Characterisation of Arabidopsis myosin recruitment

Plant research has an increasingly important global impact on society; in the areas of agriculture/sustainable crops, biofuels (fuel from plant biomass/material), and using them as tools for synthesising pharmaceuticals. Unlike mammals, plants are (1) sessile and have developed adaptive responses to cope with changes in the environment, and (2) are photoautotrophic and therefore synthesise all the necessary macromolecules which have to be efficiently sent to the relevant compartments/destinations in the cell to meet requirements for growth and development. Cells contain intricate scaffolds (cytoskeleton) upon which compartments (organelles) move along with the aid of molecular motors (myosins) to reach their destination in the cell. This process is analogous to the intricate road network throughout the country; vehicles travelling along the motorway is similar to the 'super highway' in cells where cytoplasmic streaming takes place and organelles can move at impressive speeds up to 5-6 microns/second (analogous to an Olympic sprinter running 40kmph), and the smaller byroads and streets are indicative of slower organelle movement where they appear to meander around in a small area within the cell. Organelle movement in the cell is very dynamic and appears almost chaotic and random. However, this apparent disorder is altered upon pathogen invasion where certain organelles appear to cluster around the site of invasion. Also, certain metabolic pathways span several organelles (e.g. photorespiration and the glyoxylate cycle), and these organelles appear to lie close to one another. It was suggested that location/movement of organelles in these instances is to allow a quick and efficient release of components to combat invasion / metabolism, but this has never been experimentally proven. Our model organism is Arabidopsis which contains 17 myosin genes. Over the past 2 years we have shown that mutant forms of six myosin motors appear to indiscriminately control the movement of all types of organelle. By using microscopy of living cells which have fluorescently tagged organelles and mutant forms of myosin, we will be able to understand which parts of the myosin are required for (1) recruitment to the organelle surface and (2) effect movement. A screen for the proteins (signals) involved in recruiting myosins to the organelle surface and mediating the motors' effect on organelle movement will also be undertaken. We have already successfully developed several analytical tools to map and quantify movement of different types of organelles which will be used in these studies. By understanding how organelle movement is controlled at the molecular level, we will in future be able to start dissecting the role of specific types of organelle movement for plant growth, adaptation and development. This fundamental research has several implications including the potential to generate plants which are better able to adapt to changes in the environment and pathogen attack, and will therefore result in increased crop yields, vital for the increasing world population and demand on food and biofuel in the changing global climate.

The aim of this proposal is to build upon the applicants' recent success in publishing a comprehensive study of the effects of all 17 Arabidopsis myosins on organelle movement, and the effect of XIK on ER network remodelling. This was achieved through the development of high speed scanning, Volocity tracking software and cumulative distribution frequency plots for comparing whole population dynamics, and a novel analytical tool, persistency mapping, to quantify effects on ER network dynamics in collaboration with Dr. L. Griffing. This proposal will use these tools to determine how myosins MYA2 and XIK are recruited to the surface of specific classes of organelle to mediate movement. By using a combination of live cell imaging and transient expression of MYA2 / XIK tail domain fusions, the effects on organelle movement and recruitment will be monitored and the tail domains of both myosins will be mapped. Novel XIK tail interactors will be identified through a two pronged approach, a commercial yeast-2-hybrid screen and STREP tagging. Potential interaction between MYA2 and RabC2a and RabD1 will be tested in vivo, followed by interaction studies to determine GTP dependency and role in recruitment and movement of organelles. All interactions will be confirmed through FRET-FLIM, a well established technique in the lab carried out at the Central Laser Facility at the Rutherford Appleton Labs., Harwell and by bimolecular complementation.

Plant organelles display a range of dynamic behaviours, yet the role(s) of these movements are far from understood. Recently, it was proposed that a decrease in organelle movement perturbs plant growth and development, and organelle positioning alters upon pathogen attack and changes in heavy metal levels. Therefore, genetic tools which can perturb the movement of certain classes of organelle over another will not only provide model systems for studying the role of organelle movement under different environmental stimuli, but could also provide economic benefits in terms of increasing plant productivity and yield through genetic engineering. In the event that any immediately exploitable IP arises from the project Oxford Brookes Research and Business Development Office has staff specifically dedicated to identifying potential for commercialisation and has a working agreement with ISIS Innovation (Oxford University) who mentor any spin out activity. Dr. Sparkes has led several successful collaborations; the fundamental characterization of the role of 17 Arabidopsis myosin genes on organelle movement (Plant Physiology 2009), and the development of a novel persistency mapping tool to characterize and quantify the role of the cytoskeleton and myosin XIK on ER remodeling with Dr. L. Griffing (Plant Cell 2009). The proposal outlined will build upon this success and generate more novel analytical tools which will benefit the scientific community. The PI was awarded a fellowship in October 2009 and so is at an early stage in her career. She has supervised undergraduate, postgraduate and an MSc student which has resulted in publications including a recent article in Plant Cell on ER shaping proteins. The PI has therefore demonstrated that she can provide mentorship and support to achieve successful end goals with those under her supervision. The work produced from the proposal will be disseminated to the scientific community through publications and seminars, and conveyed to the wider audience through out reach activities currently in place within the Plant Cell Biology group at Oxford Brookes University (e.g. demonstration of live cell imaging of plants during the Oxford Science Festival, running short imaging workshops and demonstrating imaging equipment to schools).