How are mono-oriented chromosome-microtubule attachments protected to prevent errors in mitosis and associated cellular ageing?

如何保护单向染色体微管附着以防止有丝分裂和相关细胞衰老的错误？

• 批准号: BB/W002698/1
• 负责人: Viji Draviam
• 金额: $ 51.47万
• 依托单位: Queen Mary University of London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW002698%2F1
• 关键词: How; mono; oriented; chromosome; microtubule

When a mother cell divides into two, its chromosomes are pulled apart into two equal sets by rope-like microtubules. Errors in chromosome-microtubule attachment can result in the loss or gain of chromosomes, leading to irregular chromosome numbers in cells - a hallmark of animal and human infertility and several premature ageing syndromes. To precisely pinpoint why chromosome numbers are incorrect in some diseases, a clear molecular understanding of how microtubules capture and pull chromosomes apart is essential.Chromosome-microtubule attachment is mediated by a macromolecular structure - the kinetochore - made of nearly 100 proteins. The Draviam group reported a protein complex Astrin-SKAP that is recruited to kinetochores soon after the formation of correct chromosome-microtubule attachments, and the complex is required for maintaining correct attachments. How Astrin senses attachment status and how it stabilises them are not known. These will be addressed to explain how Astrin ensures the accurate segregation of chromosomes.By combining methods in structural biology (Pickersgill lab) and evolutionary biology (Martin-Duran lab), the Draviam group showed that Astrin interacts with an outer-kinetochore protein, HEC1, and Astrin delivers an enzyme PP1-phosphatase. Astrin-mediated delivery of PP1 is carefully scheduled to selectively stabilise correct attachments. In other words, Astrin works like a 'messenger' arriving selectively at correctly attached kinetochores to deliver a 'tool' that is needed to stabilise the attachments. This means that determining how Astrin-HEC1 and Astrin-PP1 interactions are controlled will unravel how cells ensure proper chromosome-microtubule attachments and prevent chromosome missegregation.This project is timely as it takes advantage of a Super-resolution microscope (funded by BBSRC) to track dynamic changes at the outer kinetochore, at the highest spatial resolution possible. First, Astrin's arrival at kinetochores will be correlated with nanoscale structural changes at the outer-kinetochore to learn about changes specific to correct attachments. Second, the regions of Astrin essential for HEC1 or PP1 interaction will be determined, and mutants of Astrin that cannot bind to HEC1 or PP1 will be expressed in cells to study how Astrin senses attachments and how cells schedule Astrin-PP1 interaction to ensure the accurate segregation of chromosomes.To further strengthen the research program, two collaborations have been planned: (i) Pull-downs using Owenia embryo lysates to take clues from evolutionarily conserved Astrin interactions.(ii) Computational modelling of protein structure to take clues from HEC1 and PP1 crystal structures for designing Astrin interaction mutants.Astrin mutants that disrupt chromosome-microtubule attachment and chromosome segregation accuracy or timing will be studied for the extent to which they promote cellular ageing, either immediately (within hours) or in the long-term (in days), by tracking markers for stress, DNA damage and repair.This will be the first nano-scale study of dynamic changes at the outer-kinetochore which protect correct attachments and prevent chromosome missegregation. Simultaneous single-cell tracking of attachment defects, segregation inaccuracy and premature ageing make this project unique and invaluable for isolating mitotic errors that cause ageing. This knowledge can help build biomarkers to predict and track premature ageing in animals and humans.Fundamental discoveries made here about microtubule-mediated pulling or pushing of chromosomes will be widely useful for other microtubule-mediated processes in our body. For instance, neuronal growth, spindle rotation, immune signalling and cell migration are all reliant on regulatory switches to sense and stabilise microtubules in different parts of the cell. Thus the study will be broadly useful to understand force generation mechanisms within cells.

当母细胞分裂成两个时，它的染色体被绳状微管拉成两组相等的部分。染色体-微管连接的错误可能导致染色体的丢失或获得，导致细胞中的染色体数量不规则--这是动物和人类不孕不育以及几种早衰综合征的标志。为了准确地找出一些疾病中染色体数量不正确的原因，对微管如何捕获和拉开染色体的分子理解是至关重要的。染色体-微管附着是由近100种蛋白质组成的大分子结构-动粒-介导的。Draviam小组报告了一种蛋白质复合体Astrin-skap，它在正确的染色体-微管连接形成后不久就被招募到动点，而该复合体是维持正确连接所必需的。阿斯特林如何感知依恋状态以及如何稳定它们尚不清楚。通过结合结构生物学(Pickersgill实验室)和进化生物学(Martin-Duran实验室)的方法，Draviam小组表明Astrin与外部动粒蛋白HEC1相互作用，Astrin提供一种PP1-磷酸酶。Astrin介导的PP1的传递经过精心安排，以选择性地稳定正确的附着。换句话说，阿斯特林的工作原理就像一名“信使”，有选择地到达正确连接的动控中心，提供稳定连接所需的“工具”。这意味着确定Astrin-HEC1和Astrin-PP1相互作用的控制方式将揭开细胞如何确保正确的染色体-微管连接和防止染色体错误分离的谜团。这个项目是及时的，因为它利用超分辨率显微镜(由BBSRC资助)以尽可能高的空间分辨率跟踪外部着丝粒的动态变化。首先，阿斯特林到达动点将与外部动点的纳米级结构变化相关联，以了解特定于正确连接的变化。其次，将确定与HEC1或PP1相互作用所必需的Astrin区域，并将不能与HEC1或PP1结合的Astrin突变体在细胞中表达，以研究Astrin如何感知连接以及细胞如何调度Astrin-PP1相互作用，以确保染色体的准确分离。为进一步加强研究计划，已经计划了两项合作：(I)使用Owenia胚胎裂解物从进化上保守的Astrin相互作用中提取线索。(Ii)从HEC1和PP1晶体结构中获得设计Astrin相互作用突变体的线索的蛋白质结构的计算模拟。将通过跟踪应激、DNA损伤和修复的标记，立即(在几小时内)或在长期(以天为单位)研究破坏染色体-微管连接和染色体分离准确性或时机的Astrin突变体促进细胞衰老的程度。这将是第一次对外部动粒动态变化的纳米尺度研究，这些变化保护正确的连接并防止染色体错误分离。对附着缺陷、分离不准确和过早老化的同时单细胞跟踪使该项目成为分离导致衰老的有丝分裂错误的独特和宝贵的项目。这些知识可以帮助建立生物标记物来预测和跟踪动物和人类的过早衰老。这里关于微管介导的染色体拉或推的基础性发现将广泛用于我们身体中其他微管介导的过程。例如，神经元生长、纺锤体旋转、免疫信号和细胞迁移都依赖于调节开关来感知和稳定细胞不同部分的微管。因此，这项研究将对理解细胞内的力产生机制有广泛的帮助。