The Structural Basis of Microtubule Nucleation by gamma-TuSC

描述（由申请人提供）：中心体在组织微管（MT）细胞骨架中起着基本作用。中心体功能缺陷导致细胞分裂过程中染色体分离错误，在癌变的起始和癌变的进展中起重要作用。本提案中概述的研究将通过关注所有真核生物中MT成核的关键异四聚体分子复合物的结构来寻求理解微管成核的机制。提出了EM单粒子重建方法和x射线晶体学相结合的方法来确定酵母管4和/或果蝇γ -微管蛋白小复合物的三维结构。EM工作的目标将是确定tub4复合物的整体组织，发现哪些γ -微管蛋白表面暴露，并绘制spd 10结合的位置。高分辨率晶体学研究将提供有关复合物结构、γ -微管蛋白构象和核苷酸作用的前所未有的信息。了解中心体微管成核位点的机制、结构、组成和调控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.