Regulation of Cytokinesis in Fission Yeast

描述(申请人提供)：这个项目的长期目标是了解胞质分裂是如何调节的，以及胞质分裂是如何与其他有丝分裂事件相协调的，例如纺锤体的形成和染色体分离。为了确保遗传物质的适当分离，细胞分裂必须在适当的时间开始，如果染色体没有得到适当的分离，必须有某种机制来推迟细胞质分裂。如果做不到这一点，可能会导致异常的染色体分离、非倍体和癌症。许多实验室的大量研究表明，细胞周期控制的基本机制在酵母和人类细胞之间高度保守。在分裂酵母S.pombe中，一个被称为SIN的保守信号网络在后期触发胞质分裂的启动。我们发现，这个网络对于协调细胞和核分裂以确保细胞维持基因组稳定至关重要。SIN的关键成分包括名为CDC7p、Sidlp和Sid2p的三种蛋白激酶。在本文提出的研究中，我们将阐明SIN在协调晚期有丝分裂事件中的分子机制。我的具体目标是：(1)描述胞质分裂信号启动所需的CDC7p、Sidlp和Sid2p蛋白激酶之间的精确分子相互作用。(2)确定Sid2p亚细胞定位到Spb、细胞分裂部位和微管所需的结构域，确定Sid2p与Mob L p和CDC 1 L p相互作用所需的结构域，了解Mob L p如何促进Sid2p激酶活性，并通过对Sid2p-Mob L p蛋白复合体的生化纯化来鉴定Sid2p-Mob L p相互作用蛋白。(3)研究Sus1突变抑制SIN突变的机制，克隆Sus1+基因，研究Sus1p与SIN之间的分子相互作用。(4)鉴定Dma1p泛素连接酶在抑制SIN和防止细胞在有丝分裂纺锤体缺陷时退出有丝分裂和分裂中的作用，确定是否必须抑制SIN以维持纺锤体检查点，确定Dma1p是否具有抑制C1p1p维持CDc2p酪氨酸去磷酸化状态的功能，以响应纺锤体检查点，识别被Dma1p抑制的SIN的成分，并通过生化纯化Dma1p蛋白复合体来鉴定潜在的Dma1p靶点和相互作用的蛋白质。

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.