The Structure and Regulation of Microtubule Nucleation by y-tubulin

中心体是细胞所必需的微管(MT)细胞骨架的主要核因子 极性，囊泡运输，纺锤体的形成和功能。而酵母S。 Cerevisiae(纺锤体极体或SPB)在形态上是独特的，一组保守的Y-微管蛋白复合体是 用于使MT组装成核。在本项目中，我们的重点是成核的组装和调节 使用广泛组合的结构方法的机械(X射线结晶学、低温EM单粒子 重建，低温电子显微镜断层扫描)以确定Y-微管蛋白复合体的结构，以及 通过定量的体外功能研究和创新动力学了解其作用机制 模特儿。以前我们发现酵母Y-微管蛋白小复合体(YTUSC)可以组装成环和 得到了环的6.5A低温电子显微镜结构，解释了MT 13次对称性的起源，并发现了 意想不到的监管和组装模式。基于我们以前的结果，我们提出了三个 调整阶段：Y-TUSC环组件限制在主轴杆体上，因为需要与 SPCL 10或Spc72，环关闭以完全匹配MT对称性，并激活Y-微管蛋白以实现高效 成核作用。拟议的实验在我们之前结果的基础上进行了扩展，长期目标是 合成AP-AP之间所有相关结构和功能相互作用的原子分辨率图 和Y-微管蛋白复合体，调节蛋白，以及这些复合体如何连接到纺锤体极体或 中心体矩阵。具体地说，我们将(I)提高酵母YTUSC的冷冻EM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。构筑物 将测定和比较与MTS结合的yTuSC环或1层非聚合酵母AP-tuBulin 从酵母SPBS的低温电子显微镜断层扫描中观察到原位封顶的MT-末端结构。(Ii)我们将使用新的 建立了FRET方法，可以有效地在体外测量环组装并确定Spcl10的哪些结构域 和Spc72是组装和发挥SpCl10/72磷酸化作用所必需的，(Iii)当需要时， 组装成环对于有效的MT成核是不够的，需要YTUSC闭合以匹配MT 对称性和变构活化。临时秘书处或其他有约束力的伙伴在这一进程中的作用将是 下定决心。

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.