Kinetochore life-histories: understanding the mechanical events that ensure error-free chromosome segregation

A fundamental challenge in modern cell biology is to understand how complex behaviours emerge from populations of molecular machines, machines that work close to the thermal energy level, thereby giving their behaviour a significant random component, whilst working independently within the context of a global communication network. Working with such systems poses significant challenges given their small size; however recent advances in light microscopy has enabled these systems to be observed and quantified with unprecedented spatial and time resolution.

The biological system we are interested in is how chromosomes are separated during cell division. Human beings are built from a single fertilised cell, the zygote. Each cell contains 46 chromosomes - the packages of genetic material (DNA), which provide the instructions for how a cell should work and how to build a human. Cells divide generating two near identical daughter cells. The chromosomes are copied prior to division and a key part of cell division is the accurate separation of these replicated chromosomes such that each daughter receives 1 and only 1 copy. Failure of a cell to receive a complete set of 46 chromosomes is a cause of multiple human diseases, including cancers and Down's syndrome. During the mechanical process of cell division, most of the time is taken up with relocating the paired chromosomes (original and copy) into a holding pattern at the equatorial plane, prior to pulling the pairs apart to either end of the cell. Clearly it is vital that we work out how chromosome separation works.

Chromosome separation is a mechanical spatial process. 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 attach to these cables, both on their sides (lateral attachment) and to the ends of these cables. The kinetochore is an extremely versatile and 'intelligent' machine, comprising sensors and motors that allow it to determine how it is attached to microtubules and how its paired sister is attached, making a sequence of informed decisions so that the paired chromosomes are relocated to the equatorial plane. The kinetochore is thus the "control centre" that decides when and where a chromosome moves. But, how does the kinetochore do this? Why and how do kinetochores make decisions, and using what information as input? 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 faithfully separated into daughter cells during cell division.

We will use state-of-the-art imaging technology (microscopes) to observe how chromosomes move in living human cells. We will then use mathematical modelling and sophisticated statistical techniques (called reverse engineering) to determine the parameters of that model from the data (one engineers what the system must be from the observations). This will allow us to infer what cues the kinetochore is using to regulate the attached microtubules. This will include forces acting on the kinetochores and internal stretch and rotation of the kinetochore, our previous study showing that the kinetochore has a structure similar to a hip joint which potentially prevents breakage of the attachment under impulse forces. Our model will only be as good as our data; thus we will use a variety of techniques to perturb the system (remove or knock-down certain components, thereby changing behaviour), and thus 'road-test' our model through a range of different situations. Through such techniques we will achieve greater biological understanding.

Chromosome congression is a complex, highly orchestrated spatial mechanical process, the kinetochore being central to brokering and regulating forces that position chromosomes within the cell. Kinetochores are multi-protein complexes that navigate the microtubule spindle by regulating their attachment to microtubules, demonstrating an ability to self-organise to the metaphase plate (at the equatorial plane) through sensory and mechanical cues, although the exact signals are unknown. Multiple, layered, processes are involved, including chromosome capture, lateral movement along microtubules, lateral to end-on conversion, maturation of microtubule attachments and error correction, whilst mal-attachment states generate signals that regulate the SAC and delay anaphase. Elucidating the mechanisms and signals that control this range of processes is the primary aim of this proposal. We propose a state-of-the-art combined experimental and computational study, utilising the latest high resolution live-cell imaging (light sheet) and experimental techniques (CRISPR, correlative serial block-face scanning electron microscopy) with sophisticated computational algorithms to automatically annotate trajectories for their attachment state based on their dynamic signature. We will use endogenously tagged reporters for the first time, with 2 and 3 channel live cell microscopy to quantify both kinetochore conformation and protein recruitment, thereby ascertaining how kinetochore maturation, inter-kinetochore forces and intra-kinetochore stress determine kinetochore function that ultimately leads to high fidelity segregation. By detecting state switching at high resolution throughout congression, we will determine the causal factors for late congressing chromosomes, and crucially the risk factors for segregation errors at anaphase. We will validate our results by utilising a range of perturbations, and develop a data driven mathematical model of chromosome congression and segregation.

Economic and societal impact.
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; 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 chemotherapeutic 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.