The regulation of dynein mechanochemistry in vivo

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.

Cytoplasmic dynein is the principal minus end-directed microtubule motor in animal cells, responsible for transporting a wide range of essential cargoes towards the cell centre. Biophysical studies have been performed using purified dynein, but the relevance of these to the in vivo activity of the motor remains unclear. Specifically, we have found that dynein can move early endosomes, labelled with GFP-Rab5, at rates of up to 8 um/sec, almost 10 times as fast as the rate of purified dynein in vitro. Our preliminary observations indicate that even single dynein motors can move endosomes at these speeds. In addition, we find that individual GFP-Rab5-labelled endosomes can change speed during a dynein-driven run, suggesting that the motor activity is subject to rapid alterations. We aim to extend this work to examine in far greater detail how dynein moves endocytic organelles in vivo. Our principal technical innovation will be to load endosomes or lysosomes with endocytic tracer conjugated to gold particles, and then image these using bright-field microscopy and imaging rates of 10,000 frames/sec. These acquisition conditions will allow us to resolve dynein-driven motility into individual motor steps. Using this information, we will address the following aims: 1. To examine in detail the mechanochemical properties of dynein-driven endosome movement in vivo. Previous studies have shown that dynein can generate step sizes of between 8 and 32 nm. We will examine whether changes in endosome speed during a run correlate with alterations to dynein's step size and/or frequency. 2. To test whether dynein acting as a single copy motor can move endosomes at high speeds in vivo, or whether multiple motors are required to achieve these speeds. 3. To perform a systematic analysis of a group of dynein interactors, Lis1, Nde, Ndel1, ZW10 and BicD, to determine how they work together to support fast, long-range dynein-driven movement.

There are potential long term benefits to health and bionanotechnology in the long term from the knowledge that will be obtained through this research. Dyneins have a wide range of pharmaceutical applications e.g. successful gene therapy requires fast active transport to the nucleus to deliver the DNA. Dynein transport is used naturally by viruses to avoid the body's defenses and could be targeted for improved synthetic carriers. Medical industry may be interested in ways to treat hereditary diseases associated with cytoplasmic dynein dysfunction e.g. retinitis pigmentosa, Lissencephaly (smooth brain disease), motor neuron disease, perhaps by being able to stimulate dynein activity. In addition, the findings from this research may be significant for the bionanotechnology field, if they enable the development of more robust motors. The primary means of informing these communities of our work is through publication in the scientific literature. In addition, the PI, Co-Is and RAs will all be expected to play an active role in disseminating information to increase the impact of the research. They will present their work at both national and international conferences. Waigh has existing contacts with Malvern Instruments and Unilever Colworth. Development of robust intracellular particle tracking techniques is a key enabling technology for a wide range of modern biotechnology applications. A huge amount of information can be mined from parallelized intracellular tracking experiments (1000s of particles tracked simultaneously in a single cell) and it is believed that they will become a standard systems biology tool for exploring single cell physiology. It is expected that the imaging community will find these methods of use. While they will learn of these advances through publication, Allan also expects to be able to share this information through the Eurobioimaging an UK Bioimaging networks that are just being established. Waigh is a committee member of the Biological Physics and Polymer Physics groups at the Institute of Physics. He has organized meetings on biopolymers, photonic instrumentation and general life sciences topics. Further meetings will be organized on cellular biophysics during the duration of the grant. The analysis software will be made freely available via the internet. Waigh has previously released the PolyParticleTracker software in this way. Any findings that could be of commercial interest will be developed via University of Manchester Intellectual Property Ltd. The function of microtubule motors is a topic that will be of general interest to the public, mainly because of the immediate visual impact of the work. Allan and Woodman have links with the Manchester Museum, who organise science days regularly. Both the Woodman and Allan laboratories have recently assisted with school science activities. Woodman has recently written articles about their research in Biological Sciences Review, a journal aimed at school students. The Faculty encourages staff to communicate their work. The most appropriate route will be communicating with schools as part of our outreach programmes, which are organised by the Faculty's outreach officer. Students visit our laboratories in order to find out about research. FLS has press officers who will help us promote our research to the general public via the media.