Protecting chromosome number: how cells establish, monitor and maintain chromosome-microtubule interaction?

所有的植物、动物和人类都必须通过细胞分裂来补充死亡或受伤的细胞，其中一个母细胞分裂成两个新细胞。在细胞分裂过程中，染色体被绳状微管捕获，并分成两组。这个过程中的错误可能导致过早衰老，不孕不育和癌症。我们的目标是在分子水平上了解细胞如何准确分裂，因为这是预测和解决细胞分裂缺陷所必需的。为了确保染色体的正确分离，微管必须在一个特殊的位置捕获染色体-动粒-由近100种不同的蛋白质组成。就像发动机的各个部分一起工作一样，动粒蛋白质共同建立了染色体-微管相互作用。研究动粒如何工作的一种有效方法是使用精确靶向突变破坏单个动粒蛋白的一小部分，然后使用显微镜观察其对染色体分离过程的影响。PI和她的团队已经使用这种方法发现了几种动粒蛋白的作用，因此将在本研究中继续使用这种方法。拟议的研究利用了该团队在可视化人类细胞中称为末端转换的过程方面的国际领先地位。在末端转换过程中，沿着微管壁沿着捕获的人类动粒被带到微管的末端。虽然很容易沿沿着微管壁捕获动粒，但最终所有动粒必须拴系到微管末端。只有当着丝粒与微管末端结合时，微管才能施加力将染色体拉开。微管壁结合的动粒如何变成微管末端结合的动粒尚不清楚。为了解决这个有趣的难题，该小组将破坏末端转换所需的三种蛋白质，并研究它们的影响，如下所述。Ndc 80是一种具有Velcro样尾巴的动粒蛋白，当磷酸化时（添加小磷酸基团）会阻止动粒-微管相互作用。ndc 80尾部磷酸化可以被调节以确定一个动粒是否应该结合到微管的端壁或端部，但这还没有在人类细胞中进行过测试。为了验证这一点，将Ndc 80尾部不允许磷酸化的突变引入细胞，并使用超分辨率显微镜观察其影响-这是德国合作者实验室最近建立的一种尖端工具。最近，该小组发现了两种末端转换的主要调节剂：Aurora-B激酶和BubR 1结合的PP 2A磷酸酶，它们可以分别添加或去除磷酸基团。改变他们的动粒定位破坏微管末端相互作用，但根本原因尚不清楚，这将是explored.Finally，Ndc 80或BubR 1突变体，'陷阱'的动粒在一个特定的步骤的结束上的转换过程是重要的和优雅的工具，查询其他分子的变化与该特定步骤的动粒是'困'在。例如，可以探测监测附着的检查点蛋白或稳定附着的微管末端相关蛋白的定位。因此，除了解释Ndc 80或BubR 1的作用外，这项工作还将提供分子工具来探索染色体分离的整个过程。这项研究与BBSRC的研究优先事项直接相关：在整个生命过程中更好的健康。缺乏BubR 1的小鼠由于染色体数目不规则而表现出过早衰老。类似地，具有马赛克杂色非整倍性（换句话说，染色体数目不规则）的患者表现出过早衰老，并且这些患者经常缺乏BubR 1或BubR 1的部分。通过对染色体分离过程的分子理解，这项工作将支持未来开发与不规则染色体数目相关的各种疾病的预测标记或药物靶点。

All plants, animals and humans must replenish dying or injured cells through cell division; where a mother cell divides into two new cells. During cell division, chromosomes are captured by rope-like microtubules and pulled apart into two sets. Errors in this process can lead to premature ageing, infertility, and cancers. We aim to understand how cells divide accurately, at a molecular level, as this is needed to predict and tackle defects in cell division.To ensure proper separation of chromosomes, microtubules must capture chromosomes at a special site - the kinetochore - made of nearly 100 different proteins. Like the parts of an engine that work together, proteins of the kinetochore jointly establish the chromosome-microtubule interaction. A powerful way to study how a kinetochore works is by disrupting small parts of individual kinetochore proteins, using precisely targeted mutations, and then observing its impact on the process of chromosome segregation using microscopy. The PI and her group have discovered the role of several kinetochore proteins using this approach, and hence will continue to use this approach in this study.The proposed study takes advantage of the group's international lead in visualizing a process called end-on conversion in human cells. During end-on conversion, human kinetochores captured along the walls of microtubules are brought to the ends of microtubules. While it is easy to capture kinetochores along microtubule-walls, ultimately all kinetochores must be tethered to microtubule-ends. Only when kinetochores are bound to microtubule-ends, microtubules can impart forces that pull chromosomes apart. How a microtubule-wall bound kinetochore becomes a microtubule-end bound kinetochore is unclear. To solve this intriguing puzzle, the group will disrupt three proteins needed for end-on conversion and study their impact as detailed below.Ndc80 is a kinetochore protein with a Velcro-like tail which when phosphorylated (addition of small phosphate groups) prevents kinetochore-microtubule interaction. Ndc80 tail phosphorylation may be modulated to specify whether a kinetochore should bind to microtubule -wall or -end - but this has not been tested in human cells so far. To test this, mutations in Ndc80 tail that disallow phosphorylation will be introduced into cells and its impact will be observed using super-resolution microscopy - a cutting-edge tool recently built in a collaborator's lab in Germany.Recently, the group discovered two master regulators of end-on conversion: Aurora-B kinase and BubR1-bound PP2A phosphatase, which can add or remove phosphate groups, respectively. Altering their kinetochore localization disrupts microtubule-end interaction; but the underlying reason is not clear and this will be explored.Finally, Ndc80 or BubR1 mutants that 'trap' the kinetochore in a particular step of the end-on conversion process are important and elegant tools to query other molecular changes associated with that particular step the kinetochore is 'trapped' in. For example, checkpoint proteins that monitor attachment or microtubule-end associated proteins that stablilise attachment can be probed for their localization. Thus, in addition to explaining the role of Ndc80 or BubR1, this work will also provide molecular tools to explore the process of chromosome segregation as a whole.This study is directly relevant to BBSRC's research priority: better health across the life-course. Mice lacking BubR1 show premature ageing due to cells with irregular chromosome numbers. Similarly, patients with Mosaic Variegated Aneuploidy (in other words, irregular chromosome numbers) show premature ageing and these patients frequently lack either BubR1 or parts of BubR1. By contributing to a molecular understanding of the chromosome segregation process this work will support future development of predictive markers or drug targets for a variety of disorders linked to irregular chromosome numbers.