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

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.

The generation and re-organisation of actin filaments is central to many cell processes, and is closely linked to motility, morphology and membrane trafficking. Understanding how actin filaments are generated from actin monomers is critical if we are to determine how actin can be recruited and function at specific sites within healthy cells, and how this processes is altered in cancerous cells or during pathogen invasion. We have used the model organism, Saccharomyces cerevisiae to investigate formation of actin filaments de novo. We have shown that Las17, the homologue of the Wiskott Aldrich Syndrome protein (WASP) can function in the absence of the well-characterized nucleator Arp2/3 to drive actin nucleation and polymerization. The actin nucleating functionality resides in the central poly-proline region, and disrupting this function by mutation of specific prolines causes severe defects in actin organization. We have also shown that mammalian WASP and N-WASP are able to generate filaments in the absence of Arp2/3.
Through this study we will elucidate the molecular mechanism of Arp2/3-independent actin nucleation by WASP family proteins. Through a series of well developed, integrated biochemical and cell biological approaches we will determine fundamental properties of the polyproline region and gain insight into how it interacts with actin to support filament nucleation. We will also increase understanding of regulation of Las17/WASP in actin organization and membrane trafficking through analysis of phosphorylation and its interaction with SH3 domain-containing proteins. This is an ambitious project, but entirely achievable due to generation of relevant tools and expertise. We consider that the project will provide important advances in our conceptual understanding of how WASP family proteins, and possibly other proteins with polyproline stretches, are able to organize and nucleate actin in cells.

This project will tackle our fundamental understanding of critical cell processes making it highly significant in the wider economic and societal arena. Our preliminary work in making relevant, quantitative observations and generating many tools for the study will allow us to make rapid progress and to gain substantial insights in this important area of research in a relatively short timescale.
(A) Potential Beneficiaries
Beneficiaries of the research will be academics, health professionals, industry, schools and the wider community
(B) How might they benefit?
(i) Academic beneficiaries will be researchers in the areas of yeast and mammalian cell biology as well as structural biologists, geneticists and modellers. PIs and post-docs will present work at relevant meetings and this will be backed up by publications including reviews to make the work accessible to a wider audience at a range of academic levels. Work at this level enhances the reputation and competitiveness of UK science, which is directly related to wealth and economic output of the higher education industry. Timescale 18 months+.
(ii) Health related disciplines will benefit from this study. There is potential to influence understanding about cancer in terms of cell motility mechanisms, immune disorders (Wiskott Aldrich syndrome) and pathogen invasion which often involves actin cytoskeleton. It is critical that we understand the pathways affected in these disorders so that any therapeutics can be targeted more specifically. Improved understanding of these diseases will impact on treatments and therefore directly on patients and wider society. Timescale for increased understanding in fields relevant to at least some of the diseases 18+ months.
(iii) Industry. Fungal diseases are hard to treat and most drugs are fungostatic rather than fungotoxic. There is a significant interest in anti-fungal drugs by industry as the diseases are widespread and are associated with high mortality. Identification of new targets therefore has the potential to yield new drug targets. Any development that allows new drugs to be developed would be a marked benefit to the economy. Our phosphorylation analysis and understanding of regulatory events for endocytosis may be of particular interest to industry. Timescale is difficult to judge, though, relevant industries could be contacted to explore collaborative possibilities within 24 months.
In addition, postdocs and students from the lab enter industry and carry out much of the Research and Development in such arenas. For this reason, our students/post-docs in Sheffield are encouraged to become critical and independent thinkers and, through the Think Ahead programme, to consider their wider range of skills and how they might be applied in a range of workplace environments .
(iv) Schools. The future of science depends on enthusiastic young scientists. The best way to achieve this is to provide stimulating scientific based activities for school children. The main applicant is involved in visits to local schools to give talks and run activities. I am also involved in Departmental open days and UCAS visits during which time I explain projects in the department to parents and prospective students. Timescale: schools are visited at least every year. Clearly impact is mostly longer term. However, feedback from students on open days has been very positive particularly with respect to the scientific displays and their final decision to apply to Sheffield for their degree.
(v) Wider society continues to show either apathy or even fear of science. One way in which this can be addressed is through a completely different approach such as the arts. In terms of translating science in art, KA has established collaborations with a ceramic artist and an illustrator in Cardiff to develop ways to portray aspects of cell structure in these distinct media.