Elucidating the mechanism of microtubule depolymerisation by Kip3D kinesin

Your body has a skeleton that gives you strength and support and allows you to move. The cells of your body also need strength and support, so cells also have a skeleton called the cytoskeleton. The cytoskeleton is built from 3 different types of fibre-like structures: one of these is called the microtubule. The aim of this project is to understand some of the functions that microtubules perform in cells, especially in cells that are multiplying in number. This is a process called mitosis. It is important to understand as much about mitosis as we can because it is very important for human health in lots of different ways. We all start life as just one cell and it is only after cells multiply billions of times that there are enough cells to make a complete human body. When mitosis goes wrong, the human body may not develop properly and this can cause birth defects and severe health problems. Also, when cells multiply out of control, cancer can occur. The more we understand about how cells multiply and what enzymes are important for controlling this multiplication, the better we will be able to find ways to stop mitosis from going wrong. The microtubule cytoskeleton is different from the human skeleton because it can change shape depending on what the cell needs to do or be. For example, a cell that is going through mitosis has a very different shape and behaviour from a cell that is not. Cells use different types of enzymes to control the shape of the microtubule cytoskeleton. In this project, we want to understand how an enzyme called Kip3D controls microtubule shape. Kip3D is an microscopic motor that uses the energy from ATP to do work when it binds to microtubules. Kip3D uses the energy of ATP to pull microtubules to pieces (depolymerisation). This is especially important in mitosis when the organisation of the microtubule cytoskeleton is constantly changing to ensure that mitosis is successful. We will use an electron microscope - a very powerful kind of microscope that takes pictures at very high magnification - to see what Kip3D looks like when it interacts with microtubules. Looking at the structure of Kip3D bound to microtubules will help us understand how the Kip3D molecules use ATP to depolymerise microtubules. When we understand how individual molecules of Kip3D work, we will understand better how Kip3D functions in a multiplying cell this and will help us find new ways to treat human diseases like cancer.

The dynamic nature of the microtubule cytoskeleton is crucial to its cellular role. Pure polymerised tubulin is intrinsically dynamic but the cell employs multiple factors to ensure that microtubule growth and shrinkage is precisely coordinated with cellular events. This complex coordination is impressively demonstrated during mitosis, when the many components of the microtubule-based mitotic spindle must accurately separate the cell's replicated genome. In particular, microtubule depolymerisation is catalysed by members of the kinesin superfamily of ATP-dependent microtubule motors. The best understood of these are the kinesin-13s. These motors depolymerise microtubules from their ends by using the energy from binding ATP to bend the terminal tubulin dimers with which they interact. This brings about release of the tubulin dimer and thus, microtubule depolymerisation. In vivo studies of several kinesin-8 family members suggest that these motors are also microtubule depolymerisers, but the molecular mechanism by which this is achieved is unknown. The aim of this project is to elucidate the microtubule depolymerising mechanism of the essential human mitotic kinesin-8 family member, Kip3D. We will take two main experimental approaches to understand how Kip3D harnesses the energy of ATP when it interacts with its microtubule substrate: 1) we will use a cosedimentation assay to evaluate how ATP binding and hydrolysis by the motor is coupled to affinity changes for its microtubule substrate. We will use nucleotide analogues to mimic different stages of the motor ATPase cycle. Kinesins are force-generating motors and the force-generating steps of the Kip3D ATPase cycle are likely to be coupled to high affinity states. 2) we will use cryo-electron microscopy and image reconstruction methods to calculate the structure of the motor with its microtubule substrate in different nucleotide states (again using nucleotide analogues) to enable us to visualise the conformational changes which occur in the motor as ATP is bound and hydrolysed. We will also compare the Kip3D-microtubule complex with the Kip3D interaction with curved tubulin oligomers, formed in the presence of the drug dolastatin. We will use both the cosedimentation assay and cryo-electron microscopy methods to examine this interaction. It is thought that these oligomers mimic the flexibility of microtubule ends, the site of microtubule depolymerisation. Characterisation of the affinity of Kip3D for a bent tubulin conformation and examination of the structure of this interaction will provide us with additional insight into the specific nature of the depolymerisation step. Integration of both biochemical and structural information will enable us to understand how Kip3D depolymerises microtubules. We will take a divide-and-conquer approach to understanding the molecular mechanism of the entire Kip3D molecule and will begin by examining the structure and function of the Kip3D motor core. Although the motor cores represents the most highly conserved domains in all kinesins, they frequently contain family-specific residues that define the functions of the entire molecule. Information about the structure and function of the Kip3D motor core will be used to design point mutations that test hypotheses about the key residues involved in ATP binding, motor-tubulin interaction and the residues that define the depolymerisation activity. We will then be able to examine larger Kip3D constructs to understand motor function in the context of the whole polypeptide.