Investigating the Regulation and Structural Features of the conserved Membrane-Binding F-BAR Protein Cdc15

 描述（由申请人提供）：细胞复制的最后一步是子细胞的物理分离或胞质分裂。动物细胞使用基于肌动蛋白和肌球蛋白的收缩环（CR）进行分裂。CR组装、收缩和拆卸受到严格调控以保护基因组互补。胞质分裂失败或胞质分裂的不适当位置或时机可导致细胞死亡或非整倍性，这是一种促进肿瘤发生的状态。这个提议的目的是利用真核分裂酵母裂殖酵母来增强对胞质分裂的分子理解。S.粟酒裂殖酵母是研究胞质分裂的极好模式生物，因为该过程从酵母到动物细胞在进化上是保守的。此外，S. Pombe提供了许多技术优势，包括基因缺失收集和容易实施的正向和反向遗传学、生物化学和显微镜。在S. Pombe已经允许大规模筛选以鉴定另外的细胞动力学蛋白，其中许多蛋白在高等真核生物中是保守的。该研究探讨了一种重要的细胞动力学蛋白Cdc 15的调控和分子机制。Cdc 15是Pombe Cdc 15同源（PCH）/ FCH-Bin/Amphiphysin/Rvs（F-BAR）家族的创始成员，该家族包括通过F-BAR结构域将膜连接到通过额外功能结构域重塑的肌动蛋白细胞骨架的蛋白质。Cdc 15的N-末端F-BAR结构域将Cdc 12募集到分裂位点并结合膜，而其非必需的C-末端SH 3结构域与许多参与胞质分裂稳健完成的蛋白质相互作用。cdc 15的功能是通过磷酸化>33个丝氨酸或苏氨酸来调节细胞周期，其中大部分发生在无序的中心区域。磷酸化位点与不同的激酶共有序列相匹配，表明多种信号传导途径会聚在Cdc 15上。在目标1中，激酶和磷酸酶调节Cdc 15将通过筛选基因缺失或温度敏感性突变体来确定。质谱法，诱变，遗传分析，生物化学和显微镜的组合将被用来表征特定残基的磷酸化状态（去）由一个单一的激酶或磷酸酶磷酸化影响Cdc 15活性。该建议的第二个目的是采用蛋白质组学，结构功能分析，显微镜，生物化学和遗传实验来测试的假设，即无序的中心区域结合一个或多个蛋白质参与胞质分裂。这些目标的综合结果将确定时间和空间调节动物细胞胞质分裂的信号转导通路，并将更好地表征其完整性是成功的胞质分裂所必需的分子机制。这些知识将为理解细胞动力学缺陷引起的疾病的发病机制提供基础性的见解。

 DESCRIPTION (provided by applicant): The final step in cell replication is the physical separation of daughter cells, or cytokinesis. Animal cells divide using an actin- and myosin-based contractile ring (CR). CR assembly, constriction, and disassembly are tightly regulated to protect the genomic complement. Cytokinesis failure or inappropriate location or timing of cytokinesis can lead to cell death or aneuploidy, a state that promotes tumorigenesis. The goal of this proposal is to enhance the molecular understanding of cytokinesis using the eukaryotic fission yeast Schizosaccharomyces pombe. S. pombe is an excellent model organism to study cytokinesis because this process is evolutionarily conserved from yeast to animal cells. Furthermore, S. pombe offers many technical advantages including a gene deletion collection and easy implementation of forward and reverse genetics, biochemistry, and microscopy. Facile genetic manipulation in S. pombe has allowed for large-scale screens to identify additional cytokinetic proteins, many of which are conserved in higher eukaryotes. The proposed research investigates the regulation and molecular mechanism of an essential cytokinetic protein, Cdc15. Cdc15 is the founding member of the Pombe Cdc15 Homology (PCH) / FCH-Bin/Amphiphysin/Rvs (F-BAR) family, which comprises proteins that link membranes via F-BAR domains to actin cytoskeleton remodeling through additional functional domains. Cdc15's N-terminal F-BAR domain recruits the formin Cdc12 to the division site and binds membranes, while its non-essential C-terminal SH3 domain interacts with numerous proteins involved in robust completion of cytokinesis. Cdc15 functions are cell cycle regulated by phosphorylation of >33 serines or threonines, most of which occur in the disordered central region. The phosphorylated sites match diverse kinase consensus sequences, suggesting that multiple signaling pathways converge on Cdc15. In Aim 1, kinases and phosphatases that regulate Cdc15 will be identified by screening gene deletion or temperature-sensitive mutants. A combination of mass spectrometry, mutagenesis, genetic analysis, biochemistry and microscopy will be used to characterize how the phosphorylation status of the specific residues (de)phosphorylated by a single kinase or phosphatase affects Cdc15 activity. The second aim of this proposal employs proteomics, structure-function analysis, microscopy, biochemistry, and genetic experiments to test the hypothesis that the disordered central region binds one or more proteins that participate in cytokinesis. The combined results of these aims will identify signalin pathways that temporally and spatially regulate animal cell cytokinesis and will better characterize the molecular machinery whose integrity is essential for successful cytokinesis. This knowledge will provide foundational insight for understanding the pathogenesis of disorders resulting from cytokinetic defects.