Maintenance of chromosomal ploidy during the rapid cell divisions of the early embryo requires a precise coordination of chromosome segregation and cytoplasmic partitioning (cytokinesis) such that the contractile ring does not assemble before the onset of anaphase. Recent studies in both yeast and animal cells have identified a mitotic checkpoint that monitors kinetochore attachment to the mitotic spindle and regulates the onset of sister chromatid segregation and CDK1 inactivation (mitotic exit). In fission yeast, the mitotic checkpoint also regulates a signaling cascade termed the Septation Initiation Network (SIN) that regulates the initiation of cytokinesis. The temporal regulation of cytokinesis in animal cells is not well characterized, but studies in sea urchin embryos suggest that the signaling pathways regulating contractile ring placement and assembly are subject to the mitotic checkpoint but independent of mitotic exit. In an effort to understand how the mitotic checkpoint coordinates mitosis and cytokinesis in higher eukaryotes, a terminal component of the SIN pathway (Mob1) has been identified and characterized in sea urchin embryos. The lines of experimentation described in this application seek to combine molecular, biochemical and live cell analyses to assess the role of the Septation Initiation Network in regulating the terminal events of cell division. The first Specific Aim of this proposal seeks to identify the urchin homolog of Sid2 kinase, examine its association with Mob1 and characterize its activity during the cell cycle.
Specific Aim 2 will study the dynamics of Mob1 and Sid2 association with the spindle poles in vitro and in vivo.
Specific Aim 3 will assess the roles of Mob1 and Sid2 in directing the terminal events of the cell cycle in vivo by expressing dominant-negative forms of Sid2 and Mob1, and studying their effects on mitotic exit and cytokinesis using live cell fluorescence imaging. Lastly, constitutively activated Sid2 will be introduced into living cells to test whether uncoupling SIN from the mitotic checkpoint can result in unregulated cytokinesis. These efforts should lead to a clearer understanding of how mitosis and cytokinesis is coordinated, and confirm whether the strategies employed by unicellular fungi to regulate septation are conserved in higher eukaryotes.
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