Mechanistic Analysis of Cytokinesis in Eukaryotes

真核生物细胞分裂的机制分析

• 批准号: 10224222
• 负责人: Erfei Bi
• 金额: $ 44.78万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10224222
• 关键词: Actins; Actomyosin; Address; Affect; Anemia; Aneuploidy; Animal Model; Animals; Architecture; Biochemical Genetics; Biological Models; Biology; Biosensor; C2 Domain; Cell model; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Coupled; Cytokinesis; Defect; Deposition; Development; Disease; Electron Microscopy; Enzymatic Biochemistry; Enzymes; Eukaryota; Excision; Exocytosis; Extracellular Matrix; Failure; Family; Filament; Fission Yeast; Genomic Instability; Goals; Guanine Nucleotide Exchange Factors; Hela Cells; Human; Image; Label; Life; Malignant Neoplasms; Mammalian Cell; Mediating; Microfilaments; Microscopy; Molecular; Monitor; Myosin ATPase; Myosin Type II; Neurons; Optics; Organism; Phosphorylation; Platinum; Positioning Attribute; Process; Production; Protein Isoforms; Proteins; Recombinants; Regulation; Research; Resolution; Role; Saccharomyces cerevisiae; Saccharomycetales; Site; System; Tail; Testing; Trans-Activators; Transglutaminases; Vesicle; Yeasts; base; design; glycosyltransferase; human disease; imaging modality; in vivo; innovation; member; monomer; non-muscle myosin; novel; protein complex; rab GTP-Binding Proteins; reconstruction; spatiotemporal; vesicle transport

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.

项目摘要/摘要： 细胞分裂对于所有生物体的发育和生存至关重要。胞质分裂缺陷导致 非整倍体和基因组不稳定，从而导致癌症、神经元疾病等严重疾病 疾病和贫血。因此，胞质分裂的机制研究不仅对于理解胞质分裂很重要 基本过程的基本原则，也可用于设计治疗人类疾病的新策略。 动物和真菌细胞的细胞分裂需要收缩细胞的时空协调功能 肌动球蛋白环（AMR）、靶向囊泡融合和局部细胞外基质（ECM）重塑。这是 比以前想象的要复杂得多。在此应用程序中，我们将解决三个主要问题 关于使用芽殖酵母和哺乳动物细胞的这一基本过程的未解答的问题 模型，目的是剖析酵母的深层机制并探索酵母的进化保守性 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（例如肌动蛋白和 IQGAP）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中同步 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 检验我们的假设，即肌球蛋白丝组装受重链磷酸化以及 反式作用因子，例如使用生化、遗传、定量实时成像和其他切割技术的 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 以及分部现场的 ECM 改造。具体来说，我们将检验我们的假设，即酵母的尾部 肌球蛋白-II 通过与 囊泡相关鸟嘌呤核苷酸交换因子 (GEF) Sec2，用于 Rab GTPase Sec4。然后， 肌球蛋白相关蛋白复合物（Inn1、Hof1 和 Cyk3）通过 C2 结构域促进囊泡融合 Inn1，并激活 ECM 的货物酶 Chs2（糖基转移酶家族 2 的成员） 通过 Cyk3 的转谷氨酰胺酶样结构域进行重塑（即酵母中隔膜的形成）。 发现（目标 1）和假设驱动（目标 2 和 3）研究预计将产生新颖的 胞质分裂的概念和机制超出了特定模式生物的范围。