Mechanistic Analysis of Cytokinesis in Eukaryotes

 描述(申请人提供)：胞质分裂对所有生物体的发育和生存都是必不可少的。胞质分裂的缺陷会导致非整倍体和基因组的不稳定，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一个基本过程的基本原理，而且对于设计治疗人类疾病的新策略都是重要的。动物和真菌细胞的胞质分裂需要收缩肌球蛋白环(AMR)、靶向囊泡融合和局部细胞外基质(ECM)重塑的协调功能。尽管一个多世纪以来对胞质分裂进行了广泛的研究，但在任何系统中，AMR的基本结构仍然未知。肌球蛋白-II的定位和细丝组装在胞质分裂过程中是如何调节的也是不清楚的。越来越多的证据表明，ECM重塑不仅对酵母细胞，而且对动物细胞的胞质分裂都是至关重要的。然而，人们对其潜在机制仍知之甚少。我们建议使用发芽酵母和哺乳动物细胞作为我们的实验模型来解决胞质分裂中的这些关键问题。在目标1中，我们将确定发芽酵母中的AMR结构，并建立其定量模型。这个模型将开辟新的途径来解决细胞质分裂中的广泛问题，例如关于环收缩机制的问题以及关于环的组装、调节和功能的问题。我们还将研究哺乳动物细胞中的AMR，以确定这一核心细胞动力学结构的保守程度。在目标2中，我们将验证我们的假设，即IQGAP在萌芽酵母和哺乳动物细胞胞质分裂过程中作为肌球蛋白定位和细丝组装的双重调节因子发挥作用。在目标3中，我们将检验我们的假设，即AMR相关蛋白Inn1通过其C2结构域与SNARS相互作用，促进分裂部位的小泡融合，从而增加货物酶Chs2(几丁质合成酶-II)的局部浓度，随后该酶通过其转谷氨酰胺酶样域被Cyk3激活，以促进细胞质分裂期间的隔膜形成(相当于动物细胞内细胞外基质的重塑)。这项拟议的研究是创新的，因为将应用各种尖端技术来产生关于胞质分裂核心机制的新信息和概念。

 DESCRIPTION (provided by applicant): Cytokinesis is essential for development and survival of all organisms. Defects in cytokinesis cause aneuploidy and genomic instability, and thereby contribute 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 concerted functions of a contractile actomyosin ring (AMR), targeted vesicle fusion, and localized extracellular matrix (ECM) remodeling. Despite extensive studies of cytokinesis over a century, the basic architecture of the AMR remains unknown in any system. It is also unclear how myosin-II localization and filament assembly are regulated during cytokinesis. Increasing evidence suggests that ECM remodeling is critical for cytokinesis not only for yeast cells but also for animal cells. However, the underlying mechanisms remain poorly understood. We propose to address these key questions in cytokinesis using both budding yeast and mammalian cells as our experimental models. In Aim 1, we will determine the AMR structure in budding yeast and then develop a quantitative model for it. This model will open new avenues to address broad questions in cytokinesis, e.g. those regarding the mechanism of ring constriction as well as those concerning the assembly, regulation, and function of the ring. We will also examine the AMR in mammalian cells to establish the degree of conservation in this core cytokinetic structure. In Aim 2, we will test our hypothesis that IQGAP functions as a dual regulator of myosin localization and filament assembly during cytokinesis in both budding yeast and mammalian cells. In Aim 3, we will test our hypothesis that the AMR-associated protein Inn1 interacts with SNAREs via its C2 domain to facilitate vesicle fusion at the division site, thereby increasing the local concentration of the cargo enzyme Chs2 (chitin synthase-II), which is subsequently activated by Cyk3 via its transglutaminase-like domain to promote septum formation (equivalent of ECM remodeling in animal cells) during cytokinesis. The proposed research is innovative, as diverse and cutting-edge technologies will be applied to generate new information and concepts regarding the core mechanisms of cytokinesis.