The Structure and Regulation of Microtubule Nucleation by y-tubulin

y-微管蛋白对微管成核的结构和调控

• 批准号: 8668220
• 负责人: DAVID A. AGARD
• 金额: $ 19.04万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8668220
• 关键词: Address; Behavior; Binding; Bioinformatics; Biological Assay; Cell Polarity; Centrosome; Collaborations; Complement; Complex; Cryoelectron Microscopy; Crystallography; Cytoskeleton; Disulfides; Drosophila genus; Electrons; Face; Fluorescence Resonance Energy Transfer; Goals; In Situ; In Vitro; Kinetics; Link; Maps; Measurement; Measures; Microtubules; Minus End of the Microtubule; Modeling; Molecular Models; Mutagenesis; Mutation; Nuclear; Phase; Phosphorylation; Post-Translational Protein Processing; Process; Recombinant Proteins; Regulation; Resolution; Role; Saccharomyces cerevisiae; Side; Structure; Test Result; Testing; Tubulin; Vesicle; Yeasts; base; crosslink; detector; gamma Tubulin; genetic regulatory protein; improved; in vivo; innovation; insight; mimetics; molecular modeling; particle; protein complex; reconstitution; reconstruction; research study; spindle pole body; stoichiometry; tomography; trafficking

The centrosome is the principal nucleator of the microtubule (MT) cytoskeleton, which is required for cell polarity, vesicle trafficking, and spindle formation and function. While the analogous structure in the yeast S. cerevisiae (the spindle pole body or SPB) is morphologically distinct, a conserved set of Y-tubulin complexes is used to nucleate MT assembly. In this Project we focus on the assembly and regulation of the nucleating machinery using a broad combination of structural approaches (x-ray crystallography, cryoEM single particle reconstruction, cryoEM Tomography) to determine the structures of Y-tubulin complexes in vitro and in situ, and to understand their mechanism of action through quantitative in vitro functional studies and innovative kinetic modeling. Previously we discovered that yeast Y-tubulin small complex (YTUSC) can assemble into rings and obtained a 6.5A cryoEM structure of the rings, explaining the origins of MT 13-fold symmetry and discovering unexpected modes of regulation and assembly. Based on our previous results we propose that there are three phases of regulation: Y-TUSC ring assembly restricted to the spindle pole body by requiring interactions with Spcl 10 or Spc72, ring closure to fully match MT symmetry and, activation of the Y-tubulins for efficient nucleation. The proposed experiments expand upon our previous results with the long-term goal of synthesizing an atomic resolution picture of all the relevant structural and functional interactions between aP- and Y-tubulin complexes, regulatory proteins, and how these complexes are linked to the spindle pole body or centrosome matrix. Specifically we will (i) improve the resolution of our cryoEM reconstruction of yeast YTUSC rings and, collaborate with the Bioinformatics Core to generate a complete pseudo-atomic structure. Structures of yTuSC rings bound to MTs or 1 layer of non-polymerizing yeast ap-tubulin will be determined and compared to structures of in situ capped MT minus ends from cryoEM tomography of yeast SPBs. (ii) We will use a newly developed FRET assay to efficiently measure ring assembly in vitro and determine what domains of Spcl 10 and Spc72 are required for assembly and the role of Spcl 10/72 phosphorylation, (iii) While necessary, assembly into rings is insufficient for potent MT nucleation, with a need for both YTUSC closure to match MT symmetry and an allosteric activation. The role of PTMs or other binding partners in this process will be determined.

中心体是微管细胞骨架的主要成核剂，是细胞生长和分化的必需物质。 极性、囊泡运输和纺锤体的形成和功能。而酵母S. 酿酒酵母（纺锤体极体或SPB）在形态上是不同的，一组保守的Y-微管蛋白复合物是 用于使MT组装成核。在这个项目中，我们专注于成核的组装和调节， 机器使用结构方法的广泛组合（x射线晶体学，cryoEM单粒子 重建，cryoEM断层扫描），以确定体外和原位Y-微管蛋白复合物的结构，以及 通过定量的体外功能研究和创新的动力学研究来了解它们的作用机制 建模以前我们发现酵母Y-微管蛋白小复合物（YTUSC）可以组装成环， 获得了环的6.5A cryoEM结构，解释了MT 13重对称的起源，并发现 意想不到的调节和组装模式。根据我们以前的结果，我们提出，有三个 调节阶段：Y-TUSC环组件因需要与 Spcl 10或Spc 72，环闭合以完全匹配MT对称性，并且激活Y-微管蛋白以有效地调节MT对称性。 成核所提出的实验扩展了我们以前的结果，其长期目标是 合成aP之间所有相关结构和功能相互作用的原子分辨率图像， 和Y-微管蛋白复合物，调节蛋白，以及这些复合物如何连接到纺锤体极体或 中心体基质具体而言，我们将（i）提高我们的酵母YTUSC的cryoEM重建的分辨率 环，并与生物信息学核心合作，生成一个完整的伪原子结构。结构 将测定并比较与MT结合的yTuSC环或1层非聚合酵母ap-tubulin 从酵母SPB的cryoEM断层扫描原位加帽的MT负末端的结构。(ii)我们将使用一个新的 开发了FRET分析，以有效地测量体外环组装，并确定Spcl 10的哪些结构域 和Spc 72是组装所需的，并且Spc 1 10/72磷酸化的作用。（iii）虽然必要， 组装成环不足以实现有效的MT成核，需要两个YTUSC闭合以匹配MT 对称性和变构激活。PTM或其他结合伙伴在这一过程中的作用将是 测定