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 (MT) 细胞骨架的主要成核剂，是细胞正常运转所必需的。 极性、囊泡运输以及纺锤体的形成和功能。而酵母 S. 中的类似结构。 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，我们重点关注成核的组装和调节 使用结构方法的广泛组合（X 射线晶体学、冷冻电镜单粒子 重建、冷冻电镜断层扫描）以确定体外和原位 Y-微管蛋白复合物的结构，以及 通过定量体外功能研究和创新动力学了解其作用机制 造型。之前我们发现酵母Y-微管蛋白小复合体（YTUSC）可以组装成环并 获得了环的 6.5A 冷冻电镜结构，解释了 MT 13 重对称性的起源并发现 意想不到的调节和组装模式。根据我们之前的结果，我们建议有以下三种 调节阶段：Y-TUSC 环组件通过需要与 Spcl 10 或 Spc72，环闭合以完全匹配 MT 对称性，并且激活 Y 微管蛋白以实现高效 成核。拟议的实验扩展了我们之前的结果，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片 和 Y-微管蛋白复合物、调节蛋白，以及这些复合物如何与纺锤体极体或 中心体基质。具体来说，我们将 (i) 提高酵母 YTUSC 冷冻电镜重建的分辨率 环，并与生物信息学核心合作生成完整的伪原子结构。结构 将确定并比较与 MT 或 1 层非聚合酵母 ap-微管蛋白结合的 yTuSC 环 酵母 SPB 冷冻电镜断层扫描中原位加帽 MT 负端的结构。 (ii) 我们将使用新的 开发了 FRET 测定法，可有效测量体外环组装并确定 Spcl 10 的哪些结构域 和 Spc72 是组装和 Spcl 10/72 磷酸化作用所必需的，(iii) 必要时， 组装成环不足以实现有效的 MT 成核，需要 YTUSC 闭合来匹配 MT 对称性和变构激活。 PTM 或其他具有约束力的合作伙伴在此过程中的作用将是 决定。