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

项目摘要/摘要 染色体分离的错误会导致非整倍体，这是癌症的一个标志。有丝分裂保真度的崩溃 与肿瘤分期和患者耐药有关。确定防止出现错误的机制 染色体分离，并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 以降低或增加癌症中的分离错误率。 着丝粒将染色体连接到纺锤体微管上。它分离染色体和 监测他们的微管连接，稳定正确的连接和破坏不稳定的连接。我们 现在知道几乎所有哺乳动物的动粒蛋白，其中许多在癌症中表达异常。多么 动粒是否能检测并纠正附着错误，而在癌症中却无法做到这一点？关于紧张局势的想法 从双向信号正确的依恋是几十年前，起源于尼克拉斯的开创性实验在 蝗虫精母细胞。然而，动粒是如何监控张力和强有力地整合信息的 通过它的许多结合的微管来调节附着稳定性尚不清楚。在很大程度上，这是由于 在对细胞内的动粒施加张力，在定量调节动粒成分方面的挑战， 以及实时成像短暂的纠错事件。我们的实验室最近已经解决了这些问题 挑战，为我们回答这些问题提供了独特的定位。值得注意的是，两个候选动粒蛋白 已被提出用于感觉张力，激酶AurKB和微管聚合酶chTOG，以及 两者在癌症中的表达是失调的，主要的微管结合蛋白Hec1也是如此。 在这里，我们测试了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性假设， 结合了高分辨率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，我们检验了AurKB和chTOG感知张力的假设。我们 使用微针直接向动粒微管施力，测量附着稳定性 反应，并评估这些蛋白质的异常表达如何改变癌症中的这种反应。在目标2中，我们 测试微管是否独立地或协同地对连接提示做出反应的模型 张力，并检验Hec1在癌细胞中过表达导致超稳定附着的假设 要正确纠正这一点可能更具挑战性。我们通过定量调整动粒微管来实现这一点。 使用混合Hec1突变体的结合能力，并使用光标记法测量微管附着寿命。 在定义纠正有丝分裂错误的关键机制以及它们在癌症中是如何修改的过程中，我们 期望在癌细胞中识别适应的纠错机制。例如，一些癌细胞可能 纠错不足，导致非整倍体，或有改进的纠错以弥补 额外的染色体。在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。

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.