Error Correction in Mammalian Mitosis: Defining Physical Cues and Integration Mechanisms

哺乳动物有丝分裂中的错误纠正：定义物理线索和整合机制

• 批准号: 10447001
• 负责人: Megan Kaiulani Chong
• 金额: $ 4.21万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10447001
• 关键词: 3-Dimensional; Affinity; Aneuploidy; Binding; Biological Assay; Breast; Breast Cancer Cell; Cancer Etiology; Cell division; Cells; Chromosomal Instability; Chromosome Segregation; Chromosomes; Color; Congenital Abnormality; Crowding; Cues; Development; Drug resistance; Ensure; Event; Fiber; Grasshoppers; Heart; Image; Individual; Inherited; Kinetochores; Laboratories; Lead; MCF10A cells; MDA MB 231; Malignant Neoplasms; Mammalian Cell; Mammals; Measures; Microtubule Stabilization; Microtubules; Mitosis; Mitotic; Modeling; Molecular; Monitor; Normal Cell; Oncogenic; Patients; Phosphotransferases; Plant Roots; Polymerase; Positioning Attribute; Proteins; Resolution; Role; Signal Transduction; Sister; Spermatocytes; Structure; Testing; Therapeutic; Time; Tumor stage; Up-Regulation; Work; aurora B kinase; cancer cell; cancer therapy; daughter cell; experimental study; improved; insight; live cell imaging; mutant; new therapeutic target; novel; novel therapeutics; overexpression; prevent; protein expression; real-time images; response; segregation; sensor; therapy development; tool; tumor

Project Summary/Abstract Errors in chromosome segregation give rise to aneuploidy, a hallmark of cancer. Breakdown of mitotic fidelity correlates with both tumor stage and patient drug resistance. Identifying mechanisms that prevent errors in chromosome segregation, and determining how they go wrong in cancer, are essential to developing therapies to either decrease or increase segregation error rates in cancer. The kinetochore attaches chromosomes to spindle microtubules. It segregates chromosomes and monitors their microtubule attachments, stabilizing correct attachments and destabilizing incorrect ones. We now know nearly all mammalian kinetochore proteins, and many have dysregulated expression in cancer. How does the kinetochore detect and correct attachment errors, and fail to do so in cancer? The idea that tension from bi-orientation signals correct attachments is decades-old, originating in Nicklas' pioneering experiments in grasshopper spermatocytes. Yet, how the kinetochore monitors tension and robustly integrates information across its many bound microtubules to regulate attachment stability is not known. In large part, this is due to challenges in applying tension on kinetochores inside cells, in quantitatively tuning kinetochore composition, and in imaging short-lived error correction events in real-time. Our laboratory has recently overcome these challenges, uniquely positioning us to answer these questions. Notably, two candidate kinetochore proteins have been proposed for sensing tension, the kinase AurKB and microtubule polymerase chTOG, and the expression of both is dysregulated in cancer, as is that of the main microtubule binder Hec1. Here, we test defining hypotheses on how normal and cancer cells detect and correct mitotic errors, combining high resolution 3D live-cell imaging, state-of-the-art physical perturbations, and molecular tools in normal and breast cancer cells. In Aim 1, we test the hypothesis that AurKB and chTOG sense tension. We use microneedles to directly apply force to kinetochore-microtubules, measure how attachment stability responds, and assess how these proteins' dysregulated expression alters this response in cancer. In Aim 2, we test models for whether microtubules respond independently or cooperatively to attachment cues such as tension, and test the hypothesis that Hec1 overexpression in cancer cells leads to hyper-stable attachments that may be more challenging to properly correct. We do so by quantitatively tuning kinetochore microtubule binding capacity using mixed Hec1 mutants, and measuring microtubule attachment lifetime using photomarks. In defining critical mechanisms for correcting mitotic errors, and how they are modified in cancer, we expect to identify adapted mechanisms of error correction in cancer cells. For example, some cancer cells may be deficient in error correction, leading to aneuploidy, or have improved error correction to compensate for extra chromosomes. Mechanisms uniquely or preferentially employed in cancer would offer a new therapeutic window.

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。有丝分裂保真度的破坏 与肿瘤分期和患者耐药性相关。识别防止错误的机制 染色体分离以及确定它们在癌症中如何出错对于开发治疗方法至关重要 减少或增加癌症的分离错误率。 着丝粒将染色体附着在纺锤体微管上。它分离染色体并 监控它们的微管附着，稳定正确的附着并破坏不正确的附着。我们 现在我们知道几乎所有哺乳动物动粒蛋白，其中许多在癌症中表达失调。如何 动粒是否能够检测并纠正附着错误，但在癌症中却无法做到这一点？紧张的想法 来自双向信号的正确依恋已有数十年历史，起源于尼克拉斯的开创性实验 蝗虫精母细胞。然而，动粒如何监测张力并稳健地整合信息 穿过其许多结合的微管来调节附着稳定性尚不清楚。这在很大程度上是由于 对细胞内动粒施加张力、定量调整动粒组成的挑战， 以及实时成像短暂的纠错事件。我们的实验室最近克服了这些 挑战，使我们能够以独特的方式回答这些问题。值得注意的是，两个候选动粒蛋白 已提出用于传感张力的激酶 AurKB 和微管聚合酶 chTOG，以及 两者的表达在癌症中均失调，主要微管结合物 Hec1 的表达也失调。 在这里，我们测试关于正常细胞和癌细胞如何检测和纠正有丝分裂错误的定义假设， 结合高分辨率 3D 活细胞成像、最先进的物理扰动和分子工具 正常细胞和乳腺癌细胞。在目标 1 中，我们测试 AurKB 和 chTOG 感知张力的假设。我们 使用微针直接对动粒微管施加力，测量附着稳定性 反应，并评估这些蛋白质的失调表达如何改变癌症中的这种反应。在目标 2 中，我们 测试微管是否独立或合作地响应依恋线索的模型，例如 张力，并检验癌细胞中 Hec1 过度表达导致超稳定附着的假设 正确纠正这可能更具挑战性。我们通过定量调节动粒微管来做到这一点 使用混合 Hec1 突变体的结合能力，并使用光标记测量微管附着寿命。 在定义纠正有丝分裂错误的关键机制以及它们在癌症中如何被修改时，我们 期望确定癌细胞中纠错的适应性机制。例如，一些癌细胞可能 错误纠正不足，导致非整倍体，或者改进了错误纠正来补偿 额外的染色体。癌症中独特或优先使用的机制将提供一种新的治疗方法 窗户。