The Structural Basis of Microtubule Nucleation by gamma-TuSC

描述(申请人提供)：中心体在组织微管(MT)细胞骨架中起着重要作用。中心体功能缺陷会导致细胞分裂过程中染色体分离的错误，并在癌症的发生和发展中发挥重要作用。这项提案中概述的研究将通过关注所有真核生物中构成MT成核的关键异四聚体分子复合体的结构来寻求了解微管成核的机制。提出了一种结合EM单粒子重建方法和X射线结晶学来确定酵母Tub4和/或果蝇γ-微管蛋白小复合体的三维结构的方法。EM工作的目标将是确定Tub4复合体的整体组织，发现哪些伽马微管表面暴露，并绘制SPD 10结合的位置。高分辨率的结晶学研究将提供关于该复合体的结构、γ-微管蛋白的构象以及核苷酸的作用的前所未有的信息。了解中心体中微管成核位置的机制、结构、组成和调节将为中心体缺陷如何导致癌症提供重要的见解。

DESCRIPTION (provided by applicant): The centrosome plays a fundamental role in organizing the microtubule (MT) cytoskeleton. Defects in centrosome function lead to errors in chromosome segregation during cell division and play a major role in both the initiation of carcinogenesis and in the progression of cancer. The studies outlined in this proposal will seek to understand the mechanisms of microtubule nucleation by focusing on the structure of a key hetero-tetrameric molecular complex that underlies MT nucleation in all eukaryotes. A combination of EM single particle reconstruction methods and X-ray crystallography is proposed to determine the 3D structure of the yeast tub4 and/or the drosophila gamma-tubulin small complex. The goal of the EM work will be to determine the overall organization of the tub4 complex and discover which gamma-tubulin surfaces are exposed, and to map the location where spd 10 binds. High resolution crystallographic studies will provide unprecedented information on the structure of the complex, the conformation of gamma-tubulin, and the role of nucleotide. Understanding the mechanism, structure, composition, and regulation of microtubule nucleation sites in the centrosome will provide significant insight into how centrosome defects can lead to cancer.