Microtubule Polymerization and Depolymerization Mechanisms by Conserved Proteins

本研究的目的是了解微管(MT)聚合酶和 解聚酶在调节MT+末端中的作用在指导阶段，我获得了广泛的专业知识 在单分子全内反射荧光显微镜下研究MT的作用机制 聚合酶、XMAP215/Dis1蛋白和细胞质连接物相关蛋白(CLASPS)。这些 研究表明，TOG结构域在调节MT聚合中起着基础性作用。我的研究表明 CLASP和XMAP215 TOG结构域招募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聚合或解聚MT末端的活性。我也达到了 XMAP215酵母同源基因Stu2的中分辨结构与A结合的广泛进展 微管蛋白二聚体的冷冻电子显微镜(Cryo-EM)和单粒子图像分析。和以前一样 在最初的建议中，我的独立阶段的研究计划将作为一个 加州大学戴维斯分校的助理教授，将利用TIRF显微镜的组合 和结构生物学的方法。1)我将探讨更深层次的问题，即 XMAP215和CLASP蛋白中的TOG结构域。2)研究MT解聚酶的结构 (Kinesins-13和8)与微管蛋白二聚体形成络合物，使用冷冻-EM和X射线结晶学。我也会 用TIRF探讨保守的MT解聚酶特性在MT解聚机制中的作用 显微镜。3)重建MT聚合酶与MT聚合酶之间的拮抗和/或相互作用 用多色TIRF显微镜和低温电子显微镜分析解聚酶。后一项研究的目的是探索这一点 拮抗作用将间期缓慢的MT动力学改变为有丝分裂开始时的快速MT动力学。我也会 研究有丝分裂酶的磷酸化对MT聚合酶活性的影响 解聚它是如何影响这种对抗的。来自生物物理和结构研究的假说 在独立阶段提出的将使用分裂酵母作为模型系统在体内进行验证 微管动力学，与弗雷德·张的实验室(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).