The regulation of dynein mechanochemistry in vivo

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Cells contain a system of filaments, called microtubules, that are made up of polymers of a protein call tubulin. Microtubules act as tracks within the cell for the transport of small structures from one region to another. This delivery system relies on proteins that 'walk' along microtubules, acting as minute motors. These motors can be attached to a variety of different structures in the cell, or 'cargoes', in much the same way that a railway engine can pull passenger coaches or freight wagons. This grant application addresses the function of a motor called dynein. Dynein moves cargoes from the peripheral regions of a cell towards the cell centre. Such transport is especially important in nerve cells, where the distance from the periphery to the centre can be very long indeed; even with this active transport system, this journey will take 2.5 days for material carried all the way from the tip of a nerve cell in your big toe to the cell body in your spinal cord. For this reason, mutations that may only mildly affect dynein function lead to neurodegenerative diseases. It is important to note, however, dynein is important for the function of all cells in the body. Since full dynein activity is so vital for cell function, this grant will investigate what is needed for the motor unit to work at maximum efficiency. Dynein itself is very complicated, being made up of several different proteins. Other proteins are needed to link dynein to its cargoes, and to assemble a functional motor unit. Altogether, the unit contains the engine that drives the dynein motor, the means to link dynein both to the microtubule track and to the cargoes, and the means to change gears so that dynein can move at a range of speeds. The grant will test the hypothesis that specific proteins that interact with dynein within the motor unit are important, either for cargo attachment or gearing. Testing this hypothesis requires an experimental system in which dynein activity can be followed in minute detail. For this reason, we will exploit the fact that dynein moves a particular set of cargoes called endosomes. These are tiny bags made up of lipid and protein that are used to deliver important material such as nutrients from outside the cell to the cell centre. For this reason they must be transported rapidly in an inward fashion, using dynein. One critical advantage of using endosomes as a model cargo is that they can be visualized quite easily, since they can be loaded with materials that can be seen under the microscope. One such material is gold. Our laboratories are equipped with cameras that image at astonishingly high frames rates (10,000 frames per second and over). This allows us to follow gold-labelled endosomes moving using dynein in such detail that individual strokes, or steps, of the motor's engine can be identified. It is thought that the length of these steps, or their frequency, influences how fast the cargo moves. We are in a position to test this directly, by seeing how they change as endosomes speed up or slow down. In addition, by manipulating the copy number and function of the proteins thought to affect cargo binding or gearing of the motor, we will test whether these proteins do influence endosome speed, and if so, whether they exert these effects by modulating step length or frequency.