RhoA GTPase inhibition organizes contraction during epithelial morphogenesis.
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ABSTRACT:
SUBMITTER: Mason FM
PROVIDER: S-EPMC5004446 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Rap1, a Ras-like small GTPase, plays a crucial role in cell-matrix adhesive interactions, cell-cell junction formation, cell polarity and migration. The role of Rap1 in vertebrate organ development and tissue architecture, however, remains elusive. We addressed this question in a mouse lens model system using a conditional gene targeting approach. While individual germline deficiency of either Rap1a or Rap1b did not cause overt defects in mouse lens, conditional double deficiency (Rap1 cKO) prior to lens placode formation led to an ocular phenotype including microphthalmia and lens opacification in embryonic mice. The embryonic Rap1 cKO mouse lens exhibited striking defects including loss of E-cadherin- and ZO-1-based cell-cell junctions, disruption of paxillin and ?1-integrin-based cell adhesive interactions along with abnormalities in cell shape and apical-basal polarity of epithelium. These epithelial changes were accompanied by increased levels of ?-smooth muscle actin, vimentin and N-cadherin, and expression of transcriptional suppressors of E-cadherin (Snai1, Slug and Zeb2), and a mesenchymal metabolic protein (Dihydropyrimidine dehydrogenase). Additionally, while lens differentiation was not overtly affected, increased apoptosis and dysregulated cell cycle progression were noted in epithelium and fibers in Rap1 cKO mice. Collectively these observations uncover a requirement for Rap1 in maintenance of lens epithelial phenotype and morphogenesis.
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Project description:Ultraviolet radiation is the main cause of DNA damage to melanocytes and development of melanoma, one of the most lethal human cancers, which leads to metastasis due to uncontrolled cell proliferation and migration. These phenotypes are mediated by RhoA, a GTPase overexpressed or overactivated in highly aggressive metastatic tumors that plays regulatory roles in cell cycle progression and cytoskeleton remodeling. This work explores whether the effects of UV on DNA damage, motility, proliferation, and survival of human metastatic melanoma cells are mediated by the RhoA pathway. Mutant cells expressing dominant-negative (MeWo-RhoA-N19) or constitutively active RhoA (MeWo-RhoA-V14) were generated and subjected to UV radiation. A slight reduction in migration and invasion was observed in MeWo and MeWo-RhoA-V14 cells but not in MeWo-RhoA-N19 cells, which presented inefficient motility and invasiveness associated with stress fibers fragmentation. Proliferation and survival of RhoA-deficient cells were drastically reduced by UV compared to cells displaying normal or high RhoA activity, suggesting increased sensitivity to UV. Loss of RhoA activity also caused less efficient DNA repair, with elevated levels of DNA lesions such as strand breaks and cyclobutane pyrimidine dimers (CPDs). Thus, RhoA mediates genomic stability and represents a potential target for sensitizing metastatic tumors to genotoxic agents.
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Project description:In neonatal ventricular cardiomyocytes (NVCM), decreased contractile activity stimulates sarco-endoplasmic reticulum Ca(2+)-ATPase2a (SERCA2a), analogous to reduced myocardial load in vivo. This study investigated in contracting NVCM the role of load-dependent RhoA-ROCK signaling in SERCA2a regulation. Contractile arrest of NVCM resulted in low peri-nuclear localized RhoA levels relative to contracting NVCM. In arrested NVCM, ROCK activity was decreased (59%) and paralleled a loss in F-actin levels. Y-27632-induced ROCK inhibition in contracting NVCM increased SERCA2a messenger RNA expression by 150%. This stimulation was transcriptional, as evident from transfections with the SERCA2a promoter. A reciprocal effect of Y-27632 treatment on the promoter activity of atrial natriuretic factor was observed. SERCA2a transcription was not altered by co-transfection of the RhoA-ROCK-dependent serum response factor (SRF) alone or in combination with myocardin. Furthermore, GATA4, another ROCK-dependent transcription factor, induced rather than repressed SERCA2a transcription. This study shows that contractile activity suppresses SERCA2a gene expression via RhoA-ROCK-dependent transcription modulation. This modulation is likely to be accomplished by a transcription factor other than SRF, myocardin, or GATA4.
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Project description:The antagonism between the GTPases Rac1 and RhoA controls cell-to-cell heterogeneity in isogenic populations of cells in vitro and epithelial morphogenesis in vivo. Its involvement in the regulation of cell-to-cell heterogeneity during epidermal morphogenesis has, however, never been addressed. We used a quantitative cell imaging approach to characterize epidermal morphogenesis at a single-cell level during early elongation of Caenorhabditis elegans embryos. This study reveals that a Rac1-like pathway, involving the Rac/Cdc42 guanine-exchange factor β-PIX/PIX-1 and effector PAK1/PAK-1, and a RhoA-like pathway, involving ROCK/LET-502, control the remodeling of apical junctions and the formation of basolateral protrusions in distinct subsets of hypodermal cells. In these contexts, protrusions adopt lamellipodia or an amoeboid morphology. We propose that lamella formation may reduce tension building at cell-cell junctions during morphogenesis. Cell-autonomous antagonism between these pathways enables cells to switch between Rac1- and RhoA-like morphogenetic programs. This study identifies the first case of cell-to-cell heterogeneity controlled by Rac1/RhoA antagonism during epidermal morphogenesis.
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Project description:Signalling by the GTPase RhoA, a key regulator of epithelial cell behaviour, can stimulate opposing processes: RhoA can promote junction formation and apical constriction, and reduce adhesion and cell spreading. 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 recruitment, 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.
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Project description:The vertebrate heart undergoes early complex morphologic events in order to develop key cardiac structures that regulate its overall function (Fahed et al., 2013). Although many genetic factors that participate in patterning the heart have been elucidated (Tu and Chi, 2012), the cellular events that drive cardiac morphogenesis have been less clear. From a chemical genetic screen to identify cellular pathways that control cardiac morphogenesis in zebrafish, we observed that inhibition of the Rho signaling pathways resulted in failure to form the atrioventricular canal and loop the linear heart tube. To identify specific Rho proteins that may regulate this process, we analyzed cardiac expression profiling data and discovered that RhoU was expressed at the atrioventricular canal during the time when it forms. Loss of RhoU function recapitulated the atrioventricular canal and cardiac looping defects observed in the ROCK inhibitor treated zebrafish. Similar to its family member RhoV/Chp (Tay et al., 2010), we discovered that RhoU regulates the cell junctions between cardiomyocytes through the Arhgef7b/Pak kinase pathway in order to guide atrioventricular canal development and cardiac looping. Inhibition of this pathway resulted in similar underlying cardiac defects and conversely, overexpression of a PAK kinase was able to rescue the loss of RhoU cardiac defect. Finally, we found that Wnt signaling, which has been implicated in atrioventricular canal development (Verhoeven et al., 2011), may regulate the expression of RhoU at the atrioventricular canal. Overall, these findings reveal a cardiac developmental pathway involving RhoU/Arhgef7b/Pak signaling, which helps coordinate cell junction formation between atrioventricular cardiomyocytes to promote cell adhesiveness and cell shapes during cardiac morphogenesis. Failure to properly form these cell adhesions during cardiac development may lead to structural heart defects and mechanistically account for the cellular events that occur in certain human congenital heart diseases.
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Project description:The Rho GTPase Cdc42 is overexpressed and hyperactivated in breast cancer, and several studies have described mechanisms by which it may promote tumor formation and progression. However, little is known about the role of Cdc42 during normal mammary epithelial cell (MEC) morphogenesis. Here we aimed to define the precise role for Cdc42 during primary mammary acinus formation in vitro. For these studies, MECs were isolated from Cdc42fl/fl conditional knockout mice, transduced with Adeno-cre-GFP virus to delete Cdc42 or Adeno-GFP control virus, and effects on morphogenesis were investigated using a three-dimensional (3D) culture assay. Interestingly, markedly fewer mammary acini developed in Cdc42 deficient cultures, and the acini that formed were significantly smaller and disorganized. Cellular proliferation and survival were reduced in the Cdc42 deficient acini. However, control and knockout MECs cultured as monolayers displayed similar cell cycle profiles, suggesting that Cdc42 is important for MEC proliferation in the context of 3D polarity. Overexpression of cyclin D1, which promotes cell cycle progression downstream of Cdc42, failed to rescue the defect in acinus size. Furthermore, lumen formation and apical-basal polarity were disrupted, and mitotic spindle orientation and Cdc42/aPKC polarity complex defects likely contributed to these phenotypes. Studies using dominant negative Cdc42 and siRNa to knockdown Cdc42 in MDcK and Caco-2 cell lines undergoing cystogenesis in 3D cultures revealed critical roles for Cdc42 in spindle orientation, polarity and lumen formation. Our studies, using complete knockout in primary epithelial cells, demonstrate that Cdc42 is not only an important regulator of polarity and lumen formation; it is also essential for proliferation and survival, which are key cellular processes that drive MEC morphogenesis in vitro and in vivo.
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Project description:BACKGROUND:Epithelial cells of developing embryonic organs, such as salivary glands, can display substantial motility during branching morphogenesis. Their dynamic movements and molecules involved in their migration are not fully characterized. RESULTS:We generated transgenic mice expressing photo-convertible KikGR and tracked the movements of individual cells highlighted by red fluorescence in different regions of developing salivary glands. Motility was highest for outer bud epithelial cells adjacent to the basement membrane, lower in inner bud cells, and lowest in duct cells. The highly motile outer cells contacting the basement membrane were pleomorphic, whereas inner cells were rounded. Peripheral cell motility was disrupted by antibodies inhibiting ?6+?1 integrins and the nonmuscle myosin II inhibitor blebbistatin. Inner bud cell migration was unaffected by these inhibitors, but their rate of migration was stimulated by inhibiting E-cadherin. CONCLUSIONS:Cell motility in developing salivary glands was highest in cells in contact with the basement membrane. The basement membrane-associated motility of these outer bud cells depended on integrins and myosin II, but not E-cadherin. In contrast, motility of inner bud cells was restrained by E-cadherin. These findings identify the importance of integrin-dependent basement membrane association for the morphology, tissue organization, and lateral motility of morphogenetic epithelial cells.
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Project description:The formation and maintenance of cell-cell junctions, both under physiological and pathological conditions, requires the targeting and trafficking of junctional proteins. Proteins of the syntaxin (Stx)-family localize to a variety of subcellular membranes and contribute to intracellular transport of cargo by regulating vesicle fusion events at these sites. Unlike plasma membrane localized Stxs, the roles of endosome- and Golgi-localized stx proteins in epithelial morphogenesis are less understood. Here we show that Stx16- an endosome- and Golgi-localized target-membrane soluble N-ethylmaleimide attachment protein receptor (t-SNARE) that plays a role in membrane trafficking between these compartments - is essential for lumen development. In cultured Madin Darby Canine Kidney (MDCK) cells, Stx16 was selectively upregulated as sparsely plated cells attained confluency. Stx16-depleted confluent monolayers consistently showed lower transepithelial resistance than control monolayers, and failed to maintain endogenous and ectopically expressed E-cadherin at the adherens junctions due to decreased recycling. We further found that whereas cysts formed by MDCK cells cultured in Matrigel have a single hollow lumen, those formed by stx16-depleted counterparts had multiple lumens, due to abnormal orientiation of the mitotic spindle. Finally, a similar role for stx16 function in vivo is indicated by our analysis of pronephric-duct development in zebrafish expressing the claudinB:lynGFP transgene; lack of stx16 function in this structure (in stx16-morphant embryos) led to the development of enlarged, torturous pronephric ducts with more than one lumen. Taken together, our in vitro and in vivo studies establish a role for Stx16 in maintaining the integrity of cell-cell junctions, and thereby in morphogenesis of the kidney epithelial lumen.
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Project description:We previously identified Fam65b as an atypical inhibitor of the small G protein RhoA. Using a conditional model of a Fam65b-deficient mouse, we first show that Fam65b restricts spontaneous RhoA activation in resting T lymphocytes and regulates intranodal T cell migration in vivo. We next aimed at understanding, at the molecular level, how the brake that Fam65b exerts on RhoA can be relieved upon signaling to allow RhoA activation. Here, we show that chemokine stimulation phosphorylates Fam65b in T lymphocytes. This post-translational modification decreases the affinity of Fam65b for RhoA and favors Fam65b shuttling from the plasma membrane to the cytosol. Functionally, we show that the degree of Fam65b phosphorylation controls some cytoskeletal alterations downstream active RhoA such as actin polymerization, as well as T cell migration in vitro. Altogether, our results show that Fam65b expression and phosphorylation can finely tune the amount of active RhoA in order to favor optimal T lymphocyte motility.
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