The polarity protein Yurt associates with the plasma membrane via basic and hydrophobic motifs embedded in its FERM domain.

The subcellular distribution of the polarity protein Yurt (Yrt) is subjected to a spatio-temporal regulation in Drosophila melanogaster embryonic epithelia. After cellularization, Yrt binds to the lateral membrane of ectodermal cells and maintains this localization throughout embryogenesis. During terminal differentiation of the epidermis, Yrt accumulates at septate junctions and is also recruited to the apical domain. Although the mechanisms through which Yrt associates with septate junctions and the apical domain have been deciphered, how Yrt binds to the lateral membrane remains as an outstanding puzzle. Here, we show that the FERM domain of Yrt is necessary and sufficient for membrane localization. Our data also establish that the FERM domain of Yrt directly binds negatively charged phospholipids. Moreover, we demonstrate that positively charged amino acid motifs embedded within the FERM domain mediates Yrt membrane association. Finally, we provide evidence suggesting that Yrt membrane association is functionally important. Overall, our study highlights the molecular basis of how Yrt associates with the lateral membrane during the developmental time window where it is required for segregation of lateral and apical domains.

Direct Phosphorylation of SRC Homology 3 Domains by Tyrosine Kinase Receptors Disassembles Ligand-Induced Signaling Networks.

Phosphotyrosine (pTyr) signaling has evolved into a key cell-to-cell communication system. Activated receptor tyrosine kinases (RTKs) initiate several pTyr-dependent signaling networks by creating the docking sites required for the assembly of protein complexes. However, the mechanisms leading to network disassembly and its consequence on signal transduction remain essentially unknown. We show that activated RTKs terminate downstream signaling via the direct phosphorylation of an evolutionarily conserved Tyr present in most SRC homology (SH) 3 domains, which are often part of key hub proteins for RTK-dependent signaling. We demonstrate that the direct EPHA4 RTK phosphorylation of adaptor protein NCK SH3s at these sites results in the collapse of signaling networks and abrogates their function. We also reveal that this negative regulation mechanism is shared by other RTKs. Our findings uncover a conserved mechanism through which RTKs rapidly and reversibly terminate downstream signaling while remaining in a catalytically active state on the plasma membrane.

Girdin is a component of the lateral polarity protein network restricting cell dissemination.

Epithelial cell polarity defects support cancer progression. It is thus crucial to decipher the functional interactions within the polarity protein network. Here we show that Drosophila Girdin and its human ortholog (GIRDIN) sustain the function of crucial lateral polarity proteins by inhibiting the apical kinase aPKC. Loss of GIRDIN expression is also associated with overgrowth of disorganized cell cysts. Moreover, we observed cell dissemination from GIRDIN knockdown cysts and tumorspheres, thereby showing that GIRDIN supports the cohesion of multicellular epithelial structures. Consistent with these observations, alteration of GIRDIN expression is associated with poor overall survival in subtypes of breast and lung cancers. Overall, we discovered a core mechanism contributing to epithelial cell polarization from flies to humans. Our data also indicate that GIRDIN has the potential to impair the progression of epithelial cancers by preserving cell polarity and restricting cell dissemination.

A Fanci knockout mouse model reveals common and distinct functions for FANCI and FANCD2.

Fanconi Anemia (FA) clinical phenotypes are heterogenous and rely on a mutation in one of the 22 FANC genes (FANCA-W) involved in a common interstrand DNA crosslink-repair pathway. A critical step in the activation of FA pathway is the monoubiquitination of FANCD2 and its binding partner FANCI. To better address the clinical phenotype associated with FANCI and the epistatic relationship with FANCD2, we created the first conditional inactivation model for FANCI in mouse. Fanci -/- mice displayed typical FA features such as delayed development in utero, microphtalmia, cellular sensitivity to mitomycin C, occasional limb abnormalities and hematological deficiencies. Interestingly, the deletion of Fanci leads to a strong meiotic phenotype and severe hypogonadism. FANCI was localized in spermatocytes and spermatids and in the nucleus of oocytes. Both FANCI and FANCD2 proteins co-localized with RPA along meiotic chromosomes, albeit at different levels. Consistent with a role in meiotic recombination, FANCI interacted with RAD51 and stimulated D-loop formation, unlike FANCD2. The double knockout Fanci-/- Fancd2-/- also showed epistatic relationship for hematological defects while being not epistatic with respect to generating viable mice in crosses of double heterozygotes. Collectively, this study highlights common and distinct functions of FANCI and FANCD2 during mouse development, meiotic recombination and hematopoiesis.

Girdin-mediated interactions between cadherin and the actin cytoskeleton are required for epithelial morphogenesis in Drosophila.

E-cadherin-mediated cell-cell adhesion is fundamental for epithelial tissue morphogenesis, physiology and repair. E-cadherin is a core transmembrane constituent of the zonula adherens (ZA), a belt-like adherens junction located at the apicolateral border in epithelial cells. The anchorage of ZA components to cortical actin filaments strengthens cell-cell cohesion and allows for junction contractility, which shapes epithelial tissues during development. Here, we report that the cytoskeletal adaptor protein Girdin physically and functionally interacts with components of the cadherin-catenin complex during Drosophila embryogenesis. Fly Girdin is broadly expressed throughout embryonic development and enriched at the ZA in epithelial tissues. Girdin associates with the cytoskeleton and co-precipitates with the cadherin-catenin complex protein α-Catenin (α-Cat). Girdin mutations strongly enhance adhesion defects associated with reduced DE-cadherin (DE-Cad) expression. Moreover, the fraction of DE-Cad molecules associated with the cytoskeleton decreases in the absence of Girdin, thereby identifying Girdin as a positive regulator of adherens junction function. Girdin mutant embryos display isolated epithelial cell cysts and rupture of the ventral midline, consistent with defects in cell-cell cohesion. In addition, loss of Girdin impairs the collective migration of epithelial cells, resulting in dorsal closure defects. We propose that Girdin stabilizes epithelial cell adhesion and promotes morphogenesis by regulating the linkage of the cadherin-catenin complex to the cytoskeleton.

Yurt, Coracle, Neurexin IV and the Na(+),K(+)-ATPase form a novel group of epithelial polarity proteins.

The integrity of polarized epithelia is critical for development and human health. Many questions remain concerning the full complement and the function of the proteins that regulate cell polarity. Here we report that the Drosophila FERM proteins Yurt (Yrt) and Coracle (Cora) and the membrane proteins Neurexin IV (Nrx-IV) and Na(+),K(+)-ATPase are a new group of functionally cooperating epithelial polarity proteins. This 'Yrt/Cora group' promotes basolateral membrane stability and shows negative regulatory interactions with the apical determinant Crumbs (Crb). Genetic analyses indicate that Nrx-IV and Na(+),K(+)-ATPase act together with Cora in one pathway, whereas Yrt acts in a second redundant pathway. Moreover, we show that the Yrt/Cora group is essential for epithelial polarity during organogenesis but not when epithelial polarity is first established or during terminal differentiation. This property of Yrt/Cora group proteins explains the recovery of polarity in embryos lacking the function of the Lethal giant larvae (Lgl) group of basolateral polarity proteins. We also find that the mammalian Yrt orthologue EPB41L5 (also known as YMO1 and Limulus) is required for lateral membrane formation, indicating a conserved function of Yrt proteins in epithelial polarity.

Crumbs controls epithelial integrity by inhibiting Rac1 and PI3K.

Drosophila Crumbs (Crb) and its mammalian ortholog CRB3 control epithelial polarity through poorly understood molecular mechanisms. Elucidating these mechanisms is crucial, because the physiology of epithelia largely depends on the polarized architecture of individual epithelial cells. In addition, loss of CRB3 favors tumor cell growth, metastasis and epithelial to mesenchymal transition (EMT). Using Drosophila embryos, we report that Rac1 sustains PI3K signaling, which is required for Rac1 activation. Crb represses this positive-feedback loop. Notably, this property confers to Crb its ability to promote epithelial integrity in vivo, because attenuation of either Rac1 or PI3K activity rescues the crb mutant phenotype. Moreover, inhibition of Rac1 or PI3K results in Crb-dependent apical membrane growth, whereas Rac1 activation restricts membrane localization of Crb and interferes with apical domain formation. This illustrates that Crb and the Rac1-PI3K module are antagonists, and that the fine balance between the activities of these proteins is crucial to maintain epithelial organization and an appropriate apical to basolateral ratio. Together, our results elucidate a mechanism that mediates Crb function and further define the role of PI3K and Rac1 in epithelial morphogenesis, allowing for a better understanding of how distinct membrane domains are regulated in polarized epithelial cells.

EPH receptor tyrosine kinases phosphorylate the PAR-3 scaffold protein to modulate downstream signaling networks.

EPH receptors (EPHRs) constitute the largest family among receptor tyrosine kinases in humans. They are mainly involved in short-range cell-cell communication events that regulate cell adhesion, migration, and boundary formation. However, the molecular mechanisms by which EPHRs control these processes are less understood. To address this, we unravel EPHR-associated complexes under native conditions using mass-spectrometry-based BioID proximity labeling. We obtain a composite proximity network from EPHA4, -B2, -B3, and -B4 that comprises 395 proteins, most of which were not previously linked to EPHRs. We examine the contribution of several BioID-identified candidates via loss-of-function in an EPHR-dependent cell-segregation assay. We find that the signaling scaffold PAR-3 is required for cell sorting and that EPHRs directly phosphorylate PAR-3. We also delineate a signaling complex involving the C-terminal SRC kinase (CSK), whose recruitment to PAR-3 is dependent on EPHR signals. Our work describes signaling networks by which EPHRs regulate cellular phenotypes.

The FERM protein Yurt is a negative regulatory component of the Crumbs complex that controls epithelial polarity and apical membrane size.

The Crumbs (Crb) complex is a key regulator of epithelial cell architecture where it promotes apical membrane formation. Here, we show that binding of the FERM protein Yurt to the cytoplasmic domain of Crb is part of a negative-feedback loop that regulates Crb activity. Yurt is predominantly a basolateral protein but is recruited by Crb to apical membranes late during epithelial development. Loss of Yurt causes an expansion of the apical membrane in embryonic epithelia and photoreceptor cells similar to Crb overexpression and in contrast to loss of Crb. Analysis of yurt crb double mutants suggests that these genes function in one pathway and that yurt negatively regulates crb. We also show that the mammalian Yurt orthologs YMO1 and EHM2 bind to mammalian Crb proteins. We propose that Yurt is part of an evolutionary conserved negative-feedback mechanism that restricts Crb complex activity in promoting apical membrane formation.

Emerging role for epithelial polarity proteins of the Crumbs family as potential tumor suppressors.

Defects in apical-basal polarity regulation are associated with tissue overgrowth and tumorogenesis, yet the molecular mechanisms linking epithelial polarity regulators to hyperplasia or neoplasia remain elusive. In addition, exploration of the expression and function of the full complement of proteins required for the polarized architecture of epithelial cells in the context of cancer is awaited. This paper provides an overview of recent studies performed on Drosophila and vertebrates showing that apical polarity proteins of the Crumbs family act to repress tissue growth and epithelial to mesenchymal transition. Thus, these proteins emerge as potential tumor suppressors. Interestingly, analysis of the molecular function of Crumbs proteins reveals a function for these polarity regulators in junctional complexes stability and control of signaling pathways regulating proliferation and apoptosis. Thereby, these studies provide a molecular basis explaining how regulation of epithelial polarity is coupled to tumorogenesis.

Pak1 and PP2A antagonize aPKC function to support cortical tension induced by the Crumbs-Yurt complex.

The Drosophila polarity protein Crumbs is essential for the establishment and growth of the apical domain in epithelial cells. The protein Yurt limits the ability of Crumbs to promote apical membrane growth, thereby defining proper apical/lateral membrane ratio that is crucial for forming and maintaining complex epithelial structures such as tubes or acini. Here, we show that Yurt also increases Myosin-dependent cortical tension downstream of Crumbs. Yurt overexpression thus induces apical constriction in epithelial cells. The kinase aPKC phosphorylates Yurt, thereby dislodging the latter from the apical domain and releasing apical tension. In contrast, the kinase Pak1 promotes Yurt dephosphorylation through activation of the phosphatase PP2A. The Pak1-PP2A module thus opposes aPKC function and supports Yurt-induced apical constriction. Hence, the complex interplay between Yurt, aPKC, Pak1, and PP2A contributes to the functional plasticity of Crumbs. Overall, our data increase our understanding of how proteins sustaining epithelial cell polarization and Myosin-dependent cell contractility interact with one another to control epithelial tissue architecture.

Drosophila convoluted/dALS is an essential gene required for tracheal tube morphogenesis and apical matrix organization.

Insulin-like growth factors (IGFs) control cell and organism growth through evolutionarily conserved signaling pathways. The mammalian acid-labile subunit (ALS) is a secreted protein that complexes with IGFs to modulate their activity. Recent work has shown that a Drosophila homolog of ALS, dALS, can also complex with and modulate the activity of a Drosophila IGF. Here we report the first mutations in the gene encoding dALS. Unexpectedly, we find that these mutations are allelic to a previously described mutation in convoluted (conv), a gene required for epithelial morphogenesis. In conv mutants, the tubes of the Drosophila tracheal system become abnormally elongated without altering tracheal cell number. conv null mutations cause larval lethality, but do not disrupt several processes required for tracheal tube size control, including septate junction formation, deposition of a lumenal/apical extracellular matrix, and lumenal secretion of Vermiform and Serpentine, two putative matrix-modifying proteins. Clearance of lumenal matrix and subcellular localization of clathrin also appear normal in conv mutants. However, we show that Conv/dALS is required for the dynamic organization of the transient lumenal matrix and normal structure of the cuticle that lines the tracheal lumen. These and other data suggest that the Conv/dALS-dependent tube size control mechanism is distinct from other known processes involved in tracheal tube size regulation. Moreover, we present evidence indicating that Conv/dALS has a novel, IGF-signaling independent function in tracheal morphogenesis.

Essential function of Drosophila Sec6 in apical exocytosis of epithelial photoreceptor cells.

Polarized exocytosis plays a major role in development and cell differentiation but the mechanisms that target exocytosis to specific membrane domains in animal cells are still poorly understood. We characterized Drosophila Sec6, a component of the exocyst complex that is believed to tether secretory vesicles to specific plasma membrane sites. sec6 mutations cause cell lethality and disrupt plasma membrane growth. In developing photoreceptor cells (PRCs), Sec6 but not Sec5 or Sec8 shows accumulation at adherens junctions. In late PRCs, Sec6, Sec5, and Sec8 colocalize at the rhabdomere, the light sensing subdomain of the apical membrane. PRCs with reduced Sec6 function accumulate secretory vesicles and fail to transport proteins to the rhabdomere, but show normal localization of proteins to the apical stalk membrane and the basolateral membrane. Furthermore, we show that Rab11 forms a complex with Sec5 and that Sec5 interacts with Sec6 suggesting that the exocyst is a Rab11 effector that facilitates protein transport to the apical rhabdomere in Drosophila PRCs.

Adenoviral protein E4orf4 interacts with the polarity protein Par3 to induce nuclear rupture and tumor cell death.

The tumor cell-selective killing activity of the adenovirus type 2 early region 4 ORF4 (E4orf4) protein is poorly defined at the molecular level. Here, we show that the tumoricidal effect of E4orf4 is typified by changes in nuclear dynamics that depend on its interaction with the polarity protein Par3 and actomyosin contractility. Mechanistically, E4orf4 induced a high incidence of nuclear bleb formation and repetitive nuclear ruptures, which promoted nuclear efflux of E4orf4 and loss of nuclear integrity. This process was regulated by nucleocytoskeletal connections, Par3 clustering proximal to nuclear lamina folds, and retrograde movement of actin bundles that correlated with nuclear ruptures. Significantly, Par3 also regulated the incidence of spontaneous nuclear ruptures facilitated by the downmodulation of lamins. This work uncovered a novel role for Par3 in controlling the actin-dependent forces acting on the nuclear envelope to remodel nuclear shape, which might be a defining feature of tumor cells that is harnessed by E4orf4.

Oligomerization of the FERM-FA protein Yurt controls epithelial cell polarity.

Drosophila melanogaster Yurt (Yrt) and its mammalian orthologue EPB41L5 limit apical membrane growth in polarized epithelia. EPB41L5 also supports epithelial-mesenchymal transition and metastasis. Yrt and EPB41L5 contain a four-point-one, ezrin, radixin, and moesin (FERM) domain and a FERM-adjacent (FA) domain. The former contributes to the quaternary structure of 50 human proteins, whereas the latter defines a subfamily of 14 human FERM proteins and fulfills unknown roles. In this study, we show that both Yrt and EPB41L5 oligomerize. Our data also establish that the FERM-FA unit forms an oligomeric interface and that multimerization of Yrt is crucial for its function in epithelial cell polarity regulation. Finally, we demonstrate that aPKC destabilizes the Yrt oligomer to repress its functions, thereby revealing a mechanism through which this kinase supports apical domain formation. Overall, our study highlights a conserved biochemical property of fly and human Yrt proteins, describes a novel function of the FA domain, and further characterizes the molecular mechanisms sustaining epithelial cell polarity.

Mouse Crumbs3 sustains epithelial tissue morphogenesis in vivo.

The human apical protein CRB3 (Crb3 in mouse) organizes epithelial cell polarity. Loss of CRB3 expression increases the tumorogenic potential of cultured epithelial cells and favors metastasis formation in nude mice. These data emphasize the need of in vivo models to study CRB3 functions. Here, we report the phenotypic analysis of a novel Crb3 knockout mouse model. Crb3-deficient newborn mice show improper clearance of airways, suffer from respiratory distress and display perinatal lethality. Crb3 is also essential to maintain apical membrane identity in kidney epithelial cells. Numerous kidney cysts accompany these polarity defects. Impaired differentiation of the apical membrane is also observed in a subset of cells of the intestinal epithelium. This results in improper remodeling of adhesive contacts in the developing intestinal epithelium, thereby leading to villus fusion. We also noted a strong increase in cytoplasmic ß-catenin levels in intestinal epithelial cells. ß-catenin is a mediator of the Wnt signaling pathway, which is overactivated in the majority of colon cancers. In addition to clarifying the physiologic roles of Crb3, our study highlights that further functional analysis of this protein is likely to provide insights into the etiology of diverse pathologies, including respiratory distress syndrome, polycystic kidney disease and cancer.

Rac1 controls epithelial tube length through the apical secretion and polarity pathways.

The morphometric parameters of epithelial tubes are critical to the physiology and homeostasis of most organs. In addition, many human diseases are associated with tube-size defects. Here, we show that Rac1 limits epithelial tube elongation in the developing fly trachea by promoting Rab5-dependent endocytosis of the apical determinant Crumbs. Rac1 is also involved in a positive feedback loop with the septate junction protein Coracle. Thereby, Rac1 precludes paracellular diffusion and contributes to the septate junction-dependent secretion of the chitin-modifying enzymes Vermiform and Serpentine, which restrict epithelial tube length independently of Crumbs. Thus, Rac1 is a critical component of two important pathways controlling epithelial tube morphogenesis.

CRB3A Controls the Morphology and Cohesion of Cancer Cells through Ehm2/p114RhoGEF-Dependent Signaling.

The transmembrane protein CRB3A controls epithelial cell polarization. Elucidating the molecular mechanisms of CRB3A function is essential as this protein prevents the epithelial-to-mesenchymal transition (EMT), which contributes to tumor progression. To investigate the functional impact of altered CRB3A expression in cancer cells, we expressed CRB3A in HeLa cells, which are devoid of endogenous CRB3A. While control HeLa cells display a patchy F-actin distribution, CRB3A-expressing cells form a circumferential actomyosin belt. This reorganization of the cytoskeleton is accompanied by a transition from an ameboid cell shape to an epithelial-cell-like morphology. In addition, CRB3A increases the cohesion of HeLa cells. To perform these functions, CRB3A recruits p114RhoGEF and its activator Ehm2 to the cell periphery using both functional motifs of its cytoplasmic tail and increases RhoA activation levels. ROCK1 and ROCK2 (ROCK1/2), which are critical effectors of RhoA, are also essential to modulate the cytoskeleton and cell shape downstream of CRB3A. Overall, our study highlights novel roles for CRB3A and deciphers the signaling pathway conferring to CRB3A the ability to fulfill these functions. Thereby, our data will facilitate further investigation of CRB3A functions and increase our understanding of the cellular defects associated with the loss of CRB3A expression in cancer cells.

A bidirectional antagonism between aPKC and Yurt regulates epithelial cell polarity.

During epithelial cell polarization, Yurt (Yrt) is initially confined to the lateral membrane and supports the stability of this membrane domain by repressing the Crumbs-containing apical machinery. At late stages of embryogenesis, the apical recruitment of Yrt restricts the size of the apical membrane. However, the molecular basis sustaining the spatiotemporal dynamics of Yrt remains undefined. In this paper, we report that atypical protein kinase C (aPKC) phosphorylates Yrt to prevent its premature apical localization. A nonphosphorylatable version of Yrt dominantly dismantles the apical domain, showing that its aPKC-mediated exclusion is crucial for epithelial cell polarity. In return, Yrt counteracts aPKC functions to prevent apicalization of the plasma membrane. The ability of Yrt to bind and restrain aPKC signaling is central for its role in polarity, as removal of the aPKC binding site neutralizes Yrt activity. Thus, Yrt and aPKC are involved in a reciprocal antagonistic regulatory loop that contributes to segregation of distinct and mutually exclusive membrane domains in epithelial cells.