Structure Function Studies of the Spindle-Pole Body in Saccharomyces Cerevisiae

描述（由申请人提供）：本提案的长期目标是建立微管组织中心（MTOCs）基础的分子框架。它们是复杂的大分子组合，在多种细胞功能中发挥重要作用，包括染色体分离、细胞质分裂、受精、细胞运动和细胞内运输。鉴于MTOCs在细胞分裂中的关键作用，MTOCs组装和功能的缺陷在疾病，特别是癌症和出生缺陷中具有重要意义就不足为奇了。本研究的重点是出芽酵母酵母（Saccharomyces cerevisiae）的纺锤杆体（SPB），它长期以来一直是理解MTOCs的范例。目的是提供这种复杂的多层结构如何组装和调节的高分辨率图像。这将通过确定SPB组分及其大分子组件的三维结构来完成。这些结构的生物学意义将在体内进行测试，重点是磷酸化在SPB组装中的作用。为此，我们开发了一种表达含有卷曲线圈的SPB蛋白的策略。确定了中间层2的一个组分的三维结构，并获得了第二组分的晶体。这为研究构成SPB的蛋白质复合物的结构和功能奠定了基础。本提案的具体目标是：1)建立中间层2 （IL2）的底层架构。这是一种形成二维晶体阵列的结构，它定义了整个SPB的横向组织。2)了解中央空斑的组织，它嵌入核膜中，并在SPB的核侧和细胞质侧之间建立连续性。3)确定连接白细胞介素2和外部斑块的中间层1内的分子框架。4)利用这些结构信息作为研究SPB在体内组装和调控的基础。本研究属于高风险、高回报的研究提案。它是高风险的，因为它关注的是由大量独特的蛋白质-蛋白质相互作用形成的巨大大分子组装。同时，由于对这组蛋白质的结构所知甚少，该提议的回报也很高。因此，对这些蛋白的任何研究进展都将极大地促进我们对MTOCs的理解。本文研究SPB结构的策略通常对促进其他大型大分子组装体（如中心体和纤毛）的研究有用。公共卫生相关性：本提案的目标是建立微管组织中心的三维模型。这些细胞器在所有真核生物的细胞分裂中发挥重要作用，并在癌症和出生缺陷中具有重要意义。在这里获得的基本知识将对理解疾病的分子基础有很大的价值。

DESCRIPTION (provided by applicant): The long term goal of this proposal is to establish the molecular framework that underlies Microtubule Organizing Centers (MTOCs). These are complex macromolecular assemblies that play essential roles in a wide variety of cellular functions including chromosome segregation, cytokinesis, fertilization, cell motility and intracellular trafficking. Given their critical role in cell division, it is not surprising that defects in the MTOCs assembly and function have important implications in disease, particularly in cancer and birth defects. The focus of this proposal is the spindle pole body (SPB) of the budding yeast Saccharomyces cerevisiae, which has long served as the paradigm for understanding MTOCs. The aim is to provide a high resolution picture of how this intricate multilayer structure assembles and is regulated. This will be accomplished by determining the three-dimensional structures of the SPB components and their macromolecular assemblies. The biological implications of these structures will be tested in vivo with an emphasis on the role of phosphoryation in SPB assembly. To this end, we have developed a strategy for expressing proteins of the SPB that contain coiled coils. The three dimensional structure of one component of the intermediate layer 2 has been determined and crystals for a second component have been obtained. This establishes the foundation for investigating the structure and function of the protein complexes that constitute the SPB. The specific aims for this proposal are: 1) to establish the underlying architecture of the intermediate layer 2 (IL2). This is a structure that forms a two-dimensional crystalline array which defines the lateral organization of the entire SPB. 2) to understand the organization of the central plaque, which is embedded in the nuclear envelope and establishes continuity between the nuclear and cytoplasmic sides of the SPB. 3) to define the molecular framework within the intermediate layer 1 which bridges the IL2 and the outer plaque. 4) to utilize the structural information as the basis for investigating the in vivo assembly and regulation of the SPB. This study falls into the high-risk high-payoff category of research proposals. It is high risk because it focuses on an enormous macromolecular assembly that is formed by a large number of unique protein-protein interactions. At the same time, the proposal is high payoff because little is known about the structure of this group of proteins. Thus any progress on these proteins will significantly advance our understanding of MTOCs. The strategies developed here to study the architecture of the SPB should be generally useful in facilitating the studies of other large macromolecular assemblies such as the centrosomes and cilia. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to establish a three dimensional model for the Microtubule Organizing Centers. These organelles play essential roles in cell division in all eukaryotes and have important implications in cancer and birth defects. The fundamental knowledge gained here will be of great value in understanding the molecular basis of disease.