Structure-based mechanism of the augmin complex in promoting branching microtubule nucleation and spindle assembly

项目概述：减数分裂和有丝分裂纺锤体的快速和有组织的组装是必不可少的， 确保在细胞分裂期间染色体的适当分离。大多数纺锤体微管 是通过分支微管成核产生的。在这一微管成核途径中， 微管从预先存在的微管以浅角度成核， 微管的扩增。Augmin，一个大的杂八聚体复合物，在整个 真核生物，是必不可少的分支微管成核。它既需要招募成核， 因子到预先存在的微管，并通过以锐角定向新生微管， 母亲，以确保主轴极性保持。然而，augmin如何实现这两个功能呢？ 仍然知之甚少，这既是因为我们缺乏对Augmin直接约束力的完整了解， 因为我们没有关于Augmin复合物的高分辨率结构信息。我 将使用单粒子冷冻电子显微镜，重建实验， 纯化的蛋白质和离体非洲爪蟾减数分裂卵提取物测定，以确定Augmin 复合物使分支微管成核并建立正确的微管分支 主轴中的几何形状。除了揭示纺锤体这一关键方面的机制之外， 对augmin复合物的结构和功能的深入了解也将为我们提供一个 这是理解细胞内其他复杂微管成核位点是如何组织的基础。 除了给我生物学背景，我还需要追求我最终的科学兴趣， 微管细胞骨架如何与囊泡运输合作，这项拟议的研究计划将 我还在两个关键的技术领域培训我，我将需要建立自己的独立研究 组其中第一个是基于TIRF的分析，用于研究单个分子的微管成核 水平使用非洲爪蟾减数分裂卵提取物。这种体外系统是一种非常强大的工具， 允许精确地消耗或替换蛋白质组分，以定量地确定它们的 有助于微管成核，弥合自下而上重建方法之间的差距 和活细胞成像。我与Petry博士合作的主要动机之一就是接受这方面的培训。 法第二种技术是单粒子技术，我将在整个提案中广泛使用它 冷冻电子显微镜，特别是在解决扩展和动态分子的结构。在 学习这种技术，我将受益于我的共同赞助人Nieng Yan博士和 普林斯顿最前沿的充满活力的结构生物学社区。最后，我会向博士学习。 Petry和Yan博士如何成功地领导一个研究小组，无畏地利用广泛的 技术和学科来回答关键的科学问题。

Project Summary: Rapid and organized assembly of meiotic and mitotic spindles is essential for ensuring proper segregation of chromosomes during cell division. A majority of spindle microtubules are generated via branching microtubule nucleation. In this microtubule nucleation pathway, new microtubules are nucleated from pre-existing microtubules in shallow angles, which causes exponential amplification of microtubules. Augmin, a large hetero-octameric complex conserved throughout eukaryotes, is essential for branching microtubule nucleation. It is required both to recruit nucleation factors to the pre-existing microtubule and, by orienting the nascent microtubule at an acute angle to the mother, to ensure that spindle polarity is maintained. Yet how augmin carries out these two functions remains poorly understood, both because we lack a complete picture of augmin's direct binding partners and because we have no high resolution structural information about the augmin complex. I will use a combination of single particle cryo-electron microscopy, reconstitution experiments using purified proteins, and ex vivo Xenopus laevis meiotic egg extract assays to determine how the augmin complex enables branching microtubule nucleation and establishes correct microtubule branching geometry in the spindle. In addition to revealing the mechanism that underlies this key aspect of spindle formation, insight into the structure and function of the augmin complex will also provide us with a foundation to understand how other complex microtubule nucleation sites are organized within the cell. In addition to giving me the biological background I need to pursue my ultimate scientific interest of how the microtubule cytoskeleton cooperates with vesicle trafficking, this proposed research plan will also train me in two key technical areas that I will need to establish my own independent research group. The first of these is a TIRF-based assay to study microtubule nucleation at the single molecule level using Xenopus laevis meiotic egg extract. This ex vivo system is an incredibly powerful tool that allows precise depletion or replacement of protein components to quantitatively determine their contribution to microtubule nucleation, bridging the gap between bottom-up reconstitution approaches and live-cell imaging. One of my primary motivations for working with Dr. Petry was to be trained in this method. The second technique, which I will use extensively throughout this proposal, is single particle cryo-electron microscopy, especially in solving the structures of extended and dynamic molecules. In learning this technique, I will benefit from the mentorship of my co-sponsor Dr. Nieng Yan and the cutting-edge vibrant structural biology community at Princeton as a whole. Finally, I will learn from Dr. Petry and Dr. Yan how to successfully lead a research group that fearlessly leverages a broad range of techniques and disciplines to answer critical scientific questions.