Elucidating the mechanism of endocytic invagination and scission

Cells are the basic unit of life and all organisms are composed of one or more cells. Cells need to interact with their environment to ensure that they respond correctly to signals that come from their surroundings. The majority of this interaction takes place through the proteins that lie on its surface. Endocytosis is an essential process in most eukaryotic cells. It involves a small amount of the outer (plasma) membrane of the cell being pulled inwards into the cell until some of this membrane pinches off to form a little sphere called a vesicle. This vesicle will contain fluid from outside the cell and, within its membrane it will contain proteins that were on the surface. A cell may want to remove these proteins from the surface because they are damaged, or because they can bind or respond to signals from outside that the cell no longer wants, or needs to respond to. Endocytosis is a very important way for a cell to control what is on its surface. Some pathogens or toxins can bind to proteins on the cell surface and trigger endocytosis. In this way these inappropriate substances can gain entry to the cell. Defects in the endocytic process have also been detected early in some neurological disorders such as Alzheimers.
Research in the Ayscough laboratory uses a simple one-celled organism Saccharomyces cerevisiae (bakers yeast) as a model system. Many processes are known to happen in the same way in this cell-type and in cells of more complex organisms such as mammals. We are particularly interested in the interplay between three types of protein that we, and others, have shown are critical in the inward movement of the membrane and its pinching off (scission) to form a vesicle. These proteins are called, dynamins, amphiphysins and actin. They are proposed to be involved in endocytosis but the exact step at which they function has been difficult to elucidate. One reason for this, is that much work on the relevant mammalian proteins has been performed only with purified proteins. It is not always easy to then translate this data into a physiological context. Manipulating the various mammalian systems has not always been straightforward and some experiments can take months to perform. Yeast provides a more simple situation to investigate, and we can study things within the context of the whole organism. We use imaging of fluorescently tagged proteins to investigate how the proteins of interest move in the cell. We can determine when the proteins localise to sites of endocytosis and how long they stay there. This imaging needs to be very sensitive as the endocytic sites are only fractions of a micron in size. Furthermore, the actual membrane invagination and scission events occur on a seconds timescale. Using yeast we can readily investigate the effect of changing just single amino acids within the dynamin or amphiphysin proteins. As well as using live cell imaging we are trying to generate synthetic systems, using pure proteins to reproduce the events that we have studied in the cells. Understanding how to manipulate membranes might be important in the future to generate functioning synthetic cells. Our approach will give new insights into how the proteins work at the molecular level. In turn, this will inform approaches in other, more complex systems studying these proteins in the context of both healthy and diseased cell types.

Endocytosis is an essential eukaryotic cell process that is required to regulate cell surface composition so allowing cells to interact appropriately with their environment. Recent advances in our understanding of endocytosis has indicated it may play an important role in various diseases including Alzheimers, Huntington's, epilepsy and cancer, highlighting the need to understand more about the complex mechanisms involved. Research in yeast has been central to our understanding of the mechanism of membrane invagination at the onset of endocytosis. A recent detailed analysis of endocytic sites in mammalian cells has demonstrated that the spatiotemporal organisation of protein complexes during invagination and scission are very similar to that in yeast. The growing realisation of the importance of actin-driven invagination in certain mammalian cell types, and our recent demonstration of the interplay between yeast dynamin and amphiphysins in endocytosis, will allow us to identify underlying principles, and draw widely applicable conclusions about this fundamental cell process.
The work outlined here will use an in vivo, manipulable yeast system and, based on current assays we will develop relevant complementary in vitro synthetic models for invagination and scission. We have already generated many tools such as plasmids and mutant yeast strains, and will make rapid progress in our analysis to determine the importance of actin and lipid interactions with dynamin. We will also work towards generating models and computer simulations to generate predictions about the role of key proteins that will then be tested within the system. Overall this programme of work will lead to genuine mechanistic insight into the fundamental processes of membrane invagination and scission. The outcomes will be relevant to a wide range of scientists working in membrane trafficking and, within a wider sphere will be important for our ability to manipulate membranes in synthetic systems.

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, 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 epilepsy, neuromuscular disorders (Charcot Marie Tooth disorder and centronuclear myopathy), neurodegenerative disorders, (Alzheimers and Huntingtons), and cancer. It is critical that we understand the pathways affected in these disorders so that any therapeutics 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 this 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 schools to give talks and run activities. I am also involved in Departmental open days and UCAS visits during which time I explain projects 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. Preliminary meetings have been made and there is a timescale: to submit an Arts Fund application to Wellcome in Jan2012.