Drosophila myosin IB (Myo1B) is one of two class I myosins in the Drosophila genome. In the larval and adult midgut enterocyte, Myo1B is present within the microvillus (MV) of the apical brush border (BB) where it forms lateral tethers between the MV membrane and underlying actin filament core. Expression of green fluorescent protein-Myo1B tail domain in the larval gut showed that the tail domain is sufficient for localization of Myo1B to the BB. A Myo1B deletion mutation exhibited normal larval gut physiology with respect to food uptake, clearance, and pH regulation. However, there is a threefold increase in terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive enterocyte nuclei in the Myo1B mutant. Ultrastructural analysis of mutant midgut revealed many perturbations in the BB, including membrane tethering defects, MV vesiculation, and membrane shedding. The apical localization of both Dinged (fascin) and Moesin is impaired. BBs isolated from mutant and control midgut revealed that the loss of Myo1B causes the BB membrane and underlying cytoskeleton to become destabilized. Myo1B mutant larvae also exhibit enhanced sensitivity to oral infection by the bacterial pathogen Pseudomonas entomophila, and severe cytoskeletal defects are observed in the BB of proximal midgut epithelial cells soon after infection. Resistance to P. entomophila infection is restored in Myo1B mutant larvae expressing a Myo1B transgene. These results indicate that Myo1B may play a role in the local midgut response pathway of the Imd innate immune response to Gram-negative bacterial infection (Hegan, 2007).
These results demonstrate a role for Myo1B in maintaining the structural integrity of the BB domain in the larval midgut enterocyte and in providing resistance against oral infection by bacterial pathogens. Although Drosophila Myo1B is structurally most similar to vertebrate Myo1c, it seems to be the functional homologue of vertebrate Myo1a, because both of these myosins form lateral links that tether the MV membrane to the underlying actin core in the gut epithelium. A subset of the consequences of loss of Myo1B are reminiscent of the perturbations of the BB domain observed in the Myo1a knockout (KO) mouse, although there are distinct differences between the two organisms. On the whole, the mild alterations in BB structure in the Myo1B mutant enterocyte, including irregular MV morphology and membrane shedding were less dramatic than that observed in the Myo1a KO mouse. For example, in the mouse KO, many microvilli have a vesiculated, sausage-link appearance resulting from severing of the MV core actin filaments by villin. This is presumably due to elevated intramicrovillar Ca2+ resulting from impaired Ca2+ buffering due to the absence of Myo1a-associated calmodulin light chains. In contrast, Quail, the Drosophila homologue of villin (note that Quail lacks filament severing and capping activity), is not expressed in midgut, although other members of the gelsolin family of actin capping and severing proteins may be expressed based on the detection of villin immunogens in the midgut of Manduca larvae. It will be important in future studies to determine whether the Myo1B-associated light chains (presumably calmodulin) play a similar role to mouse Myo1a in contributing to cytosolic Ca2+ homeostasis. For example, Ca2+ cytotoxicity could contribute to the elevated apoptosis observed in the Myo1B mutant (Hegan, 2007).
Although the perturbations of the midgut BB are relatively mild in the Myo1B mutant compared with the Myo1a KO mouse BB, Myo1B may play an even more critical role than mouse Myo1a in stabilizing the BB membrane cytoskeleton, based on the fragility of the Myo1B mutant BB in response to mechanical lysis. In contrast to the mouse Myo1a KO, few, if any normal looking BBs can be recovered from the Myo1B mutant gut. This destabilization may be due to the additive effects of the loss of actin-membrane tethering by both Myo1B and Dmoesin, together with the diminished filament bundling activity by singed. It is also possible that due to the small number of class I myosins expressed in the Drosophila enterocyte, Myo1B may have a larger repertoire of functions than Myo1a in the vertebrate enterocyte, in which at least three other Myo1s are also expressed (Myo1b, c, and e), whereas in Drosophila, Myo1A is the only other Myo1 present - a myosin that has been shown to have antagonistic functions with Myo1B in determination of left-right asymmetry. Indeed, in the mouse Myo1a KO enterocyte, myosin Ic is ectopically recruited to the BB from the basolateral domain, effecting partial rescue of the loss of lipid-raft-associated membrane proteins, including sucrase isomaltase. In contrast, Drosophila Myo1A remains in the subapical domain in the Myo1B mutant (Hegan, 2007).
The reduced apical localization of Dmoesin in the Myo1B mutant midgut is of particular interest given the critical roles Moesin plays in establishment of epithelial cell polarity. The absence of Myo1B mechanochemical linkages to the membrane could have an indirect impact on Moesin-membrane interactions due to altered biophysical and/or compositional changes in the BB membrane. Conversely, there may be direct interactions between these two membrane-actin linker proteins. One important question for future studies will be to determine whether loss of Myo1B results in reduced levels of active, phosphorylated Moesin (Hegan, 2007).
In contrast to Myo1B and Moesin, Singed does not exhibit tight localization to the BB domain in most regions of the gut. Nevertheless, Singed could play a key role in MV actin bundle structure, a role that could be modulated by Myo1B actin-membrane interactions. Singed is required for actin bundle assembly in nurse cells during oogenesis, yet Singed exhibits diffuse cytoplasmic localization rather than bundle association characteristic of other components such as Quail that also contribute to bundle formation. Moreover, the loss of apical localization in epithelial cells of the proventriculus and diminishment of association with invaginations of the cuprophilic cells in the Myo1B mutant indicate that Myo1B motor linkages between filaments and the membrane can also contribute to the localization/recruitment of Singed to actin bundles (Hegan, 2007).
In contrast to the vertebrate BB membrane, the physiological roles of the enterocyte in nutrient absorption and digestion in the Drosophila midgut are not well understood at the molecular level. However, the presumed absorptive and secretory functions required for lumenal nutrient digestion and nutrient uptake are not discernibly affected in the absence of Myo1B given that larval growth is not severely impaired and the complex regulation of lumenal pH along the length of the midgut is normal. However, loss of Myo1B does cause a threefold increase in the number of apoptotic enterocytes, suggesting that there is an elevated level of stress in these cells. Similar results were seen in the Myo1a KO mouse. Thus, although both Myo1B mutant larvae and Myo1a KO mice exhibit normal growth rates, indicative of adequate nutrient absorption under ideal laboratory feeding and living environments, the increased cellular stress in these mutants may have significant consequences under less than ideal and more realistic environments (Hegan, 2007).
The cortical mechanisms that drive the series of mitotic cell shape transformations remain elusive. This paper identifies two novel networks that collectively control the dynamic reorganization of the mitotic cortex. Moesin, an actin/membrane linker, integrates these two networks to synergize the cortical forces that drive mitotic cell shape transformations. The Pp1-87B/Slik phosphatase restricts high Moesin activity to early mitosis and down-regulates Moesin at the polar cortex, after anaphase onset. Overactivation of Moesin at the polar cortex impairs cell elongation and thus cytokinesis, whereas a transient recruitment of Moesin is required to retract polar blebs that allow cortical relaxation and dissipation of intracellular pressure. This fine balance of Moesin activity is further adjusted by Skittles and Pten, two enzymes that locally produce phosphoinositol 4,5-bisphosphate and thereby, regulate Moesin cortical association. These complementary pathways provide a spatiotemporal framework to explain how the cell cortex is remodeled throughout cell division (Roubinet, 2011).
These findings unravel how, by integrating two regulatory networks, Moe activity provides a spatiotemporal framework to control cell shape transformations during division (see Model of the spatiotemporal regulation of Moe activity throughout the successive steps of the cell cycle). The increase in cortical rigidity that drives cell shape remodeling at the interphase/mitosis transition involves a Pp1-87B/Slik molecular switch that timely regulates Moe phosphorylation (Roubinet, 2011).
PI(4,5)P2 was further identified as a spatial cue that controls Moe distribution at the cortex. This latter aspect coordinates the spatial balance in cortical stiffness/contractility that is required for anaphase cell elongation and cytokinesis. It is proposed that the concerted action of these two regulatory networks ensures the proper series of mitotic cell shape transformations required for the fidelity of cell division (Roubinet, 2011).
A global increase in cortical actomyosin forces generate cell rounding at mitosis entry. These forces are transmitted to the plasma membrane through the activation of ERM proteins. At mitosis exit, both cortical contractions and ERM activity must be down-regulated to allow cells to go back to their interphase shape. In Drosophila cells, the Slik kinase was shown to activate Moe at mitosis entry (Carreno, 2008; Kunda, 2008). This study identifies the Pp1-87B phosphatase as essential for Moe inactivation after cytokinesis and in interphase (Roubinet, 2011).
Although Slik homogenously associates with the cell cortex in both interphase and early mitosis, Pp1-87B is cytoplasmic in interphase and relocalizes to the spindle in pro/metaphase. An attractive model would be that together with a 'constitutive' cortical association of the Slik activator in interphase and pro/metaphase, intracellular redistribution of the Pp1-87B inhibitor represents an efficient way to restrict high levels of Moe phosphorylation to mitosis entry. During anaphase, Pp1-87B concentrates near the chromosomes migrating toward the polar cortex, whereas Slik accumulates at the cleavage furrow. In this model, redistribution of both Pp1-87B and Slik after the anaphase onset contributes to enrich Moe at the equator and to decrease it at poles. Finally, relocalization of Pp1-87B in the cytoplasm after cytokinesis would contribute to relax the cortex for the next interphase by maintaining low Moe activity. A growing number of evidence supports that Pp1 phosphatases play important roles in the temporal control of cell division. Pp1-87B being required for mitotic spindle morphogenesis, this phosphatase could contribute to synchronize cell shape control operated through Moe regulation to chromosome segregation. Although additional investigations will be required to unravel how the activity and distribution of Pp1-87B and Slik are regulated, these results indicate that the Slik/Pp1-87B switch represents an important control of Moe activity during the cell cycle (Roubinet, 2011).
The results show that local levels of PI(4,5)P2 provide an additional mechanism to regulate Moe function at the cortex of dividing cells. Several studies have established a role of PI(4,5)P2 in the localization of ERM proteins in polarized processes of differentiated cells. This study provides evidence that during mitosis, PI(4,5)P2-rich membrane domains act as a spatial cue that regulates both Moe distribution and activation at the cortex (Roubinet, 2011).
The distribution of PI(4,5)P2 at the plasma membrane is tightly regulated during mitosis. As in mammalian cells, it was found that PI(4,5)P2 is actively enriched at the equator of anaphase Drosophila S2 cells, suggesting that equatorial accumulation of PI(4,5)P2 is a feature shared by most animal cells. Although a previous study did not detect PI(4,5)P2 enrichment at the cleavage furrow of Drosophila spermatocytes, whether this is caused by an intrinsic difference between mitosis and meiosis or by experimental limitations in vivo remains to be established. However, how this dynamic localization is regulated remained unknown. This study shows that the equatorial enrichment of PI(4,5)P2 relies, at least in part, on the enzymatic activity of Skittles and Pten. During cytokinesis, the equatorial accumulation of PI(4,5)P2 plays a role in cleavage furrow formation and ingression, through controlling the activity and/or recruitment of several components of the contractile ring. PI(4,5)P2 hydrolysis is also necessary for maintaining cleavage furrow stability and efficient cytokinesis. The current findings extend the functional repertoire of PI(4,5)P2 during mitosis to the control of local properties of the mitotic cortex, which are required for polar relaxation and cell elongation. Through functional screenings, novel regulators of cell division were identified among the entire set of enzymes implicated in phosphoinositide biosynthesis. Two main pathways regulate PI(4,5)P2 levels in mitotic cells, and their alterations provoke similar cortical disorganization. The first pathway involves the Pten tumor suppressor, a PI(3,4,5)P3 3-phosphatase. Pten was shown to accumulate at the septum of dividing yeast cells, as well as at the cleavage furrow in Dictyostelium discoideum. The results of living Drosophila cells show a progressive delocalization of Pten from the polar cortex to the equator after anaphase onset, suggesting that Pten dynamics rely on mechanisms conserved throughout evolution. Furthermore, depletion of Pten leads to a significant enrichment of PI(3,4,5)P3 at the cortex, especially at the cleavage furrow. These results show that Pten uses PI(3,4,5)P3 to spatially control PI(4,5)P2 levels at the mitotic cortex (Roubinet, 2011).
The second pathway relies on Skittles, a PI(4)P 5-kinase that plays a major role in regulating the levels and localization of PI(4,5)P2 during mitosis. Skittles switches from an isotropic cortical distribution in pro/metaphase to equatorial enrichment after the anaphase onset. Depletion of Skittles results in a phenotype similar to the mitotic cortical defects observed after inducible PI(4,5)P2 hydrolysis. It was also found that CG10260, a phosphoinositide 4-kinase, contributes to the organization of the mitotic cortex. Genetics screens have identified a role for phosphoinositide 4-kinases in the division of budding and fission yeast as well as for cytokinesis of male spermatocytes in flies. CG10260 is involved in PI(4)P synthesis, the major substrate of Skittles to produce PI(4,5)P2. Together, these data show that Skittles acts as a key regulator of PI(4,5)P2 levels and Moe activation at the mitotic cortex. Interestingly, Skittles is required for Moe activation in Drosophila oocytes, suggesting that this enzyme plays a broad role in the regulation of ERM proteins (Roubinet, 2011).
An important question is how Skittles and Pten are enriched at the equator in anaphase. It has been reported that activated RhoA stimulates a PI(4)P 5-kinase activity and promotes PI(4,5)P2 synthesis in mammalian cells. During anaphase, activated RhoA localizes at the equatorial cortex, where it could recruit and/or activates Skittles to promote PI(4,5)P2 production. This anisotropy in PI(4,5)P2 distribution might be in turn reinforced by the localized activity of Pten, whose membrane association is itself dependent on PI(4,5)P2. Together, the activity of Skittles and Pten could therefore provide a feed-forward regulatory loop of local PI(4,5)P2 levels at the cortex of dividing cells (Roubinet, 2011).
The metaphase/anaphase transition is characterized by a break in cortical symmetry, with concomitant relaxation of the polar cortex and contraction of the equator. The anisotropic distribution of Moe participates in coordinating this differential in cortical tension. Overactivation of Moe impairs cell elongation and causes cytokinesis failure, suggesting that the polar cortex is too rigid for cell division. Accumulation of F-actin at the cleavage furrow can be attributed, at least in part, to a cortical flow of F-actin filaments from polar regions to the equator. Overactivation of Moe at the poles could block this actin cortical flow, through an excessive bridging of the actin cytoskeleton with the plasma membrane, leading to an abnormal stiffness of the polar cortex. Therefore, redistribution of activated Moe from the polar cortex to the equator participates in polar relaxation, anaphase cell elongation, and cytokinesis fidelity (Roubinet, 2011).
Contraction of the equatorial actomyosin ring increases the cytoplasmic pressure exerted on the plasma membrane. Relaxation of the polar cortex is thus required to dissipate this extra pressure by increasing the cellular volume, a process that was proposed to involve short-lived polar blebs. These polar blebs were recently found to play important roles during cell division. Perturbation of their dynamics triggers anaphase spindle rocking and destabilization of cleavage furrow positioning. Although recent studies have addressed how cortical blebs are regulated in interphase, understanding of the signalization that controls dynamics of cortical blebs in mitosis has poorly progressed since pioneering studies. The results show that a transient recruitment of Moe at the mitotic bleb membrane is required for efficient polar bleb retraction, as are the functions of the Moe positive regulators Slik, Skittles, and Pten. Active Moe contributes to cortical bleb organization because alteration of Moe function (or regulation) disrupts actin organization and efficient bleb retraction. This leads to disorganization of the mitotic cortex, characterized by giant blebs that continue growing in an unregulated manner. Therefore, although a global decrease in Moe activity at the polar cortex contributes to cell elongation and cytokinesis, transient and local association of Moe at the rim of polar blebs is important for their retraction. If the binding of Moe to PI(4,5)P2 is required at both the equator and bleb membrane, the influence of the Slik kinase on Moe activation appears different between these two regions of the anaphase cortex. Although Slik depletion abolishes Moe recruitment to polar blebs, remnants of cortical Moe are still visible at the equator, likely as a result of high PI(4,5)P2 levels at the furrow (Roubinet, 2011).
Although these mechanisms synergistically contribute to the cortical contractility at the equator, they also allow cortical relaxation at the polar cortex through control of transient anaphase blebs. It is proposed that this dual mechanism of Moe regulation is exploited by animal cells to ensure proper cell division (Roubinet, 2011).
A key element in the regulation of subcellular branching and tube morphogenesis of the Drosophila tracheal system is the organization of the actin cytoskeleton by the ERM protein Moesin. Activation of Moesin within specific subdomains of cells, critical for its interaction with actin, is a tightly controlled process and involves regulatory inputs from membrane proteins, kinases and phosphatases. The kinases that activate Moesin in tracheal cells are not known. This study shows that the Sterile-20 like kinase Slik, enriched at the luminal membrane, is necessary for the activation of Moesin at the luminal membrane and regulates branching and subcellular tube morphogenesis of terminal cells. The results reveal the FGF-receptor Breathless as an additional necessary cue for the activation of Moesin in terminal cells. Breathless-mediated activation of Moesin is independent of the canonical MAP kinase pathway (Ukken, 2014).
Semaphorin family proteins are well-known axon guidance ligands. Recent studies indicate that certain transmembrane Semaphorins can also function as guidance receptors to mediate axon-axon attraction or repulsion. The mechanisms by which Semaphorin reverse signaling modulates axon-surface affinity, however, remain unknown. This study reveals a novel mechanism underlying upregulation of axon-axon attraction by Semaphorin-1a (Sema1a) reverse signaling in the developing Drosophila visual system. Sema1a promotes the phosphorylation and activation of Moesin (Moe), a member of the ezrin/radixin/moesin family of proteins, and downregulates the level of active Rho1 in photoreceptor axons. It is proposed that Sema1a reverse signaling activates Moe, which in turn upregulates Fas2-mediated axon-axon attraction by inhibiting Rho1 (Hsieh, 2014).
Several observations regarding the Dmoesin sequence and localization suggest that, like other ERM proteins, it may bind to filamentous actin. Of particular note is a region of N35 amino acids at the very COOH terminus of Drosophila Moesin, a sequence that is highly conserved in vertebrate moesin, radixin, and ezrin. In ezrin, this region has been shown to bind filamentous actin, suggesting that the COOH terminus of Drosophila Moesin may serve a similar function. In addition, there is a striking colocalization of F-actin and Moesin in the apical buds of the precellularized embryo. Interestingly, the other localizations that have been noted for Moesin are also similar to those of filamentous actin, namely in the region of the adherens junctions, the apical membrane, and associated with the basolateral membrane. Finally, a previous study provides evidence that the COOH- terminal half of Moesin interacts with cytoskeletal actin when expressed in yeast (Edwards, 1994). Taken together, these results suggest that Dmoesin binds cytoskeletal actin and functions similarly to the vertebrate ERM proteins (McCartney, 1996).
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 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. Crumbs, ßHeavy-spectrin, and Moesin are all coimmunoprecipitated from embryos, confirming the existence of a multimolecular complex. It is proposed that Crumbs stabilizes the apical SBMS via Moesin and actin, leading to reinforcement of the ZA and effectively coupling epithelial morphogenesis and cell polarity (Médina, 2002).
The Crumbs-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 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 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, 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, 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 can both coimmunoprecipitate Crumbs. Furthermore, the capping assay and embryo expression evidence provide in vivo support for this result. Not only will Moesin 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 forms a bridge between Crumbs and the SBMS. A functional test of this relationship must wait until mutations in the Moesin locus become available. Thus, the current data, although highly suggestive, do not formally distinguish between the possibility of a Moesin bridge between Crumbs and the SBMS, and the existence of two separate complexes with direct interaction between Crumbs and ßH or Moesin 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 one protein network; a Moesin/Spectrin/actin-based network. However, it is unclear at present whether Moesin/ßH/actin coexists 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 (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 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 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).
Recent studies in Drosophila retina indicate that absorption of light causes the translocation of signaling molecules and actin from the photoreceptor's signaling membrane to the cytosol, but the underlying mechanisms are not fully understood. Since ezrin-radixin-moesin (ERM) proteins are known to regulate actin-membrane interactions in a signal-dependent manner, the role of Drosophila Moesin (Moe), the unique D. melanogaster ERM, was examined in response to light. Illumination of dark-raised flies triggers the dissociation of Moe from the light-sensitive Transient receptor potential (TRP) and TRP-like channels, followed by the migration of Moe from the membrane to the cytoplasm. Furthermore, light-activated migration of Moe results from the dephosphorylation of a conserved threonine in Moe. The expression of a Moe mutant form that impairs this phosphorylation inhibits Moe movement and leads to light-induced retinal degeneration. Thus, these data strongly suggest that the light- and phosphorylation-dependent dynamic association of Moe to membrane channels is involved in maintenance of the photoreceptor cells (Chorna-Ornan, 2005).
In dark-raised flies, Moe interacts with both the TRP and TRPL channels, as evidenced by reciprocal coimmunoprecipitation experiments. In contrast, virtually no Moe-TRP and -TRPL complexes are coimmunoprecipitated from illuminated eyes, thus providing strong evidence for Moe binding to the photoreceptor-specific channels primarily in the dark. Furthermore, the results show that light induces dissociation of Moe from TRP and TRPL channels followed by movement of Moe from the rhabdomere membranes to the cytoplasm. Since there is increasing evidence to suggest that functions of invertebrate TRPs are conserved in their mammalian counterparts, these findings might provide new insights for characterizing vertebrate TRP functions. Interestingly, TRPC3 is part of a multimolecular signaling complex containing Ezrin, PLCþ1, and Galphaq/11 that is involved in Ca2+-mediated regulation of channel activity and cytoskeletal reorganization. In addition, it has been shown that the ERM adaptor EBP50/Na+/H+ exchanger regulatory factor associates with PLCþ, TRPC4, and TRPC5 and regulates channel activity and subcellular localization. Altogether, these data strongly suggest that TRP-ERM interactions are an evolutionarily conserved mechanism with important functional properties. The ability to modify Moe binding to TRPs in vivo using illumination should constitute an invaluable tool for investigating the molecular mechanisms regulating this interaction (Chorna-Ornan, 2005).
In this study, the critical role of T559 phosphorylation on Moe activation was extended through the demonstration that dissociation of the Moe from the channel proteins upon illumination depends on T559 dephosphorylation. Accordingly, specific antibodies for the phosphorylated T559 form of Moe immunoprecipitate the TRP channel of dark-raised flies, but not of illuminated flies. Moreover, monospecific TRP antibody immunoprecipitates the phosphorylated form of Moe only in dark-raised flies. These results strongly suggest that only the phosphorylated form of Moe binds TRP. This finding further suggests that light induces dephosphorylation of Moe, leading to dissociation of Moe from the channel proteins, followed by its movement to the cell body (Chorna-Ornan, 2005).
Using WT and transgenic flies that express Moe-GFP fusion proteins, the light-induced movement of Moe from the rhabdomere to the cell body was directly visualized, through confocal imaging of fixed and living retinae. The critical role of T559 phosphorylation on light-induced Moe movement in vivo was further demonstrated through the use of two mutant forms of Moe, in which T559 was replaced by alanine or aspartate residues. The fact that light-activated movement of Moe is blocked in the T559A mutant that remains localized primarily to the soluble fraction of the cell body strongly supports the conclusion that phosphorylation of T559 is crucial for binding of Moe to the channel proteins. Although the T559A mutation kept Moe in its inactive cytosolic state, the T559D phosphomimetic mutation was expected to keep Moe constitutively active. Although some T559D Moe was also found in the cytosol, a significant fraction of T559D Moe was indeed found in the membrane fraction that remains associated with the rhabdomeres after illumination. In addition, both T559A and T559D mutations block the light-dependent movement of Moe. How could nontrafficking forms of Moe (Moe T559A and T559D) lead to light-induced retinal degeneration when expressed in an otherwise WT background? T559 phosphomutants of the Moe protein have been shown to perturb the role of endogenous Moe in actin organization and Oskar localization during oogenesis. This study found consistently that expression of MoeT559A-GFP and MoeT559D-GFP impairs the ability of endogenous Moe to move upon illumination. Since ERM proteins are capable of homotypic interaction (usually as dimers), Moe T559A and Moe T559D can titrate either WT Moe or other functional partners. Therefore, the light-induced degeneration observed upon Moe T559A and Moe T559D expression can be explained by this reduction of endogenous Moe traffic (Chorna-Ornan, 2005).
Altogether, these findings indicate that the rhabdomeric localization of Moe requires its open (active) state, which is achieved either by phosphorylation or by the T559D phosphomimetic mutation. These results further support that light-induced dephosphorylation triggers the movement of Moe to the cytosol, and when this reaction is impaired by mutations, the light dependent movement of Moe is blocked (Chorna-Ornan, 2005).
Light-induced Moe movement has implications for the translocation of signaling molecules, cytoarchitectural changes, and maintenance of the photoreceptor cells. Recent studies have demonstrated reversible light-induced reorganization of the actin cytoskeleton of the microvilli and translocation of the TRPL channel from the rhabdomere to the cell body in time scales comparable to that of light-induced Moe movement. Therefore, the light-induced movement of Moe is likely involved in the control of the aforementioned processes (Chorna-Ornan, 2005).
Interestingly, genetic elimination of either signaling protein PLCß (norpA) or TRP prevents the light-induced movement of Moe. These mutations are known to either block (norpA), or to strongly reduce (trp), the light-induced Ca2+ entry into the photoreceptor cells. The effect of light on Moe movement could thus be mediated via Ca2+-induced dephosphorylation of Moe; e.g., by activation of a Ca2+-dependent phosphatase. PLCß-mediated hydrolysis of PIP2 (which is highly enriched in rhabdomere membranes) might also participate in the release of Moe into the cytoplasm upon illumination; several works have shown the positive effect of PIP2 binding on ERM protein activation, membrane localization, and binding to their partners. The data accumulated in this study indicate the existence of a tight link between light reception and the Moe-mediated reorganization of the rhabdomere cytoarchitecture (Chorna-Ornan, 2005).
Although the elucidation of the full spectrum of the physiological functions of the light-induced Moe movement now awaits further works, it is suggested that these light-induced changes are necessary for the functional maintenance of photoreceptor cells. Photoreceptors are vulnerable cells because of their prolonged interaction with light. The peculiar organization of the rhabdomere in the form of very long (and tightly packed) microvilli makes it difficult for housekeeping mechanisms to operate in the rhabdomere. Light-activated reorganization of actin, along with cytoarchitectural changes, may allow the housekeeping function to operate and/or to participate in the down-regulation of signaling mechanisms triggered by light reception (Chorna-Ornan, 2005).
Epithelial tissues maintain a robust architecture during development. This fundamental property relies on intercellular adhesion through the formation of adherens junctions containing E-cadherin molecules. Localization of E-cadherin is stabilized through a pathway involving the recruitment of actin filaments by E-cadherin. This study identifies an additional pathway that organizes actin filaments in the apical junctional region (AJR) where adherens junctions form in embryonic epithelia. This pathway is controlled by Bitesize (Btsz), a synaptotagmin-like protein that is recruited in the AJR independently of E-cadherin and is required for epithelial stability in Drosophila embryos. On loss of btsz, E-cadherin is recruited normally to the AJR, but is not stabilized properly and actin filaments fail to form a stable continuous network. In the absence of E-cadherin, actin filaments are stable for a longer time than they are in btsz mutants. Two polarized cues have been identified that localize Btsz: phosphatidylinositol (4,5)-bisphosphate, to which Btsz binds; and Par-3. Btsz binds to the Ezrin-Radixin-Moesin protein Moesin, an F-actin-binding protein that is localized apically and is recruited in the AJR in a btsz-dependent manner. Expression of a dominant-negative form of Ezrin that does not bind F-actin phenocopies the loss of btsz. Thus, these data indicate that, through their interaction, Btsz and Moesin may mediate the proper organization of actin in a local domain, which in turn stabilizes E-cadherin. These results provide a mechanism for the spatial order of actin organization underlying junction stabilization in primary embryonic epithelia (Pilot, 2006).
Homotypic binding of the cell-adhesion molecule E-cadherin (E-cad) at the adherens junctions of epithelial cells organizes the formation of multiprotein complexes, composed in part of the ß-catenin and alpha-catenin proteins, and their dynamic interaction with actin filaments (F-actin). F-actin is required to stabilize E-cad-ß-catenin-alpha-catenin complexes. Moreover, E-cad regulates its own stability through the organization of actin filaments through alpha-catenin: alpha-catenin binds Formin (also known as Diaphanous) and suppresses branching by competing with Arp2/3 (Drees, 2005). When epithelia form through the mesenchymal-epithelial transition, the sites of initial cell contact serve as spatial landmarks for the recruitment of E-cad-ß-catenin-alpha-catenin complexes during the formation of adherens junctions. In primary embryonic epithelia, however, adherens junctions do not form through specific cell contacts, and the spatial cues positioning the adherens junctions in the AJR are less characterized and may be different. The identification of such spatial cues and the mechanisms whereby these cues organize structural, cytoskeletal components associated with the formation and/or stabilization of adherens junctions is an important challenge (Pilot, 2006).
This problem was addressed in the early Drosophila embryo. Formation, stabilization and remodelling of adherens junctions occur in a tightly and genetically controlled sequence involving e-cad (or shotgun), armadillo (or ß-catenin), par-6 , par-3 (or bazooka, baz), aPKC, crumbs and others. A microarray-based RNA interference (RNAi) screen of epithelial morphogenesis identified btsz, a gene previously known to control growth in adult flies (Serano, 2003), as a regulator of embryonic epithelial integrity. In embryos injected with double-stranded RNA (dsRNA) probes specific for btsz (hereafter called btszRNAi embryos), gastrulation is severely affected and the epithelium fails to extend properly. Defects are either strong or medium; that is, they are visible at the beginning of gastrulation or about 15 min later, respectively. The defects are penetrant (80%) and dose dependent. Four different, nonoverlapping probes produce these defects and embryos were not affected with control probes (Pilot, 2006).
Btsz is the only Drosophila member of the synaptotagmin-like protein (SLP) family characterized by the presence of tandem carboxy-terminal C2 boxes. btsz encodes several isoforms (Serano, 2003). In early embryos, btsz1 was not detected but btsz2 and btsz3 are expressed together with btsz0, another isoform not previously reported. At least one of these isoforms is maternally and zygotically provided. The most efficient dsRNA probes used recognizes all three maternally and zygotically expressed isoforms. These isoforms were strongly reduced in btszRNAi embryos, suggesting that RNAi produces a severe btsz loss-of-function phenotype (Pilot, 2006).
Two btsz alleles have been described (Serano, 2003): btszK13-4 introduces a deletion in the amino terminus of btsz2 (residues 501-1,494), btszJ5-2 corresponds to a frameshift mutation that introduces a stop codon at amino acid 390, which leads to a truncation in Btsz0 and Btsz2, and probably the absence of Btsz3. btszK13-4 homozygous female escapers can be recovered and were crossed to heterozygous btszK13-4 or btszJ5-2 males. Although many embryos were not fertilized, those that were reached cellularization and showed epithelial defects during gastrulation: 26% of btszK13-4/btszK13-4 and 46% of btszK13-4/btszJ5-2 embryos. btszJ5-2 germline clones do not produce eggs and btszJ5-2 is lethal. Trans-heterozygous embryos were examined with a deficiency removing the btsz locus (Df(3R)Exel6275, called Dfbtsz): 12% of embryos from crosses of Dfbtsz/btszK13-4 females and wild-type males showed epithelial defects. This proportion went up to 39% when males were heterozygous btszK13-4/+. It is concluded that btsz is zygotically and maternally required. Whereas RNAi targeted all three btsz isoforms, btszK13-4 left intact a large fraction of Btsz2 and Btsz0, probably explaining the weaker penetrance of phenotypes in btszK13-4 (26%) or btszK13-4/btszJ5-2 (46%) mutants, as compared with btszRNAi embryos (80%). Notably, despite its essential role in the formation of epithelia in early embryos, the recovery of adult escapers suggests that btsz may be dispensable in adult epithelia (Pilot, 2006).
Overexpression of a btsz2 isoform lacking the 3' untranslated region (UTR) rescues the phenotypes produced by an RNAi probe targeting the 3' UTR of all btsz isoforms. Overexpression of btsz2 more robustly rescues the btszRNAi phenotype than does btsz3 overexpression, suggesting that btsz2 has a prominent role. The injection of morpholino antisense oligonucleotides (morpholinos) specific to each btsz isoform confirmed this: a control morpholino showed no defect, a mix of btsz0, btsz2 and btsz3 morpholinos caused penetrant defects (92%), and a btsz2-specific morpholino alone caused defects in 73% of embryos. Experimental focus was therefore placed on Btsz2, a 286-kDa protein (2,645 residues) (Pilot, 2006).
The expression of a Glu-epitope-tagged variant of Btsz2 (Btsz2-Glu) was strongly reduced in btszRNAi embryos. The epithelium failed to maintain its regular morphology in btszRNAi embryos, btsz mutants and btsz morphants. Although cellularization proceeds similarly in btszRNAi and control embryos, at the onset of gastrulation the epithelium collapses and becomes multilayered in btszRNAi and btszK13-4/btszJ5-2 mutant embryos, as compared with controls. A similar phenotype was observed in e-cadRNAi embryos. Thus, btsz controls the stable architecture of primary embryonic epithelia (Pilot, 2006).
These data suggested that btsz might regulate the formation of adherens junctions. In contrast to the wild type, in which E-cad is uniformly present at the adherens junctions, E-cad expression is heterogeneous and the adherens junctions appears severely fragmented in btsz mutants and btszRNAi embryos. Time-lapse recordings of E-cad fused to green fluorescent protein (GFP) showed that adherens junctions forms properly in the AJR of btszRNAi embryos but that, subsequently, E-cad-GFP expression disappears, suggesting a defect in the stabilization but not targeting of E-cad. E-cad-GFP, or endogenous E-cad, disappears in small patches at cell contacts, pointing to defects in actin organization. Indeed, the actin belt in the AJR is fragmented in btszRNAi embryos. Tested were performed to see whether actin organization or the E-cad-ß-catenin-alpha-catenin complexes was the primary cause of the disassembly of adherens junctions in btszRNAi embryos. In e-cadRNAi embryos, in which E-cad was undetectable in the nascent AJR, the actin belt is not considerably affected during early gastrulation and clearly less affected than in btszRNAi embryos at the same stage. Subsequently, however, F-actin was disorganized in e-cadRNAi embryos. This suggests that Btsz is part of an E-cad-independent pathway controlling actin organization in the AJR and consequently junction stability (Pilot, 2006).
Next, Btsz2 localization was examined. Btsz2-Glu is a functional protein that rescues the btszRNAi phenotype. Btsz2-Glu was previously reported to localize apically in follicular epithelial cells (Serano, 2003). In early embryos, Btsz2-Glu is detected at the AJR together with E-cad from the end of cellularization until about 30 min into gastrulation. Subsequently, Btsz2-Glu was found in a subapical compartment. At these early stages, E-cad colocalizes with Par-3 (also known as Baz). Therefore the possible role of E-cad and Par-3/Baz in Btsz2 localization in the AJR was addressed. In e-cadRNAi embryos, the recruitment of E-cad in the AJR is blocked and Btsz2 is normal; by contrast, in par-3/bazRNAi embryos Btsz2 is largely cytoplasmic, like PatJ, another marker of AJR at this stage. Btsz2 is thus a target of the early polarity marker Par-3/Baz, which is required for E-cad localization in the AJR (Pilot, 2006).
The role of the two C2 boxes (C2AB) in the localization of Btsz2 was tested. Purified glutathione S-transferase (GST)-tagged C2AB binds to phosphatidylinositol mono- and bisphosphate species in a Ca2+-dependent fashion in vitro. The in vivo relevance of this binding was assessed. A tagged form of Btsz2 lacking the C2 boxes (Btsz2-DeltaC2-HA) expressed in gastrulating embryos was cytoplasmic and failed to localize at the AJR. Conversely, an epitope-tagged form of C2AB (C2AB-HA) localizes at the plasma membrane in gastrulating embryos. Notably, C2AB is polarized and concentrates in the apical surface and in the AJR. Of all the phosphoinositides that C2AB binds in vitro, phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2) is the most abundant at the plasma membrane, suggesting that PtdIns(4,5)P2 could be required for Btsz2 localization in the AJR. Injection of cellularizing embryos with neomycin, a compound that binds and inhibits PtdIns(4,5)P2, resulted in epithelial defects similar to btsz, par-3 or e-cadRNAi, and inhibits the recruitment of Btsz2 at the plasma membrane and the AJR. A fusion between GFP and the pleckstrin homology (PH) domain of phospholipase Cdelta (PLCdelta), which specifically binds PtdIns(4,5)P2, localizes apically and in the AJR, similar to the Btsz C2 boxes. It is concluded that PtdIns(4,5)P2 is a polarized spatial cue required for localization of Btsz in the AJR, and hence for adherens junction stability, together with Par-3/Baz (Pilot, 2006).
How does localized Btsz organize F-actin in the AJR? A large-scale two-hybrid screen identified an interaction between Btsz and Moesin, the only Drosophila member of the Ezrin-Radixin-Moesin (ERM) family of F-actin binding proteins that has been implicated in various aspects of epithelial polarity. This interaction was confirmed and Btsz was shown to bind to the third F3 subdomain of Moesin. A minimal region in Btsz that binds Moesin was narrowed down. This interaction occurred in GST pull-down assays of Drosophila S2 cell lysates and embryonic extracts. The functional relevance of this interaction was assessed. Moesin and the phosphorylated active form of Moesin, which binds F-actin, localizes in early embryos in the apical surface and in the AJR, together with Btsz2 and E-cad, suggesting that the interaction between Btsz and Moesin may spatially define a domain of actin organization in the AJR required to stabilize E-cad. In agreement with this, in btszRNAi and btsz mutant embryos, Moesin localization was diminished in the AJR as compared with controls, and E-cad and Moesin segregated in distinct domains as E-cad progressively disappeared (Pilot, 2006).
Whether Moesin is required for epithelial stability was tested in early embryos. Moesin has a major maternal contribution and is a very stable protein. Moreover, females whose germline is mutant for moesin do not lay eggs. Thus, no phenotype was identified using either various moesin mutant alleles or RNAi. Therefore a dominant-negative construct of Ezrin, a mammalian Moesin orthologue that lacks the C-terminal actin-binding domain and acts as a dominant-negative in Drosophila (EzrinDN, containing residues 1-280) was expressed in early embryos . Embryos expressing EzrinDN during gastrulation showed epithelial defects (41% of embryos) similar to btsz mutants. In particular, cellularization was normal, adherens junctions formed properly, but E-cad was no longer present homogeneously around the AJR (Pilot, 2006).
These results shed light on the mechanisms underlying the spatial control of actin filament and the stability of the adherens junctions in the Drosophila primary embryonic epithelium. In Btsz, an E-cad independent pathway has been identified that participates in F-actin organization in the AJR, together with Moesin. Btsz and Moesin bind and colocalize in the AJR in a btsz-dependent fashion, and expression of a mutant form of Ezrin that does not bind F-actin disrupts adherens junctions stability similar to loss of btsz. Notably, this work identifies upstream polarity cues (Par-3/Baz and PtdIns(4,5)P2) that control the spatial order of actin organization at the AJR through the localization of Btsz. The fact that PtdIns(4,5)P2 acts as a key regulator of epithelial polarity in the early embryo raises the issue of how PtdIns(4,5)P2 metabolism is spatially regulated in epithelial cells. The observation that Par-3 binds PTEN, which converts PtdIns(3,4,5)P3 into PtdIns(4,5)P2, suggests that Par-3/Baz may be part of this process. Thus a key intermediate between polarity cues and structural elements of adherens junctions important for embryonic epithelial stability has been identified. Five SLPs and two SLP-related (Slac2) proteins are close orthologues of Btsz in mammals. It would be worth investigating their potentially conserved role in the dynamic organization of actin at adherens junctions in embryonic epithelia (Pilot, 2006).
The Rho-kinases are widely utilized downstream targets of the activated Rho GTPase that have been directly implicated in many aspects of Rho-dependent effects on F-actin assembly, acto-myosin contractility, and microtubule stability, and consequently play an essential role in regulating cell shape, migration, polarity, and division. The single closely related Drosophila Rho-kinase ortholog, DRok, is required for several aspects of oogenesis, including maintaining the integrity of the oocyte cortex, actin-mediated tethering of nurse cell nuclei, 'dumping' of nurse cell contents into the oocyte, establishment of oocyte polarity, and the trafficking of oocyte yolk granules. These defects are associated with abnormalities in DRok-dependent actin dynamics and appear to be mediated by multiple downstream effectors of activated DRok that have previously been implicated in oogenesis. DRok regulates at least one of these targets, the membrane cytoskeletal cross-linker DMoesin, via a direct phosphorylation that is required to promote localization of DMoesin to the oocyte cortex. The collective oogenesis defects associated with DRok deficiency reveal its essential role in multiple aspects of proper oocyte formation and suggest that DRok defines a novel class of oogenesis determinants that function as key regulators of several distinct actin-dependent processes required for proper tissue morphogenesis (Verdier, 2006).
DRok has been previously implicated as an effector of the DRho1 GTPase in the regulation of planar cell polarity in the eye and in the wing, downstream of Frizzled-Dishevelled signals. In germline cells, DRok and DRho1 mutants exhibit some overlapping actin defects; e.g., the oocyte cortex exhibits a more diffuse F-actin distribution in both Drok2 GLCs and Rho1 loss-of-function egg chambers in which Rho1 levels have been reduced in a heterozygous mutant Rho1 and wimp background. In addition, wild-type oocytes injected with the Rho-inhibitory C3 toxin exhibit the same ooplasmic streaming defects as Drok2 mutant oocytes, strongly suggesting that the germline clone phenotypes reflect the disruption of DRho1-DRok signaling in germ cells (Verdier, 2006).
Previous analysis of the polarity of Dmoe GLC oocytes indicated that DMoesin is specifically required for the localization of posterior determinants such as oskar mRNA and Oskar protein but not the formation of the dorso-ventral axis nor the anterior pole. DMoesin appears to function in maintaining posterior polarity by anchoring actin to the membrane cortex which in turn anchors microtubule-delivered oskar mRNA and its protein product Oskar to the posterior pole. Similarly, in Drok2 GLCs, the localization of anterior (bicoid) or dorso-ventral determinants (gurken) is not altered although most oskar mRNA is found mislocalized within the ooplasm starting at stage 9. While the establishment of oocyte polarity generally depends upon microtubule cytoskeleton organization, it has been reported that Dmoe null mutations do not disrupt the microtubule cytoskeleton and do not perturb its polarity. Similarly, this study observed that microtubules in Drok2 mutant oocytes appear normal. Taken together with the fact that some oskar mRNA remains anchored at the posterior tip of the oocyte plasma membrane in Drok2 GLCs, as is seen in Dmoe GLCs, this indicates that oskar mislocalization, and consequently, the alteration of posterior polarity in Drok2 GLCs is not due to an abnormally organized microtubule cytoskeleton. Moreover, unlike other germline clone mutants with oocyte polarity defects, such as chic or capu, the Drok2 and Dmoe polarity defects most likely reflect the incapacity of the disorganized subcortical actin cytoskeleton to properly anchor oskar at the posterior membrane of the oocyte (Verdier, 2006).
The similarity between the oskar polarity phenotype of Drok2 and Dmoe GLCs is also consistent with a likely role for DMoesin as an essential DRok substrate that mediates its effects on the formation of posterior polarity and further supports the functional significance of a signaling pathway from DRok to DMoesin to the actin cytoskeleton in oocyte development. Moreover, the proper localization of Gurken, as defined by the position of the oocyte nucleus, which migrates in a microtubule-dependent manner from the posterior to the anterior and then to the antero-dorsal side of the oocyte starting at stage 8, is consistent with the presence of a grossly normal appearing microtubule cytoskeleton in DRok-deficient oocytes (Verdier, 2006).
A majority of mutations resulting in egg chambers with dorso-ventral axis patterning defects, such as dorsal appendages aberrations, have been associated with genes encoding components of the Gurken-EGFR signaling pathway. Cross-signaling between the oocyte and the surrounding follicle cells at the antero-dorsal side of the oocyte has been extensively studied and involves the binding of the secreted Gurken ligand to the EGFR present on the apical membrane of follicle cells and subsequent activation of downstream signaling to control the formation of follicle cell-derived dorsal structures. Although Gurken localization is correct in Drok2 mutant oocytes, loss of Gurken secretion (in 80% of the Drok2 mutant oocytes) in the intercellular space between the oocyte membrane and the follicle apical membranes indicates the likelihood of altered communication between the oocyte and surrounding follicle cells, possibly resulting in a disruption of the EGFR signaling pathway leading to dorsal appendage defects. The observed requirement for DRok in Gurken secretion may reflect a well established role of Rho signaling in the control of vesicular trafficking and secretion. However, it remains possible that the apparent absence of Gurken secretion into the intercellular space reflects a consequence of the observed disruption of oocyte plasma membrane integrity (Verdier, 2006).
The unexpected observation in time-lapse confocal microscopy studies that most autofluorescent yolk granules in Drok2 mutant oocytes or Rho-inhibitory C3 transferase-treated wild-type egg chambers accumulate at the oocyte membrane suggests a role for DRho1 and DRok in early vitellogenesis. Vitellogenesis is a process that begins around stage 8 and is defined by the co-secretion of vitelline membrane and yolk material by the surrounding follicle cells leading to the eventual formation of chorionic structures of the egg and normal oocyte growth, respectively. After their secretion, yolk proteins are internalized into the oocyte through endocytosis and are swirled around the ooplasm at later stages, when microtubule-dependent streaming occurs. The high concentration of yolk granules at the oocyte membrane from early vitellogenesis underlies a possible defect in endocytosis of the yolk granules. Together with the fact that C3-treated egg chambers and Drok2 GLCs exhibit an identical yolk granule phenotype, this suggests that DRok mediates Rho1's role in the trafficking of yolk granules at the oocyte plasma membrane. In addition, nurse cells also normally accumulate yolk material and transfer it to the oocyte. The detection of yolk granules moving to the plasma membrane of Drok2 mutant oocytes or oocytes in C3-treated egg chambers after they are deposited by the nurse cells is an intriguing phenotype that has not been previously reported and may reflect a trafficking defect in the ooplasm. Further studies to examine molecular components of the endocytic machinery will be required to develop a better understanding of the roles of Rho1 and DRok in yolk granule trafficking within the ooplasm. Notably, it is also conceivable that alteration of oocyte plasma membrane integrity through disruption of actin cytoskeleton organization in most Drok2 GLCs, as was observed, could exert a secondary effect on the endocytosis of yolk granules (Verdier, 2006).
Because of the yolk granule phenotype in Drok2 GLCs in early oogenesis, it is not possible to visualize microtubule cytoskeleton dynamics at later stages in time-lapse confocal microscopy. Thus, it is difficult to determine whether Drok2 mutant oocytes would undergo normal or premature ooplasmic streaming at stages 10b–11. As a functional relationship between actin and microtubule cytoskeletons has been suggested based on findings with several mutants with oogenesis defects, it is quite conceivable that the abnormalities of the actin cytoskeleton in Drok2 mutant oocytes could affect microtubule cytoskeleton dynamics. Indeed, it has been demonstrated that some aspect of the actin cytoskeleton normally represses microtubule-based streaming within the oocyte. Thus, it is possible that the accumulation of yolk granules near the plasma membrane of Drok2 mutant oocytes reflects a combination of trafficking/endocytosis defects and actin cytoskeleton perturbation-induced alteration of microtubule cytoskeleton dynamics in the ooplasm during early oogenesis (Verdier, 2006).
The oocyte volume in Drok2 GLCs is frequently smaller than that seen in wild-type oocytes, before the rapid phase of cytoplasmic transport takes place. This suggests a possible defect in the slow phase of cytoplasmic transport. It has been previously reported that transport of some particles towards the oocyte during stages 7–10A depends upon a proper acto-myosin network. In addition, sqhAX3 GLCs exhibit a similar oocyte size defect. sqhAX3 is a loss-of-function mutation in the sqh locus which codes for the Drosophila ortholog of myosin light chain of myosin II. Taken together with the fact that DRok has been shown to phosphorylate Sqh in vivo, these data suggest that DRok mediates, via regulation of Sqh, some aspects of the acto-myosin contractility involved in cytoplasmic transport from early stages of oogenesis (Verdier, 2006).
The observation of dumpless-like oversized nurse cells in most of Drok2 GLCs also supports a role for DRok in the rapid phase of cytoplasmic transport at stages 10B–11 of oogenesis. Unlike other classes of dumpless mutants including chickadee, singed or quail, failure of rapid cytoplasmic transport from the Drok2 mutant nurse cells to the oocyte does not result from the obstruction of the ring canals by unanchored nurse cell nuclei, suggesting that Drok constitutes a distinct class of dumpless-like mutants. In addition, in sqhAX3 GLCs, dumpless nurse cells are associated with a lack of acto-myosin contractility by nurse cells, as revealed by mislocalization of myosin II and by absence of the perinuclear organization of actin filaments bundles in the nurse cells. Therefore, sqhAX3 mutant nurse cells cannot contract properly to expulse their cytoplasm through otherwise weakly damaged ring canals. Drok2 and sqhAX3 mutant nurse cells do not share the same actin filament phenotype; Drok2 mutant nurse cells exhibit a more dramatic phenotype associated with absence of radial filaments and disorganization of cortical actin. It is, however, likely that DRok and Sqh are part of the same signaling pathway that regulates acto-myosin contractility in nurse cells; it has already been shown that DRok phosphorylates Sqh in Drosophila development. Moreover, the severity of the Drok2 mutant F-actin phenotypes may reflect DRok's potential to engage multiple distinct downstream substrates, of which Sqh is only one. Significantly, the actin-binding protein, adducin, is also reportedly a direct substrate for mammalian Rho-kinases, and the Drosophila Adducin ortholog, Hts, is a major component of ring canals. Thus, it is possible that the observed defects in ring canal morphology in Drok2 GLCs involve abnormal regulation of adducin by DRok. However, it is difficult to determine whether this ring canal phenotype contributes to the dumpless-like nurse cell phenotype observed in Drok2 GLCs (Verdier, 2006).
The observation that nurse cell nuclei are substantially increased in size in Drok2 GLCs suggests a possible involvement of DRok in increased endoreplication of the nurse cells. The Rho-related Rac and Cdc42 GTPases have previously been associated with endoreplication in porcine aortic endothelial (PAE) cells, although Rho has not been implicated thus far. Interestingly, this nurse cell nuclei phenotype has not been observed in other previously described GLC mutants of other actin cytoskeleton-regulating signaling components that exhibit oogenesis defects. Thus, chic as well as sqhAX3 GLCs reveal cytokinesis defects associated with the presence of multinucleated nurse cells. In addition, the majority of sqhAX3 mutant egg chambers harbor less than 15 nurse cells (64% of sqhAX3 mutant egg chambers have less than 7 nurse cells), a phenotype that is not shared by Drok2 mutant nurse cells. These findings also suggest that Drok2 defines a new category of oogenesis mutants that affect the actin cytoskeleton (Verdier, 2006).
Both Dmoe and Drok2 GLCs exhibit similar actin defects in the oocyte, associated with a loose uneven cortical actin distribution and the presence of actin clumps in the ooplasm and near the cortex. Moreover, phospho-DMoesin levels are decreased at the cortex or mislocalized within the ooplasm of Drok2 GLCs and the conserved kinase domain of Rho-kinase phosphorylates DMoesin on threonine 559 in vitro. A potential mechanism for the DRok-DMoesin signal in this setting is that DRok controls actin reorganization through phosphorylation of DMoesin, which has been previously shown to cross-link actin to the plasma membrane when phosphorylated on T559 at the oocyte cortex. However, the detection of some phospho-DMoesin in the Drok2 GLCs indicates that the critical T559 residue can be phosphorylated by other kinases in the oocyte. Indeed, direct phosphorylation of T559 of mammalian Moesin by protein kinase C (PKC)-θ has been shown in vitro. In addition, mammalian Rho-kinase and PAK have been reported to both phosphorylate the very conserved T508 residue of LIM-kinase in vitro. Therefore, phosphorylation of the conserved T559 residue of Moesin by additional kinases might also occur in Drosophila, highlighting the complexity of cross-talk within developmental signaling pathways (Verdier, 2006).
The observation that Drok2 mutant oocytes are morphologically more affected than Dmoe mutant oocytes with regard to the deformed plasma membrane also suggests that to exert its functions at the oocyte cortex, DRok is not only signaling to DMoesin but probably also to additional downstream targets that cooperate with DMoesin in the maintenance of the cortical actin cytoskeleton. The strong phenotype associated with the deformed oocyte plasma membrane, which separates dramatically from the apical plasma membranes of the follicle cell layer in most Drok2 GLCs, raises an intriguing question about DRok's apparent role in an adhesive process. That specific phenotype has not been previously reported in studies of other oogenesis mutants associated with defective adhesion between the oocyte and the surrounding follicle cells. Previous reports regarding such adhesion largely address cross-signaling between the apical Notch receptor and the germline-derived putative secreted and transmembrane proteins, Brainiac and Egghead, respectively, in which germline loss of either Brainiac or Egghead results in loss of epithelial apico-basal polarity and accumulation of follicular epithelial cells in multiple layers around the oocyte, but does not lead to a physical separation between the oocyte and the follicle cells membranes. The unique phenotype of Drok2 GLCs could reflect a role for DRok in mediating a distinct signaling pathway from the oocyte to regulate its shape and its adherence to the surrounding follicle cells. Alternatively, the aberrant morphology of the nurse cells, which appear to 'push' against the oocyte without contracting, might produce a mechanical stress on the oocyte itself that prevents it from remaining apposed to the follicle cell layer. Notably, it was found that the follicle cells themselves also appear to require DRok function for the maintenance of their shape, and it is possible that their ability to signal to the oocyte is also affected by DRok deficiency (Verdier, 2006).
In summary, the single closely related Drosophila Rho-kinase ortholog, DRok, is required for several aspects of oogenesis, including maintaining the integrity of the oocyte cortex, actin-dependent tethering of nurse cell nuclei, 'dumping' of nurse cell contents into the oocyte, establishment of oocyte polarity, and the trafficking of oocyte yolk granules. It is likely that several previously identified direct phosphorylation targets of DRok, including DMoesin, Sqh (myosin light chain), and Hts (adducin), which have each been implicated in various aspects of oogenesis, mediate at least some of the functions of DRok in developing egg chambers. These findings indicate an essential role for Rho-DRok signaling via multiple DRok effectors in several distinct aspects of oogenesis (Verdier, 2006).
Organization of the plasma membrane in polarized epithelial cells is accomplished by the specific localization of transmembrane or membrane-associated proteins, which are often linked to cytoplasmic protein complexes, including the actin cytoskeleton. This study identified Sip1 as a Drosophila orthologue of the ezrin-radixin-moesin (ERM) binding protein 50 (EBP50; also known as the Na+/H+ exchanger regulatory factor NHERF1). In mammals, EBP50/NHERF1 is a scaffold protein required for the regulation of several transmembrane receptors and downstream signal transduction activity. In Drosophila, loss of Sip1 leads to a reduction in Slik kinase protein abundance, loss of Moesin phosphorylation and changes in epithelial structure, including mislocalization of E-cadherin and F-actin. Consistent with these findings, Moesin and Sip1 act synergistically in genetic-interaction experiments, and Sip1 protein abundance is dependent on Moesin. Co-immunoprecipitation experiments indicate that Sip1 forms a complex with both Moesin and Slik. Taken together, these data suggest that Sip1 promotes Slik-dependent phosphorylation of Moesin, and suggests a mechanism for the regulation of Moesin activity within the cell to maintain epithelial integrity (Hughes, 2010).
Previous studies of EBP50/NHERF1 suggested a key role for this protein in regulating transmembrane protein localization and retention at the plasma membrane. In this study, advantage was taken of the genetic tools available in Drosophila to identify and characterize some of the in vivo functions of the EBP50/NHERF1 orthologue Sip1. Searches of the Drosophila genome suggest that Sip1 is the single fly orthologue of EBP50/NHERF1, whereas in mammals there are two paralogues, EBP50/NHERF1 and NHERF2. Thus studies in Drosophila should have fewer potential genetic redundancy problems than those in mammalian systems (Hughes, 2010).
Previous studies of EBP50/NHERF1 suggested a key role for this protein in regulating transmembrane protein localization and retention at the plasma membrane. This study has taken advantage of the genetic tools available in Drosophila to identify and characterize some of the in vivo functions of the EBP50/NHERF1 orthologue Sip1. Searches of the Drosophila genome suggest that Sip1 is the single fly orthologue of EBP50/NHERF1, whereas in mammals there are two paralogues, EBP50/NHERF1 and NHERF2. Thus studies in Drosophila should have fewer potential genetic redundancy problems than those in mammalian systems (Hughes, 2010).
Collectively, the data suggest a novel activity for Sip1, namely that it facilitates Slik-dependent phosphorylation and activation of Moesin. Both genetic and biochemical evidence support this conclusion. Clonal analysis indicates that Sip1 is required for normal subcellular localization and abundance of Slik and normal phosphorylation of Moesin without affecting overall Moesin abundance. In addition, Sip1 appears to be upregulated in slik mutant cells, suggesting that Slik and Sip1 have an interdependent relationship. Co-immunoprecipitation experiments in pupae and in cultured S2 cells indicated that Sip1 forms complexes with both Slik and Moesin, although at the moment it is unclear whether all three proteins are present in a single complex. Taken together, these results suggest that Sip1 functions to bring Slik and Moesin in close proximity, possibly by acting as a scaffold that links these proteins together and leading to phosphorylation of Moesin. Although yeast two-hybrid data indicate that Moesin and Sip1 interact directly, this study has not distinguished between the possibilities of direct versus indirect interactions with Slik. It is interesting to note that EBP50/NHERF1 has been shown to form a complex with Ste20-related kinase SLK, the mammalian orthologue of Slik, via the adapter protein PDZK1 (Hughes, 2010).
One potential problem with this model is that previous work has suggested that EBP50/NHERF1 is unable to bind the folded, inactive form of ezrin, raising the question of how Sip1 can facilitate activation of Moesin if it cannot bind the inactive, folded form. A possible answer to this question comes from recent observations that ERMs are regulated by several factors that might operate in a step-wise fashion. Specifically, previous studies have suggested either a two-step mechanism involving binding of PIP2 to the FERM domain, followed by phosphorylation of the conserved C-terminal Thr, or a 'rheostat'-like mechanism that allows for several intermediate states of unfolding and activation. Proposed components of the rheostat mechanism include binding of the FERM domain to PIP2 and phosphorylation, not only of the C-terminal Thr, but of other residues as well. Thus, it is proposed that interaction of a Sip1-Slik complex with Moesin probably requires previous partial unfolding of Moesin through one or more of these mechanisms (Hughes, 2010).
Although a role for EBP50/NHERF1 in promoting ERM phosphorylation has not been proposed previously, several aspects of the current findings and conclusions are consistent with previous studies in mammalian systems. Sip1 binds Moesin in both two-hybrid and co-immunoprecipitation analyses, as has been observed for EBP50/NHERF1 in mammalian cells. Consistent with this result, loss of Moesin and/or ezrin results in mislocalization of Sip1/EBP50 in both the fly and in the mouse. In Ebp50 mutant mice, there is a decrease in phosphorylated ERM proteins in the kidney and intestinal epithelia. EBP50 is suggested to be necessary to stabilize active phosphorylated ERM proteins at the apical membrane, but this observation would also be consistent with the model whereby EBP50/Sip1 promotes ERM phosphorylation. Assuming that this is true, what remains unclear is the identity of the responsible kinase in mammalian cells. Although Rho kinase has been proposed to phosphorylate this residue, subsequent studies have raised questions about this proposal, and no alternative kinase has been identified. The overall conservation of ERMs and Sip1/EBP50/NHERF1 between flies and humans strongly suggests that there is an as yet unidentified sterile 20 kinase family member involved in ERM phosphorylation in mammalian cells (Hughes, 2010).
The requirement of Sip1 for promotion of Moesin activity is also supported by the observation that reduction of the Rho1 gene dosage partially suppresses the lethality of the Sip1 mutant animal. Previously, it has been shown that Moesin regulates epithelial integrity via downregulation of Rho1 signaling. Similarly, Slik hypomorphic defects are rescued by a reduced Rho1 gene dosage, suggesting that Slik regulates Rho1 signaling via phosphorylation and activation of Moesin. Taken together, these results suggest that Sip1, Slik and Moesin function synergistically to negatively regulate Rho1 pathway activity, and emphasize the importance of the functional interaction between Sip1 and Moesin for proper epithelial morphogenesis (Hughes, 2010).
Support for this model comes from the genetic interaction that were observed between Sip1 and Moe. Co-expression of an activated Moe transgene strongly enhances the effects of Sip1 expression, whereas expression of the inactive, non-phosphorylatable MoeT559A allele suppresses Sip1 hypermorphic phenotypes. These results are consistent with Sip1 and Moe acting synergistically. Furthermore, the observation that the non-phosphorylatable T559A allele suppresses Sip1 expression phenotypes could suggest that co-expression of this form inactivates Sip1, possibly through direct interaction, although it was not possible to demonstrate increased complex formation in co-immunoprecipitation experiments. In addition, the strong synergistic genetic interactions between Sip1 and the phosphomimetic form of MoeT559D suggests that Sip1 either interacts specifically with phosphorylated Moesin, or promotes Moesin phosphorylation (Hughes, 2010).
It is interesting to note that either reduced or increased levels of Sip1 expression have dramatic effects on epithelial integrity. Loss of function Sip1 clones in follicle cell epithelia result in reorganization of the actin cytoskeleton and loss of E-cadherin localization, strongly suggesting that adherens junction integrity is affected. In addition, ectopic expression of Sip1 in the adult wing leads to the formation of vesicles, which is indicative of defects in cell adhesion. In mammalian cells it has also been shown that reduced ezrin levels result in the loss of cell adhesion complexes. This suggests that the production of Sip1 protein must be tightly regulated within epithelial cells to regulate epithelial integrity, probably through regulation of the interaction between Slik and Moesin (Hughes, 2010).
Strong effects of loss of Sip1 function were consistently observed within the follicle cell epithelia, but not in the columnar epithelial cells of third imaginal wing discs, indicating a differential cell requirement for Sip1 activity. This might be linked to changes in cell morphology, because follicle epithelial cells are initially cuboidal, but undergo dramatic changes in cell shape beginning at stage 9. At this point, the follicle cells located over the growing oocyte migrate posteriorly and become columnar, whereas the follicle cells over the nurse cells become squamous. The majority of the defects that were observed in the follicle cells, whether located over the oocyte or nurse cells, occur at or following, stage 9. These changes in cell type would require alteration in both cell adhesion and the organization of the actin cytoskeleton, which contribute to the force required for the migration. Thus, these cells might be particularly susceptible to perturbations that affect regulation of cytoskeletal dynamics or linkage between the plasma membrane and the cytoskeleton. Another, not mutually exclusive, factor is that follicle cells are no longer mitotically active at the stages observed, whereas the wing imaginal discs undergo mitosis throughout their development. Perhaps there are independent mechanisms for regulating the interactions with Slik and Moesin in actively dividing cells. A third possibility is that an unknown protein acts redundantly to Sip1 to regulate the activity of Slik and Moesin in the third instar imaginal disc tissue (Hughes, 2010).
A crucial question that remains to be answered regarding Sip1, Moesin and Slik is how their activities are co-regulated in developing epithelia. Previous work clearly suggests that regulation of Slik kinase activity is important in developing epithelial cells. The results presented in this study indicate that the effect of Slik on Moesin activity, and therefore on epithelial integrity, could be regulated by Sip1. Furthermore, because previous studies have shown that Slik also regulates the tumour suppressor Merlin, these results raise the possibility that Sip1 could control proliferation in epithelial tissues. Since it is now possible to study the EBP50/NHERF family in Drosophila, it should be possible to identify other gene pathways that are involved in the activity and localization of Sip1 (Hughes, 2010).
The signalling activities of Merlin and Moesin, two closely related members of the protein 4.1 Ezrin /Radixin/Moesin family, are regulated by conformational changes. These changes are regulated in turn by phosphorylation. The same sterile 20 kinase-Slik co-regulates Merlin or Moesin activity whereby phosphorylation inactivates Merlin, but activates Moesin. Thus, the corresponding coordinate activation of Merlin and inactivation of Moesin would require coordinated phosphatase activity. Drosophila protein phosphatase type 1 β (Flapwing) fulfils this role, co-regulating dephosphorylation and altered activity of both Merlin and Moesin. Merlin or Moesin are detected in a complex with Flapwing both in-vitro and in-vivo. Directed changes in flapwing expression result in altered phosphorylation of both Merlin and Moesin. These changes in the levels of Merlin and Moesin phosphorylation following reduction of flapwing expression are associated with concomitant defects in epithelial integrity and increase in apoptosis in developing tissues such as wing imaginal discs. Functionally, the defects can be partially recapitulated by overexpression of proteins that mimic constitutively phosphorylated or unphosphorylated Merlin or Moesin. These results suggest that changes in the phosphorylation levels of Merlin and Moesin lead to changes in epithelial organization (Yang, 2012).
The results suggest that Flw would act antagonistically to the kinase Slik during the coordinate regulation of Mer, acting as a tumour suppressor protein, and Moe, required to maintain epithelial integrity. If Flw acts as a coordinate regulatory phosphatase for Mer and/or Moe, it would be expected that Flw is in a protein complex with both Mer and Moe, and this was found to be true. A reproducible increase was found in the ratio of dephosphorylated to phosphorylated Mer isoforms when flw is overexpressed, and a decrease in this ratio was found when flw expression is reduced. In addition, four distinct Mer phosphorylation isoforms were detected. Supporting these observations, the over-expression of flw increases the amount of dephosphorylated Mer signal present as compared to the wild type tissue. Flw also affects the phosphorylation of Moe. The amount of phosphorylated Moesin protein is reduced when flw is over-expressed as compared to when flw expression is reduced. Thus, Flw appears to be a phosphatase specific for both Mer and Moe (Yang, 2012).
Most importantly, using functional assays in whole animals, Flw mediated regulation of Mer and Moe has clear effects on both Mer and Moe protein localization to the plasma membrane and on epithelial organization. There is a higher intensity of staining of both Mer and phosphorylated Moe associated with the plasma membrane upon reduction of flw expression. When the levels of other typical apical domain markers as well as basolateral markers were examined by maximum intensity projection analysis, it was found that maximum projections from larval wing discs show increased brightness of p-ERM, F-actin and anti-Armadillo, within the cells in which flw expression is reduced, whereas the septate junction marker anti-Coracle staining is not changed in intensity over the whole disc. This suggests that as a result of changes in Mer and Moe phosphorylation there are changes in links to the actin cytoskeleton and adherens junctions where both Mer and Moe play roles in wild type cells. Previous studies have demonstrated that phosphorylated Mer is more tightly associated with the plasma membrane. In agreement with data from Drosophila, mammalian cells also show increased plasma membrane association of a phosphomimic form of moesin or the related protein ezrin whereas dephosphorylated ERM proteins are less associated with the plasma membrane. Following flw knockdown in selected cells in the wing epithelium, cells within the boundary between cells with reduced flw expression levels and cells with wild type flw expression levels undergo the greatest amount of change in terms of epithelial integrity. The loss of polarity leads to increased apoptosis in these cells. These effects are observed when flw expression is reduced in only a few cells such as using the ptc Gal4 driver or in the entire dorsal compartment of the wing such as using the apterous Gal4 driver. The cells along the boundary region appear to fold inwards and detach from the rest of epithelium. This is likely the direct result of the difference in adhesion between cells that have reduced flw expression and cells which express wild type levels of Flw protein. As Mer and Moe appear to be direct targets of Flw, and both Mer and Moe have roles in adhesion, the changes in the adhesion of wing epithelium upon reduction of flw are likely a result of changes in Mer and Moe phosphorylation and thus activity. The combination of excess active Moe and excess inactive Mer would affect the balance between maintenance and loss of stabilization of adherens junctions leading to the changes in adhesion and deformation of the wing epithelia that were observed. These adhesion differences could account for the formation of the large folds along the boundary of the ptc expression domain, since cells of similar adhesion are more likely to adhere to themselves (Yang, 2012).
The deformation of the wing imaginal tissue appears to be progressive, since in pre-pupal wing discs (10 h after pupariation) deep holes are observed that extend from the apical surface basally indicating that cells at the apical surface have left the epithelium and are forming balls of cells basally within the disc. In further support of the results, the loss of sds22, a PP1 regulatory subunit, in clonal analysis shows that in large clones in wing discs there is infolding of the mutant tissue with cells being extruded from the epithelium. Cells with loss of function Sds22 also exhibit Moe hyper-phosphorylation. Notably, this is reminiscent of what was observe with reduction of flw expression and overexpression of phosphomimic or nonphsophorylatable Mer or Moe (Yang, 2012).
While a likely cause of some of the changes seen in functional assays are due to changes in Mer and Moe phosphorylation as a result of changes in flw expression, the possibility remains that the level of analysis and resolution of the functional assays in both larval and pupal imaginal wing discs may be insufficient to clearly show subtle differences in the subcellular localization on the membrane of Mer, Moe and apical markers. Thus, it cannot be concluded that the defects associated with flw are due solely to defects in Mer and Moe activity (Yang, 2012).
The ability to partially recapitulate the loss of flw phenotype in ptc expressing cells by the over-expression of either a phosphomimic or nonphsophorylatable Mer or Moe also strongly suggests that this phenotype is, in part, due to the differences in the ratios of active Mer or Moe to inactive Mer or Moe which lead to the corresponding changes in apical epithelial integrity, in third instar discs. This is exemplified by the observation that often with overexpression of either the phosphomimic or nonphosphorylatable Mer or Moe, the formation of a fold is most apparent at the edge of ptc expression at the boundary where the difference in the expression of Mer or Moe within the ptc expressing cells and the neighbouring wild type cells would be greatest. In this way it is not unexpected that the overall effect on the tissue deformation and adhesion is the same with phosphomimic or nonphosphorylatable Mer or Moe, although it is possible that the underlying causes are different due to the predicted opposite activities of the transgenes (Yang, 2012).
Within or directly beside the edge of the ptc expression domain in wing imaginal discs, significantly more cells stain positively for activated Caspase 3. This suggests that cells in these affected domains are undergoing increased levels of apoptosis. These phenotypes are again reminiscent of what is observed in loss of function clones of Sds22, which exhibit an increase in the number of apoptotic cells in the wing discs (Yang, 2012).
It was also demonstrated that Flw binds to the scaffold protein Sip1. It functions with the kinase Slik to regulate Moe activity to maintain epithelial cell integrity. Therefore, the findings suggest that Mer, Moe, Flw, and Sip1 function within a protein complex. This coordinate incorporation within a regulated protein complex is necessary to coordinate cellular response to changing epithelial integrity. This might also explain why the overexpression of flw does not have a strong effect on epithelial integrity. If Mer and Moe need to be part of a complex with Flw and Sip1 in order to regulate epithelial integrity and proliferation, then expression of excess phosphatase outside the complex would have no effect on tissue morphology and growth. In contrast, loss of the phosphatase would have a direct effect since there would be reduced levels of functional protein complex (Yang, 2012).
Future studies are required to determine additional members of this regulatory complex, such as the likely candidates Sds22 and MYPT-75D. The similarity in phenotypes between Sds22 mutant cells and the results of knockdown of flw function would also support a role of Sds22 to interact with Flw in regulating Moe function (Yang, 2012).
This study has shown that the Mer and Moe proteins are direct targets of the catalytic subunit of the PP1 phosphatase Flw. This identifies another important player in the regulation of both Mer and Moe in Drosophila. This is the first identification of a phosphatase coordinately regulating both Mer and Moe activity in vivo. What remains to be determined is how Flw is targeted to regulate Mer and Moe function and what downstream pathways may be affected by these interactions (Yang, 2012).
RhoA, a small GTPase, regulates epithelial integrity and morphogenesis by controlling filamentous actin assembly and actomyosin contractility. Another important cytoskeletal regulator, Moesin (Moe), an ezrin, radixin, and moesin (ERM) protein, has the ability to bind to and organize cortical F-actin, as well as the ability to regulate RhoA activity. ERM proteins have previously been shown to interact with both RhoGEF (guanine nucleotide exchange factors) and RhoGAP (GTPase activating proteins), proteins that control the activation state of RhoA, but the functions of these interactions remain unclear. This study demonstrates that Moe interacts with an unusual RhoGAP, Conundrum (Conu; CG34104), and recruits it to the cell cortex to negatively regulate RhoA activity. In addition, this study shows that cortically localized Conu can promote cell proliferation and that this function requires RhoGAP activity. Surprisingly, Conu's ability to promote growth also appears dependent on increased Rac activity. These results reveal a molecular mechanism by which ERM proteins control RhoA activity and suggest a novel linkage between the small GTPases RhoA and Rac in growth control (Neisch 2013).
Although previous studies have implicated ERM proteins in the regulation of RhoA activity, the molecular mechanisms underlying this function have remained unclear. This work shows that a previously uncharacterized RhoGAP, Conu, physically and functionally interacts with Moe. Consistent with its sequence similarity to other RhoGAP proteins, Conu was found to have GAP activity for Rho1 in vitro and negatively regulates Rho1 in vivo. The data further suggest that Moe promotes Conu's RhoGAP activity, and therefore negatively regulates Rho1 by recruiting Conu to the plasma membrane (Neisch 2013).
Surprisingly, the data suggest that Conu also functions to positively regulate Rac1 activity. Although Conu's ability to promote proliferation is dependent on its RhoGAP activity, this alone is not sufficient to cause overproliferation. Two lines of evidence indicate that this additional function involves positively regulating Rac1. First, coexpression of Conu enhances a rough-eye phenotype associated with Rac1 expression in the eye. This effect is not dependent on Conu GAP activity, indicating that it is not the result of cross-talk between different Rho family small GTPases. Second, Conu acts synergistically with the small GTPase Arf6 in causing overproliferation. Previous studies have shown that Arf6 promotes activation of Rac1 at the plasma membrane. Consistent with the idea that Arf6 promotes Rac1 activity, coexpression of Arf6 strongly enhances the Rac1 eye phenotype. These results suggest that Conu functions to negatively regulate Rho1 activity and positively regulate Rac1 activity, and that the combination of these two effects promotes overproliferation when Conu is activated (Neisch 2013).
Conu's closest orthologue in the mammalian genome is ARHGAP18, also known as MacGAP, with which it shares 40% sequence similarity. ARHGAP18 has been shown to have GAP activity for the Rho1 homologue RhoA. ARHGAP18 was also recently found to promote tumor formation and cell proliferation when ectopically expressed in mammary epithelia, consistent with the observation that Conu promotes proliferation in Drosophila epithelial tissues. Little is currently known about the details of ARHGAP18 function in mammalian cells, but it is interesting to note that ARHGAP18 is also required for cell spreading, a function that is associated with Rac1 activation in many cells. It is also notable that Conu and ARHGAP18 share a region of homology near the N-terminus (aa 45-90) that is separate from the GAP domain and has similarity to the SAM (sterile alpha motif) kazrin repeat 2 domain. SAM domains serve as oligomerization or protein-protein interaction motifs, so it is possible that this domain mediates interactions with one or more Rac1 regulatory proteins. However, preliminary results indicate that this domain alone does not enhance Rac1 in the eye. It will be interesting to determine whether ARHGAP18 also regulates Rac1 activity, and whether both functions are also involved in growth control in mammals (Neisch 2013).
Despite the overproliferation phenotypes observed from activated Conu, loss-of-function conu mutations are viable and lack obvious imaginal disk phenotypes. Functional redundancy with another RhoGAP seems a likely explanation for this result, because the Drosophila genome encodes 21 RhoGAP proteins in addition to Conu. Thus the loss of a single RhoGAP, such as Conu, may have only a very minor effect on overall Rho1 activity in the imaginal epithelium. In addition, the phenotypes exhibited by Moe mutants, which are much more severe, are probably due to Moe's ability to negatively regulate Rho1 activity via Conu, together with its ability to stabilize the apical cell cortex through interactions with F-actin. Indeed, it is speculated that the severe epithelial defects observed in Moe mutants are the combined result of increased apical actomyosin contractility caused by increased RhoA activity and decreased cortical stability caused by the loss of Moe's membrane-cytoskeletal cross-linking function. In this model, loss of either function alone would have relatively minor effects, but the combined defect would result in the severe cortical disorganization that has been described for Moe mutants. A critical future line of investigation is to identify other Rho1 regulatory proteins that function with Moe to regulate the activity of Rho1 in the apical domain (Neisch 2013).
A remaining question is how does Conu contribute to growth control in the imaginal disks? The answer to this important question is unclear, but the data suggest that decreased Rho1 activity functions synergistically with Rac1 in this process. Recent studies in Drosophila suggest that Jun N-terminal kinase (JNK) activation downstream of Rac1 activity can promote increased proliferation and metastatic activity in the presence of activated Ras (Brumby, 2011). Although attempts to suppress Conu-mediated overgrowth by inhibiting the JNK pathway gave ambiguous results, increased expression of the puc-lacZ reporter for JNK activity was observed in cells expressing activated Conu, consistent with a role of JNK pathway activation in Conu-mediated proliferation. In contrast, other signaling pathways involved in growth control (Notch, TGFβ, Wnt, Hippo, and Hedgehog) were unaffected. Ras activation is thought to protect cells from the proapoptotic effects of JNK pathway activation, and it is possible that decreased Rho1 activity caused by Conu activation has similar effects, especially given that it has been shown that Rho1 is proapoptotic in these tissues. While further studies will be required to more firmly establish the mechanistic basis of Conu function in tissue growth, these findings of a role for Moe and Conu in this process highlight the importance of precise regulation of the apical cell cortex and Rho family small GTPases in growth control (Neisch 2013).
Ezrin, Radixin, and Moesin (ERM) proteins play important roles in many cellular processes including cell division. Recent studies have highlighted the implications of their metastatic potential in cancers. ERM's role in these processes is largely attributed to their ability to link actin filaments to the plasma membrane. This paper, shows that the ERM protein Moesin directly binds to microtubules in vitro and stabilizes microtubules at the cell cortex in vivo. Two evolutionarily conserved residues in the FERM (4.1 protein and ERM) domains of ERMs were identified that mediated the association with microtubules. This ERM-microtubule interaction was required for regulating spindle organization in metaphase and cell shape transformation after anaphase onset but was dispensable for bridging actin filaments to the metaphase cortex. These findings provide a molecular framework for understanding the complex functional interplay between the microtubule and actin cytoskeletons mediated by ERM proteins in mitosis and have broad implications in both physiological and pathological processes that require ERMs (Solinet, 2013).
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).
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