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；在这种情况下，一个母细胞分裂成两个新的细胞。在细胞分裂过程中，染色体被绳状微管捕获，并被分成两组。这一过程中的错误可能会导致过早衰老、不孕和癌症。我们的目标是在分子水平上了解细胞是如何准确分裂的，因为这是预测和解决细胞分裂中的缺陷所必需的。为了确保染色体的正确分离，微管必须捕获由近100种不同蛋白质组成的特殊位置的染色体-着丝粒。就像发动机的各个部分一起工作一样，动粒的蛋白质共同建立了染色体-微管的相互作用。研究动粒如何工作的一个有效方法是破坏单个动粒蛋白的一小部分，使用精确的靶向突变，然后用显微镜观察它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，因此将在这项研究中继续使用这种方法。拟议的研究利用了该小组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优势。在端接转换过程中，沿着微管壁捕获的人类动点被带到微管的末端。虽然沿着微管壁很容易捕捉到动点，但最终所有的动点都必须系在微管末端。只有当动点与微管末端结合时，微管才能传递将染色体分开的力。微管壁结合的动粒是如何变成微管末端结合的动粒的尚不清楚。为了解决这一耐人寻味的难题，该小组将破坏末端转化所需的三种蛋白质，并详细研究它们的影响。Ndc80是一种动粒核心蛋白，尾巴类似尼龙搭扣，当磷酸化(添加小磷酸基团)时，阻止动粒-微管相互作用。NDC80尾部的磷酸化可能被调节，以指定动粒是否应该结合到微管-壁或末端-但这一点到目前为止还没有在人类细胞中进行测试。为了测试这一点，Ndc80尾部不允许磷酸化的突变将被引入细胞，并将使用超分辨率显微镜来观察其影响--这是最近在德国的一个合作者实验室建立的一种尖端工具。最近，该小组发现了两个末端转化的主要调节因子：Aurora-B激酶和BubR1结合的PP2A磷酸酶，它们分别可以添加或移除磷酸基团。改变它们的动粒定位会扰乱微管-末端的相互作用；但潜在的原因尚不清楚，这将被探索。最后，在末端转换过程的特定步骤中‘捕获’动粒的Ndc80或BubR1突变体是重要而优雅的工具，可以查询与该动粒被‘困’在其中的特定步骤相关的其他分子变化。例如，监测附着的检查点蛋白或稳定附着的微管末端相关蛋白可以被探测其定位。因此，除了解释Ndc80或BubR1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体过程。这项研究与BBSRC的研究重点直接相关：改善整个生命过程的健康。缺乏BubR1的小鼠由于染色体数量不规则而表现出过早衰老。同样，马赛克杂色非整倍体(换句话说，染色体数目不规则)的患者显示出过早衰老，这些患者经常缺乏BubR1或部分BubR1。通过促进对染色体分离过程的分子理解，这项工作将支持未来针对与染色体数目不规则的各种疾病相关的预测性标记或药物靶标的发展。

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.