Elucidating the mechanism of endocytic invagination and scission
Lead Research Organisation:
University of Sheffield
Department Name: Molecular Biology and Biotechnology
Abstract
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.
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.
Technical Summary
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.
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.
Planned Impact
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.
(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.
Publications
Urbanek AN
(2015)
Function and interactions of the Ysc84/SH3yl1 family of actin- and lipid-binding proteins.
in Biochemical Society transactions
Palmer SE
(2015)
A Charge Swap mutation E461K in the yeast dynamin Vps1 reduces endocytic invagination.
in Communicative & integrative biology
Palmer SE
(2015)
A dynamin-actin interaction is required for vesicle scission during endocytosis in yeast.
in Current biology : CB
Rzepnikowska W
(2017)
Amino acid substitution equivalent to human chorea-acanthocytosis I2771R in yeast Vps13 protein affects its binding to phosphatidylinositol 3-phosphate.
in Human molecular genetics
Moustaq L
(2016)
Insights into dynamin-associated disorders through analysis of equivalent mutations in the yeast dynamin Vps1.
in Microbial cell (Graz, Austria)
Smaczynska-De Rooij I
(2023)
Phosphorylation Regulates the Endocytic Function of the Yeast Dynamin-Related Protein Vps1
in Molecular and Cellular Biology
Alpadi K
(2013)
Dynamin-SNARE interactions control trans-SNARE formation in intracellular membrane fusion.
in Nature communications
Smaczynska-De Rooij II
(2019)
Mutation of key lysine residues in the Insert B region of the yeast dynamin Vps1 disrupts lipid binding and causes defects in endocytosis.
in PloS one
Aghamohammadzadeh S
(2014)
An Abp1-dependent route of endocytosis functions when the classical endocytic pathway in yeast is inhibited.
in PloS one
Urbanek AN
(2015)
Distinct Actin and Lipid Binding Sites in Ysc84 Are Required during Early Stages of Yeast Endocytosis.
in PloS one
Description | We have discovered that the yeast dynamin protein binds to actin and that this interaction is important for its function in endocytosis. FIndings published in Current Biology 2015 We have shown that Vps1 is phosphorylated and that this modification is important for its function in endocytosis. FIndings published in Molecular Cellular Biology 2015. We haved defined a domain important for lipid binding and have shown that it binds to a small subset of lipids in membranes. This outcome is published in PLoS One 2019 We have investigated Vps1 as a model for understanding disease mutations in dynamin. Publication accepted, Microbial Cell March 2016. |
Exploitation Route | Our demonstration that generation of mutations in Vps1 equivalent to those causing disease in mammalian dynamins might help in understanding how the mechanism of dynamin function is affected when other mutations are discovered. Our demonstration of links between Vps1 and actin in endocytosis can further understanding of the biophysical mechanisms of endocytic scission |
Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |
Description | BBSRC White Rose DTP |
Amount | £60,000 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2017 |
End | 09/2021 |
Description | Polish Collaboration |
Organisation | Polish Academy of Sciences |
Department | Institute of Biochemistry and Biophysics |
Country | Poland |
Sector | Public |
PI Contribution | We have hosted a research student fom the Polish lab for 4 months and have trained the student in a number of techniques. We have continued to share outcomes and to collaborate including undertaking experiments according to relevant expertise in areas of actin and membrane traffickign in S.cerevisiae |
Collaborator Contribution | The student has visited and worked on a project related to some of our work. The Polish lab has made many mutant strains and undertaken extensive genetic analysis. |
Impact | 1. Soczewka P, Kolakowski D, Smaczynska-de Rooij I, Rzepnikowska W, Ayscough KR, Kaminska J, Zoladek T. (2019). Yeast-model-based study identified myosin- and calcium-dependent calmodulin signalling as a potential target for drug intervention in chorea-acanthocytosis. Dis. Model Mech. 12(1). PMID:30635263 2. Rzepnikowska W, Flis K, Kaminska J, Grynberg M, Urbanek AN, Ayscough KR, Zoladek T. (2017) Amino acid substitution equivalent to human chorea-acanthocytosis I2771R in yeast Vps13 protein affects its binding to phosphatidylinositol 3-phosphate. Hum Mol. Genet 26:1497-1510. PMID 28334785. 3. Kaminska J, Rzepnikowska W, Polak A, Flis C, Soczewka P, Bala K, Sienko M, Grynberg M, Kaliszewski P, Urbanek A, Ayscough K, Zoladek T. (2016) Phosphatidylinositol-3-phosphate regulates response of cells to proteotoxic stress. Intl J. Biochem. Cell Biol. 79; 494-504. |
Start Year | 2013 |
Description | Structural aspects of WASP nucleated actin filaments |
Organisation | University of Sheffield |
Department | Department of Human Communication Sciences |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We have initiated a study in collaboration with Professor Per Bullough to undertake a study of the actin filaments generated by WASP family members |
Collaborator Contribution | We have identified a novel actin nucleation machinery and are now interested in understanding the mechanism of this nucleation. We will undertake the biochemical protein purification and preparation for the analysis. We have just obtained studentship funding for this collaboration |
Impact | Output - BBSRC White Rose DTP studentship |
Start Year | 2017 |
Description | School visit. Sheffield |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | On the latest visit 90 pupils listened to a short talk about microbes and then investigated the presence of bacteria on their hands and the effect of handwashing on this. This workshop has been run by myself in all of the years indicated and also by postdocs in different local schools including widening participation schools School keen to repeat and broaden activity. Neighbouring school also interested in liaising more closely with scientists. |
Year(s) Of Engagement Activity | 2006,2008,2010,2011,2012,2013,2014,2015 |