BIOLOGICAL OVERVIEW

Two prominent characteristics of epithelial cells, apical-basal polarity and a highly ordered cytoskeleton, depend on the existence of precisely localized protein complexes associated with the apical plasma membrane and on a separate machinery that regulates the spatial order of actin assembly. ERM (ezrin, radixin, moesin) proteins have been proposed to link transmembrane proteins to the actin cytoskeleton in the apical domain, suggesting a structural role in epithelial cells, and they have been implicated in signalling pathways. The sole Drosophila ERM protein Moesin functions to promote cortical actin assembly and apical-basal polarity. As a result, cells lacking Moesin lose epithelial characteristics and adopt invasive migratory behaviour. These data demonstrate that Moesin facilitates epithelial morphology not by providing an essential structural function, but rather by antagonizing activity of the small GTPase Rho. Thus, Moesin functions in maintaining epithelial integrity by regulating cell-signalling events that affect actin organization and polarity. Furthermore, these results show that there is negative feedback between ERM activation and activity of the Rho pathway (Speck, 2003).

ERM proteins, highly related members of the larger protein 4.1 superfamily, can exist in an active or inactive conformation. The inactive conformation is brought about by head-to-tail interactions between the FERM (four-point-one, ERM) domain and the carboxy terminus. In this state, ERM actin-binding and membrane association sites are not accessible. A conformational change induced by Rho pathway activation produces the active ERM form. Mammalian ERM proteins bind actin and cell-type-dependent transmembrane proteins such as CD44, CD43 and several I-CAMs. Genetic tests of hypotheses regarding ERM protein involvement in signalling and cytoskeletal support have been impeded by functional redundancies between these three proteins in mammals. In contrast, Drosophila has only one ERM gene, Moesin, enabling such analyses to be performed (Speck, 2003).

To test Moesin function in vivo, a transposable element insertion in the Moesin gene, P{lacW}l(1)G0323, was identified. Males hemizygous for this insertion display late larval lethality. This lethality can be rescued by a duplication of region 8B, Dp(1;Y)619, as well as by a complementary DNA-based Moesin+ transgene. Taken together, these results indicate that l(1)G0323 disrupts Moesin function but does not affect any other essential genes, and is hereafter referred to as Moe^G0323 (Speck, 2003).

To determine the extent to which Moesin function is disrupted by this allele, Moesin protein level and distribution were examined in Moe^G0323 mutants. Moesin protein is not detected in imaginal discs from Moe^G0323 hemizygous larvae by immunofluorescence or immunoblotting. Furthermore, animals trans-heterozygous for the Moe^G0323 allele and a deficiency of the 8B region, Df(1)lz-90b24, display the same lethal period and have phenotypes identical to Moe^G0323 hemizygous and homozygous mutant larvae, establishing that Moe^G0323 is genetically null (Speck, 2003).

To investigate Moesin function in the morphogenesis of epithelial structures the imaginal discs were examined from Moe^G0323 hemizygous larvae. In wild-type epithelial cells, filamentous actin associates with the cell cortex and accumulates in the apical domain, where it largely coincides with Moesin. In Moe^G0323 cells, F-actin undergoes a marked redistribution: it accumulates in ectopic sites within cells and becomes depleted from the apical region. To demonstrate that actin is reduced in the apical domain, a Moe⁺ transgene was examined only in the posterior compartment of the mutant disc, generating a 'half-rescued' disc. The rescued cells in the posterior compartment have a wild-type phenotype, and have significantly more filamentous actin in the apical domain than adjacent Moe^G0323 cells (Speck, 2003).

The imaginal epithelium normally consists of a single layer of tall columnar cells with an obvious apical-basal polarity. In Moe^G0323 imaginal discs, many cells are situated basal to the epithelial monolayer. These cells do not express the junctional marker E-Cadherin or other epithelial polarity markers, including ß-Catenin, Coracle, Crumbs or Discs lost. These data suggest that in Moe^G0323 organisms, epithelial cells lose intercellular junctions and epithelial polarity, and are extruded basally from the epithelium (Speck, 2003).

To determine whether cells lacking Moesin not only lose epithelial morphology but also adopt migratory behaviour, a subset of cells within the imaginal epithelium was marked by expressing green fluorescent protein (GFP) under control of the region-specific patched-Gal4 driver. In a wild-type wing imaginal disc, marked cells stay within a continuous stripe along the anterior-posterior boundary. In a Moe^G0323 disc, cells expressing GFP under control of patched-Gal4 are found away from the stripe, suggesting that these cells have dispersed. Furthermore, these cells are able to invade adjacent regions of the disc comprising cells that still retain epithelial morphology. Thus, Moe^G0323 cells not only lose epithelial markers and intercellular adhesion, but also become motile and show invasive behavior, suggesting that cells lacking Moesin undergo an epithelial-to-mesenchymal transition (Speck, 2003).

Rho-induced phosphorylation of a C-terminal threonine has been shown to relieve the inhibitory intramolecular association and generate the active conformation of the ERM molecule (Matsui, 1998; Shaw, 1998; Simons, 1998). This residue is conserved in Drosophila Moesin at amino acid position 559. To test the role of Thr 559 phosphorylation in Moesin activation, two mutant cDNA constructs were generated: Moe(T559D), a phosphomimetic form, and Moe(T559A), a form that cannot be phosphorylated at this residue. Moe^G0323 hemizygous males are rescued to viability by transgenes expressing Moe(T559D), but not by Moe(T559A), implying that Moe(T559D) is an active form whereas Moe(T559A) is inactive (Speck, 2003).

The phosphomimetic form of ERM proteins has constitutively active properties because it may be locked in the open conformation (Gautreau, 2000). To examine the effects of ERM activation, Moe(T559D) was overexpressed under region-specific Gal4 drivers in wild-type imaginal discs. A marked upregulation of cortical F-actin was observed in cells that express Moe(T559D). By contrast, little or no effect was seen with Moe⁺ and Moe(T559A) overexpression. The accumulation of cortical actin induced by the gain-of-function mutant is complementary to the reduction of apical actin seen in the Moesin loss-of-function background, indicating that Moesin controls assembly of cortical actin (Speck, 2003).

The pleiotropic nature of the Moe^G0323 phenotype suggests an involvement in a signalling pathway that controls cell adhesion and motility. Given the crucial function carried out by the Rho family of small GTPases in regulating these processes and the evidence for Rho regulation of ERM function (Shaw; 1998; Hirao; 1996; Mackay, 1997) the relationship between Moesin and Rho signalling in vivo was examined by manipulating the genetic dose of Rho1, a Drosophila RhoA homolog, in a Moe^- background. The null Rho172R allele was used to reduce by half maternal and zygotic Rho function. Halving the dose of Rho1 not only ameliorates the Moe^G0323 disc epithelium organization and actin localization phenotypes, but also strongly suppresses Moe^G0323 lethality. A similar suppression of lethality was observed with other Rho1 alleles and when the dose of Drok, a Rho effector kinase, was reduced, and a weaker effect was observed with another downstream effector of Rho1 signalling, the non-muscle myosin-II heavy chain gene zipper). The observed suppression was not due to enhanced Moesin protein production or stability. Taken together, these results suggest that Moesin functions antagonistically to activity of the Rho pathway in regulating epithelial polarity and integrity (Speck, 2003).

The ultrastructure was examined of wild-type and Moe^G0323 epithelial cells as well as that of Moe^G0323 cells that are also heterozygous for Rho172R. In transmission electron micrographs, wild-type epithelial cells have apical finger-like microvilli that tend to congregate near the areas of contact between adjacent cells. In the Moesin mutant, the microvilli are replaced by large apical protrusions that are often of irregular shape. Reduction of Rho activity partially suppresses this Moe^- phenotype, and results in cells that lack the protrusions but still display abnormally large microvillus-like structures. These results suggest that Moesin may have a Rho-independent role in maintaining apical integrity that is not essential for viability (Speck, 2003).

If Moesin normally functions as an antagonist of Rho pathway activity, then the effects of Rho pathway hyperactivation should be similar to the Moe loss-of-function phenotype. To test this hypothesis, whether Rho1 overexpression phenotypes in wing imaginal discs mimic those seen in Moe loss-of-function mutants was investigated. Cells overexpressing Rho1 mislocalize F-actin and lose epithelial characteristics. As with Moe^G0323 cells, Rho1-expressing cells in the blade region of the wing imaginal disc lose junctional markers and drop basally. These cells also assume a mesenchymal character, become motile and invade between wild-type epithelial cells. Similar, but more disruptive phenotypes were observed when a constitutively active form of Rho1, Rho1V14, was overexpressed in wild-type imaginal discs. Therefore, Rho1 overexpression strongly resembles Moe loss of function, suggesting that Moesin and Rho1 exert opposite effects on the epithelium (Speck, 2003).

To test further the relationship of Moesin with the Rho pathway, whether a reduction in Moe function would suppress a phenotype associated with downregulation of Rho1 was investigated. Rho1 has been shown to function downstream of dishevelled (dsh) in the planar cell polarity (PCP) pathway that regulates the polarity and number of hairs generated by each wing blade cell. Each wild-type wing cell produces a single hair; however, multiple wing hairs result when Rho pathway function is impaired. In flies that are mutant for the dsh1 allele (which inactivates the PCP but not the wingless-signalling function of dishevelled), double-hair-producing cells occurred at a frequency of 6.3%. Removal of a single dose of Moe suppresses the number of cells with double hairs in dsh¹ mutants to 1.2%, suggesting that Rho pathway function is upregulated in response to reduction in Moesin activity (Speck, 2003).

These genetic results in Drosophila suggest that Moesin functions antagonistically to Rho pathway activity. To distinguish between an effect on Rho itself and effects on a downstream component of the pathway, and to extend these results from Drosophila to mammalian cells, the effect of reducing ERM function in mammalian LLC-PK1 epithelial cells was examined. Use was made of an ERM truncation, ezrin^Act, that has dominant-negative properties in flies and reduces function of all three ERM proteins in these cells (as assayed by phospho-ERM staining, a marker for ERM activation (Hayashi, 1999). Expression of ezrin^Act resulted in increased levels of Rho activity, whereas expression of wild-type ezrin caused either no effect, or slightly decreased the level of Rho activity. These results indicate that ERM proteins negatively regulate Rho pathway function by altering the activation state of Rho itself, rather than a downstream component of the pathway (Speck, 2003).

The phenotypic analysis presented in this study clearly demonstrates that Moesin function is required to maintain apical-basal polarity, epithelial integrity, and correct actin distribution. This is consistent with Moesin's association with the apical membrane in epithelial cells and the ability to bind filamentous actin (McCartney; 1996; Edwards, 1997). These results also show that Moesin promotes epithelial morphology by functioning antagonistically to Rho pathway activity, rather than by providing structural linkage between the plasma membrane and the cytoskeleton. Moe^- defects, including lethality, are strongly suppressed when Rho pathway activity is reduced, suggesting that Moesin does not have essential structural functions. Furthermore, the diverse Moe^- phenotypes -- decreased cortical actin accumulation, loss of epithelial polarity, and acquisition of migratory cell behavior -- can all be attributed to increased Rho pathway activity. Consistent with this model for Moesin function, increased Rho activity has been linked to invasive and metastatic phenotypes in mammalian epithelial cells. Because ERM proteins are known downstream effectors of Rho signalling, the results further suggest the existence of a negative feedback loop between ERM proteins and the Rho pathway. This negative feedback mechanism may be important in inhibiting migratory and invasive cellular behaviors that characterize metastatic cells (Speck, 2003).

The results with mammalian epithelial cells suggest that the effect of ERM function on Rho signalling occurs upstream to or at the level of Rho itself. Previous studies have detected direct binding between ERM proteins and two known regulators of Rho function, RhoGDI (Rho GDP dissociation inhibitor: Takahash, 1997) and Dbl GEF (guanine nucleotide exchange factor for Rho: Takahashi, 1998). Although one of these studies suggested that ERM proteins function to promote Rho activation in vitro (Takahashi, 1997), the in vivo significance of these interactions has not yet been examined. Elucidation of the functional significance of these interactions may be essential to the further understanding of the relationship between ERM proteins and Rho pathway activity. Of note, the proposed negative feedback loop between ERM and Rho parallels the antagonistic relationship between the ERM-related tumour suppressor Merlin and another Rho-family GTPase Rac (Shaw, 2001), and suggests that ERM and Merlin may have similar roles in regulating Rho-family signalling (Speck, 2003).

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

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

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

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

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

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

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

STRIPAK regulates Slik localization to control mitotic morphogenesis and epithelial integrity

Proteins of the ezrin, radixin, and moesin (ERM) family control cell and tissue morphogenesis. A previous study reported that moesin, the only ERM in Drosophila, controls mitotic morphogenesis and epithelial integrity. This study also found that the Pp1-87B phosphatase dephosphorylates moesin, counteracting its activation by the Ste20-like kinase Slik. To understand how this signaling pathway is itself regulated, a genome-wide RNAi screen was conducted, looking for new regulators of moesin activity. Slik was identified as a new member of the striatin-interacting phosphatase and kinase complex (STRIPAK). The phosphatase activity of STRIPAK reduces Slik phosphorylation to promote its cortical association and proper activation of moesin. Consistent with this finding, inhibition of STRIPAK phosphatase activity causes cell morphology defects in mitosis and impairs epithelial tissue integrity. These results implicate the Slik-STRIPAK complex in the control of multiple morphogenetic processes (De Jamblinne, 2020).

Cell morphogenesis is an important process by which cells adapt their shapes to achieve different functions. Filaments of the actin and microtubule cytoskeletons play important roles during this process. Actin filaments apply forces to the cortex to contribute to plasma membrane remodeling, whereas microtubules are important for targeted trafficking and signaling. Proteins of the ezrin, radixin, and moesin (ERM) family link actin filaments and microtubules to the plasma membrane. They regulate important cellular processes, such as cell division, migration, and epithelial organization. The phosphorylation of a conserved threonine residue in the C-terminus of ERMs promotes their activation. ERMs cycle between an active conformation at the plasma membrane and an inactive form in the cytosol. An intramolecular interaction between the N-terminal FERM domain and C-terminal tail (C-terminal ERM association domain [CERMAD]) inactivates ERMs. Their activation involves a conformational switch through a multistep mechanism: (1) Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), a phospholipid of the plasma membrane, recruits ERMs to the cortex and slightly opens the molecules; (2) this preopening allows access to LOK, a sterile 20 (Ste20)-like Ser/Thr protein kinase that wedges between the FERM and CERMAD domains to complete full ERM opening; and (3) the kinase phosphorylates the regulatory threonine of ERMs to stabilize their open conformation. This opening unmasks the actin-binding site in the CERMAD and the microtubule-interacting site in the FERM domain and allows ERMs to link actin and microtubule filaments at the plasma membrane to regulate cell morphogenesis (De Jamblinne, 2020).

Previous work has shown that moesin controls epithelial integrity and mitotic morphogenesis. In Drosophila wing imaginal discs, it was discovered that Slik, the Drosophila orthologue of mammalian SLK and LOK, phosphorylates moesin to control epithelial tissue integrity. Cells lacking either Slik or moesin undergo an epithelial-to-mesenchymal transition (EMT), sort basally out of the wing disc epithelium, and undergo apoptosis (De Jamblinne, 2020).

In Drosophila S2 cells in culture, it was shown that moesin controls mitotic cell shape and organization of the mitotic spindle. Mitosis entry is characterized by an approximately threefold increase in moesin phosphorylation. Slik is responsible for the basal level of moesin phosphorylation in interphase and for the specific activation of moesin in mitosis. In metaphase, phosphorylated moesin (p-moesin) spreads around the cortex and increases global cortical rigidity by coupling actin forces to the plasma membrane. This promotes rounding of cells. In addition, moesin-microtubule interactions regulate mitotic spindle organization. In anaphase, the Ser/Thr phosphatase Pp1-87B dephosphorylates moesin at the poles and favors p-moesin accumulation at the equator. This redistribution drives cell elongation and cytokinesis. After abscission, Pp1-87B dephosphorylates moesin to allow cortex relaxation. Either inhibition or overactivation of moesin has damaging effects in mitosis. Overactivation of moesin overstiffens the polar cortex in anaphase, preventing cell elongation and cytokinesis. Conversely, moesin or Slik double-stranded RNA (dsRNA) depletion renders the cortex too soft; cells do not control mitotic morphogenesis properly and present abnormal cortical blebs(De Jamblinne, 2020).

In addition to Pp1-87B and Slik, this study also found that the PtdIns-4-kinase CG10260 and the PtdIns(4)P 5-kinase Skittles regulate localized PtdIns(4,5)P2 production at the plasma membrane to control moesin localization. However, it is still unknown how the enzymes of the signaling network activating moesin are themselves regulated. To this end, an unbiased genome-wide dsRNA screen was performed in Drosophila S2 cells and p-moesin levels were measured. Striatin-interacting phosphatase and kinase complex (STRIPAK) were found to be a positive regulator of moesin phosphorylation. At the core of the STRIPAK complex, there is a protein Ser/Thr phosphatase 2A (hereafter referred to as 'PP2A_STRIPAK'). PP2A_STRIPAK is a heterotrimeric enzyme that consists of a PP2A catalytic C subunit (Mts, in Drosophila), a PP2A scaffold A subunit (PP2A-29B), and a striatin regulatory B subunit (Cka). On top of this core, striatin-interacting proteins known as STRIPs (Strip) help to scaffold Ser/Thr kinases of the Ste20-like family (De Jamblinne, 2020).

STRIPAK controls the activity of its associated kinases, either by regulating phosphorylation of regulatory residues or by recruiting them to specific sites in the cell. Thereby, STRIPAK affects cell proliferation and survival, cytoskeletal regulation, and vesicle trafficking. This study shows that Slik is a new STRIPAK-associated kinase. PP2A_STRIPAK was found to promote Slik association with the cortex by regulating its phosphorylation status. Finally, PP2A_STRIPAK was shown to control Slik and moesin functions to regulate mitotic morphogenesis and epithelial integrity. These results place STRIPAK as a critical upstream regulator of cell morphogenesis through its effects on Slik and, ultimately, moesin (De Jamblinne, 2020).

This study discovered that dSTRIPAK dephosphorylates Slik to favor its association with the cortex. This regulation of Slik cortical association occurs both in interphase and in metaphase. Importantly, loss of Slik cortical association correlates with a decreased activation of moesin, the substrate of Slik throughout the cell cycle. Finally, dSTRIPAK was shown to control moesin-related biological functions such as mitotic morphogenesis and epithelial integrity during development (De Jamblinne, 2020).

dSTRIPAK is a multimolecular complex that functionally bridges PP2A phosphatase activity with kinases of the Ste20-like family. PP2A_STRIPAK was shown to inhibit the catalytic activity of its associated kinases. For instance, PP2A_STRIPAK dephosphorylates key regulatory residues of the activation loop of Hippo and Mst3/4. This study found a new functional interaction between PP2A_STRIPAK and an Ste20-like kinase. PP2A_STRIPAK regulates Slik phosphorylation to control its association with the cortex and thereby regulates its activity toward moesin. PP2A_STRIPAK could indirectly regulate an upstream regulator of Slik or directly dephosphorylate this Ste20-like kinase. The latter hypothesis is favored because this study found that Slik associates with Cka and Strip, two major components of PP2A_STRIPAK (De Jamblinne, 2020).

It is unlikely that PP2A_STRIPAK dephosphorylates the regulatory residues of the activation loop of Slik, because no change of its kinase activity was detected upon depletion of key components of PP2A_STRIPAK. Focus was then placed on 21 other potential phosphosites, and PP2A_STRIPAK was found to favor association of Slik with the cortex by regulating some of these phosphosites. Replacing these potentially phosphorylated residues by nonphosphorylatable alanine either within the NCD or within the CTD of Slik is sufficient to promote the cortical enrichment of these two phosphodeficient mutants and to prevent their regulation by PP2A_STRIPAK. Importantly, both these mutants rescue or partially rescue the decrease in moesin phosphorylation observed after PP2A_STRIPAK depletion. Although these experiments demonstrate that global phosphorylation governs the association of Slik with the cortex, the sites regulated by PP2A_STRIPAK remain to be precisely identified (De Jamblinne, 2020).

It was previously found that the CTD of Slik is necessary and sufficient for its association with the cell cortex. Interestingly, this domain is polybasic (with a theoretical isoelectric point of ~9.6 compared with Slik ~6.1). As previously demonstrated for other signaling proteins, these positive charges could promote the association of Slik with the negatively charged inner leaflet of the plasma membrane. It is proposed that the global negative charge brought by phosphorylation promotes the dissociation of Slik with the cell cortex by electrostatic repulsion with the negatively charged plasma membrane. In this model, a cycle of phosphorylation-dephosphorylation of Slik serves as an electrostatic switch that controls Slik cortical association. It has previously been reported that Slik can be phosphorylated by other not yet identified kinases. Consistent with this, it was found that OA treatment promotes phosphorylation of a Slik kinase dead mutant even in the absence of endogenous Slik. Therefore the hypothesis is favored that one or more other kinases regulate the association of Slik with the cortex. Current efforts aim to identify which kinase(s) could promote the dissociation of Slik from the plasma membrane (De Jamblinne, 2020).

PP2A phosphatase activity plays important roles during mitosis. Independently of STRIPAK, PP2A regulates mitotic exit by dephosphorylating Cdk1 substrates when associated with the B55 regulatory subunit (Drosophila Tws). When associated with the B56 regulatory subunit (Drosophila Wdb), PP2A controls spindle organization by counteracting several kinases, such as Aurora-B or Plk1. However, only a few studies reported a role of PP2A_STRIPAK during mitosis. In mammalian cells, PP2A_STRIPAK regulates abscission by controlling Mink1 activity, and depletion of STRIP or striatin promotes cytokinesis failures. This study has discovered that PP2A_STRIPAK controls mitotic morphogenesis. Mitotic cell morphogenesis requires that moesin be activated at mitosis onset. PP2A_STRIPAK was found to control enrichment of Slik at the cortex of metaphase cells and subsequent moesin activation (De Jamblinne, 2020).

Moesin and Slik play an important role in promoting epithelial integrity in vivo, although the details of their regulation in this context are also poorly understood. In mitotic epithelial cells, p-moesin accumulates at the basolateral membrane and is needed for planar spindle orientation. Moesin-depleted cells frequently undergo mitoses perpendicular to the plane of the epithelium, with the daughter cells undergoing EMT and delaminating and ultimately being eliminated by apoptosis. This appears to be a major cause of the loss of epithelial tissue integrity in moesin mutants. Several observations implicate dSTRIPAK in promoting moesin activation and epithelial tissue integrity. dSTRIPAK-depleted cells undergo EMT, delaminate, and are eliminated by apoptosis, particularly those located in the wing pouch. In dSTRIPAK-depleted cells in the wing hinge region that maintained their epithelial phenotype, reduced levels were observed of p-moesin, a readout for Slik activity. This was not just correlative; the ability of overexpressed Slik to phosphorylate moesin depended on dSTRIPAK. Finally, dSTRIPAK-depleted cells showed a redistribution of Slik protein, with a reduction of protein levels particularly prominent in the basal half of the cells. Together, these results support the finding that dSTRIPAK is a general regulator of Slik localization to promote the morphogenetic functions of moesin. It is noted that the phenotypic consequences of interfering with dSTRIPAK were more severe than those observed upon depletion of Slik, with strong dSTRIPAK depletion causing early lethality. This suggests that dSTRIPAK regulates other targets, consistent with its known role in downregulating the growth-inhibitory function of Hippo. Hippo signaling is particularly important for growth of the wing blade, which may explain why depletion of dSTRIPAK components had a stronger effect in the wing pouch than in hinge regions (De Jamblinne, 2020).

In conclusion, this study identified that PP2A_STRIPAK regulates the localization of Slik by phosphorylation to control moesin activation. This work places STRIPAK as a critical upstream regulator of moesin function for mitotic morphogenesis and epithelial integrity (De Jamblinne, 2020).

Moesin is involved in polarity maintenance and cortical remodelling during asymmetric cell division

An intact actomyosin network is essential for anchoring polarity proteins to the cell cortex and maintaining cell size asymmetry during asymmetric cell division of Drosophila neuroblasts. However, the mechanisms that control changes in actomyosin dynamics during asymmetric cell division remain unclear. This study finds that the actin-binding protein, Moesin, is essential for neuroblast proliferation and mitotic progression in the developing brain. During metaphase, phosphorylated Moesin (p-Moesin) is enriched at the apical cortex and loss of Moesin leads to defects in apical polarity maintenance and cortical stability. This asymmetric distribution of p-Moesin is determined by components of the apical polarity complex and Slik kinase. During later stages of mitosis, p-Moesin localization shifts more basally, contributing to asymmetric cortical extension and myosin basal furrow positioning. These findings reveal Moesin as a novel apical polarity protein that drives cortical remodelling of dividing neuroblasts, which is essential for polarity maintenance and initial establishment of cell size asymmetry (Abeysundara, 2017).

Studies investigating ERM function have highlighted the importance of the ERM proteins in regulating the mechanical properties of the cell cortex. This study provides new insight into the role of Moesin in organizing the cortex of cells that establish intrinsic polarity and undergo asymmetric cell division (ACD) in vivo. When Moesin was knocked down in Insc-expressing cells, the larval CNS was reduced in size due to a decrease in the proportion of dividing NBs throughout larval development. Expressing Moe^dsRNA using the Insc-GAL4 driver affected overall larval development and resulted in larval lethality. However, viable progeny were obtained when Moesin levels were reduced using other NB-GAL4 drivers, asense-GAL4 and worniu-GAL4. When upstream activation sequence-GFP (UAS-GFP) was expressed using the different NB GAL4 drivers, GFP mRNA expression was ~5 fold greater using Insc-GAL4 compared with asense- or worniu-GAL4. Thus, the differences in viability are likely due to the increased strength of Insc-GAL4. Recent studies that identified the Hippo pathway as an essential regulator of NB quiescence also used Insc-GAL4 in their analyses. As it cannot be excluded that the reduced proportion of mitotic NBs may partially be due to impaired cell cycle reentry or an overall delay in larval development, the analysis focussed on the mitotic NBs that had exited quiescence. It was confirmed that defects in mitotic progression and polarity maintenance were observed at both early and late stages of the Moesin knockdown and in the late hypomorphic mutants, demonstrating a functional requirement of Moesin within the larval NBs (Abeysundara, 2017).

Proper regulation and function of the ERM proteins are required during cell division in both flies and mammals . In Drosophila S2 cells, the increased and uniform distribution of p-Moesin at the metaphase cortex enhanced cortical rigidity and cell rounding, proposed to be essential for stable spindle positioning. Drosophila Moesin was also shown to bind and stabilize microtubules at the cortex of cultured cells. Thus, an asymmetric ERM distribution during metaphase would be predicted to influence spindle position and orientation accordingly. In human colorectal Caco2 cells, polarized ezrin locally stabilized actin, providing a physical platform for astral microtubule-mediated centrosome positioning during interphase. HeLa cells cultured on L-shaped micropatterns also displayed restricted ERM activation at the cell cortex adjacent to the adhesive substrate, which was essential for LGN/NuMA polarization and guiding spindle orientation. In Drosophila wing imaginal epithelial cells, p-Moesin was enriched at the basal cortex of mitotic cells and the loss of Moesin led to defects in planar spindle orientation and recruitment of the pericentriolar material marker, Centrosomin. Thus, a role for Moesin in guiding spindle orientation and centrosome behaviour has been well documented. In Drosophila NBs, this study found that p-Moesin was apically enriched at the metaphase cortex, although the mitotic spindle has been reported to be symmetric and centrally located during metaphase. Thus, apical p-Moesin is likely not involved in generating spindle asymmetry during metaphase. The possibility of its involvement in preparing for the establishment of an asymmetric spindle during anaphase cannot be excluded. Furthermore, the loss of Moesin affected spindle orientation in only a small proportion of NBs, and the localization of the Drosophila LGN orthologue, Pins, was largely unaffected in Moesin knockdown NBs during metaphase. Thus, Moesin does not appear to play a prominent role in regulating spindle orientation in NBs. However, Moesin may affect the localization or activity of interacting partners downstream of Pins such as Mud or the heterotrimeric G protein subunit Gαi. Alternatively, the loss of both Moesin and Pins may cause more severe defects in spindle orientation and cell size asymmetry. Thus, future studies examining the loss of both Moesin and Pins may reveal a role for Moesin in maintaining centrosome positioning and spindle orientation in NBs (Abeysundara, 2017).

This study found that overall NB cell size was reduced in the Moesin knockdown. The reduced size of interphase NBs during early larval stages (48 h ALH) suggests that Moesin may be involved in NB enlargement prior to NB exit from quiescence. NB reactivation also appeared impaired in the ventral nerve cords of Moesin knockdown larvae. Previous studies have implicated Insulin/PI3K signaling in NB growth and reactivation during early larval stages. Further examination of these signaling pathways in the Moesin knockdown NBs are required to determine the mechanisms underlying its potential role in NB enlargement and reactivation. Of the NBs that had exited quiescence, a large proportion of mitotic defective NBs were observed during early and late larval stages. These NBs were not round and may reflect the importance of Moesin in cell rounding during early mitosis, as previously shown in Drosophila cell culture. Alternatively, the mitotic defective NBs may represent a population of NBs that have failed to undergo cell division. As the loss of Moesin also resulted in a reduced proportion of mitotic NBs undergoing each stage of mitosis, it is proposed that Moesin is essential for cell shape changes and mitotic progression during ACD (Abeysundara, 2017).

ERM proteins localize to the apical cortex of a wide variety of polarized cells and are essential for maintaining the apical identity and surface properties of epithelial tissues across multiple organisms. By binding directly to filamentous actin and linking membrane-associated proteins to the underlying actin cytoskeleton, the ERM proteins localize to numerous actin-rich structures. Thus, it is possible that the apical p-Moesin represents areas rich in actin filaments at the NB cortex. Although the actin cytoskeleton is important for cortical tethering of polarity complexes in NBs, filamentous actin does not display an asymmetric distribution. Thus, apical p-Moesin may correlate with enhanced cortical stability at the apical cortex necessary for polarity maintenance and integrity (Abeysundara, 2017).

Confirming a role for p-Moesin in stabilizing cortical actin, it was found that Bazooka and aPKC crescents were not observed in a proportion of Moe^dsRNA NBs undergoing prophase and actin appeared discontinuous at the cell cortex. As Bazooka and aPKC polarity is established by prophase, prior to the polar enrichment of p-Moesin, it is concluded that Moesin is involved in polarity maintenance rather than establishment. Similarly, in the Mus musculus and Caenorhabditis elegans intestinal epithelium, ERM proteins are involved in apical membrane assembly and integrity but do not appear to be required for polarity establishment. During metaphase, a proportion of Moe^dsRNA NBs lacked both Par-6 and aPKC polar crescents. However, the majority of Moe^dsRNA NBs displayed Bazooka and Pins polar crescents at the metaphase cortex. In the absence of Par-6 and aPKC, apical domains consisting of Bazooka, Inscuteable, Pins, and Discs large are still able to form. Thus, Moesin may be specifically maintaining Par-6/aPKC polarity during metaphase but have little effect on other apical polarity proteins such as Bazooka and Pins. Furthermore, the aPKC polar domain was disorganized, and cortical blebbing was observed in the Moe^G0323 mutant NBs. Thus, Moesin regulates the integrity and maintenance of the apical domain, likely through affecting cortical stability during ACD (Abeysundara, 2017).

The complex spatiotemporal regulation of Moesin activity during mitosis has been demonstrated in symmetrically dividing S2 cells and requires the coordinated activities of PP1-87B phosphatase, Slik kinase, and regulators of phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) levels at the cell cortex. This study showed that Slik was uniformly distributed at the NB cell cortex. As Slik is regulated by phosphorylation, it is possible that the phosphorylated Slik is asymmetrically distributed in mitotic NBs. Furthermore, Slik was found to be essential for NB proliferation and polarity maintenance, likely through regulating Moesin phosphorylation at the NB cortex. The loss of Flapwing and PP1-87B phosphatases did not alter the apical enrichment of p-Moesin in metaphase NBs. Future studies examining other phosphatases and regulators of PI(4,5)P2 levels at the NB cortex are essential for further understanding Moesin regulation during ACD (Abeysundara, 2017).

In addition to Slik kinase, this study found that known apical polarity proteins (Cdc42, Par-6, aPKC, Lgl, and Pins) are important for the proper apical enrichment of p-Moesin during metaphase. As Moesin is also important for maintenance of the apical domain, these findings support a mutually dependent interaction among the apical polarity proteins that has been extensively reported in NBs. Components of the apical polarity complexes also mediate spindle asymmetry and asymmetric cortical extension during anaphase, leading to the generation of unequal-sized daughter cells. Similarly, this study found that Moesin was important for initial positioning of an asymmetric basal furrow during anaphase (Abeysundara, 2017).

In Drosophila NBs, a cortical polarity-induced pathway, consisting of Pins and the heterotrimeric G-proteins, is essential for apical cortical extension and formation of a Myosin-induced basal furrow, independent of the mitotic spindle. This study found that the relative fluorescent intensity (FI) of p-Moesin was reduced at the apical cortex during anaphase when compared with metaphase NBs. Furthermore, the loss of Moesin resulted in the absence of p-Myosin at the basal cortex, affecting basal furrow positioning during anaphase. In Drosophila S2 cells, reduced p-Moesin at the cell poles was shown to lead to cortical relaxation and membrane elongation. Thus, it is proposed that p-Moesin regulation at the apical cortex is important for asymmetric cortical extension and furrow positioning during early anaphase, likely along with Pins and the heterotrimeric G-proteins. However, Moesin also appeared to influence Myosin-mediated cortical contractility during metaphase as well. This study showed that with the loss of Moesin, p-Myosin and Rok-GFP displayed a nonuniform distribution at the metaphase cortex, revealing unstable actomyosin dynamics and a delay in anaphase onset. Although no observable differences were found in cortical Rho1 localization at the metaphase cortex, future studies using alternative biosensor approaches may allow for more precise visualization and analysis of Rho1 signaling. In addition, further investigation of the mechanical properties of cultured NBs will provide great insight into how Moesin function influences the mitotic cortex in the absence of physical constraint or external cues. While this work was under review, another group showed that Rok and Protein Kinase N are involved in the precise spatiotemporal regulation of Myosin flow during the establishment of physical asymmetry. Given the current findings, it will be interesting to further examine how Moesin precisely regulates Myosin dynamics, along with the other components of the polarity-induced cleavage furrow positioning pathway (Abeysundara, 2017).

GENE STRUCTURE

cDNA clone length - 2418

Bases in 5' UTR - 247

Exons - 9

Bases in 3' UTR - 343

PROTEIN STRUCTURE

Amino Acids - 575

Structural Domains

Moesin shares 58% overall identity with both human moesin and human ezrin, 57% identity with mouse radixin, and only 41% identity with human merlin. Designation of this gene as Moesin, rather than as ezrin or radixin, is based primarily on its lack of the polyproline tract characteristic of the latter two proteins. furthermore, Moesin shares a greater degree of identity in the C-terminal divergent region with human moesin (26%) than with human ezrin (25%) or mouse radixin (22%) (McCartney, 1996).

Analysis of the Drosophila genome identified a single gene encoding an ERM protein, which had been previously cloned and named Moesin. However, when compared with human ERMs, Drosophila Moesin has strongest conservation with Radixin in the FERM domain, with Ezrin in the C-terminal tail, and shares with mammalian Moesin the absence of a polyproline track that is found in both Ezrin and Radixin. To gain further insight into the evolution of ERMs, sequence conservation of ERM proteins was analyzed from various species. Each available ERM protein from vertebrates seems to be a true orthologue of Ezrin, Radixin or Moesin, respectively, suggesting that three ERM genes exist in these species. By contrast, in common with Drosophila, ERMs from Caenorhabditis elegans and the snail Biomphalaria glabrata are equally related to Ezrin, Radixin and Moesin, thus suggesting that the presence of three ERM genes is restricted to deuterostomes. However, Merlin/NF2 proteins from protostomes and deuterostomes are more related to each other than to ERM, indicating that divergence between NF2 and ERM preceded the separation of these two taxa. Within the C-terminal tail, Drosophila Moesin also contains residues characteristic of Ezrin, Radixin or Moesin, and the threonine involved in ERM activation is conserved in Drosophila Moesin (position 559). Thus, Drosophila Moesin seems to be a prototypical ERM protein, sharing both common and specific features with all three vertebrate ERMs (Polesello, 2002).

date revised: 25 October 2004

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