Defining factors that ensure unidirectionality of endocytosis

Cells are the basic unit of life and all organisms are composed of one or more cells. These cells are covered in proteins that are embedded in their cell surface. This protein coat helps cells to perform certain functions such as responding to chemical messages. The coat also lets other cells recognise it. An analogy would be recognising groups of people according to the clothes they wear. However, sometimes a message that a cell receives tells it to behave differently. It therefore needs to remove elements of its protein coat. One of the ways that many cells do this is by a process of internalising some of the proteins from the coat. This protein internalisation is achieved by a process called endocytosis. The surface of the cell folds inwards in small portions and then pinches off inside the cell. This forms a small vesicle that can then carry the protein away inside the cell. Often the proteins get broken down and their most basic parts are released for re-use by the cell. In this way the parts of the coat that are not needed can be removed. At the same time, this process is often balanced by the addition of new proteins, which respond to different messages, to the cell surface. If the proteins are not removed properly because endocytosis is not functioning correctly, the cell could have proteins at the surface that send conflicting messages that may be detrimental to the well-being of the cell. Several diseases have been associated with defective endocytosis including Alzheimers, Huntingtons and cancer. In addition, several pathogens and toxins exploit the endocytic pathway to gain entry to cells.
This project aims to use a simple system to understand how cells can take-up proteins from their surface. We are using a model organism called yeast because the molecules involved in the endocytic process are very similar to those in more complex human cells, but the yeast cells are much easier to manipulate to investigate the processes we are interested in. In particular we are trying to determine the mechanisms that ensure that once the internalisation process has begun, that it continues to completion. We know that the endocytosis process involves more than 50 proteins. Many of them form groups or complexes with one another, and then components of these groups interact with a single factor that is called Las17/WASP. We aim to test the idea that this particular protein acts as a ratchet in the endocytic process. By closely regulating the interactions of this one protein, the cell can ensure that interactions take place sequentially. Overall, we think that this might be the mechanism that ensures key stages of endocytosis proceed in a linear fashion rather than starting and then reversing or aborting before the invagination has completed and the membrane has pinched off inside the cell. We will use a wide range of techniques both inside and outside of the cell environment to investigate the interactions of Las17/WASP and to determine how these interactions are regulated to ensure endocytosis functions correctly.

Endocytosis is a highly regulated and essential process required to regulate cell surface composition. Advances in our understanding of endocytosis has indicated that it may play an important role in various diseases including Alzheimers, Huntington's, Dent's disease, epilepsy and cancer. Clathrin-mediated endocytosis is a well-characterised pathway in both yeast and mammalian cells. During endocytosis plasma membrane invaginates into the cell resulting in the production of a vesicle that is then able to fuse with endosomes and enter the endolysosomal membrane system. Live cell imaging has revealed that the endocytic process involves a choreographed pathway with sequential and transient assembly, followed by disassembly of the proteins that localize to endocytic sites. It is considered that the stages of coat assembly (early), invagination (mid) and scission/inward movement (late) are conserved across evolution, and in many cases direct homologues of proteins are responsible for carrying out equivalent steps in the process.
The overall aim of this research is to elucidate how the Las17/WASP protein is regulated, so its many interactions take place sequentially, and to determine how this mechanism ensures unidirectionality of endocytosis. Using a multidisciplinary approach, and exploiting the versatility of the yeast model system, our work will yield significant insights into this fundamental eukaryotic process. We will combine genetic manipulation of yeast, with live cell imaging and high-resolution ultrastructural analysis to determine the contributions of key players in the assembly and disassembly of the endocytic complex. Biochemical approaches will also be used to gain a deeper mechanistic knowledge of the interactions that take place at the protein level. The complementarity of approaches, advances already made, and the fundamental importance of the proteins, make this study timely, and likely to yield high impact outputs within the lifetime of the grant.

This project will tackle a very important cell biological issue that is 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. Reviews will ensure coverage not just to those in the immediate field, but to a broader audience of biologists at a range of academic levels. Work at this level enhances the reputation of UK science and this is key to confidence in the competitiveness of UK science, which is directly related to wealth and economic output of the higher education industry. Timescale 12 months+.
(ii) Health related disciplines will benefit from this study. There is potential to influence understanding about neurodegenerative disorders, (e.g. Alzheimers and Huntingtons), epilepsy and cancer. It is critical that we understand the pathways affected in these disorders so that any therepeutics can target more specifically. Improved understanding of these diseases will impact on treatments and therefore directly on patients and wider society. There is also the potential to influence policy on such treatments. 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. 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. Timescale is difficult to judge, though, relevant industries could be contacted to explore collaborative possibilities within 24 months.
In addition, postdocs and students from labs such as mine are likely to enter industry and carry out much of the Research and Development in such arenas. For this reason, our students/post-docs area in Sheffield are encouraged to become critical and independent thinkers and 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 a STEM Ambassador and 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 some impact will be 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 a collaboration with a ceramic artist in Cardiff to develop ways to portray aspects of cell structure in this highly tactile and accessible medium. Timescale: to submit an Arts Fund application to Wellcome in October 2011.