mDia2 Induces the Actin Scaffold for the Contractile Ring and Stabilizes Its Position during Cytokinesis in NIH 3T3 Cells

Abstract

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). ...

Formins at a glance

“…2008; Harris et al, 2010), lamellipodia (Yang et al, 2007;Sarmiento et al, 2008;Block et al, 2012), stress fibers Satoh and Tominaga, 2001;Gasteier et al, 2003;Peng et al, 2003;Hotulainen and Lappalainen, 2006;Sato et al, 2006;Takeya et al, 2008), cytoplasmic actin networks used for longrange vesicle transport (Leader et al, 2002;Azoury et al, 2008;Li et al, 2008;Pfender et al, 2011;Schuh, 2011), cytokinetic actin rings (Severson et al, 2002;Watanabe et al, 2008) and phagocytic cups (Brandt et al, 2007). Formins also have essential roles in physiological processes ranging from cell motility in the immune system (Yayoshi-Yamamoto et al, 2000;Eisenmann et al, 2007;Shi et al, 2009), to gastrulation and neural tube closure (Habas et al, 2001;Sato et al, 2006;Lai et al, 2008), heart morphogenesis (Iskratsch et al, 2010;Li et al, 2011), kidney morphogenesis (Brown et al, 2010;Boyer et al, 2011a;Boyer et al, 2011b), and dendritic spine formation in neurons.…”

Rho signaling, ROCK and mDia1, in transformation, metastasis and invasion

“…Indeed, expression of an active form of mDia induces stress fibers in cultured cells, and treatment of these cells with a specific ROCK inhibitor, Y-27632 [15], causes dissolution of the bundles, leaving the cells with diffusely distributed actin filaments [16]. Requirement of ROCK and mDia in the contractile ring formation and function was also reported [17,18].…”

Cytokinesis in Metazoa and Fungi

“…Active RhoA directly stimulates the Rho kinase ROCK, which induces myosin light chain phosphorylation, filament assembly, and motor activation (Kosako et al 2000). RhoA also directly activates formins, which nucleate assembly of unbranched actin filaments (Otomo et al 2005;Watanabe et al 2008Watanabe et al , 2013. RhoA recruits anillin, a scaffold protein that can interact with RhoA, F-actin, and myosin ( Fig.…”