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.

项目概要/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，其中子细胞 有额外或缺失的染色体拷贝。主轴缺陷（例如多极）是原因之一 非整倍性，表明极完整性对于分离保真度至关重要。因此，了解如何 主轴的机械稳健性对于构建和维护适当的杆至关重要 了解纺锤体如何准确发挥作用，以及它如何在癌症等疾病中失效。 蛋白质 NuMA 和运动动力蛋白通过聚集微管负端来驱动极聚焦。 鉴于这种在极点聚焦中的作用，改变 NuMA 表达或功能可能会通过以下方式导致癌症： 多极性和非整倍性增加。事实上，NuMA 在某些癌症类型中过度表达，并且 NuMA 过度表达与多极性和非整倍性增加相关。然而，分子 NuMA 产生纺锤体力学和极完整性的机制及其在癌症中的作用， 还远不清楚。根据最近的工作和初步数据，我假设 NuMA 扮演两个角色 纺锤体力学中的不同作用，并且癌细胞中的 NuMA 破坏会影响这两种作用： 被动（非能量消耗）作用交联微管和调节运动性的作用 主动运动动力蛋白。在癌症背景下，其中一个或两个角色可能会被破坏并成为目标。 在这里，我建议通过结合分子和机械扰动来检验这个假设， 显微镜和人类中期细胞的定量图像分析。在目标 1 中，我将测试是否 以及 NuMA 如何在纺锤体力学中发挥独立于动力蛋白的被动作用。为此，我将使用 基于 PDMS 的细胞限制，以机械方式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，并将比较杆在受力下结构上如何失效。在目标 2 中， 我将确定 NuMA 如何调节动力蛋白功能来驱动主轴机械。使用函数式 NuMA/动力蛋白转运测定，我将测试 NuMA 是否以及如何调节动力蛋白力的产生 主轴。具体来说，我将比较不同 NuMA 突变体改变动力蛋白力的能力 一代，重点关注缺乏动力蛋白激活所必需的卷曲螺旋的突变体 以及阻止 NuMA 寡聚和聚集动力蛋白的突变体。最后，我将使用 这些相同的 NuMA 突变体和细胞限制来测试 NuMA 调节动力蛋白是否对于 癌纺锤体中的极机械完整性。 这些目标共同决定了必需蛋白质 NuMA 如何驱动纺锤体力学 以及 NuMA 的主动和被动作用如何导致其在癌细胞中的功能被破坏。像这样， 这项工作将使我们能够确定主轴杆故障的机制，并可能提供新的 控制癌症多极性和非整倍性的治疗策略。