Distinct actions and cooperative roles of ROCK and mDia in Rho small G protein-induced reorganization of the actin cytoskeleton in Madin-Darby canine kidney cells

Abstract

Rho, a member of the Rho small G protein family, regulates the formation of stress fibers and focal adhesions in various types of cultured cells. We investigated here the actions of ROCK and mDia, both of which have been identified to be putative downstream target molecules of Rho, in Madin-Darby canine kidney cells. The dominant active mutant of RhoA induced the formation of parallel stress fibers and focal adhesions, whereas the dominant active mutant of ROCK induced the formation of stellate stress fibers and focal adhesions, and the dominant active mutant of mDia induced the weak formation of parallel stress fibers without affecting the formation of focal adhesions. In the presence of C3 ADP-ribosyltransferase for Rho, the dominant active mutant of ROCK induced the formation of stellate stress fibers and focal adhesions, whereas the dominant active mutant of mDia induced only the diffuse localization of actin filaments. These results indicate that ROCK and mDia show distinct actions in reorganization of the actin cytoskeleton. The dominant negative mutant of either ROCK or mDia inhibited the formation of stress fibers and focal adhesions, indicating that both ROCK and mDia are necessary for the formation of stress fibers and focal adhesions. Moreover, inactivation and reactivation of both ROCK and mDia were necessary for the 12-O-tetradecanoylphorbol-13-acetate-induced disassembly and reassembly, respectively, of stress fibers and focal adhesions. The morphologies of stress fibers and focal adhesions in the cells expressing both the dominant active mutants of ROCK and mDia were not identical to those induced by the dominant active mutant of Rho. These results indicate that at least ROCK and mDia cooperatively act as downstream target molecules of Rho in the Rho-induced reorganization of the actin cytoskeleton.

Figure 1

Structures of ROCK, mDia, and their mutants. (A) Structural domains of mDia are schematically illustrated at the top, and full-length and four mutants are represented by the thick lines below. Numbers indicate amino acid residues of the N and C termini of each mutant. RBD, Rho-binding domain; FH1, FH domain 1; FH2, FH domain 2. (B) Structural domains of ROCK are schematically illustrated at the top, and three mutants are represented by the thick lines below. Numbers indicate amino acid residues of the N and C termini of each mutant. KD, kinase domain; CCD, coiled coil–forming amphiphatic α-herical domain; RBD, Rho-binding domain; PH, pleckstrin homology domain; CRD, cysteine-rich zinc-finger domain; X, positions of mutations with amino acid numbers.

Figure 2

Effect of the dominant active mutant of Rho, ROCK, or mDia on the actin cytoskeleton in MDCK cells. pEF-BOS-myc-V14RhoA (a–f), pCAG-myc-ROCK-Δ1 (g–l), or pEF-BOS-myc-mDia-ΔN (m–r) was microinjected into the nuclei of MDCK cells. At 10 h after the microinjection, the cells were fixed and triple stained with rhodamine-phalloidin (a, g, and m), the anti-vinculin mAb (b, h, and n), and the anti-myc pAb (c, i, and o) or double stained with rhodamine-phalloidin (d, e, j, k, p, and q) and the anti-myc mAb (f, l, and r). (a–d, g–j, and m–p) Confocal microscopic analysis at the basal levels. (e, f, k, l, q, and r) Confocal microscopic analysis at the junctional levels. The results shown are representative of three independent experiments. Bars, 10 μm. Arrows in a, d, and e indicate the sites where stress fibers coalesced in the V14RhoA-expressing cells. Arrowheads in g, j, and k indicate the sites where stress fibers coalesced in the ROCK-Δ1-expressing cells.

Figure 3

Effect of coexpression of C3 with the dominant active mutant of ROCK or mDia on the actin cytoskeleton in MDCK cells. pEF-BOS-myc-C3 (a–c), pEF-BOS-myc-C3 plus pCAG-myc-ROCK-Δ1 (d–f), and pEF-BOS-myc-C3 plus pEF-BOS-myc-mDia-ΔN (g–i) were microinjected into the nuclei of MDCK cells. At 10 h after the microinjection, the cells were fixed and double stained with rhodamine-phalloidin (a, b, d, e, g, and h) and the anti-myc mAb (c, f, and i). (a, c, d, f, g, and i) Confocal microscopic analysis at the basal levels. (b, e, and h) Confocal microscopic analysis at the junctional levels. The results shown are representative of three independent experiments. Bars, 10 μm. Arrowheads in d and e indicate the sites where stress fibers coalesced in the cells coexpressing C3 and ROCK-Δ1.

Figure 4

Effect of the dominant negative mutant of ROCK or mDia on the actin cytoskeleton, E-cadherin, and the ERM family in MDCK cells. (A) pCAG-myc-ROCK-KDIA (a–c) and pEF-BOS-myc-mDia-ΔRBDΔC (d–f) were microinjected into the nuclei of MDCK cells. At 10 h after the microinjection, the cells were fixed and double stained with rhodamine-phalloidin (a, b, d, and e) and the anti-myc mAb (c and f). (a and d) Confocal microscopic analysis at the basal levels. (b, c, e, and f) Confocal microscopic analysis at the junctional levels. (B) pCAG-myc-ROCK-KDIA (a–d) and pEF-BOS-myc-mDia-ΔRBDΔC (e–h) were microinjected into the nuclei of MDCK cells. At 10 h after the microinjection, the cells were fixed and double stained with the ECCD-2 anti-E-cadherin mAb (a and e) and the anti-myc mAb (b and f) or with the anti-ERM family mAb (c and g) and the anti-myc mAb (d and h) and analyzed at the junctional levels using confocal microscopy. The results shown are representative of three independent experiments. Bars, 10 μm. Arrowheads in A, b, indicate the sites where cell–cell adhesion is disrupted. Arrowheads in B, a, indicate the sites where the staining of E-cadherin at the cell–cell adhesion sites disappeared.

Figure 5

Effect of coexpression of ROCK and mDia on the actin cytoskeleton in MDCK cells. pCAG-myc-ROCK-Δ1 plus pEF-BOS-myc-mDia-ΔN were microinjected into the nuclei of MDCK cells. At 10 h after the microinjection, the cells were fixed and double stained with rhodamine-phalloidin (a and b) and the anti-myc mAb (c) or triple stained with rhodamine-phalloidin (d), the anti-vinculin mAb (e), and the anti-myc pAb (f). (a, d, e, and f) Confocal microscopic analysis at the basal levels. (b and c) Confocal microscopic analysis at the junctional levels. The results shown are representative of three independent experiments. Bars, 10 μm.

Figure 6

Effect of ROCK and mDia mutants on the TPA-induced reorganization of the actin cytoskeleton in MDCK cells. pEF-BOS-myc-V14RhoA (a–d), pEF-BOS-myc-C3 (e and f), pCAG-myc-ROCK-Δ1 (g–j), pCAG-myc-ROCK-KDIA (k and l), pEF-BOS-myc-mDia-ΔN (m–p), or pEF-BOS-myc-mDia-ΔRBDΔC (q and r) were microinjected into the nuclei of MDCK cells. At 10 h after the microinjection, the cells were stimulated with 100 nM TPA for 15 min (a, b, g, h, m, and n) or 2 h (c–f, i–l, and o–r) and fixed. The cells were double stained with rhodamine-phalloidin (a, c, e, g, i, k, m, o, and q) and the anti-myc mAb (b, d, f, h, j, l, n, p, and r) and analyzed at the basal levels using confocal microscopy. The results shown are representative of three independent experiments. Bars, 10 μm.