Mechanistic Analysis of Kinesin-14 Motility and Regulation for Bipolar Spindle Assembly

项目摘要：在有丝分裂细胞分裂过程中，复制的染色体必须准确分离 转化为两个子细胞，以确保所有真核生物的正常发育和生长。染色体中的错误 种族隔离可能导致非整倍体，这是癌症的一个标志，以及一系列相关的健康问题。结果, 生物医学研究的一个主要前沿是了解支配组装和 维持有丝分裂纺锤体，这是一种基于微管的双极机器，负责准确的染色体 有丝分裂期间的分离。已经确定的是，双极有丝分裂的正确组装和维持 纺锤体需要许多基于微管的动蛋白马达(有丝分裂动蛋白)的协调作用。然而， 单个有丝分裂激动素的机制及其受配对蛋白的调节和协调 主轴的装配和维护仍然知之甚少。和平研究所的长期目标是重组 从体外双极有丝分裂纺锤体中分离纯化蛋白，并全面剖析其机制、调控和 有丝分裂动蛋白在纺锤体组装和维护中的协调。这个项目的重点是有丝分裂。 Kinesin-14s，已知与kinesin-5s形成拮抗对以驱动双极纺锤体组装。这个 PI已经为这个项目建立了几个基础发现，包括：(1)有丝分裂激动素-14KlpA Nidulans(及其在黑曲霉中的直系同源物)在 单个微管，以及(2)相同的KlpA与两个保守的伙伴蛋白在复合体中切换为 在单个微管上进行负端定向运动。后一项发现代表了第一组 体外研究表明，有丝分裂激动素-14依赖于伴侣蛋白获得负末端导向的进行性 能动性。在这些发现的基础上，该项目旨在填补两个主要的公开问题，这两个问题是关键 了解双极纺锤体组装中Kinesin-14如何对抗Kinesin-5：(1)分子基础是什么 单个微管上潜在的正端导向的动蛋白-14运动？(2)动蛋白-14s是怎样的？ 受伙伴蛋白调节以驱动双极纺锤体组装？该项目使用了创新的组合 多种技术，包括单分子全内反射荧光显微镜、暗场 纳米粒子跟踪、双色高精度荧光跟踪、非天然氨基的基因掺入 酸，以及高分辨率的光学捕获。这一项目的结果不仅将显著拓宽目前的 了解Kinesin的运动机制，但也提供了对Kinesin-14的机制的理解 两极主轴装配中的调节。此外，这项工作将是迈向长期目标的垫脚石。 对有丝分裂纺锤体中动蛋白的机制、调节和协调有完整的了解 组装和维持，也为有丝分裂动蛋白及其伴侣蛋白如何 可以作为治疗干预的靶点。

Project Summary: During mitotic cell division (mitosis), replicated chromosomes must be accurately segregated into two daughter cells to ensure normal development and growth in all eukaryotes. Errors in chromosome segregation can lead to aneuploidy, a hallmark of cancer and a host of associated health problems. As a result, a major frontier in biomedical research is to understand the mechanisms that govern the assembly and maintenance of the mitotic spindle, a microtubule-based bipolar machine responsible for accurate chromosome segregation during mitosis. It is well established that proper assembly and maintenance of a bipolar mitotic spindle requires the coordinated action of many microtubule-based kinesin motors (mitotic kinesins). However, the mechanisms of individual mitotic kinesins and their regulation by partner proteins and coordination during spindle assembly and maintenance remain poorly understood. The long-term goal of the PI is to reconstitute bipolar mitotic spindles in vitro from purified proteins and to fully dissect the mechanisms, regulation and coordination of mitotic kinesins in spindle assembly and maintenance. The focus of this project is on mitotic kinesin-14s, which are known to form an antagonistic pair with kinesin-5s to drive bipolar spindle assembly. The PI has established several foundational findings for this project, including that (1) the mitotic kinesin-14 KlpA from Aspergillus nidulans (and its ortholog in Aspergillus niger) exhibits processive plus-end-directed motility on single microtubules, and (2) the same KlpA in complex with two well-conserved partner proteins switches to processive minus-end-directed motility on single microtubules. The latter discovery represents a first set of in vitro studies showing that a mitotic kinesin-14 depends on partner proteins to gain processive minus-end-directed motility. Building on these findings, this project is aimed at filling two major open questions that are key to understanding how kinesin-14 opposes kinesin-5 in bipolar spindle assembly: (1) What is the molecular basis underlying processive plus-end-directed kinesin-14 motility on single microtubules? (2) How are kinesin-14s regulated by partner proteins to drive bipolar spindle assembly? This project uses an innovative combination of multiple techniques, including single-molecule total internal reflection fluorescence microscopy, dark field nanoparticle tracking, two-color high-precision fluorescence tracking, genetic incorporation of unnatural amino acids, and high-resolution optical trapping. Results from this project will not only markedly broaden current understanding of kinesin motility mechanisms but also provide a mechanistic understanding of kinesin-14 regulation in bipolar spindle assembly. Furthermore, this work will be a stepping stone toward the long-term goal of a complete understanding of the mechanisms, regulation and coordination of kinesins in mitotic spindle assembly and maintenance, and also provide new insights into how mitotic kinesins and their partner proteins can be targeted for therapeutic interventions.