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.

项目总结/摘要 染色体分离错误导致非整倍体，这是癌症的标志， 有多余或缺失的染色体拷贝。主轴极缺陷，如多极化，是一个原因 的非整倍体，表明极点的完整性是至关重要的分离保真度。因此，了解如何 主轴的机械坚固性出现，以建立和保持适当的极点是至关重要的， 了解纺锤体是如何准确地发挥作用的，以及它是如何在癌症等疾病中失效的。 蛋白质NuMA和马达动力蛋白通过聚集微管负末端来驱动极聚焦。 鉴于这种在极聚焦中的作用，改变NuMA表达或功能可能会导致癌症， 增加多极性和非整倍性。事实上，NuMA在某些癌症类型中过表达， NuMA过表达与多极性和非整倍性增加相关。然而，分子 NuMA引起纺锤体力学和磁极完整性的机制，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， 在纺锤体力学中有不同的作用，癌细胞中的NuMA破坏影响这两种作用： 被动（非能量消耗）作用交联微管和调节运动的作用 主动运动动力蛋白在癌症背景下，其中一种或两种作用都可以被破坏和靶向。 在这里，我建议通过结合分子和机械扰动来测试这一假设， 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发挥动力蛋白独立的被动作用。为此，我将使用 基于PDMS的细胞限制，以机械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，并将比较如何杆结构下的力量失败。在目标2中， 我将确定NuMA如何调节动力蛋白功能以驱动主轴机械。使用功能 NuMA/动力蛋白转运试验，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，重点是缺乏卷曲螺旋怀疑动力蛋白激活所必需的突变体 以及防止NuMA寡聚化和使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，以测试NuMA调节动力蛋白是否是必需的， 癌症纺锤体的磁极机械完整性。 总之，这些目标将决定必需蛋白质NuMA如何驱动纺锤体力学 以及NuMA的主动和被动作用如何影响其在癌细胞中的功能。因此，在本发明中， 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 控制癌症中多极和非整倍性的治疗策略。