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Active RhoA (active + rhoa)
Selected AbstractsRhoA/ROCK and Cdc42 regulate cell-cell contact and N-cadherin protein level during neurodetermination of P19 embryonal stem cellsDEVELOPMENTAL NEUROBIOLOGY, Issue 3 2004Isabel Laplante Abstract RhoGTPases regulate actin-based signaling cascades and cellular contacts. In neurogenesis, their action modulates cell migration, neuritogenesis, and synaptogenesis. Murine P19 embryonal stem cells differentiate to neurons upon aggregation in the presence of retinoic acid, and we previously showed that RhoA and Cdc42 RhoGTPases are sequentially up-regulated during neuroinduction, suggesting a role at this very early developmental stage. In this work, incubation of differentiating P19 cells with C3 toxin resulted in decreased aggregate cohesion and cadherin protein level. In contrast, C3 effects were not observed in cells overexpressing recombinant dominant active RhoA. On the other hand, C3 did not affect cadherin in uninduced cells and their postmitotic neuronal derivatives, respectively expressing E- and N-cadherin. RhoA is thus influential on cell aggregation and cadherin expression during a sensitive time window that corresponds to the switch of E- to N-cadherin. Cell treatment with Y27632 inhibitor of Rho-associated-kinase ROCK, or advanced overexpression of Cdc42 by gene transfer of a constitutively active form of the protein reproduced C3 effects. RhoA-antisense RNA also reduced cadherin level and the size of cell aggregates, and increased the generation of fibroblast-like cells relative to neurons following neuroinduction. Colchicin, a microtubule disrupter, but not cytochalasin B actin poison, importantly decreased cadherin in neurodifferentiating cells. Overall, our results indicate that the RhoA/ROCK pathway regulates cadherin protein level and cell-cell interactions during neurodetermination, with an impact on the efficiency of the process. The effect on cadherin seems to involve microtubules. The importance of correct timing of RhoA and Cdc42 functional expression in neurogenesis is also raised. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 289,307, 2004 [source] RhoE stimulates neurite-like outgrowth in PC12 cells through inhibition of the RhoA/ROCK-I signallingJOURNAL OF NEUROCHEMISTRY, Issue 4 2010Raquel Talens-Visconti J. Neurochem. (2010) 112, 1074,1087. Abstract Neurite formation involves coordinated changes between the actin cytoskeleton and the microtubule network. Rho GTPases are clearly implicated in several aspects of neuronal development and function. Indeed, RhoA is a negative regulator of neurite outgrowth and its effector Rho-kinase mediates the Rho-driven neurite retraction. Considering that RhoE/round protein (Rnd3) acts antagonistically to RhoA and it is also able to bind and inhibit rho kinase-I (p160ROCK) , ROCK-I, it is tempting to speculate a role of RhoE in neurite formation. We show for the first time that, in the absence of nerve growth factor (NGF), RhoE induces neurite-like outgrowth. Our results demonstrate that over-expression of RhoE decreases the activity of RhoA and reduces the expression of both ROCK-I and the phosphorylated myosin light chain phosphatase (MLCPp). Conversely, over-expression of either active RhoA or ROCK-I abolishes the RhoE-promoted neurite outgrowth, suggesting that RhoE induces neurite-like formation through inhibition of the RhoA/ROCK-I signalling. We also show that Rac and Cdc42 have a role in RhoE-induced neurite outgrowth. Finally, the present data further indicate that RhoE may be involved in the NGF-induced neurite outgrowth in PC12 cells, as depletion of RhoE by siRNA reduces the neurite formation induced by NGF. These findings provide new insights into the molecular mechanism implicated in neuronal development and may provide novel therapeutic targets in neurodegenerative disorders. [source] The cyclic GMP-protein kinase G pathway regulates cytoskeleton dynamics and motility in astrocytesJOURNAL OF NEUROCHEMISTRY, Issue 1 2007Mariela Susana Borán Abstract We have previously demonstrated that inflammatory compounds that increase nitric oxide (NO) synthase expression have a biphasic effect on the level of the NO messenger cGMP in astrocytes. In this work, we demonstrate that NO-dependent cGMP formation is involved in the morphological change induced by lipopolysaccharide (LPS) in cultured rat cerebellar astroglia. In agreement with this, dibutyryl-cGMP, a permeable cGMP analogue, and atrial natriuretic peptide, a ligand for particulate guanylyl cyclase, are both able to induce process elongation and branching in astrocytes resulting from a rapid, reversible and concentration-dependent redistribution of glial fibrillary acidic protein (GFAP) and actin filaments without significant change in protein levels. These effects are also observed in astrocytes co-cultured with neurons. The cytoskeleton rearrangement induced by cGMP is prevented by the specific protein kinase G inhibitor Rp-8Br-PET-cGMPS and involves downstream inhibition of RhoA GTPase since is not observed in cells transfected with constitutively active RhoA. Furthermore, dibutyryl-cGMP prevents RhoA-membrane association, a step necessary for its interaction with effectors. Stimulation of the cGMP-protein kinase G pathway also leads to increased astrocyte migration in an in vitro scratch-wound assay resulting in accelerated wound closure, as seen in reactive gliosis following brain injury. These results indicate that cGMP-mediated pathways may regulate physio-pathologically relevant responses in astroglial cells. [source] Overexpression of the partially activated ,IIb,3D723H integrin salt bridge mutant downregulates RhoA activity and induces microtubule-dependent proplatelet,like extensions in Chinese hamster ovary cellsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 7 2009E. SCHAFFNER-RECKINGER Summary.,Background: We have recently reported a novel mutation in the ,3 subunit of the platelet fibrinogen receptor (,IIb,3D723H) identified in a patient with dominantly inherited macrothrombocytopenia, and we have shown that this mutation promotes a new phenotype in Chinese hamster ovary (CHO) cells, characterized by fibrinogen-dependent, microtubule-driven proplatelet-like cell extensions. Results: Here we demonstrate that the partially activated ,IIb,3D723H or ,IIb,3D723A salt bridge mutants, but not fully activated ,IIb,3 mutants, cause this phenotype. Time-lapse videomicroscopy clearly differentiated these stable microtubule-driven and nocodazole-sensitive extensions from common dynamic actin-driven pseudopodia. In addition, overexpression of a mitochondrial marker confirmed their functional role in organelle transport. Comparative immunofluorescence analysis of the subcellular localization of ,IIb,3, the focal adhesion proteins talin or vinculin and actin revealed a similar membrane labeling of CHO cell extensions and CD34+ -derived megakaryocyte proplatelets. Mutant ,IIb,3D723H signaling was independent of Src, protein kinase C or phosphoinositide 3-kinase, but correlated with decreased RhoA activity as compared with wild-type ,IIb,3 signaling, reminiscent of integrin signaling during neurite outgrowth. Accordingly, overexpression of constitutively active RhoA in CHO ,IIb,3D723H cells prevented protrusion formation on fibrinogen. Most interestingly, RhoA/ROCK inhibition was necessary, but not sufficient, and integrin activity was additionally required to induce CHO cell extension formation. Conclusions: CHO ,IIb,3D723H cell protrusions and megakaryocyte proplatelets, like neuronal cell neurites, result from a common integrin-dependent signaling pathway, promoting strongly decreased RhoA activity and leading to microtubule-driven formation of cytoplasmic extensions. [source] | ||||||||||