Mechanistic Analysis of Cytokinesis in Eukaryotes

项目概要/摘要： 胞质分裂对所有生物的发育和生存至关重要。胞质分裂缺陷导致 非整倍性和基因组不稳定性，从而导致严重的疾病，如癌症，神经元疾病， 疾病和贫血。因此，胞质分裂的机制研究不仅对于理解细胞分裂的机制是重要的， 基本过程的基本原则，而且还用于设计治疗人类疾病的新策略。 在动物和真菌细胞中的胞质分裂需要一个收缩因子的时空协调功能。 肌动球蛋白环（AMR）、靶向囊泡融合和局部细胞外基质（ECM）重塑。是 比以前认识到的复杂得多。在本申请中，我们将解决三个主要问题， 关于这一使用芽殖酵母和哺乳动物细胞的基本过程， 模型，目的是解剖酵母中的深层机制，并探索酵母中的进化保守性。 哺乳动物细胞在目标1中，我们将确定AMR的架构。具体来说，我们将研究如何 肌球蛋白-II及其相关蛋白如肌动蛋白和IQGAP被组织在细胞的收缩环中 使用铂复制电子显微镜（PREM）结合免疫金在胞质分裂时同步化 标记以及超分辨率随机光学重建显微镜（STORM）。在目标2中，我们 验证我们的假设，即肌球蛋白丝的组装是由重链磷酸化以及 反式作用因子，如IQGAP，使用生化，遗传，定量实时成像和其他切割- 如上所述的边缘成像方法。在目标3中，我们将确定AMR如何引导胞吐作用 以及分裂部位的ECM重塑具体来说，我们将测试我们的假设，酵母菌的尾巴 肌球蛋白-II通过与 囊泡相关的鸟嘌呤核苷酸交换因子（GEF），Sec 2，用于Rab GT3 Sec 4。然后， 肌球蛋白相关蛋白复合物（Inn 1、Hof 1和Cyk 3）通过C2结构域促进囊泡融合， Inn 1，并激活货物酶Chs 2，糖基转移酶家族2的成员，用于ECM 通过Cyk 3的转氨酶样结构域重塑（即酵母中的隔膜形成）。 发现（目标1）和假设驱动（目标2和3）研究预计将产生新的 细胞质分裂的概念和机制超出了特定的模式生物。

Project Summary/Abstract: Cytokinesis is essential for development and survival of all organisms. Defects in cytokinesis cause aneuploidy and genomic instability, thereby contributing to serious diseases such as cancer, neuronal disorders, and anemia. Thus, mechanistic study of cytokinesis is important not only for understanding the basic principles of a fundamental process but also for designing new strategies to treat human diseases. Cytokinesis in animal and fungal cells requires spatiotemporally coordinated functions of a contractile actomyosin ring (AMR), targeted vesicle fusion, and localized extracellular matrix (ECM) remodeling. It is much more complex than previously appreciated. In this application, we will address three major unanswered questions regarding this fundamental process using both budding yeast and mammalian cell models, with the goal of dissecting deep mechanisms in yeast and exploring evolutionary conservation in mammalian cells. In Aim 1, we will determine the architecture of the AMR. Specifically, we will examine how myosin-II and its associated proteins such as actin and IQGAP are organized in the contractile ring from cells synchronized at cytokinesis using platinum-replica electron microscopy (PREM) coupled with immuno-gold labeling as well as super-resolution stochastic optical reconstruction microscopy (STORM). In Aim 2, we will test our hypothesis that myosin filament assembly is regulated by heavy chain phosphorylation as well as by trans-acting factors such as IQGAP using biochemical, genetic, quantitative live imaging, and other cutting- edge imaging methods as described above. In Aim 3, we will determine how the AMR guides exocytosis and ECM remodeling at the division site. Specifically, we will test our hypothesis that the tail of the yeast myosin-II positions and unloads vesicles from the transport machinery at the division site by interacting with the vesicle-associated guanine-nucleotide-exchange factor (GEF), Sec2, for the Rab GTPase Sec4. Then, the myosin-associated protein complex (Inn1, Hof1, and Cyk3) promotes vesicle fusion via the C2 domain of Inn1, and activates the cargo enzyme Chs2, a member of the glycosyltransferase family 2, for ECM remodeling (i.e. septum formation in yeast) via the transglutaminase-like domain of Cyk3. The discovery (Aim 1) and hypothesis-driven (Aims 2 and 3) research is expected to generate novel concepts and mechanisms of cytokinesis that are beyond specific model organisms.