Protecting chromosome number: how cells establish, monitor and maintain chromosome-microtubule interaction?

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.

Accurate segregation of chromosomes requires proper chromosome-microtubule attachment. Chromosome missegregation can cause aneuploidy that is linked to premature ageing and breeding problems, relevant to several BBSRC research priorities: agricultural production, animal health and healthy ageing across the lifecourse.

Chromosome-microtubule attachment is established by a macromolecular kinetochore. Kinetochores are captured along microtubule walls (lateral attachment) and then tethered to microtubule ends (end-on attachment) through a multi-step end-on conversion process. Molecular details of end-on conversion - how lateral attachments are formed, monitored and finally converted into end-on attachments? - remain unclear. To uncover them, high-resolution microscopy tools developed in the Draviam group, which allowed the first visualisation of end-on conversion in human cells, super-resolution microscopy (EMBL, Germany) and electrically tunable lens technology (Keio University, Japan) will all be combined to study chromosome segregation at the highest spatial and temporal resolution possible.

Building on the group's recent success in identifying master regulators of the end-on conversion process, the first goal is to identify their precise roles and compare these to map a temporal sequence of molecular steps that underpin end-on conversion. Next, to determine how cells monitor and stabilise attachments, kinetochore recruitment dynamics of spindle checkpoint proteins and microtubule-end associated proteins will be quantified in two ways: first, during end-on conversion and second, in mutants that 'trap' kinetochores at distinct end-on conversion steps. These studies will generate new molecular knowledge on mechanisms that ensure the accurate segregation of chromosomes. It will also inform a variety of other processes powered by microtubule-mediated forces namely, cell migration, leukocyte extravasation and wound healing - all relevant to healthy ageing.

Scientific impact: The main impact will be scientific as the project aims to uncover how a fundamental cellular process is regulated at the molecular level. Chromosome segregation regulators are evolutionarily conserved and are important to maintain correct chromosome number and prevent aneuploidy-related disorders throughout life; thus, a molecular understanding of the segregation process will have a wide scientific impact across various BBSRC research priority areas from plant and animal breeding to healthy ageing.

Researcher career development: The PDRA and members of the Draviam and collaborator's groups will have an exceptional opportunity to learn, use and extend cutting-edge microscopy techniques. With the microscopy market predicted to grow annually at least by 23%, until 2022, across biology fields, in particular, and natural sciences, in general, the interdisciplinary training gained during the course of this project will prepare the researchers for an excellent future career.

Translational opportunity in ageing research: Steady increase in ageing human population is a grand societal challenge. Incidence of aneuploidy is strongly correlated with ageing, but the molecular reasons for aneuploidy associated with ageing are not fully understood restricting biomarker development. The cell biology work planned here will complement a separate collaborative project with Prof Sun's group at Beijing Institute for Genomics (BIG) to identify frequently recurring mutations in aneuploid tissues, including cancers. Thus, the work proposed will bring together high-throughput (genome sequencing) and high-resolution (cell biology) technologies to help advance research relevant to aneuploidy associated with ageing.

Commercial impact: High-speed imaging using electrically tunable lens is still a niche research area due to the implementation steps that are often too technical for end-users in cell biology laboratories. By working with IMSOL (microscope vendor), the group is working towards building a computational software tool to bring ETLs as part of the mainstream market. The work proposed will use ETLs and will serve as an additional proof of concept to the one we have already published.