Regulation of Cytokinesis in Fission Yeast

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DESCRIPTION (provided by applicant): The long-term goal of this project is to understand how cytokinesis is regulated, and how cytokinesis is coordinated with other mitotic events such as spindle formation and chromosome segregation. To ensure proper segregation of genetic material, cell division must initiate at an appropriate time, and there must be some mechanism to delay cytokinesis if the chromosomes have not been properly segregated. Failure to do so could lead to abnormal chromosome segregation, anueploidy, and cancer. A large number of studies from many labs have shown that the basic mechanisms of cell cycle control are highly conserved between yeast and human cells. A conserved signaling network called the SIN in the fission yeast S. pombe functions to trigger initiation of cytokinesis at the end of anaphase. We have found that this network is crucial for coordinating cell and nuclear division to ensure that cells maintain genomic stability. Key components of the SIN include three-protein kinase called Cdc7p, Sidlp, and Sid2p. In the studies proposed here, we will elucidate the molecular mechanisms by which the SIN functions in coordinating late mitotic events. My specific aims are: (1) To characterize the precise molecular interactions between the Cdc7p, Sidlp, and Sid2p protein kinases required for signaling initiation of cytokinesis. (2) To define domains of Sid2p required for subcellular localization to the SPB, cell division site, and microtubules, to define domains of Sid2p required for interaction with Mob l p and Cdc 1 l p, to understand how Mob l p functions to promote Sid2p kinase activity, and to identify Sid2p-Mob l p interacting proteins by biochemical purification of Sid2p-Mob l p protein complexes. (3) To characterize the mechanism by which the sus1 mutation suppresses mutations in the SIN, to clone the sus1 + gene and to characterize the molecular interactions between Sus1p and the SIN. (4) To characterize the role of the Dma1p ubiquitin ligase in inhibiting the SIN and preventing cells from exiting mitosis and dividing if the mitotic spindle is defective, to determine if it is essential for the SIN to be inhibited to maintain the spindle checkpoint, to determine if Dma1p functions to inhibit C1p1p to maintain Cdc2p in a tyrosine dephosphorylated state in response to the spindle checkpoint, to identify components of the SIN that are inhibited by Dma1p, and to identify potential Dma1p targets and interacting proteins by biochemical purification of Dma1p protein complexes.