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

描述(由申请人提供)：该提案的长期目标是建立微管组织中心(MTOCs)的分子框架。这些是复杂的大分子组件，在广泛的细胞功能中发挥重要作用，包括染色体分离、胞质分裂、受精、细胞运动和细胞内运输。鉴于它们在细胞分裂中的关键作用，MTOCs的组装和功能缺陷在疾病中具有重要意义也就不足为奇了，特别是在癌症和出生缺陷中。这项建议的重点是发芽酵母的纺锤极身体(SPB)，它长期以来一直是理解MTOCs的范例。其目的是提供这种复杂的多层结构如何组装和监管的高分辨率图像。这将通过确定SPB组件及其大分子组装的三维结构来实现。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，并获得了第二个组分的晶体。这为研究构成SPB的蛋白质复合体的结构和功能奠定了基础。该提议的具体目标是：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，其定义了整个SPB的横向组织。2)了解中央斑块的组织结构，它嵌入在核膜中，建立了SPB核质两侧之间的连续性。3)确定连接IL2和外部斑块的中间层1内的分子框架。4)利用结构信息作为研究SPB在体内组装和调控的基础。这项研究属于高风险、高回报的研究建议类别。它的风险很高，因为它集中在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上。与此同时，这项提议的回报很高，因为人们对这组蛋白质的结构知之甚少。因此，这些蛋白质的任何进展都将极大地促进我们对MTOCs的理解。在此开发的研究SPB结构的策略通常对促进其他大分子组件的研究是有用的，例如中心体和纤毛。公共卫生相关性：该提案的目标是为微管组织中心建立一个三维模型。这些细胞器在所有真核生物的细胞分裂中扮演着重要的角色，并在癌症和出生缺陷方面具有重要的意义。在这里获得的基本知识将对理解疾病的分子基础具有重要价值。

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.