mDia proteins are mammalian homologues of Drosophila diaphanous and belong to the formin family proteins that catalyze actin nucleation and polymerization. Although formin family proteins of nonmammalian species such as Drosophila diaphanous are essential in cytokinesis, whether and how mDia proteins function in cytokinesis remain unknown. Here we depleted each of the three mDia isoforms in NIH 3T3 cells by RNA interference and examined this issue. Depletion of mDia2 selectively increased the number of binucleate cells, which was corrected by coexpression of RNAi-resistant full-length mDia2. mDia2 accumulates in the cleavage furrow during anaphase to telophase, and concentrates in the midbody at the end of cytokinesis. Depletion of mDia2 induced contraction at aberrant sites of dividing cells, where contractile ring components such as RhoA, myosin, anillin, and phosphorylated ERM accumulated. Treatment with blebbistatin suppressed abnormal contraction, corrected localization of the above components, and revealed that the amount of F-actin at the equatorial region during anaphase/telophase was significantly decreased with mDia2 RNAi. These results demonstrate that mDia2 is essential in mammalian cell cytokinesis and that mDia2-induced F-actin forms a scaffold for the contractile ring and maintains its position in the middle of a dividing cell. INTRODUCTIONCytokinesis is the final step in cell division that physically separates a dividing cell into two. In a somatic cell, separation, i.e., cleavage occurs in the middle of a dividing cell between the two spindle poles to ensure each set of segregated chromosomes inherited to each daughter cell. Although cytokinesis is a multistep process under coordinated control of cell cycle progression, cytoskeletal dynamics, and vesicle transport, the actomyosin-based constriction by the contractile ring that is constructed in the equatorial region of a dividing cell is recognized as a major driving force for physical separation till abscission (Balasubramanian et al., 2004). However, how the position of the contractile ring, i.e., cleavage plane, is determined and maintained through cytokinesis and how and where actin filaments are produced and assembled with myosin and other molecules into the contractile ring in mammalian cells remain largely unknown.The small GTPase Rho functions in several organisms and several lines of cultured mammalian cells as a molecular switch linking nuclear division and cytokinesis; Rho is activated in anaphase to telophase and induces the contractile ring in dividing cells (Mabuchi et al., 1993;Piekny et al., 2005;Narumiya and Yasuda, 2006). In mammalian cells, the GTPbound, activated form of Rho acts on two downstream effectors to induce actomyosin bundles; one is ROCK/Rhokinase that activates myosin for cross-linking of anti-parallel actin filaments, and the other is mammalian homolog of Drosophila diaphanous (mDia) protein that induces actin filaments by catalyzing actin nucleation and polymerization (Watanabe et al., 1997;Sagot et al., 2002). ...

During G2 phase of cell cycle, centrosomes function as a scaffold for activation of mitotic kinases. Aurora-A is first activated at late G2 phase at the centrosome, facilitates centrosome maturation, and induces activation of cyclin B-Cdk1 at the centrosome for mitotic entry. Although several molecules including HEF1 and PAK are implicated in centrosomal activation of Aurora-A, signaling pathways leading to Aurora-A activation at the centrosome, and hence mitotic commitment in vertebrate cells remains largely unknown. Here, we have used Clostridium difficile toxin B and examined the role of Rho GTPases in G2/M transition of HeLa cells. Inactivation of Rho GTPases by the toxin B treatment delayed by 2 h histone H3 phosphorylation, Cdk1/cyclin B activation, and Aurora-A activation. Furthermore, PAK activation at the centrosome that was already present before the toxin addition was significantly attenuated for 2 h by the addition of toxin B, and HEF1 accumulation at the centrosome that occurred in late G2 phase was also delayed. These results suggest that Rho GTPases function in G2/M transition of mammalian cells by mediating multiple signaling pathways converging to centrosomal activation of Aurora-A.