Action Of RhoA Research Articles

Anthrax toxin receptor 2a controls mitotic spindle positioning

Oriented mitosis is essential during tissue morphogenesis. The Wnt/planar cell polarity (Wnt/PCP) pathway orients mitosis in a number of developmental systems, including dorsal epiblast cell divisions along the animal-vegetal (A-V) axis during zebrafish gastrulation. How Wnt signalling orients the mitotic plane is, however, unknown. Here we show that, in dorsal epiblast cells, anthrax toxin receptor 2a (Antxr2a) accumulates in a polarized cortical cap, which is aligned with the embryonic A-V axis and forecasts the division plane. Filamentous actin (F-actin) also forms an A-V polarized cap, which depends on Wnt/PCP and its effectors RhoA and Rock2. Antxr2a is recruited to the cap by interacting with actin. Antxr2a also interacts with RhoA and together they activate the diaphanous-related formin zDia2. Mechanistically, Antxr2a functions as a Wnt-dependent polarized determinant, which, through the action of RhoA and zDia2, exerts torque on the spindle to align it with the A-V axis.

Constitutively active RhoA inhibits proliferation by retarding G 1 to S phase cell cycle progression and impairing cytokinesis

The actions of RhoA in cytoskeletal regulation have been extensively studied. RhoA also contributes to proliferation and oncogenic transformation by less well-characterized means. Elevated RhoA signalling has been associated with human cancer; through increased RhoA expression, mutation or elevated expression of activating Rho guanine-nucleotide exchange factors (GEFs), or from deletion or decreased expression of inhibitory Rho GTPase-activating proteins (GAPs). Unlike the Ras oncogene, constitutively-activated GTPase-deficient RhoA mutants have not been identified in tumours. To investigate the effects of active RhoA on proliferation, we generated Swiss3T3 cells that inducibly express wild-type RhoA or GTPase-deficient active V14RhoA. We found that V14RhoA inhibited cell proliferation by retarding entry into the DNA synthetic cell cycle phase and blocking successful completion of cytokinesis, resulting in an increased incidence of binucleate cells. These effects were associated with inhibition of mitogen-induced activation of the MAPK pathway, and suppression of several proteins involved in mitosis, including anillin, ECT2 and cyclin B1 which would be expected to result in reduced activation of endogenous RhoA at the cell equator. Accumulation of active RhoA protein in the midbody of cells in telophase was inhibited in V14RhoA-expressing cells, suggesting that RhoA inactivation must occur prior to re-activation. Defective cytokinesis was also associated with prominent actin structures in V14RhoA-expressing cells, which might be incompatible with equatorial furrowing. Using super-resolution imaging based on single-molecule switching, we have significantly improved the resolution of active RhoA in midbodies. These results indicate that constitutively-active RhoA antagonizes several cellular activities that contribute to proliferation, highlighting the importance for cycling between GTP/GDP-bound states.

Regulation of mitochondria distribution by RhoA and formins

The distribution of mitochondria is strictly controlled by the cell because of their vital role in energy supply, regulation of cytosolic Ca2+ concentration and apoptosis. We employed cultured mammalian CV-1 cells and Drosophila BG2-C2 neuronal cells with enhanced green fluorescent protein (EGFP)-tagged mitochondria to investigate the regulation of their movement and anchorage. We show here that lysophosphatidic acid (LPA) inhibits fast mitochondrial movements in CV-1 cells acting through the small GTPase RhoA. The action of RhoA is mediated by its downstream effectors: formin-homology family members mDia1 in mammalian cells and diaphanous in Drosophila. Overexpression of constitutively active mutant forms of formins leads to dramatic loss of mitochondrial motility and to their anchorage to actin microfilaments. Conversely, depletion of endogenous diaphanous protein in BG2-C2 cells by RNA interference (RNAi) stimulates the mitochondrial movement. These effects are not simply explained by increased cytoplasm viscosity resulting from an increased F-actin concentration since stimulators of Arp2/3-dependent actin polymerization and jasplakinolide do not cause inhibition. The observed effects are highly specific to mitochondria since perturbations of diaphanous or mDia1 have no effect on movement of other membrane organelles. Thus, mitochondrial movement is controlled by the small GTPase RhoA and this control is mediated by formins.

Activated RhoA stimulates c-fos gene expression in myocardial cells.

Rho regulates various cell functions, including cell morphology and motility. However, the functional role of Rho on the signaling pathway in myocardial cells (MCs) is unknown. In the present study, we attempted to explore the mode of Rho action for c-fos gene expression in MCs. Expression of the c-fos promoter/enhancer linked to the luciferase reporter gene (c-fos luciferase) was stimulated by the wild type of RhoA and the point-mutated active form of RhoA (RhoA Val14) but not the biologically inactive effector domain mutant of RhoA. Rho GDP dissociation inhibitor inhibited the action of RhoA on c-fos luciferase expression. The deletion analysis revealed that the c-fos serum response element (SRE) and the 12-O-tetradecanoylphorbol-13-acetate response element (TRE) mainly account for c-fos luciferase expression by RhoA Val14. The c-fos SRE mutant, which contains an intact binding site for the serum response factor but lacks the ternary complex factor binding site, was activated by RhoA Val14. The action of RhoA Val14 on c-fos luciferase expression was not inhibited by downregulation of protein kinase C, protein kinase C inhibitors, or tyrosine kinase inhibitors. These results indicate that activated RhoA stimulates c-fos gene expression through the c-fos SRE and TRE and that the signaling pathway from activated RhoA to the c-fos promoter/enhancer is independent of these inhibitor-sensitive pathways in MCs.