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

项目摘要：快速和有组织地组装减数分裂和有丝分裂纺锤体对于 在细胞分裂过程中确保适当的染色体分离。大多数纺锤形微管 是通过分枝微管成核产生的。在这个微管成核途径中，新的 微管是由预先存在的浅角微管成核的，这会导致指数 微管的放大。Augmin，一种贯穿始终的大型异八聚体复合体 真核生物，是分支微管成核所必需的。这两个都需要招募成核 通过将新生微管以锐角定向到预先存在的微管 母亲，以确保纺锤体的极性保持不变。然而，Augmin是如何执行这两个功能的 仍然知之甚少，这两者都是因为我们缺乏Augmin直接结合的完整图景 因为我们没有关于Augmin复合体的高分辨率结构信息。我 将结合使用单粒子冷冻电子显微镜，利用重建实验 纯化的蛋白和非洲爪哇体外减数分裂卵子提取液检测 复合体使分支微管成核，并建立正确的微管分支 主轴中的几何图形。除了揭示纺锤体这一关键方面的机制外， 形成，对增强蛋白复合体的结构和功能的洞察也将为我们提供一个 为了解细胞内其他复杂的微管成核位置是如何组织的奠定了基础。 除了给我提供生物学背景，我还需要追求我的终极科学兴趣 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 还要在两个关键技术领域对我进行培训，我将需要这两个领域来建立自己的独立研究 一群人。其中第一个是基于TIRF的分析，以研究单个分子上的微管成核。 利用非洲爪哇水平减数分裂卵提取液。这个体外系统是一个令人难以置信的强大工具， 允许精确耗尽或替换蛋白质组分，以定量确定其 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。我与佩特博士合作的主要动机之一就是接受这方面的培训 方法。第二种技术，我将在整个提案中广泛使用，是单粒子 冷冻电子显微镜，特别是在解决延伸和动态分子结构方面。在……里面 学习这项技术，我将受益于我的联合发起人牛燕博士和 作为一个整体，普林斯顿的前沿充满活力的结构生物学社区。最后，我要向博士学习。 皮特里和严博士如何成功地领导一个无畏地利用广泛范围的研究小组 回答关键科学问题的技术和学科。

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.