Dissection of the Function of Microtubule Depolymerases in Mitotic Kinetochore Dynamics

我们的研究重点是有丝分裂的分子机制，即一个细胞分裂成两个细胞的过程。这是正常细胞生长、胚胎和所有组织发育以及包括癌症在内的许多疾病的关键事件。我们对微管和着丝点之间的连接特别感兴趣，微管是帮助分离染色体的电缆，着丝点是染色体上附着在微管上的结构。这种相互作用的细节仍然是个谜。为了理解这种相互作用，我们在海洋生物实验室（伍兹霍尔，美国）与Danuser（斯克里普斯），Meraldi（苏黎世联邦理工学院）和McAinsh（居里夫人）小组（一起被称为MBL着丝粒联盟）进行了为期三个夏季的密集合作，开发了一种基于成像的新型检测方法来研究有丝分裂着丝粒力学。合作的目标是开发出我们可以在家庭实验室中使用的检测方法，并最终应用于整个社区。我们对微管动力学和着丝点力学的第一次调查结果，来自5000多个延时数据集，揭示了令人惊讶的丰富的机械和动态细节，我们现在准备提交。在这个应用中，我们建议扩展这个分析，看看微管和着丝点之间不正确的附着形成的具体情况。我们将使用我们开发的检测方法来专门关注这些附着物，并跟踪它们何时以及如何被纠正。这是描述一个基本的、完全必要的细胞分裂基本过程的关键的、基本的信息。我们将检查校正途径，并使用复杂的新软件工具来分析结果数据，以提供在纠错过程中着丝点行为的定义模型。我们将研究这种纠正对特定酶的要求，称为微管解聚合酶，并研究这些酶是如何被磷酸化调节的。这是一项有针对性的工作，利用了罕见的、密集的合作，并综合了活细胞的尖端成像和复杂的数据分析。

Our research is focussed on the molecular mechanisms of mitosis, the process by which one cell divides into two. This is a critical event in normal cell growth, the development of an embryo and all tissues, and in a number of diseases, including cancer. We are interested particularly in the connections between microtubules, the cables that help separate chromosomes, and kinetochores, the structures on chromosomes that attach to microtubules. The details of this interaction are still mysterious. To understand this interaction, we have used an intensive, three-summer collaboration based at the Marine Biological Laboratory (Woods Hole, USA) with the Danuser (Scripps), Meraldi (ETH Zurich), and McAinsh (Marie Curie) groups (together referred to as the MBL Kinetochore Consortium) to develop a novel imaging-based assay to study mitotic kinetochore mechanics. The goal of the collaboration is to develop assays that we can deliver back to our home labs, and ultimately to the community at large. The results of our first survey of effects on microtubule dynamics and kinetochore mechanics, from over 5000 timelapse datasets, have revealed a surprising richness of mechanical and dynamic details that we are now preparing for submission. In this application, we propose to extend this assay, to look at specific cases where incorrect attachments form between microtubules and kinetochores. We will use the assay we developed to focus specifically these attachments and follow when and how they are corrected. This is critical, fundamental information describing a basic, completely essential fundamental process of cell division. We will examine the correction pathway, and use sophisticated new software tools to analyse the resulting data to deliver defined models of how kinetochores behave during error correction. We will examine the requirement for specific enzymes, called microtubule depolymerases, for this correction and also look at how these enzymes are regulated by phosphorylation. This is a targeted effort, taking advantage of a rare, intensive collaboration and a synthesis of cutting edge imaging in living cells and sophisticated data analysis.