No 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). ...

Human bone marrow mesenchymal stem cells (hMSCs) have the potential to differentiate into tendon/ligament-like lineages when they are subjected to mechanical stretching. However, the means through which mechanical stretch regulates the tenogenic differentiation of hMSCs remains unclear. This study examined the role of RhoA/ROCK, cytoskeletal organization, and focal adhesion kinase (FAK) in mechanical stretch-induced tenogenic differentiation characterized by the up-regulation of tendon-related marker gene expression. Our findings showed that RhoA/ROCK and FAK regulated mechanical stretch-induced realignment of hMSCs by regulating cytoskeletal organization and that RhoA/ROCK and cytoskeletal organization were essential to mechanical stretch-activated FAK phosphorylation at Tyr397. We also demonstrated that this process can be blocked by Y-27632 (a specific inhibitor of RhoA/ROCK), cytochalasin D (an inhibitor of cytoskeletal organization) or PF 573228 (a specific inhibitor of FAK). The results of this study suggest that RhoA/ROCK, cytoskeletal organization, and FAK compose a "signaling network" that senses mechanical stretching and drives mechanical stretch-induced tenogenic differentiation of hMSCs. This work provides novel insights regarding the mechanisms of tenogenesis in a stretch-induced environment and supports the therapeutic potential of hMSCs.

Centrosomes catalyze microtubule formation for mitotic spindle assembly 1 . Centrosomes duplicate once per cell cycle in a process controlled the kinase PLK4 2 , 3 . Following chemical PLK4 inhibition, cell division in the absence of centrosome duplication generates centrosome-less cells that exhibit delayed, acentrosomal spindle assembly 4 . Whether PLK4 inhibitors can be leveraged for cancer treatment is not yet clear. Here, we show that acentrosomal spindle assembly following PLK4 inhibition depends on levels of the centrosomal ubiquitin ligase TRIM37. Low TRIM37 accelerates acentrosomal spindle assembly and improves proliferation following PLK4 inhibition, whereas high TRIM37 inhibits acentrosomal spindle assembly, leading to mitotic failure and cessation of proliferation. The Chr17q region containing the TRIM37 gene is frequently amplified in neuroblastoma and in breast cancer 5 – 8 , which renders these cancer types highly sensitive to PLK4 inhibition. TRIM37 inactivation improves acentrosomal mitosis because TRIM37 prevents PLK4 self-assembly into centrosome-independent condensates that serve as ectopic microtubule-organizing centers. By contrast, elevated TRIM37 expression inhibits acentrosomal spindle assembly via a distinct mechanism that involves degradation of the centrosomal component CEP192. Thus, TRIM37 is a critical determinant of mitotic vulnerability to PLK4 inhibition. Linkage of TRIM37 to prevalent cancer-associated genomic changes, including 17q gain in neuroblastoma and 17q23 amplification in breast cancer, may offer an opportunity to use PLK4 inhibition to trigger selective mitotic failure and provide new avenues to treatments for these cancers.

Diaphanous-related formin, mDia, is an actin nucleation/polymerization factor functioning downstream of the small GTPase Rho. Although Rho is critically involved in cytokinesis, it remains elusive how Rho effectors and other regulators of cytoskeletons work together to accomplish this process. Here we focused on mDia2, an mDia isoform involved in cytokinesis of NIH 3T3 cells, and analyzed mechanisms of its localization in cytokinesis. We found that targeting of mDia2 to the cleavage furrow requires not only its binding to RhoA but also its diaphanous-inhibitory domain (DID). We then performed pulldown assays using a fragment containing the latter domain as a bait and identified anillin as a novel mDia2 interaction partner. The anillin-binding is competitive with the diaphanous autoregulatory domain (DAD) of mDia2 in its autoinhibitory interaction. A series of RNA interference and functional rescue experiments has revealed that, in addition to the Rho GTPase-mediated activation, the interaction between mDia2 and anillin is required for the localization and function of mDia2 in cytokinesis.