Microtubule Polymerization and Depolymerization Mechanisms by Conserved Proteins

描述(申请人提供)：这项研究的目的是了解微管(MT)聚合和解聚的分子机制，这是由保守的蛋白质家族介导的。这些蛋白质在MT+末端起微管蛋白聚合酶和解聚酶的作用。在哈佛医学院斯蒂芬·C·哈里森博士的指导下，我利用电子显微镜和生物化学开发了一个分子模型，研究了萌芽酵母Stu2二聚体和非洲爪哇XMAP215单体如何通过其保守的N-末端TOG结构域与微管蛋白二聚体结合。我已经用X射线结晶学确定了TOG结构域的结构，并显示出TOG桨状结构上一个狭窄的保守表面是微管结合界面。我的工作提出了一个假设，即TOG结构域如何促进MT Plus末端的微管蛋白聚合。我们目前正在研究与微管蛋白二聚体结合的TOG结构域的结构。在指导阶段，我将确定TOG结构域-微管蛋白二聚体复合体的结构，以了解TOG结构域如何结合和增强微管蛋白组装。我还打算与德国马克斯·普朗克研究所的安东尼·A·海曼和乔纳森·霍华德的实验室合作，探索不同的TOG结构域在使用显微镜方法增强MT组装方面的作用。 在独立阶段，我的目标是了解两类保守的Kinesin超家族蛋白(Kinesins-13和Kinesins-8)是如何作为MT去聚合酶介导MTS的分解的。已经提出了一个假设，即运动域如何迫使MT末端的微管蛋白弯曲，从而导致MT的解体。我打算确定它们的运动域的结构，同时与微管蛋白二聚体结合，以了解MT解聚的机制。我还打算确定保守特征在其去聚合酶活性中的作用。然后，我将探讨MT聚合酶和解聚酶在间期的拮抗和相互作用，以及它在有丝分裂开始时的调节。这种拮抗作用导致间期MT动力学缓慢，有丝分裂MT动力学快速。在间期和有丝分裂之间的过渡过程中，将探索有丝分裂激酶的磷酸化的直接作用。 细胞分裂调控的缺陷与人类基因缺陷和癌症直接相关。这项研究的结果将拓宽我们对这一调控机制的理解，并为癌症治疗提供新的靶点。

DESCRIPTION (provided by applicant): The goal of this research is to understand the molecular mechanism of microtubule (MT) polymerization and depolymerization which are mediated by conserved families of proteins. These proteins act as tubulin polymerases and depolymerases at MT plus ends. Under the mentorship of Dr. Stephen C. Harrison at Harvard Medical School, I have developed a molecular model using electron microscopy and biochemistry for how budding yeast Stu2 dimers and Xenopus XMAP215 monomers bind tubulin dimer via their conseved N-terminal TOG domains. I have determined the structure of a TOG domain using x-ray crystallography and show a narrow conserved surface on the TOG paddle-like structure is the tubulin binding interface. My work suggests a hypothesis for how TOG domains enhance tubulin polymerization at MT plus ends. We are currently studying the structure of TOG domains bound to tubulin dimer. In the mentored phase, I will determine the structure of TOG domains-tubulin dimer complex to understand how TOG domains bind and enhance tubulin assembly. I also intend to explore the role of different TOG domains in enhancing MT assembly using microscopy methodologies in collaberation with the laboratories of Anthony A Hyman and Jonathan Howard at the Max Planck institute in Germany. In the independent phase, I aim to understand how two classes of conserved kinesin superfamily proteins (kinesins-13 and 8) mediate the disassembly of MTs, acting as MT depoymerases. A hypothesis has been developed about how the motor domains may force tubulin at MT ends to curve leading to MT disassembly. I intend to determine structures for their motor domain in action, while bound to tubulin dimer to understand the mechanism of MT depolymerization. I also intend to determine the role of conservd features in their depolymerase activity. I will then explore the antagonism and interaction between MT polymerase and depolymerases in interphase and its regulation upon the onset of mitotic cell division. This antagonism leads to slow MT dynamics in interphase and fast MT dynamics in mitosis. A direct role of phosphorylation by mitotic kinases will be explored during the transition between interphase and mitosis. Defects in the regulation of cell division are directly linked to human genetic defects and cancers. The results from this study will broaden our understanding of this regulation and provide new targets for cancer therapy.