Dissection of the Function of Microtubule Depolymerases in Mitotic Kinetochore Dynamics

我们的研究集中在有丝分裂的分子机制上，有丝分裂是一个细胞分裂成两个细胞的过程。这在正常细胞生长、胚胎和所有组织的发育以及包括癌症在内的许多疾病中都是一个关键事件。我们特别感兴趣的是微管之间的连接，微管是帮助分离染色体的电缆，动点是染色体上附着在微管上的结构。这种互动的细节仍然是个谜。为了了解这种相互作用，我们在海洋生物实验室(伍兹霍尔，美国)与Danuser(斯克里普斯)、Meraldi(苏黎世理工大学)和McAinsh(Marie Curie)小组(统称为MBL Kinetochore Consortium)进行了为期三个夏天的密集合作，开发了一种新的基于成像的分析方法来研究有丝分裂动粒机制。合作的目标是开发我们可以送回我们的家庭实验室，并最终提供给整个社区的分析。我们第一次对微管动力学和动粒力学的影响的调查结果，从5000多个时间流逝的数据集，揭示了令人惊讶的丰富的力学和动力学细节，我们现在正在准备提交。在这项应用中，我们建议扩展这项测试，以观察微管和动点之间形成错误连接的特定情况。我们将使用我们开发的分析方法来特别关注这些附件，并跟踪它们何时和如何更正。这是描述细胞分裂的基本、完全必要的基本过程的关键的、基本的信息。我们将研究纠正路径，并使用复杂的新软件工具来分析产生的数据，以提供定义的模型，说明运动中枢在纠错过程中的行为。我们将研究这种纠正对特定酶的要求，称为微管解聚酶，并研究这些酶是如何通过磷酸化来调节的。这是一项有针对性的努力，利用了罕见的密集合作，以及活细胞尖端成像和复杂数据分析的综合。

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.