System-mechanics of the kinetochore: operating principles of a complex mechanochemical engine

Human beings are built from 50 trillion individual cells. Each cell contains 46 chromosomes - the packages of genetic material (DNA), which provide 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, the zygote, which 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 therefore 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: (1) greater than 80% of human solid tumors have the wrong number of chromosomes and changing chromosome number is known to cause cancer in mice. (2) Many developmental disorders are the result of mistakes in chromosome separation - a well-known example is Downs Syndrome in which cells have an extra copy of chromosome 21. (3) A large proportion of miscarriages are caused by problems in chromosome separation. Clearly, it is vital that we work out how chromosome separation works. To move a chromosome the cell makes use of molecular cables called microtubules that can grow and shrink. Each chromosome has a 'hook' called the kinetochore, which can attach to the end of a microtubule cable. As the cable grows and shrinks the chromosome can be pushed and pulled. This is a beautiful system whereby the cell can move chromosomes around inside itself. However, unlike a hook, the kinetochore is able to control how and when a microtubule cable grows and shrinks. This way the kinetochore is the 'control centre' and the 'engine room' that decides when and where a chromosome moves. Kinetochores move all the 46 chromosomes into a line at the centre of the cell. This is called metaphase. At this time the chromosomes move back-and-forth like the pendulum on a clock, and then, the chromosomes are pulled into the daughter cells. But, how does the kinetochore do this? Why and how do the chromosomes change direction? The experiments that we propose to carry out will help answer these exciting and intriguing question and therefore advance our understanding of how chromosomes are separated into daughter cells during cell division. To do this we will use state-of-the-art imaging technology (microscopes) to observe how chromosomes move in living human cells. We can then accurately measure what happens to how the chromosomes move when we remove parts of the machinery from the cell. Because this is such a complex biological problem we will use mathematics to build a model of how the system works. By combining the disciplines of biology and mathematics together we expect to make large advances in our understanding of chromosome separation.

Kinetochores are adaptive, multi-layered mechanochemical machines that assemble at the centromere of each sister chromatid and engage on their outer face with the plus ends of k-fibres, microtubule bundles that emanate from the spindle poles. We envision the kinetochore as a set of interacting springs, clutches and motors and the problem of kinetochore mechanism as one of understanding how these functional modules assemble, disassemble and interact with one another to give rise the emergent properties of the kinetochore. Incisive experiments made over 17 years ago revealed that once sister kinetochores become attached to microtubules emanating from opposite poles (biorientation), they undergo a series of oscillations - termed chromosome directional instability - prior to anaphase onset. However, neither the mechanisms nor purpose of kinetochore directional switching in human cells is well understood. We propose a parallel approach employing mathematical modeling in tandem with higher-resolution tracking experiments of both native kinetochores and kinetochores specifically depleted of specific protein components. Oscillation is a high-level emergent property, providing a read-out of functional competence and a stringent quantitative test for the accuracy of our model. Iteratively refining the model in the light of our real-world data on oscillations will enumerate and deconvolve the contributions of mechanical components of the kinetochore to its emergent behaviour. We also expect to provide the first insight into the function of chromosome oscillations in animal cells.

Economic and societal beneficiaries and impacts The general public: It is well understood that multiple human diseases are a consequence of errors in chromosome segregation. For example, mis-segregation of chromosomes during the cell divisions that generate gametes (meiosis) are responsible for a wide range of syndromes such as Down's and Turner's and are also associated with >70% of miscarriages and stillbirths. These risks worsen as women age and our work has the potential to improve our understanding of how the success of chromosome segregation diminishes with time. Mis-segregation events during mitotic cell divisions are associated with the development of cancer. Moreover, changes in chromosome number are involved with cells becoming resistant to existing chematheraputic drugs. We need to know how the process of chromosome segregation works before we can unlock new therapeutic routes to deal with these major diseases and healthcare issues. Such advances will be of direct benefit to the general population both in terms of health, well-being, and indirectly on the socio-economic state of the United Kingdom. Translational medicine: Translational medicine has established itself as discipline in which research inputs from basic and social sciences are converted into patient benefits. Given the importance of chromosome segregation in multiple human diseases, including cancer and developmental disorders our basic-scientific work will generate new knowledge that could be utilized by translational medicine. Drugs that affect kinetochore motion are in clinical trials as candidate cancer treatments. 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. In addition, we are developing state-of-the art live-cell imaging assays and computational methods, which we predict will become an important part of the drug-development and testing process. Science outreach: Science outreach is vital so that the general population is aware of scientific advances and understands how they fit into the pipeline that takes discoveries in basic science and converts them in to new therapies and health improvements.