
Given the finding that the balance between Crb stabilization and internalization/degradation is crucial for surface expression of Crb and hence polarity, future work will aim to identify the mechanisms that control this balance by regulating the rate of endocytosis, degradation and recycling by the retromer. Finding these regulators is challenging, but will give important insights into the mechanisms coordinating endocytosis and polarity, which is important to prevent tumorigenesis not only in Drosophila, but also in vertebrates (Lin, 2015).
Apical-basal polarity plays critical roles in the functions of epithelial tissues. However, the mechanisms of epithelial polarity establishment and maintenance remain to be fully elucidated. This study shows that the membrane-associated guanylate kinase (MAGUK) family protein Dlg5 is required for the maintenance of apical polarity of follicle epithelium during Drosophila oogenesis. Dlg5 localizes at the apical membrane and adherens junction (AJ) of follicle epithelium in early stage egg chambers. Specifically, the major function of Dlg5 is to promote apical membrane localization of Crumbs, since overexpression of Crumbs but not other major apical or AJ components can rescue epithelial polarity defects resulting from loss of Dlg5. Furthermore, by performing a structure-function analysis of Dlg5, it was found that the C-terminal PDZ3 and PDZ4 domains are required for all Dlg5's functions as well as its ability to localize to apical membrane. The N-terminal coiled-coil motif can be individually targeted to the apical membrane, while the central linker region can be targeted to AJ. Lastly, the MAGUK core domains of PDZ4-SH3-GUK can be individually targeted to apical, AJ and basolateral membranes (Luo, 2016).
How cell polarity is established and maintained is an important question in the fields of cell and developmental biology. During animal development, polarized cells such as epithelial cells maintain their apical-basal polarity despite undergoing dramatic shape changes and tissue remodeling during morphogenesis. Genetic screens done in developing Drosophila and C. elegans have uncovered a number of highly conserved apical and basolateral regulators essential for the establishment and maintenance of apical-basal polarity. Specifically, the Par3/Par6/aPKC complex and the Crumbs (Crb)/Stardust (Sdt)/Patj complex are thought to define and maintain the identity of apical membrane, and the Discs-large (Dlg)/Lethal giant larvae (Lgl)/Scribble (Scrib) complex delineates the basolateral membrane. Finally, the junctional complex of E-cadherin/β-catenin/α-catenin initiates and maintains the adherens junction (AJ), which divides the cortex into apical and basolateral regions. Decades of research have formed a consensus model, in which the apical-basal polarity is generated and maintained by a mutually antagonistic interaction between the apical regulators and the basolateral regulators. Recently, a mathematic modeling study done in Drosophila follicle epithelia suggested that in addition to the negative feedback between apical regulators and basolateral regulators, a positive feedback loop among apical polarity regulators is required to maintain the apical-basal polarity in epithelia. A central component of this positive feedback loop is the transmembrane protein Crb, and its apical membrane localization was thought to be the key to maintenance of apical-basal polarity (Luo, 2016).
Discs-large 5 (Dlg5) belongs to the MAGUK family, and it is highly conserved across species including human, mouse, chicken, zebra fish and Drosophila. MAGUK members also include Dlg and Sdt, which act as molecular scaffolds and are core components of the basolateral Dlg/Lgl/Scrib complex and the apical Crumb complex respectively. Dlg5 was first identified in human and was found to be expressed in placenta and in prostate gland epithelia. Since then, Dlg5 was found to interact with a variety of junctional, cytoskeletal, trafficking, and receptor molecules, including β-catenin, P55, vinexin, Girdin, Citron kinase, Syntaxin, Smoothened and TGF-β receptors. And its functions vary from inhibiting cancer cell migration to mediating receptor signaling, but most of these functions were obtained from cell culture studies. Detailed genetic analysis of Dlg5 was first done using Dlg5 knockout mice, which displayed failure of epithelial tube maintenance resulting in brain hydrocephalus and kidney cysts. These defects were likely due to disruption of apical polarity and AJ. This study focused on Dlg5's requirement of AJ function and found that Dlg5 physically interacted with the β-catenin/cadherin complex and was found together with β-catenin/cadherin complex in both the Rab11-labeled recycling vesicles and the AJ. A more recent work using the same Dlg5-/- mice found that Dlg5 was also required for lung morphogenesis. Specifically, deletion of Dlg5 resulted in loss of apical polarity markers such as aPKC, and Dlg5 was partially colocalized with aPKC in the apical membrane of the wild type lung epithelia. But how Dlg5 exerts its functions in the apical membrane and regulate apical polarity is unknown (Luo, 2016 and references therein).
In Drosophila, RNAi knockdown of dlg5 affected the cohesion and morphology of border cell clusters as well as delaying their migration. Recently, genetic analysis of Drosophila Dlg5 revealed that its mutation caused embryonic lethality and loss of germ cells in the embryonic gonad. Moreover, reduction of Dlg5 in the follicle cells in the adult ovary leads to defects in egg chamber budding, stalk cell overgrowth, ectopic polar cell induction and abnormal distribution of E-cadherin. However, detailed analysis of whether and how Drosophila Dlg5 regulates epithelial or apical polarity has not been done. This study reports that a genetic screen for follicle epithelial morphogenesis has identified the Drosophila Dlg5 as an essential player for maintenance of apical polarity by promoting Crb's apical membrane localization (Luo, 2016).
Dlg5 is specifically required for the maintenance of apical polarity and AJ of follicle epithelia during early stages of oogenesis, since both loss-of-function mutation and RNAi knockdown of dlg5 affected only apical polarity regulators and the sub-apical AJ components but not the basolateral regulators. Furthermore, the apical markers (Crb, Sdt, Patj, aPKC and Par6) were more severely reduced than the sub-apical AJ markers (Arm, E-cad and Baz) with the exception of N-cad, suggesting that the loss of apical polarity is the main cause of severe morphological defects in Dlg5-deficient follicle cells. A previous study found that loss of Crb, aPKC and Par6 did not affect the lateral localization of Dlg, whereas loss of Arm caused Dlg spreading to the apical membrane of follicle cells. This is consistent with the result that no apical spreading of Dlg was observed in dlg5 mutant clones, further confirming that loss of Dlg5 affected apical polarity more severely than the AJ function. Indeed, rescue of apical polarity defects by Crb but not Arm expression further validated this notion (Luo, 2016).
Importantly, this study demonstrates that Dlg5 positively regulates apical polarity by specifically promoting Crb's apical membrane localization, based on the following results. First, double staining revealed that Crb reduction was sometimes more severe than reduction of other apical markers (aPKC and Par6) in dlg5 mutant clones. Second, overexpression of Crb but not other apical or AJ regulators (aPKC, Par6, Arm) could completely rescue dlg5's apical polarity defects. Third, the increased membrane localization and membrane spreading of Crb as caused by blocking the Rab5-mediated endocytosis could be dramatically suppressed by dlg5 mutation. Moreover, the apical enrichment of Dlg5 in the early and mid-stage follicle epithelia (stage 1-stage 9) further suggests that Dlg5 could function at the apical region to promote Crb's membrane localization. On the other hand, Crb might conversely enhance Dlg5's localization to the apical membrane, since overexpression of Crb and hence its membrane spreading toward the basolateral region led to stronger localization of Dlg5 in the basolateral membrane. A previous study has reported that deletion of Dlg5 in mouse resulted in loss of aPKC but not Par3 (homologous with Drosophila Baz) in the developing lung epithelia and that Dlg5 was partially colocalized with aPKC, which are similar to the current findings. But how Dlg5 regulated the apical polarity during mouse lung morphogenesis was not understood. Based on the current results, it would be worthwhile to check whether murine Dlg5 promotes apical polarity by primarily regulating one of the three mammalian CRB paralogs (CRB1, CRB2, CRB3) (Luo, 2016).
As a MAGUK family member, Dlg5 is thought to function as a scaffold protein. Previous works have focused on which domains of Dlg5 physically interact with junctional and membrane-bound proteins like β-catenin, vinexin and smoothened, and trafficking regulators like syntaxin 4 in vitro or in cultured cells. But whether such domains are essential for its function and localization in vivo and which domains possess apical or AJ membrane targeting ability have not yet been addressed. Structure-function analysis demonstrates that the C-terminal fragment including MAGUK core (GUK, SH3, PDZ4) and PDZ3 is necessary but not entirely sufficient for Dlg5's functions. Furthermore, deletion of this C-terminal fragment (Δ4) caused most of Dlg5 to re-distribute to the cytoplasm, losing its membrane localization in the apical, AJ, and lateral regions. Interestingly, PDZ3 and PDZ4 (a subset of C terminal fragment) were also required for Dlg5's functions, and their deletion (Δ5) likewise resulted in the loss of apical and lateral (but not AJ) membrane localization. One interesting difference between the localization patterns of the other deletion mutants (that still possessed rescue abilities; Δ1-3, Δ6-8) and the patterns of Δ4 and Δ5 is that other deletions all retained some degree of apical localization, in contrast to the lack of localization in the apical membrane for Δ4 and Δ5. Together, these results suggest that MAGUK core and PDZ3's requirement for Dlg5's membrane localization in general and PDZ3-PDZ4's requirement for Dlg5's apical membrane localization may be critical for Dlg5's functions in the follicle cells. This is consistent with Dlg5's role in promoting Crb's apical localization. Lastly, this study found that the N-terminal coiled coil domain, the middle linker region and the MAGUK core could be individually membrane-targeted to apical, AJ and all (apical, AJ and basolateral) regions respectively (Luo, 2016).
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.