RhoA GTPase is dispensable for actomyosin regulation but is essential for mitosis in primary mouse embryonic fibroblasts

Abstract

RhoA, the founding member of mammalian Rho GTPase family, is thought to be essential for actomyosin regulation. To date, the physiologic function of RhoA in mammalian cell regulation has yet to be determined genetically. Here we have created RhoA conditional knock-out mice. Mouse embryonic fibroblasts deleted of RhoA showed no significant change in actin stress fiber or focal adhesion complex formation in response to serum or LPA, nor any detectable change in Rho-kinase signaling activity. Concomitant knock-out or knockdown of RhoB and RhoC in the RhoA(-/-) cells resulted in a loss of actin stress fiber and focal adhesion similar to that of C3 toxin treatment. Proliferation of RhoA(-/-) cells was impaired due to a complete cell cycle block during mitosis, an effect that is associated with defective cytokinesis and chromosome segregation and can be readily rescued by exogenous expression of RhoA. Furthermore, RhoA deletion did not affect the transcriptional activity of Stat3, NFκB, or serum response factor, nor the expression of the cell division kinase inhibitor p21(Cip)1 or p27(Kip1). These genetic results demonstrate that in primary mouse embryonic fibroblasts, RhoA is uniquely required for cell mitosis but is redundant with related RhoB and RhoC GTPases in actomyosin regulation.

FIGURE 1.

RhoA is dispensable for actin stress fiber and focal adhesion formation in MEFs. A, deletion of RhoA in loxp/loxp MEF cells using Cre-expressing adenovirus. Cells were infected with adeno-Cre or β-Gal virus. Cells were infected twice, and the levels of RhoA protein were evaluated by Western blotting. GA3PDH, glyceraldehyde 3-phosphate dehydrogenase. B, RhoA deletion affected expression and activity of RhoC and RhoB but not Cdc42 or Rac1 GTPases. GST-PAK1 pZ1-binding domain and GST-rhotekin Rho-binding domain were used for pulldown experiments to evaluate the respective GTPase activities 48 h after virus infection. C, actin stress formation was detected by rhodamine-phalloidin staining of the cells. Control and RhoA KO cells were compared with C3 toxin-treated WT cells (2 μg/ml for 4 h) in the F-actin network. D, focal adhesion complex proteins were examined by immunofluorescence in control and RhoA-deficient MEF cells. Cells were plated and fixed, and focal adhesion proteins were revealed using specific antibodies for p-Pax(118) (40×) or vinculin (63×) (in green). Cells were co-stained with rhodamine-phalloidin for F-actin (in red). E, Western blots for focal adhesion proteins in control and RhoA-deficient cells. F, effects of RhoA KO in MEF cells on ROCK and ROCK-mediated actomyosin activity. Cells were plated for 48–72 h before being processed for Western blotting with p-MLC and p-cofilin specific antibodies. For measuring ROCKII activity, ROCKII was immunoprecipitated with an anti-ROCKII antibody followed by an in vitro kinase activity assay using MYPT-1 polypeptide as a substrate.

FIGURE 2.

RhoA-deficient cells are impaired in proliferation due to a mitotic defect during cytokinesis. A, proliferation assays of control cells (Loxp-Gal, WT-Gal, and WT-Cre) and RhoA-deficient cells (Loxp-Cre) were carried out at a density of 1 × 105 cells/well. Cells were counted every 24 h. Cell viability was estimated by using trypan blue staining. Assays were performed in triplicate. Error bars represent S.D. B, representative cell cycle profile of RhoA-deficient cells 48 h in culture. Cells were plated at a density of 1 × 105 cells/well, fixed, and analyzed by flow cytometry following propidium iodide/7-AAD staining. Error bars represent S.D. C, nuclear morphology in RhoA-deficient cells. Control and RhoA-deficient cells were plated for 24–96 h. Cells were stained with DAPI. The percentages of cells with different nuclear phenotypes at 72 h of culture were quantified. Error bars represent S.D. D, evidence of DNA bridges between nuclei in RhoA-deficient cells. Cells were stained with DAPI to reveal nuclear morphology and were co-stained with rhodamine-phalloidin for F-actin. Arrowheads indicate DNA-bridges. E, α/β- and γ-tubulin immunostaining in RhoA-deficient cells. Cells were plated for 48 h, fixed, and incubated with antibodies for α/β-tubulin (green) and γ-tubulin (red). γ-Tubulin staining was used to determine centrosome duplication. Nuclei staining was revealed with DAPI. F, evaluation of cell cycle markers in RhoA-deficient cells. Plated cells were extracted at 72 h of culture, and Western blots were conducted for p-histone H3, cyclin D1, cyclin B1, cyclin E, cyclin A, and p-ERK2 as markers for cell cycle progression. GA3PDH, glyceraldehyde 3-phosphate dehydrogenase.

FIGURE 3.

RhoB and RhoC serve a redundant role with RhoA in regulating actomyosin activity. A, RhoA^flox/flox and WT cells were infected with MIEG3-GFP (control vector) or MIEG3-RhoA-GFP retrovirus. The transduced cells were sorted and plated for 24 h, and adeno-Cre or β-Gal adenovirus infection was carried out. Western blots (WB) for endogenous RhoA or transduced RhoA/GFP were performed. GA3PDH, glyceraldehyde 3-phosphate dehydrogenase. B, a cell proliferation assay was performed in WT, RhoA deleted, or RhoA reconstituted cells. Cells grown in triplicate plates were quantified at the indicated times. Error bars represent S.D. C, RhoB and RhoC activities in relation to that of RhoA deletion. WT and RhoC KO cells were plated, and 24 h later, endogenous RhoA was deleted by adeno-Cre treatment. Cells were extracted for RhoA, RhoB, and RhoC activity measurements by GST-rhotekin pulldown. D, RhoA/RhoC-deficient cells do not show adhesion defects. WT and RhoC KO cells were stained for vinculin, p-FAK (green), and F-actin (red). Nuclei were stained with DAPI (blue). E, effect of RhoB suppression in RhoA/RhoC-deficient cells on F-actin and focal adhesion. WT and RhoC KOs were plated, and 24 h later, they were incubated with an on-TARGETplusR control non-targeting pool or an on-TARGETplus SMARTpool mouse RhoB. Endogenous RhoA were deleted by adenovirus treatment. Cells were processed for Western blotting to determine the levels of RhoA, RhoB, and RhoC proteins. F, effect of RhoB knockdown on focal adhesion proteins and actomyosin components in RhoA/RhoC-deficient cells. Cells were treated with rhoB-specific siRNA, and the levels of p-MLC, p-cofilin, p-FAK, and p-Pax were determined by Western blotting. G, effect of RhoB knockdown on F-actin structure and focal adhesion complex in RhoA/RhoC-deficient cells. Cells were stained for p-Pax, p-FAK (green), and F-actin (red).