Epithelial cell-cell adhesion and morphogenesis require dynamic control of actin-driven membrane remodeling. The Rho guanosine triphosphatase (GTPase) Cdc42 regulates sequential molecular processes during cell-cell junction formation; hence, mechanisms must exist that inactivate Cdc42 in a temporally and spatially controlled manner. In this paper, we identify SH3BP1, a GTPase-activating protein for Cdc42 and Rac, as a regulator of junction assembly and epithelial morphogenesis using a functional small interfering ribonucleic acid screen. Depletion of SH3BP1 resulted in loss of spatial control of Cdc42 activity, stalled membrane remodeling, and enhanced growth of filopodia. SH3BP1 formed a complex with JACOP/paracingulin, a junctional adaptor, and CD2AP, a scaffolding protein; both were required for normal Cdc42 signaling and junction formation. The filamentous actin-capping protein CapZ also associated with the SH3BP1 complex and was required for control of actin remodeling. Epithelial junction formation and morphogenesis thus require a dual activity complex, containing SH3BP1 and CapZ, that is recruited to sites of active membrane remodeling to guide Cdc42 signaling and cytoskeletal dynamics.

Dividing epithelial cells need to coordinate spindle positioning with shape changes to maintain cell-cell adhesion. Microtubule interactions with the cell cortex regulate mitotic spindle positioning within the plane of division. How the spindle crosstalks with the actin cytoskeleton to ensure faithful mitosis and spindle positioning is unclear. Here we demonstrate that the tumour suppressor DLC2, a negative regulator of Cdc42, and the interacting kinesin Kif1B coordinate cell junction maintenance and planar spindle positioning by regulating microtubule growth and crosstalk with the actin cytoskeleton. Loss of DLC2 induces the mislocalization of Kif1B, increased Cdc42 activity and cortical recruitment of the Cdc42 effector mDia3, a microtubule stabilizer and promoter of actin dynamics. Accordingly, DLC2 or Kif1B depletion promotes microtubule stabilization, defective spindle positioning, chromosome misalignment and aneuploidy. The tumour suppressor DLC2 and Kif1B are thus central components of a signalling network that guides spindle positioning, cell-cell adhesion and mitotic fidelity.

Epithelial cells form sheets and tubules in various epithelial organs and establish apicobasal polarity and asymmetric vesicle transport to provide functionality in these structures. However, the molecular mechanisms that allow epithelial cells to establish polarity are not clearly understood. Here, we present evidence that the kinase activity of the receptor tyrosine kinase for collagen, discoidin domain receptor 1 (DDR1), is required for efficient establishment of epithelial polarity, proper asymmetric protein secretion, and execution of morphogenic programs. Lack of DDR1 protein or inhibition of DDR1 kinase activity disturbed tubulogenesis, cystogenesis, and the establishment of epithelial polarity and caused defects in the polarized localization of membrane-type 1 matrix metalloproteinase (MT1-MMP), GP135, primary cilia, laminin, and the Golgi apparatus. Disturbed epithelial polarity and cystogenesis upon DDR1 inhibition was caused by excess ROCK (rho-associated, coiled-coil-containing protein kinase)-driven actomyosin contractility, and pharmacological inhibition of ROCK was sufficient to correct these defects. Our data indicate that a DDR1-ROCK signaling axis is essential for the efficient establishment of epithelial polarity.

Actinomyosin activity is an important driver of cell locomotion and has been shown to promote collective cell migration of epithelial sheets as well as single cell migration and tumor cell invasion. However, the molecular mechanisms underlying activation of cortical myosin to stimulate single cell movement, and the relationship between the mechanisms that drive single cell locomotion and those that mediate collective cell migration of epithelial sheets are incompletely understood. Here, we demonstrate that p114RhoGEF, an activator of RhoA that associates with non-muscle myosin IIA, regulates collective cell migration of epithelial sheets and tumor cell invasion. Depletion of p114RhoGEF resulted in specific spatial inhibition of myosin activation at cell-cell contacts in migrating epithelial sheets and the cortex of migrating single cells, but only affected double and not single phosphorylation of myosin light chain. In agreement, overall elasticity and contractility of the cells, processes that rely on persistent and more constant forces, were not affected, suggesting that p114RhoGEF mediates process-specific myosin activation. Locomotion was p114RhoGEF-dependent on Matrigel, which favors more roundish cells and amoeboid-like actinomyosin-driven movement, but not on fibronectin, which stimulates flatter cells and lamellipodia-driven, mesenchymal-like migration. Accordingly, depletion of p114RhoGEF led to reduced RhoA, but increased Rac activity. Invasion of 3D matrices was p114RhoGEF-dependent under conditions that do not require metalloproteinase activity, supporting a role of p114RhoGEF in myosin-dependent, amoeboid-like locomotion. Our data demonstrate that p114RhoGEF drives cortical myosin activation by stimulating myosin light chain double phosphorylation and, thereby, collective cell migration of epithelial sheets and amoeboid-like motility of tumor cells.

Epithelial tight junctions regulate paracellular permeability, restrict apical/basolateral intramembrane diffusion of lipids, and have been proposed to participate in the control of epithelial cell proliferation and differentiation. Previously, we have identified ZO-1-associated nucleic acid binding proteins (ZONAB), a Y-box transcription factor whose nuclear localization and transcriptional activity is regulated by the tight junction-associated candidate tumor suppressor ZO-1. Now, we found that reduction of ZONAB expression using an antisense approach or by RNA interference strongly reduced proliferation of MDCK cells. Transfection of wild-type or ZONAB-binding fragments of ZO-1 reduced proliferation as well as nuclear ZONAB pools, indicating that promotion of proliferation by ZONAB requires its nuclear accumulation. Overexpression of ZONAB resulted in increased cell density in mature monolayers, and depletion of ZONAB or overexpression of ZO-1 reduced cell density. ZONAB was found to associate with cell division kinase (CDK) 4, and reduction of nuclear ZONAB levels resulted in reduced nuclear CDK4. Thus, our data indicate that tight junctions can regulate epithelial cell proliferation and cell density via a ZONAB/ZO-1-based pathway. Although this regulatory process may also involve regulation of transcription by ZONAB, our data suggest that one mechanism by which ZONAB and ZO-1 influence proliferation is by regulating the nuclear accumulation of CDK4.

Although development of the adult Drosophila compound eye is very well understood, little is known about development of photoreceptors (PRs) in the simple larval eye. We show here that the larval eye is composed of 12 PRs, four of which express blue-sensitive rhodopsin5 (rh5) while the other eight contain green-sensitive rh6. This is similar to the 30:70 ratio of adult blue and green R8 cells. However, the stochastic choice of adult color PRs and the bistable loop of the warts and melted tumor suppressor genes that unambiguously specify rh5 and rh6 in R8 PRs are not involved in specification of larval PRs. Instead, primary PR precursors signal via EGFR to surrounding tissue to develop as secondary precursors, which will become Rh6-expressing PRs. EGFR signaling is required for the survival of the Rh6 subtype. Primary precursors give rise to the Rh5 subtype. Furthermore, the combinatorial action of the transcription factors Spalt, Seven-up, and Orthodenticle specifies the two PR subtypes. Therefore, even though the larval PRs and adult R8 PRs express the same rhodopsins (rh5 and rh6), they use very distinct mechanisms for their specification.

Cell-cell junctions respond to mechanical forces by changing their organization and function. To gain insight into the mechanochemical basis underlying junction mechanosensitivity, we analyzed tight junction (TJ) formation between the enveloping cell layer (EVL) and the yolk syncytial layer (YSL) in the gastrulating zebrafish embryo. We found that the accumulation of Zonula Occludens-1 (ZO-1) at TJs closely scales with tension of the adjacent actomyosin network, revealing that these junctions are mechanosensitive. Actomyosin tension triggers ZO-1 junctional accumulation by driving retrograde actomyosin flow within the YSL, which transports non-junctional ZO-1 clusters toward the TJ. Non-junctional ZO-1 clusters form by phase separation, and direct actin binding of ZO-1 is required for stable incorporation of retrogradely flowing ZO-1 clusters into TJs. If the formation and/or junctional incorporation of ZO-1 clusters is impaired, then TJs lose their mechanosensitivity, and consequently, EVL-YSL movement is delayed. Thus, phase separation and flow of non-junctional ZO-1 confer mechanosensitivity to TJs.

Coordination of cell-cell adhesion, actomyosin dynamics and gene expression is crucial for morphogenetic processes underlying tissue and organ development. Rho GTPases are main regulators of the cytoskeleton and adhesion. They are activated by guanine nucleotide exchange factors in a spatially and temporally controlled manner. However, the roles of these Rho GTPase activators during complex developmental processes are still poorly understood. ARHGEF18/p114RhoGEF is a tight junction-associated RhoA activator that forms complexes with myosin II, and regulates actomyosin contractility. Here we show that p114RhoGEF/ARHGEF18 is required for mouse syncytiotrophoblast differentiation and placenta development. In vitro and in vivo experiments identify that p114RhoGEF controls expression of AKAP12, a protein regulating protein kinase A (PKA) signaling, and is required for PKA-induced actomyosin remodeling, cAMP-responsive element binding protein (CREB)-driven gene expression of proteins required for trophoblast differentiation, and, hence, trophoblast cell-cell fusion. Our data thus indicate that p114RhoGEF links actomyosin dynamics and cell-cell junctions to PKA/CREB signaling, gene expression and cell-cell fusion.

Entry into mitosis is accompanied by profound changes in cortical actomyosin organization. Here, we delineate a pathway downstream of the RhoGEF Pbl/Ect2 that directs this process in a model epithelium. Our data suggest that the release of Pbl/Ect2 from the nucleus at mitotic entry drives Rho-dependent activation of Myosin-II and, in parallel, induces a switch from Arp2/3 to Diaphanous-mediated cortical actin nucleation that depends on Cdc42, aPKC, and Par6. At the same time, the mitotic relocalization of these apical protein complexes to more lateral cell surfaces enables Cdc42/aPKC/Par6 to take on a mitosis-specific function-aiding the assembly of a relatively isotropic metaphase cortex. Together, these data reveal how the repolarization and remodeling of the actomyosin cortex are coordinated upon entry into mitosis to provide cells with the isotropic and rigid form they need to undergo faithful chromosome segregation and division in a crowded tissue environment.

Tight junctions are required for epithelial barrier formation and participate in the regulation of signalling mechanisms that control proliferation and differentiation. ZO-1 is a tight junction-associated adaptor protein that regulates gene expression, junction assembly and epithelial morphogenesis. We have previously demonstrated that the heat shock protein Apg-2 binds ZO-1 and thereby regulates its role in cell proliferation. Here, we addressed the question whether Apg-2 is also important for junction formation and epithelial morphogenesis.

Tight-junction-regulated actomyosin activity determines epithelial and endothelial tension on adherens junctions and drives morphogenetic processes; however, whether or not tight junctions themselves are under tensile stress is not clear. Here, we use a tension sensor based on ZO-1, a scaffolding protein that links the junctional membrane to the cytoskeleton, to determine if tight junctions carry a mechanical load. Our data indicate that ZO-1 is under mechanical tension and that forces acting on ZO-1 are regulated by extracellular matrix (ECM) stiffness and the junctional adhesion molecule JAM-A. JAM-A depletion stimulates junctional recruitment of p114RhoGEF/ARHGEF18, mechanical tension on ZO-1, and traction forces at focal adhesions. p114RhoGEF is required for activation of junctional actomyosin activity and tight junction integrity on stiff but not soft ECM. Thus, junctional ZO-1 bears a mechanical load, and junction assembly is regulated by interplay between the physical properties of the ECM and adhesion-regulated signaling at tight junctions.

Intercellular junctions are crucial for mechanotransduction, but whether tight junctions contribute to the regulation of cell-cell tension and adherens junctions is unknown. Here, we demonstrate that the tight junction protein ZO-1 regulates tension acting on VE-cadherin-based adherens junctions, cell migration, and barrier formation of primary endothelial cells, as well as angiogenesis in vitro and in vivo. ZO-1 depletion led to tight junction disruption, redistribution of active myosin II from junctions to stress fibers, reduced tension on VE-cadherin and loss of junctional mechanotransducers such as vinculin and PAK2, and induced vinculin dissociation from the α-catenin-VE-cadherin complex. Claudin-5 depletion only mimicked ZO-1 effects on barrier formation, whereas the effects on mechanotransducers were rescued by inhibition of ROCK and phenocopied by JAM-A, JACOP, or p114RhoGEF down-regulation. ZO-1 was required for junctional recruitment of JACOP, which, in turn, recruited p114RhoGEF. ZO-1 is thus a central regulator of VE-cadherin-dependent endothelial junctions that orchestrates the spatial actomyosin organization, tuning cell-cell tension, migration, angiogenesis, and barrier formation.

We recently reported that mutations in the widely expressed nuclear protein TOPORS (topoisomerase I-binding arginine/serine rich) are associated with autosomal dominant retinal degeneration. However, the precise localization and a functional role of TOPORS in the retina remain unknown. Here, we demonstrate that TOPORS is a novel component of the photoreceptor sensory cilium, which is a modified primary cilium involved with polarized trafficking of proteins. In photoreceptors, TOPORS localizes primarily to the basal bodies of connecting cilium and in the centrosomes of cultured cells. Morpholino-mediated silencing of topors in zebrafish embryos demonstrates in another species a comparable retinal problem as seen in humans, resulting in defective retinal development and failure to form outer segments. These defects can be rescued by mRNA encoding human TOPORS. Taken together, our data suggest that TOPORS may play a key role in regulating primary cilia-dependent photoreceptor development and function. Additionally, it is well known that mutations in other ciliary proteins cause retinal degeneration, which may explain why mutations in TOPORS result in the same phenotype.

Rho family GTPases are important regulators of epithelial tight junctions (TJs); however, little is known about how the GTPases themselves are controlled during TJ assembly and function. We have identified and cloned a canine guanine nucleotide exchange factor (GEF) of the Dbl family of proto-oncogenes that activates Rho and associates with TJs. Based on sequence similarity searches and immunological and functional data, this protein is the canine homologue of human GEF-H1 and mouse Lfc, two previously identified Rho-specific exchange factors known to associate with microtubules in nonpolarized cells. In agreement with these observations, immunofluorescence of proliferating MDCK cells revealed that the endogenous canine GEF-H1/Lfc associates with mitotic spindles. Functional analysis based on overexpression and RNA interference in polarized MDCK cells revealed that this exchange factor for Rho regulates paracellular permeability of small hydrophilic tracers. Although overexpression resulted in increased size-selective paracellular permeability, such cell lines exhibited a normal overall morphology and formed fully assembled TJs as determined by measuring transepithelial resistance and by immunofluorescence and freeze-fracture analysis. These data indicate that GEF-H1/Lfc is a component of TJs and functions in the regulation of epithelial permeability.

Mutations in the inositol polyphosphate 5-phosphatase OCRL1 cause Lowe Syndrome, leading to cataracts, mental retardation and renal failure. We noted that cell types affected in Lowe Syndrome are highly polarized, and therefore we studied OCRL1 in epithelial cells as they mature from isolated individual cells into polarized sheets and cysts with extensive communication between neighbouring cells. We show that a proportion of OCRL1 targets intercellular junctions at the early stages of their formation, co-localizing both with adherens junctional components and with tight junctional components. Correlating with this distribution, OCRL1 forms complexes with junctional components α-catenin and zonula occludens (ZO)-1/2/3. Depletion of OCRL1 in epithelial cells growing as a sheet inhibits maturation; cells remain flat, fail to polarize apical markers and also show reduced proliferation. The effect on shape is reverted by re-expressed OCRL1 and requires the 5'-phosphatase domain, indicating that down-regulation of 5-phosphorylated inositides is necessary for epithelial development. The effect of OCRL1 in epithelial maturation is seen more strongly in 3-dimensional cultures, where epithelial cells lacking OCRL1 not only fail to form a central lumen, but also do not have the correct intracellular distribution of ZO-1, suggesting that OCRL1 functions early in the maturation of intercellular junctions when cells grow as cysts. A role of OCRL1 in junctions of polarized cells may explain the pattern of organs affected in Lowe Syndrome.

Maintenance of the epithelial phenotype is crucial for tissue homeostasis. In the retina, dedifferentiation and loss of integrity of the retinal pigment epithelium (RPE) leads to retinal dysfunction and fibrosis. Transforming growth factor (TGF)-beta critically contributes to RPE dedifferentiation and induces various responses, including increased Rho signaling, up-regulation of alpha-smooth muscle actin (SMA), and cell migration and dedifferentiation. Cellular TGF-beta responses are stimulated by different signal transduction pathways: some are Smad dependent and others Smad independent. Alterations in Rho signaling are crucial to both types of TGF-beta signaling, but how TGF-beta-stimulates Rho signaling is poorly understood. Here, we show that primary RPE cells up-regulated GEF-H1 in response to TGF-beta. GEF-H1 was the only detectable Rho exchange factor increased by TGF-beta1 in a genome-wide expression analysis. GEF-H1 induction was Smad4-dependant and led to Rho activation. GEF-H1 inhibition counteracted alpha-SMA up-regulation and cell migration. In patients with retinal detachments and fibrosis, migratory RPE cells exhibited increased GEF-H1 expression, indicating that induction occurs in diseased RPE in vivo. Our data indicate that GEF-H1 is a target and functional effector of TGF-beta by orchestrating Rho signaling to regulate gene expression and cell migration, suggesting that it represents a new marker and possible therapeutic target for degenerative and fibrotic diseases.

In Drosophila epithelial cells, specification and maintenance of the zonula adherens (za) is crucial to ensure epithelial tissue integrity. This depends on the intertwined function of Bazooka (Baz), Par6-DaPKC, and the Crumbs (Crb)-Stardust (Sdt)-PATJ complex. However, the detailed molecular basis for the interplay between these factors during this process is not fully understood.

Lgl (lethal giant larvae) plays an important role in cell polarity. Atypical protein kinase C (aPKC) binds to and phosphorylates Lgl, and the phosphorylation negatively regulates Lgl activity. In this study, we identify p32 as a novel Lgl binding protein that directly binds to a domain on mammalian Lgl2 (mLgl2), which contains the aPKC phosphorylation site. p32 also binds to PKCzeta, and the three proteins form a transient ternary complex. When p32 is bound, PKCzeta is stimulated to phosphorylate mLgl2 more efficiently. p32 overexpression in Madin-Darby canine kidney cells cultured in a 3D matrix induces an expansion of the actin-enriched apical membrane domain and disrupts cell polarity. Addition of PKCzeta inhibitor blocks apical actin accumulation, which is rescued by p32 overexpression. p32 knockdown by short hairpin RNA also induces cell polarity defects. Collectively, our data indicate that p32 is a novel regulator of cell polarity that forms a complex with mLgl2 and aPKC and enhances aPKC activity.

Phagocytosis requires actin dynamics, but whether actomyosin contractility plays a role in this morphodynamic process is unclear. Here, we show that in the retinal pigment epithelium (RPE), particle binding to Mer Tyrosine Kinase (MerTK), a widely expressed phagocytic receptor, stimulates phosphorylation of the Cdc42 GEF Dbl3, triggering activation of MRCKβ/myosin-II and its coeffector N-WASP, membrane deformation, and cup formation. Continued MRCKβ/myosin-II activity then drives recruitment of a mechanosensing bridge, enabling cytoskeletal force transmission, cup closure, and particle internalization. In vivo, MRCKβ is essential for RPE phagocytosis and retinal integrity. MerTK-independent activation of MRCKβ signaling by a phosphomimetic Dbl3 mutant rescues phagocytosis in retinitis pigmentosa RPE cells lacking functional MerTK. MRCKβ is also required for efficient particle translocation from the cortex into the cell body in Fc receptor-mediated phagocytosis. Thus, conserved MRCKβ signaling at the cortex controls spatiotemporal regulation of actomyosin contractility to guide distinct phases of phagocytosis in the RPE and represents the principle phagocytic effector pathway downstream of MerTK.

Tight junction complexes are involved in the establishment and maintenance of cell polarity and the regulation of signalling pathways, controlling biological processes such as cell differentiation and cell proliferation. MarvelD3 is a tight junction protein expressed in adult epithelial and endothelial cells. In Xenopus laevis, MarvelD3 morphants present differentiation defects of several ectodermal derivatives. In vitro experiments further revealed that MarvelD3 couples tight junctions to the MEKK1-JNK pathway to regulate cell behaviour and survival. In this work, we found that MarvelD3 is expressed from early developmental stages in the exocrine and endocrine compartments of the pancreas, as well as in endothelial cells of this organ. We thoroughly characterized MarvelD3 expression pattern in developing pancreas and evaluated its function by genetic ablation. Surprisingly, inactivation of MarvelD3 in mice did not alter development and differentiation of the pancreatic tissue. Moreover, tight junction formation and organization, cell polarization, and activity of the JNK-pathway were not impacted by the deletion of MarvelD3.