Microtubule Polymerization and Depolymerization Mechanisms by Conserved Proteins

本研究的目的是了解微管（MT）聚合酶的机制， 解聚酶在调节MT+末端中的作用。在辅导阶段，1人获得了广泛的专业知识 在单分子全内反射荧光（TIRF）显微镜研究MT的机制 聚合酶、XMAP 215/Dis 1蛋白和细胞质接头相关蛋白（CLASP）。这些 研究表明TOG结构域在调节MT聚合中起重要作用。我的研究表明 由CLASP和XMAP 215 TOG结构域募集的微管蛋白二聚体促进两种独特类型的MT调节， 通过募集可溶性微管蛋白使MT末端聚合或解聚来发挥活性。我还获得了 XMAP 215酵母直系同源物Stu 2的中分辨率结构研究取得了广泛进展，Stu 2与一个 微管蛋白二聚体使用冷冻电子显微镜（Cryo-EM）和单颗粒图像分析。如前 根据最初的建议，我的独立阶段研究计划将作为一个 助理教授在加州戴维斯大学，并将利用TIRF显微镜的组合 和结构生物学方法。1)我将探讨更深层次的问题到结构和机制， XMAP 215和CLASP蛋白中的TOG结构域。2)我将研究MT解聚酶的结构 （驱动蛋白-13和8）与微管蛋白二聚体的复合物。我也会 利用TIRF探索保守的MT解聚酶特征在MT解聚机制中的作用 显微镜3)我将重新构建MT聚合酶之间的拮抗和/或相互作用， 使用多色TIRF显微镜和cryo-EM对解聚酶进行分析。后一项研究旨在探讨这一点 拮抗作用将间期的慢MT动力学改变为有丝分裂开始时的快MT动力学。我也会 研究有丝分裂激酶磷酸化对MT聚合酶活性的影响， 解聚酶如何影响这种拮抗作用。生物物理和结构研究中提出的假设 将使用裂变酵母作为模型系统在体内验证在独立阶段提出的 微管动力学，与Fred Chang实验室（哥伦比亚大学）合作。

The goal of this research is to understand the mechanisms of microtubule (MT) polymerases and depolymerases in regulating MT plus ends. During the mentored phase, 1 have gained extensive expertise in single molecule total internal reflection fluorescence (TIRF) microscopy to study mechanisms of MT polymerases, XMAP215/Dis1 proteins, and Cytoplasmic linker Associated proteins (CLASPs). These Studies indicate a fundamental role for TOG domains in regulating MT polymerization. My studies show tubulin dimers recruited by CLASP and XMAP215 TOG domains promote two unique types of MT regulatory activities by recruiting soluble tubulin either to polymerizing or depolymerizing MT ends. I have also attained extensive progress towards a medium resolution structure ofthe XMAP215 yeast ortholog, Stu2, bound to a tubulin dimer using cryo-electron microscopy (Cryo-EM) and single particle image analysis. As previously described in the original proposal, my research program for the independent phase will be carried out as an assistant professor at the University of California Davis, and will utilize a combination of TIRF microscopy and structural biology approaches. 1) I will explore deeper questions into the structures and mechanisms of TOG domains in XMAP215 and CLASP proteins. 2) I will study the structures of MT depolymerases (kinesins-13 and 8) in complex with tubulin dimers using cryo-EM andx-ray crystallography. I will also explore the role of conserved MT depotymerase features in the MT depolymerization mechanism using TIRF microscopy. 3) I will reconstitute the antagonism and/or interaction between MT polymerases and depolymerases using multi-color TIRF microscopy and cryo-EM. The latter studies aim to explore how this antagonism alters slow MT dynamics in interphase to fast MT dynamics at the onset of mitosis. I will also study the effect of phosphorylation by mitotic kinases on the activities of MT polymerases and depolymerases how it affects this antagonism. Hypotheses arising from biophysical and structural studies proposed in the independent phase will be validated in vivo using fission yeast as a model system for microtubule dynamics, in collaboration with Fred Chang's Laboratory (Columbia University).