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

双极主轴组件的 Kinesin-14 运动性和调节机制分析

• 批准号: 10206186
• 负责人: Weihong Qiu
• 金额: $ 31.57万
• 依托单位: OREGON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10206186
• 关键词: Aneuploidy; Aspergillus nidulans; Aspergillus niger; Behavior; Binding; Biomedical Research; C-terminal; Cancer Etiology; Cell division; Cells; Chromosome Segregation; Chromosomes; Color; Complex; Defect; Drosophila genus; Ensure; Eukaryota; Exhibits; Fission Yeast; Fluorescence; Fluorescence Microscopy; Foundations; Generations; Genetic; Goals; Growth and Development function; Health; Homo sapiens; Human; In Vitro; Individual; Kinesin; Label; Laboratories; Lead; Link; Maintenance; Malignant Neoplasms; Microtubules; Minus End of the Microtubule; Mitosis; Mitotic; Mitotic spindle; Molds; Molecular; Motor; N-terminal; Organism; Orthologous Gene; Outcome; Pattern; Process; Production; Proliferating; Proteins; Regulation; Resolution; Role; Slide; Structure; Tail; Techniques; Testing; Therapeutic Intervention; War; Work; base; cancer cell; cell motility; chromosome number abnormality; daughter cell; experimental study; flexibility; frontier; human disease; innovation; insight; mutant; nanoparticle; nanoscale; novel; optical traps; polypeptide; reconstitution; single molecule; targeted treatment; unnatural amino acids

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.

项目概述：在有丝分裂细胞分裂（有丝分裂）过程中，复制的染色体必须被准确地分离 分成两个子细胞，以确保所有真核生物的正常发育和生长。染色体错误 隔离可能导致非整倍体，这是癌症的标志和许多相关的健康问题。因此，在本发明中， 生物医学研究的一个主要前沿是了解控制组装的机制， 维持有丝分裂纺锤体，一个基于微管的双极机器负责准确的染色体 在有丝分裂期间分离。众所周知，双极有丝分裂细胞的正确组装和维持 纺锤体需要许多基于微管的驱动蛋白马达（有丝分裂驱动蛋白）的协调作用。然而，在这方面， 单个有丝分裂驱动蛋白的机制及其通过伴侣蛋白的调节和在有丝分裂过程中的协调 主轴装配和维护仍然知之甚少。PI的长期目标是重建 从纯化的蛋白质在体外双极有丝分裂纺锤体，并充分剖析机制，调控和 有丝分裂驱动蛋白在纺锤体组装和维持中的协调。这个项目的重点是有丝分裂 驱动蛋白-14，已知其与驱动蛋白-5形成拮抗对以驱动双极纺锤体组件。的 PI已经为该项目建立了几个基础性发现，包括：（1）有丝分裂驱动蛋白-14KlpA 来自构巢曲霉（及其在尼日尔曲霉中的直系同源物）的菌株在细胞上表现出进行性加末端定向运动， 单个微管，和（2）与两个保守的伴侣蛋白复合的相同KlpA开关， 单个微管的进行性负末端定向运动。后一个发现代表了第一组 体外研究表明，有丝分裂驱动蛋白-14依赖于伴侣蛋白获得进行性负末端导向的 能动性在这些发现的基础上，该项目旨在解决两个主要的开放问题，这两个问题是 了解驱动蛋白-14如何在双极纺锤体组装中对抗驱动蛋白-5：（1）分子基础是什么 在单个微管上潜在的进行性加末端导向的驱动蛋白14运动性？(2)驱动蛋白14 由伴侣蛋白调节来驱动双极纺锤体组装？该项目采用了创新的组合， 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，非天然氨基的遗传掺入 酸和高分辨率光学捕获。该项目的成果不仅将显着扩大目前的 了解驱动蛋白的运动机制，而且还提供了驱动蛋白-14的机制理解 双极主轴组件中的调节。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驱动蛋白的作用机制、调节和协调有了全面的了解 组装和维护，也提供了新的见解，如何有丝分裂驱动蛋白和他们的伴侣蛋白 可以作为治疗干预的目标。