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Cytokinesis, the cleavage of the cell following chromosome segregation, is the last step in a cell cycle. Cytokinesis involves the selection of the cleavage site, the assembly of the contractile ring, the ingression of the cleavage furrow, and finally the abscission of the cell. In the past few years, we have identified and characterized several important regulators of cytokinesis. Investigating the physiological function of these proteins provides mechanisms of cytokinesis in normal cells and insights on tetraploid formation in tumor cells.
A. Assembly and Ingression of the Cleavage Furrow - We found that MgcRacGAP, a GTPase activating protein for RhoA, is a substrate of APC/C (Zhao and Fang, 2005a). Although MgcRacGAP has been shown to be required for cytokinesis, the mechanism of its action remained unknown. We found that MgcRacGAP is required for the assembly of anillin and myosin into the contractile ring. In addition, MgcRacGAP is required for the localized activation of myosin through RhoA-mediated phosphorylation of the myosin regulatory light chain. Cells with MgcRacGAP RNAi failed cytokinesis without any ingression of the cleavage furrow. Paradoxically, MgcRacGAP, a GTPase activating protein, associates with ECT2, a guanine nucleotide exchange factor for RhoA, during cytokinesis and the localization of ECT2 to both the central spindle and the contractile ring depends on MgcRacGAP. Knockdown of ECT2 phenocopies that of MgcRacGAP. We conclude that MgcRacGAP controls the initiation of cytokinesis by regulating ECT2, which in turn induces the assembly of the contractile ring and triggers the ingression of the cleavage furrow.
B. Progression of the Cleavage Furrow Ingression - We found that anillin, an actin-binding protein localized at the cleavage furrow, is another substrate of APC/C required for cytokinesis (Zhao and Fang, 2005b). We showed that the levels of anillin fluctuate in the cell cycle, peaking in mitosis and dropping drastically during mitotic exit. Ubiquitination of anillin requires a destruction-box and is mediated by Cdh1, an activator of APC/C. Over-expression of Cdh1 reduces the levels of anillin, while inactivation of Cdh1-APC/C increases the half-life of anillin. Thus, anillin is a substrate of APC/C, both in vitro and in vivo. Functionally, anillin is required for the completion of cytokinesis. In anillin-knockdown cells, the cleavage furrow ingresses, but fails to complete the ingression. At late cytokinesis, the cytosol and DNA in knockdown cells undergo rapid myosin-based oscillatory movement across the furrow. During this movement, RhoA and active myosin are absent from the furrow and myosin is redistributed to cortical patches, which drives the random oscillatory movement. We conclude that anillin functions to maintain the localization of active myosin, thereby ensuring the spatial control of concerted contraction during cytokinesis.
C. Cell Abscission - We identified C10orf3/Cep55 in a genomic screen as a regulator required for the completion of cytokinesis (Zhao et al., 2006). We showed that Cep55 localizes to the mitotic spindle during prometaphase and metaphase and to the spindle midzone and midbody matrix during anaphase and cytokinesis. At the terminal stage of cytokinesis, Cep55 is required for the midbody structure and for the completion of cytokinesis. In Cep55-knockdown cells, the midbody matrix is disrupted, and the structural and regulatory components of the midbody are either absent or mislocalized. Cep55 also facilitates membrane fusion at the terminal stage of cytokinesis by controlling the localization of endobrevin, a v-SNARE required for cell abscission. Biochemically, Cep55 is a MT-associated protein that efficiently bundles MTs. In addition, Cep55 directly binds to MKLP1 in vitro and associates with the MKLP1-MgcRacGAP centralspindlin complex in vivo. Cep55 is under the control of centralspindlin, as knockdown of centralspindlin abolished the localization of Cep55 to the spindle midzone. Our study defines a cellular mechanism that links centralspindlin to Cep55, which, in turn, controls the midbody structure and membrane fusion at the terminal stage of cytokinesis.
These physiological studies provided us cellular mechanisms for the function of various cytokinesis factors. Our current focus on cytokinesis is to investigate the biochemical mechanism on how Ect2, MgcRacGAP, anillin and Cep55 function in cytokinesis and to identify additional novel regulators of cytokinesis through our functional genomic screen.