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Chalamalasetty, Ravindra babu (2006): Exploring the regulation and function of the human guanine nucleotide exchange factor Ect2 (epithelial cell transforming protein 2) in cytokinesis. Dissertation, LMU München: Faculty of Biology
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Abstract

Cytokinesis is the process that divides the cytoplasm of a parent cell into two. In animal cells, cytokinesis requires the formation of the central spindle and the contractile ring structures. The onset of cytokinesis is marked during anaphase with the specification of the division site, followed by cleavage furrow formation and ingression, midbody formation and abscission. The astral microtubules that originate from the centrosomes and the anti-parallel microtubules of the central spindle are proposed to determine the site of cleavage furrow formation (Bringmann and Hyman, 2005). The acto-myosin based contractile ring assembles at the division site and constricts the cytoplasm which is supported by the fusion of membrane vesicles to the ingressing plasma membrane. All these processes together result in the formation of two daughter cells. The small GTPase RhoA is one of the most upstream regulators of contractile ring assembly at the cortex. Rho proteins are activated by GEF’s (guanine nucleotide exchange factors) and one GEF that is required for cytokinesis is Ect2 (epithelial cell transforming protein2) (Tatsumoto et al., 1999). The Drosophila pebble (pbl) gene product is the founding member of the Ect2 protein family and has been shown to be required for cytokinesis (Lehner, 1992). In mammals, Ect2 was originally identified as a transforming protein in an expression cloning assay (Miki et al., 1993) and subsequently shown to be essential for cytokinesis. In this study, we have explored the temporal and spatial mechanisms that regulate Ect2 function. In agreement with previous studies, we show that Ect2 is a cell cycle regulated protein and is phosphorylated during mitosis. We identify a number of potentially interesting endogenous phosphorylation sites in Ect2, including potential Plk1 and Cdk1 sites. Although we have not been able to determine the function of these phosphorylation sites, their strong conservation among different species implies that they accomplish evolutionarily conserved roles.The identification of these phosphorylation sites sets the stage for future functional analyses. In complementary studies, we have shown that the central spindle and cell cortex localizations of Ect2 are facilitated by the BRCT and PH domains, respectively. The targeting of Ect2 to the central spindle is mediated by the MKlp1/MgcRacGAP and MKlp2/Aurora-B complexes. Of the two complexes, we show that Ect2 interacts and colocalizes only with the MKlp1/MgcRacGAP complex in telophase and propose that this interaction is mediated by a phosphorylation dependent docking mechanism that targets Ect2 to the central spindle. Interestingly, the displacement of Ect2 from the central spindle did not prevent cytokinesis, suggesting that localized GEF activity is not absolutely essential for cleavage furrow ingression and cytokinesis. In the second part of this thesis, we have explored the role of Ect2 during cytokinesis and show that, in Ect2 depleted cells, levels of RhoA and Citron kinase are diminished at the cleavage site, concomitant with the impairment of cleavage furrow formation and ingression. Additionally, overexpression of appropriate amino-terminal Ect2 fragments in cells also hinders cytokinesis. In these cells, RhoA and Citron kinase localize to the cortex and cleavage furrow ingression occurs, but, the subsequent abscission fails. Taken together, these results suggest that proper function of Ect2 is not only important for cleavage furrow ingression, but also for cell abscission. Finally, we investigate the overexpression phenotypes of different Ect2 truncation mutants. We show that abscission failure correlates with the persistence of amino-terminal Ect2 fragments at striking ring-like structures surrounding the midbody, indicating that completion of cell division requires the displacement of Ect2 from the contractile ring and its re-import into the reforming cell nucleus. Collectively, our data indicate that multiple mechanisms cooperate to regulate Ect2 in a spatial-temporal manner.