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

酿酒酵母纺锤体结构功能研究

• 批准号: 7892676
• 负责人: IVAN RAYMENT
• 金额: $ 2.77万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-01 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7892676
• 关键词: Architecture; Biological; Categories; Cell division; Cell physiology; Centrosome; Chromosome Segregation; Cilia; Complex; Congenital Abnormality; Cytokinesis; Defect; Disease; Eukaryota; Fertilization; Foundations; Goals; Group Structure; Investigation; Knowledge; Lateral; Malignant Neoplasms; Microtubule-Organizing Center; Molecular; Nature; Nuclear; Nuclear Envelope; Organelles; Play; Proteins; Regulation; Research Proposals; Resolution; Role; Saccharomyces cerevisiae; Saccharomycetales; Side; Structure; Tertiary Protein Structure; Testing; Time; base; cell motility; falls; high risk; in vivo; macromolecular assembly; novel strategies; protein complex; protein protein interaction; public health relevance; spindle pole body; three dimensional structure; three-dimensional modeling; trafficking; two-dimensional

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.

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（MTOC）的分子框架。这些是复杂的大分子组装体，在多种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，MTOC 组装和功能的缺陷对疾病，特别是癌症和出生缺陷具有重要影响也就不足为奇了。该提案的重点是芽殖酵母酿酒酵母的纺锤体极体（SPB），它长期以来一直是理解 MTOC 的范例。目的是提供关于这种复杂的多层结构如何组装和调节的高分辨率图片。这将通过确定 SPB 组件及其大分子组装体的三维结构来实现。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螺旋的 SPB 蛋白质的策略。已经确定了中间层2的一种组分的三维结构并且已经获得了第二种组分的晶体。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。该提案的具体目标是：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，定义了整个 SPB 的横向组织。 2) 了解中央斑块的组织，中央斑块嵌入核膜中并在 SPB 的核侧和细胞质侧之间建立连续性。 3) 定义中间层 1 内桥接 IL2 和外斑块的分子框架。 4）利用结构信息作为研究SPB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提案类别。它的风险很高，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体。同时，由于人们对这组蛋白质的结构知之甚少，因此该提议具有很高的回报。因此，这些蛋白质的任何进展都将显着增进我们对 MTOC 的理解。这里开发的研究 SPB 结构的策略应该通常有助于促进其他大分子组装体（例如中心体和纤毛）的研究。公共健康相关性：该提案的目标是为微管组织中心建立三维模型。这些细胞器在所有真核生物的细胞分裂中发挥着重要作用，并且对癌症和出生缺陷具有重要意义。这里获得的基础知识对于理解疾病的分子基础具有重要价值。