Mechanisms of spindle checkpoint silencing

This project is about understanding how cells ensure chromosomes are segregated equally to daughter cells. Human beings are built from around 100 trillion individual cells. Each cell contains 46 chromosomes into which is packaged our genetic material (DNA, deoxyribonucleic acid), which provides the instructions for how a cell should work and how a whole organism should be built. This huge number of cells originates from a single cell that is the result of fertilization of an egg with a sperm. This single cell needs to be able to divide itself to generate two new daughter cells, which then also divide to produce further cells; this process repeats until the correct number of cells are generated. Moreover, cells do not live forever and are constantly being replaced by new ones. Thus, cell division is fundamental to the existence of life.

A key part of cell division involves the accurate separation of the chromosomes into the two daughter cells - a process called mitosis. It is crucial that each daughter cell receives a complete set of chromosomes. We know that having the wrong number of chromosomes is a cause of multiple human diseases, most notably cancer where greater than 80% of human tumors have the wrong number of chromosomes. Indeed, altering chromosome number experimentally in mice has been shown to cause cancer. Secondly, many developmental disorders are the result of mistakes in chromosome separation such as Downs Syndrome, in which cells have an extra copy of chromosome 21. A large proportion of miscarriages are also caused by problems in chromosome separation. It is clearly vital that we work out how the process of chromosome segregation is controlled and why these controls are defective or over-ridden in these diseases.

Each chromosome is made up of two sister chromatids that are stuck together after the cell replicates its DNA. To separate these two sister chromatids (one to each of the two daughter cells), the cell makes use of molecular cables called microtubules. Each sister chromatid has a "hook" called the kinetochore, which can attach to the end of a microtubule cable. As the cables attached to the two sister chromatids grow and shrink the chromosomes are moved around inside the cell. This jostling motion allows the chromosomes to line up in the middle of the cell. When everything is ready, the glue joining the two sister chromatids is removed and the sister chromatids are pulled to opposite daughter cells.

But how is this process controlled? It turns out that the kinetochore also operates as the control centre for a monitoring system, called the spindle assembly checkpoint, which ensures that sister chromatids do not separate until the alignment process is complete. We have recently discovered the central players in this process but we do not yet understand how they are controlled nor how this system operates.

The experiments that we will do will help answer these exciting and intriguing questions and therefore advance our understanding of how chromosomes are separated equally into daughter cells during cell division. To do this we will use state-of-the-art imaging technology (powerful microscopes) and modern techniques in molecular genetics and biochemistry to observe how chromosomes move in living cells and how the factors involved operate. The answers to these questions will help the identification of new targets and therapies to combat disease.

The spindle assembly checkpoint is the prime cell cycle control mechanism that ensures sister chromatids are bi-oriented before anaphase takes place. Components of the checkpoint include the Mad1, Mad2, Mad3(BubR1) and Bub3 proteins and the Bub1, Mph1(Mps1) and Aurora B kinases. The checkpoint is activated when individual kinetochores are not bound to microtubules or not under tension. Upon activation at the kinetochore Mad2 and Mad3 bind to and inhibit Cdc20, an essential activator of the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase. When the checkpoint is satisfied the spindle checkpoint signal is silenced and the Cdc20-APC/C is activated. This triggers polyubiquitination of securin and cyclin, which allows the dissolution of sister chromatid cohesion and mitotic progression. Although components of the checkpoint were identified 20 years ago, their binding sites at kinetochores are largely unknown. Moreover, the molecular mechanism(s) by which the spindle checkpoint is switched off following chromosome bi-orientation are not well understood. We have recently made two important advances in this field using fission yeast as a model system. Firstly, we have found that association of type 1 phosphatase (PP1) to both the Spc7 kinetochore protein and to kinesin-8 motors are necessary to silence the spindle checkpoint. Secondly, we have found that phosphorylation of Spc7 by Mph1 recruits the Bub1 and Bub3 checkpoint proteins to kinetochores to enable spindle checkpoint signalling. We are now in an excellent position to
1. To determine how silencing of the spindle checkpoint is triggered.
2. To elucidate the critical downstream targets of PP1 that mediate checkpoint inactivation.
3. To determine whether these mechanisms are conserved in human cells.
We will use a variety of techniques including molecular genetics, biochemistry, mass spectrometry and fluorescence microscopy in both fission yeast and human cells to address these questions.

Impact summary

Our research will have a broad range of impacts, spanning the advancement of scientific knowledge, health and wellbeing, economic competitiveness, and the provision of skilled people to the workforce.

Pharmaceutical and Biotechnology industries.

Pharmaceutical and Biotechnology industries are a vital part of the UK economy employing around 250,000 people and generating billions of pounds of income each year. However, this process of drug discovery relies heavily on a strong basic-science base to provide insights into potential drug targets as well as the development of new cell-based assays and technologies. Our work into the processes of chromosome segregation will contribute to the knowledge-base. By integrating state-of-the art live-cell imaging assays with sophisticated genetics and biochemistry we hope to predict new potential targets both for early clinical diagnosis and for drug-development. This will be highly relevant not only to "big pharma", but also to regional businesses in the West Midlands participating in the Birmingham Science City Research Alliance.

Translational medicine.

Our work has the potential to contribute to significantly improved efficacy and reduced treatment costs for diseases associated with errors in chromosome segregation and changes in chromosome number (which are involved with cells becoming resistant to existing chemotherapeutic drugs). Thus our work is likely to impact positively on the economics of the health care sector over the longer-term. To facilitate translation of our research WMS has recently established a Biomedical Research Unit in reproductive health (headed by Professor Siobhan Quenby) and is advertising for a Professor of Clinical Oncology to join the Division of Biomedical Cell Biology in 2012.

Provision of skilled workforce.

Staff working on our projects will be trained in a wide range of advanced skills in molecular and cellular biology, and computational and data analysis, using the state-of-the-art techniques and training available in the Biomedical Cell Biology group at Warwick; as well as transferrable skills - Warwick University provides a wide range of transferable skills courses that are designed to maximize individual potential and employability. Thus our work will contribute to the economic competitiveness of the UK through the provision of highly skilled labour.

The general public.

We need to know how the process of chromosome segregation is controlled before we can unlock new therapeutic routes to deal with the many major diseases and healthcare issues arising from errors in chromosome segregation. Our work will contribute to such advances, which will be of direct benefit to the general population in terms of health and well-being, particularly for those social groups most at risk of syndromes such as Down's and Turner's, miscarriages and stillbirths (70% of which are associated with mis-segregation of chromosomes during cell division) and cancer. Our carefully-designed science outreach programmes will target relevant social groups to ensure they are aware of our scientific advances and understand how they fit into the pipeline that takes discoveries in basic science and converts them in to new therapies and health improvements.