Regulation and mechanism of clathrin coated vesicle formation

Lead Research Organisation: University of Sheffield
Department Name: Biomedical Science

Abstract

Cells continuously need to internalise material from the extracellular environment, to deliver material to that environment and to move material between the membrane-bound subcompartments inside the cell. Broadly the way that they do this is by packaging material into specialised areas of one membrane (the ?donor?). These specialised areas often have a protein ?coat? and they become increasingly curved or ?invaginated? and cargo molecules become concentrated in these invaginations. Eventually they pinch off from the donor membrane, forming small spherical structures called coated vesicles. The vesicle is then sent to a target ?acceptor? membrane where it fuses and delivers its contents. The whole process of forming coated vesicles is highly controlled to ensure that the selection of cargo and direction of transport is properly maintained.
Our lab has focussed its efforts on the formation of coated vesicles at the cell surface. A molecule, clathrin, is the major protein component of these coats, giving the vesicles their name. However many other molecules are also involved in forming the coat and selecting the cargo that will be included in the vesicle. Our lab uses a variety of approaches to understand the molecules involved in forming coated vesicles. In particular we use assays that allow us to reconstitute coated vesicle formation in the test-tube. These assays allow us to ask which molecules are required and how their assembly into the coat is regulated. The aim of this proposal is to understand more fully how clathrin coated vesicles are formed.
Clathrin coatedvesicles are responsible for the internalisation of many different molecules including nutrients, growth factors and hormones. They are also often hijacked by pathogens such as bacteria and viruses to gain entry into the cell. They thus play very important roles in the response of cells to their environment. If we can understand the molecular basis of this fundamental process and how it is modified depending on where a cell is located, we hope in the longer term to apply this knowledge to understanding defects in this process that contribute to disease.

Technical Summary

Clathrin coated pits and coated vesicles are the major ports of entry for a huge variety of cargo, including pathogens, and they represent the paradigm for membrane-based sorting events in higher cells. Clathrin and the AP2 adaptor complex are the major components of the coat but, by a variety of approaches, a host of other proteins has been implicated in the clathrin coated vesicle cycle. The challenge for the future lies in understanding how the interactions of all of these components are regulated in space and time to efficiently sequester and internalise cargo. The aim of this proposal is to elucidate the mechanism of coated vesicle formation. Using a multi-disciplinary approach, including reconstitution assays in permeabilised cells, intact cell assays, protein chemistry and light and electron microscopy, we aim to elucidate the molecular mechanism of coated vesicle formation. We will build on our previous findings and focus in the first instance on the role of rab5 at the plasma membrane, the regulation of cargo/adaptor interactions, the temporal requirements for individual coated pit components and the regulation of these interactions by phosphorylation.

Publications

10 25 50

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King JS (2020) Water loss regulates cell and vesicle volume. in Science (New York, N.Y.)

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Moore R (2018) Interplay of Endocytosis and Growth Factor Receptor Signalling. in Progress in molecular and subcellular biology

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Robertson AS (2009) Functions of actin in endocytosis. in Cellular and molecular life sciences : CMLS

 
Description BHF PhD studentship
Amount £103,000 (GBP)
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2011 
End 08/2017
 
Description Equipment award (co-I)
Amount £900,000 (GBP)
Funding ID G0300452 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2008 
End 10/2013
 
Description Equipment grant
Amount £175,000 (GBP)
Funding ID 093134 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2011 
End 08/2016
 
Description Expansion of the Wolfson Light Microscopy Facility
Amount £200,000 (GBP)
Funding ID Pr/ms/22261 
Organisation The Wolfson Foundation 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2020 
End 12/2020
 
Description ITN Early Career Research Network
Amount € 3,900,000 (EUR)
Funding ID BioPOl 
Organisation EU-T0 
Sector Public
Country European Union (EU)
Start 01/2015 
End 12/2018
 
Description ITN European Industrial Doctorate
Amount € 800,000 (EUR)
Organisation European Union 
Sector Public
Country European Union (EU)
Start 06/2015 
End 05/2019
 
Description PhD studenship
Amount £120,000 (GBP)
Funding ID C12332/A10681 
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2009 
End 09/2013
 
Description Research grant
Amount £650,000 (GBP)
Funding ID MR/K015753/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 01/2013 
End 12/2015
 
Description Wellcome Equipment grant (Development of light and electron microscopy facilities at University of Sheffield)
Amount £175,133 (GBP)
Funding ID 093134/Z/10/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2011 
End 08/2015
 
Description Wellcome Trust Equipment Grant (University of Sheffield Centre for BioImaging)
Amount £700,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2008 
End 02/2011
 
Description iCASE studentship
Amount £120,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2016 
End 09/2020
 
Description Krebsfest, University of Sheffield 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Several hundred people attended a range of events established to celebrate the work of the nobel laureate Sir Hans Krebs. We provided a display showing fluorescence labelling of the cytoskeleton as well as providing the opportunity for small children to paint cells on cookies.
Year(s) Of Engagement Activity 2016
URL http://krebsfest.group.shef.ac.uk/
 
Description Microscopy demonstration at University of Sheffield Researchers' Night 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Talks sparked questions from the public on the work on membrane traffic in Sheffield
1000 people attended the krebsfest where we had hands on demonstations

Enthusiastic feedback from visitors. Sparked an application to the Royal Society Summer Exhibition
Year(s) Of Engagement Activity 2014,2015,2016
URL http://krebsfest.group.shef.ac.uk/
 
Description School visits in Sheffield and the surrounding region 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact Hands-on workshop on chromatography with Years 1 and 2. Presentations on how fertilised cells develop into whole organisms.

Very positive feedback from teachers and students alike.
Year(s) Of Engagement Activity 2007,2008,2009