Signalling by the GTPase RhoA, a key regulator of epithelial cell behaviour, can stimulate opposing processes: RhoA can promote junction formation and apical constriction, as well as reduced adhesion and cell spreading1, 2. Molecular mechanisms are thus required that ensure spatially restricted and process-specific RhoA activation. For many fundamental processes, including assembly of the epithelial junctional complex, such mechanisms are still unknown. Here we show that p114RhoGEF is a junction-associated protein that drives RhoA signalling at the junctional complex and regulates tight junction assembly and epithelial morphogenesis. p114RhoGEF is required for RhoA activation at cell-cell junctions, and its depletion stimulates non-junctional Rho signalling and induction of myosin phosphorylation along the basal domain. Depletion of GEF-H1, a RhoA activator inhibited by junctional recruitment3, does not reduce junction-associated RhoA activation. p114RhoGEF associates with a complex containing myosin II, Rock II and the junctional adaptor cingulin, indicating that p114RhoGEF is a component of a junction-associated Rho signalling module that drives spatially restricted activation of RhoA to regulate junction formation and epithelial morphogenesis.

The ependymal multiciliated epithelium in the brain restricts the cerebrospinal fluid to the cerebral ventricles and regulates its flow. We report here that mice deficient for myosin IXa (Myo9a), an actin-dependent motor molecule with a Rho GTPase-activating (GAP) domain, develop severe hydrocephalus with stenosis and closure of the ventral caudal 3rd ventricle and the aqueduct. Myo9a is expressed in maturing ependymal epithelial cells, and its absence leads to impaired maturation of ependymal cells. The Myo9a deficiency further resulted in a distorted ependyma due to irregular epithelial cell morphology and altered organization of intercellular junctions. Ependymal cells occasionally delaminated, forming multilayered structures that bridged the CSF-filled ventricular space. Hydrocephalus formation could be significantly attenuated by the inhibition of the Rho-effector Rho-kinase (ROCK). Administration of ROCK-inhibitor restored maturation of ependymal cells, but not the morphological distortions of the ependyma. Similarly, down-regulation of Myo9a by siRNA in Caco-2 adenocarcinoma cells increased Rho-signaling and induced alterations in differentiation, cell morphology, junction assembly, junctional signaling, and gene expression. Our results demonstrate that Myo9a is a critical regulator of Rho-dependent and -independent signaling mechanisms that guide epithelial differentiation. Moreover, Rho-kinases may represent a new target for therapeutic intervention in some forms of hydrocephalus. INTRODUCTIONThe development and homeostasis of multicellular organisms depends on coordinated cell shape changes that are coupled with alterations in intracellular organization. The dynamic organization of the actin cytoskeleton accounts for many cell shape changes. A multitude of proteins can directly or indirectly modify the dynamics and organization of the actin cytoskeleton. Among these proteins are monomeric GTPases and the superfamily of myosin molecules. The myosin superfamily of actin-based molecular motors is subdivided into more than 30 classes (Odronitz and Kollmar, 2007). The class IX of myosin molecules includes in mammals two members, Myo9a (myr 7) and Myo9b (myr 5), that are both expressed in a number of differentially spliced variants (Bähler, 2008). The Myo9a protein, previously also called myr 7, is expressed during development and in many adult tissues, most abundantly in brain and testis (Chieregatti et al., 1998;Gorman et al., 1999). Class IX myosins comprise in addition to their myosin head domain a tail region that encompasses a C1 domain and a Rho GTPaseactivating protein (RhoGAP) domain (Reinhard et al., 1995;Chieregatti et al., 1998). The RhoGAP domain negatively regulates the monomeric Rho GTPase by accelerating its rate of GTP-hydrolysis, switching it from the active GTP-bound form to the inactive GDP-bound form. The RhoGTPases are known to be important regulators of cell morphogenesis, cell migration, and cell proliferation (Jaffe and Hall, 2005). They are inactivated under spatial and temporal con...

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation without specific permission.Epithelial tight junctions recruit different types of signalling proteins that regulate cell proliferation and differentiation. Little is known about how such proteins interact functionally and biochemically with each other. Here, we focus on the Y-box transcription factor ZONAB (zonula occludens 1-associated nucleic-acid-binding protein)/DbpA (DNA-binding protein A) and the Rho GTPase activator guanine nucleotide exchange factor (GEF)-H1/Lbc's first cousin, which are two tight-junctionassociated signalling proteins that regulate proliferation. Our data show that the two proteins interact and that ZONAB activity is Rho-dependent. Overexpression of GEF-H1 induces accumulation of ZONAB in the nucleus and activates transcription. Microtubule-affinity regulating kinase/partition-defective-1, another type of GEF-H1-associated signalling protein, remains in the cytoplasm and partially co-localizes with the exchange factor. GEF-H1 and ZONAB are required for expression of endogenous cyclin D1, a crucial RhoA signalling target gene, and GEF-H1-stimulated cyclin D1 promoter activity requires ZONAB. Our data thus indicate that GEF-H1 and ZONAB form a signalling module that mediates Rho-regulated cyclin D1 promoter activation and expression.

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