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

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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.