Mechanistic Analysis of Cytokinesis in Eukaryotes

项目摘要/摘要： 胞质分裂对所有生物体的发育和生存都是必不可少的。胞质分裂缺陷引起的 非整倍体和基因组不稳定，从而导致严重的疾病，如癌症，神经元 疾病和贫血。因此，细胞质分裂的机制研究不仅对于理解 这不仅是一个基本过程的基本原则，而且也是设计治疗人类疾病的新战略的基本原则。 动物和真菌细胞的胞质分裂需要收缩的时空协调功能 肌动球蛋白环(AMR)、靶向囊泡融合和局部细胞外基质(ECM)重塑。它是 比之前想象的要复杂得多。在本应用程序中，我们将解决三个主要问题 关于这一使用发芽酵母和哺乳动物细胞的基本过程的未解答的问题 模型，目的是剖析酵母菌的深层机制，并探索 哺乳动物细胞。在目标1中，我们将确定AMR的架构。具体地说，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在细胞的收缩环中被组织起来 铂复制电子显微镜(PREM)与免疫金联用在胞质分裂时的同步 标记以及超分辨率随机光学重建显微镜(STORM)。在目标2中，我们将 测试我们的假设，肌球蛋白细丝组装受重链磷酸化和 反式作用因子，如使用生化、遗传、定量活成像和其他切割的IQGAP- 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 以及分部的ECM改建。具体地说，我们将测试我们的假设，即酵母的尾巴 肌球蛋白II通过与以下物质相互作用定位和卸载位于分区现场的运输机械中的囊泡 Rab GTP酶Sec4的囊泡相关鸟嘌呤核苷酸交换因子Sec2。然后, 肌球蛋白相关蛋白复合体(Inn1、Hof1和Cyk3)通过C2域促进囊泡融合。 Inn1，并激活糖基转移酶家族2的成员Chs2，用于ECM 通过Cyk3的谷氨酰胺转氨酶样域重塑(即酵母中的隔膜形成)。 这项发现(目标1)和假设驱动(目标2和3)的研究有望产生新的 超越特定模式生物的细胞质分裂的概念和机制。

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.