Dissection of the Function of Microtubule Depolymerases in Mitotic Kinetochore Dynamics

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.

The resolution of mal-oriented chromosomes is a critical step in the formation of the metaphase plate. Improper syntelic or merotelic microtubule/kinetochore attachments cannot be properly segregated by the mitotic spindle and therefore can lead to aneuploidy and thus daughter cells with significant genomic defects. The goal of this project is to understand this correction process using a combination of high spatial and temporal resolution imaging and sophisticated data analysis and modeling tools. We have used an intensive, three-summer collaboration based at the Marine Biological Laboratory with three other labs to develop a novel imaging-based assay to study mitotic kinetochore mechanics in living cells. 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 key breakthrough has been the ability to automatically identify, track, and pair at least 90 separate kinetochores in each living HeLa cell. The results of our first survey of effects on microtubule dynamics and kinetochore mechanics during late prometaphase and metaphase are just being submitted, based on over 5000 timelapse data sets across 16 different small molecule and completely validated siRNAs targeting a range of kinetochore and spindle checkpoint proteins. This application represents the next step-the application and extension of the assay in our own lab to focus on microtubule/kinetochore attachments and their resolution by microtubule deploymerases. Using the pipeline we have already developed and some newly-developed analysis tools, we will record and analyse the correction of mal-orientations and determine what role each of the mitotic microtubule depolymerases plays in the process ad how this is regulated by protein phosphorylation.