Elucidating the molecular mechanism of Arp2/3-independent actin nucleation by WASP family proteins

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Cells are the basic unit of life and all organisms are composed of one or more cells. Central to the functioning of many cells, including human cells, is the internal skeleton, or cytoskeleton. This cytoskeleton is required for cells to have certain shapes that are often a necessary part of their functioning. However, unlike our own body skeleton that is static, the cytoskeleton is able to remodel itself to change cell shape, or allow a cell to move. One of the most important proteins in the cytoskeleton is called actin. It is an amazing protein because it is almost the same now as hundreds of millions of years ago, long before humans, or even vertebrates existed. Staying so similar over time is called evolutionary conservation. Proteins that are very important to cell functioning are the most highly conserved. Actin is a protein that can join together with other actin proteins to form long lines or filaments. These filaments can be organised by other proteins to form large structures that are part of the cytoskeleton. We are interested in how actin is controlled in cells and in particular, we are trying to determine how the filaments can be started from single actin proteins. This is a process called nucleation. Our study will help us understand the mechanism of this nucleation in cells. We think that it is important because actin is known to be necessary for cell movement, and this behaviour often changes in cancer cells that have become metastatic. Actin is also involved when pathogens invade our cells. One of the proteins known to be important in helping actin form new filaments is called WASP, which becomes defective in an immune-deficiency disorder called Wiskott Aldrich Syndrome. Because actin is a highly conserved protein (85% identical between yeast and humans), we have undertaken many of our studies in yeast to gain insight into fundamental aspects of actin function. Manipulating mammalian systems is not always straightforward and some experiments can take months to perform. Yeast provides a more simple system to investigate, and we can study things within the context of the whole organism as well as analysing different components individually. This complementarity of approaches is important to gain a deep understanding of a process. We also aim to exploit finding by undertaking informed experiments on mammalian proteins and cells. There are many examples of studies in yeast that have shed new light on processes in more complex organisms.Until recently it was considered that WASP and other proteins like it, work to activate a group of proteins called the Arp2/3 nucleation complex or Arp2/3. We have shown that the yeast WASP, called Las17, is able to nucleate actin without Arp2/3 present. We were also able to show that this nucleation activity is important for the function of Las17 inside cells. Importantly, we have also shown that two related mammalian proteins can generate filaments in similar conditions, suggesting that the property is conserved. We now want to learn more about the mechanism of actin nucleation by these proteins as it may underpin a new understanding of nucleation at specific sites in cells. Overall, this project is highly relevant to our understanding of key cell processes of cell organization, membrane trafficking and motility. While focused on the yeast WASP homologue our preliminary data indicates that the major findings will be of wide significance for many proteins both of the WASP family and also other proteins such as those expressed on pathogens that also drive actin filament formation. Our approach is highly complementary to, but distinct from, those used in other labs. We have already generated many tools and reagents for this work which means that we can make rapid progress towards our goals, and the outputs have the potential to be published in top international journals thus enhancing UK competitiveness in science.