Defining the mechanisms by which NuMA drives spindle mechanical robustness

定义 NuMA 驱动主轴机械稳健性的机制

• 批准号: 10677401
• 负责人: Nathan Cho
• 金额: $ 4.36万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-12-01 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10677401
• 关键词: Ablation; Affect; Aneuploidy; Automobile Driving; Binding; Biological Assay; Cell division; Cells; Chromosome Segregation; Chromosomes; Complex; Consumption; Data; Defect; Disease; Dynein ATPase; Energy consumption; Failure; Generations; Genome; Human; Image Analysis; In Vitro; Lasers; Lead; Length; Link; Malignant Neoplasms; Mechanics; Metaphase; Microtubules; Minus End of the Microtubule; Mitosis; Mitotic; Molecular; Motor; Play; Property; Proteins; Regulation; Role; Structure; System; Testing; University of Pittsburgh Cancer Institute; Walking; Work; cancer cell; cancer type; cell motility; crosslink; daughter cell; knock-down; malignant mouth neoplasm; microscopic imaging; mutant; novel therapeutic intervention; novel therapeutics; overexpression; prevent; quantitative imaging; recruit; response; segregation

Project Summary/Abstract Errors in chromosome segregation lead to aneuploidy, a hallmark of cancer where daughter cells have extra or missing chromosome copies. Spindle pole defects, such as multipolarity, are one cause of aneuploidy, indicating that pole integrity is critical to segregation fidelity. Thus, understanding how the spindle’s mechanical robustness emerges to build and maintain proper poles is crucial to understanding how the spindle accurately functions, and how it fails in disease such as cancer. The protein NuMA and the motor dynein drive pole focusing by clustering microtubule minus-ends. Given this role in pole focusing, altering NuMA expression or function could lead to cancer by increasing multipolarity and aneuploidy. Indeed, NuMA is overexpressed in certain cancer types and NuMA overexpression correlates with increased multipolarity and aneuploidy. However, the molecular mechanisms by which NuMA gives rise to spindle mechanics and pole integrity, and its role in cancer, are far from clear. Based on recent work and preliminary data, I hypothesize that NuMA plays two separate roles in spindle mechanics and that NuMA disruptions in cancer cells affect both roles: a passive (non-energy consuming) role crosslinking microtubules and a role regulating the motility of the active motor dynein. Either or both roles could be disrupted, and targeted, in a cancer context. Here, I propose to test this hypothesis by combining molecular and mechanical perturbations, microscopy, and quantitative image analysis in human metaphase cells. In Aim 1, I will test whether and how NuMA plays a dynein-independent, passive role in spindle mechanics. To do so, I will use PDMS-based cell confinement to mechanically challenge normal and cancer spindles where NuMA can and cannot interact with dynein, and will compare how poles structurally fail under force. In Aim 2, I will determine how NuMA regulates dynein function to drive spindle mechanics. Using a functional NuMA/dynein transport assay, I will test whether and how NuMA regulates dynein force generation in the spindle. Specifically, I will compare the ability of different NuMA mutants to change dynein force generation, focusing on a mutant that lacks the coiled-coil suspected necessary for dynein activation and a mutant preventing NuMA from oligomerizing and clustering dyneins together. Finally, I will use these same NuMA mutants and cell confinement to test if NuMA regulating dynein is essential for pole mechanical integrity in cancer spindles. Together, these aims will determine how the essential protein NuMA drives spindle mechanics and how NuMA’s active and passive roles contribute to its disrupted function in cancer cells. As such, this work will allow us to identify mechanisms for spindle pole failures and may provide new therapeutic strategies to control multipolarity and aneuploidy in cancer.

项目摘要/摘要 染色体分离错误会导致非整倍体，这是癌症的一个标志，在这种情况下，子代细胞 有多余或缺失的染色体副本。主轴磁极缺陷，如多极，是一个原因 非整倍体，表明极点的完整性对分离的保真度至关重要。因此，理解如何 主轴的机械坚固性对建立和维护适当的磁极至关重要 了解纺锤体是如何准确发挥作用的，以及它在癌症等疾病中是如何失败的。 蛋白NUMA和运动动力蛋白通过聚集微管负端来驱动极聚焦。 鉴于这种在极聚焦中的作用，改变NUMA的表达或功能可能通过以下方式导致癌症 多极化和非整倍体的增加。事实上，NUMA在某些癌症类型中过度表达， NUMA的过度表达与多极化和非整倍体的增加有关。然而，分子 NUMA产生纺锤力学和磁极完整性的机制，以及它在癌症中的作用， 还远远不清楚。根据最近的工作和初步数据，我假设NUMA扮演着两个角色 纺锤体机制中的单独角色和癌细胞中NUMA的破坏影响这两个角色：A 被动(非耗能)作用，交联微管和调节运动的作用 主动运动动力蛋白。在癌症的背景下，这两个角色中的任何一个都可能被破坏，并成为目标。 在这里，我建议通过结合分子和机械扰动来检验这一假设， 人中期细胞的显微镜和定量图像分析。在目标1中，我将测试 以及NUMA如何在纺锤体力学中扮演动力蛋白非依赖性的被动角色。为此，我将使用 基于PDMS的细胞限制，机械地挑战正常和癌症纺锤体，其中NUMA 可以和不能与动力蛋白相互作用，并将比较极杆在受力下的结构失效情况。在目标2中， 我将确定NUMA如何调节dynein功能来驱动主轴机械。使用泛函 NUMA/dynein转运实验，我将测试NUMA是否以及如何调节在 纺锤。具体地说，我将比较不同NUMA突变体改变动力蛋白力量的能力 产生，专注于一个突变，该突变缺乏被认为是动力蛋白激活所必需的卷曲线圈 以及防止nuA寡聚和将动力蛋白聚集在一起的突变体。最后，我将使用 这些相同的NUMA突变体和细胞限制测试NUMA调节动力蛋白是否对 癌症纺锤杆的机械完整性。 这些目标加在一起，将决定基本蛋白质NUMA如何驱动纺锤体机械 以及NUMA的主动和被动作用如何导致其在癌细胞中的功能中断。因此， 这项工作将使我们能够确定主轴磁极故障的机制，并可能提供新的 控制癌症多极化和非整倍体的治疗策略。