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

基本信息

批准号：
10674003
负责人：
Megan Kaiulani Chong
金额：
$ 4.31万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
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/10674003
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 Compensation Congenital Abnormality Crowding Cues Development Drug resistance Ensure Event Fiber Grasshoppers Heart Image Individual Inherited Kinetochores Laboratories MCF10A cells MDA MB 231 Malignant Neoplasms Mammalian Cell Mammals Measures Microtubule Stabilization Microtubules Mitosis Mitotic Modeling Molecular Monitor Needles Normal Cell Oncogenic Patients Phosphotransferases Polymerase Positioning Attribute Proteins Resolution Role Signal Transduction Sister Spermatocytes Structure Testing Therapeutic Time Tumor stage Up-Regulation Work 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

项目摘要

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.

项目摘要/摘要染色体隔离的错误导致癌症的标志性异倍倍。有丝分裂保真度的崩溃与肿瘤阶段和耐药性均相关。确定防止错误的机制染色体隔离，并确定它们在癌症中的出问题，对于开发疗法至关重要降低或增加癌症的隔离错误率。动力学将染色体连接到纺锤微管上。它分离染色体和监视其微管附件，稳定正确的附件并破坏不正确的附件。我们现在知道几乎所有哺乳动物动力学蛋白，许多蛋白质在癌症中的表达失调。如何动力学是否检测并纠正依恋误差，并且在癌症中未能做到？紧张的想法从双向方向信号中，正确的附件已有数十年历史，起源于Nicklas的开创性实验蚱hopper精子细胞。但是，动力学如何监视紧张和鲁棒的信息在其许多结合的微管中以调节附着稳定性，尚不清楚。在很大程度上，这是由于在细胞内部动力学上施加张力的挑战，在定量调整动力学组成中，并实时成像短期误差校正事件。我们的实验室最近克服了这些挑战，独特地定位我们以回答这些问题。值得注意的是，两个候选动力学蛋白已经提出了用于感应张力，激酶Aurkb和微管聚合酶CHTOG，以及两者的表达在癌症中和主要微管粘合剂HEC1的表达失调。在这里，我们检验定义假设，说明正常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AIM 1中，我们检验了Aurkb和Chtog感觉张力的假设。我们使用微针直接将力施加到动型微管上，测量固定稳定性响应，并评估这些蛋白质失调的表达如何改变癌症的这种反应。在AIM 2中，我们测试模型是微管是独立或合作对附件提示的响应的测试模型张力，并检验以下假设：癌细胞中的HEC1过表达导致超稳的附着正确纠正可能更具挑战性。我们通过定量调整动力学微管来做到这一点使用混合Hec1突变体的结合能力，并使用光域测量微管附着寿命。在定义纠正有丝分裂错误的关键机制以及如何在癌症中修饰它们时，我们期望确定癌细胞中误差校正的适应机制。例如，某些癌细胞可能缺乏误差校正，导致非整倍性或进行改进的误差校正以补偿额外的染色体。独特或优先用在癌症中使用的机制将提供新的治疗性窗户。