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: 10313117
• 负责人: Megan Kaiulani Chong
• 金额: $ 4.14万
• 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/10313117
• 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.

项目总结/摘要 染色体分离的错误导致非整倍体，这是癌症的标志。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是如何出错的，对于开发治疗方法至关重要。 来降低或增加癌症中的分离错误率。 动粒将染色体附着在纺锤体微管上。它分离染色体， 监测它们的微管连接，稳定正确的连接和不稳定的连接。我们 现在几乎知道了所有哺乳动物动粒蛋白，并且许多在癌症中表达失调。如何 动粒是否能检测并纠正附着错误，而在癌症中却不能？张力 从双向信号正确的附件是几十年的历史，起源于尼可拉斯的开创性实验， 蚱蜢精母细胞然而，动粒是如何监测紧张局势并有力地整合信息的， 通过其许多结合的微管来调节附着稳定性是未知的。在很大程度上，这是由于 对细胞内的动粒施加张力，定量调节动粒组成， 以及实时成像短暂的纠错事件。我们的实验室最近已经克服了这些 挑战，让我们有独特的定位来回答这些问题。值得注意的是，两个候选动粒蛋白 已经提出用于感测张力，激酶AurKB和微管聚合酶chTOG，以及 两者的表达在癌症中失调，主要的微管结合剂Hec 1也是如此。 在这里，我们测试定义的假设，正常细胞和癌细胞如何检测和纠正有丝分裂错误， 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， 正常和乳腺癌细胞。在目的1中，我们测试了AurKB和chTOG感知张力的假设。我们 使用微针直接向运动舞蹈微管施加力，测量附着稳定性如何 反应，并评估这些蛋白质的表达失调如何改变癌症中的这种反应。在目标2中， 测试模型是否微管独立或合作作出反应的附件线索，如 张力，并测试癌细胞中Hec 1过表达导致超稳定附着的假设 这可能更难正确纠正。我们通过定量调节动粒微管来实现 结合能力，使用混合的Hec 1突变体，并测量微管附着寿命使用photomarks。 在确定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饰时，我们 希望能在癌细胞中识别出适应性的纠错机制。例如，一些癌细胞可能 错误纠正不足，导致非整倍体，或具有改善的错误纠正以补偿 额外的染色体独特或优先用于癌症的机制将提供一种新的治疗方法， 窗口