How is vesicle uncoating achieved? Dissecting early events in clathrin disassembly by the molecular chaperone, Hsc70

Lead Research Organisation: University of Warwick
Department Name: Biological Sciences

Abstract

Cells use the movement of vesicles as their primary transport system for many important compounds. This method of transportation is termed endocytosis and vesicle trafficking . Substances are brought into the cell via endocytosis, during which a vesicle is formed from the cell membrane which encloses specific molecules. A specialised protein coat is required around the forming vesicle which helps select the contents and also directs the time and place of formation and detachment. The coat assembles and disassembles on the vesicle in a precisely controlled manner which is vital to the correct functioning of the cell. We wish to study in molecular detail how the coat is removed from the vesicle and what events are important for triggering this uncoating event. We will use a range of different physical methods to see which proteins bind at which point in time and whether their shape alters during uncoating. We will also use special mutant forms of Hsc70 which only perform certain steps along this pathway to dissect out the order of these steps and their importance in uncoating. Hence these studies will produce a picture of the initiation of uncoating of the vesicle coat and allow us to see how this intriguing molecular machine works.

Technical Summary

The aim of this project is to investigate the early events of clathrin disassembly by the molecular chaperone, Hsc70, so as to understand what events cause the uncoating of clathrin-coated vesicles. We will view the process using biophysical methods which report on changes in protein conformation, protein-substrate binding events, ATP hydrolysis and clathrin disassembly. By monitoring disassembly with these parallel techniques, we will obtain a detailed picture of clathrin disassembly which will relate to the dynamics of the vesicle uncoating process. We will use well-studied mutants of Hsc70 which are specifically impaired in certain functions of the chaperone, e.g. ATP hydrolysis, to pinpoint when, in relation to other steps, these events occur and which binding events and conformational changes are associated with a particular step. Initially we will work on the simplest system available, clathrin cages, Hsc70 and auxilin401-910. Once this has been thoroughly characterized, we will move towards the more complex cellular situation by introducing further components, such as adaptor proteins, which are essential components of clathrin-coated vesicles and have been implicated in uncoating. Thus we will determine in molecular detail the nature of the events which cause uncoating of clathrin-coated vesicles and so shed light on how the process is regulated in the cell.

Publications

10 25 50
 
Description Project grant - Quantitative analysis of the assembly and disassembly of clathrin cages
Amount £400,000 (GBP)
Funding ID BB/K003461/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2013 
End 01/2016
 
Description Royal Society International Exchange Award
Amount £6,000 (GBP)
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2012 
End 08/2013
 
Description University of Warwick Research Development Scheme
Amount £5,800 (GBP)
Organisation University of Warwick 
Sector Academic/University
Country United Kingdom
Start 01/2011 
End 01/2012
 
Title New model for mechanism of clathrin cage disassembly 
Description Our data have led to the proposal of a new model for clathrin cage disassembly by Hsc70 which has arisen as a direct result of this funding. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2009 
Provided To Others? Yes  
Impact So far it is too early to say. We have disseminated the work through a manuscript which is submitted and through talks and posters presented. 
 
Description Clathrin disassembly assay 
Organisation University of Sheffield
Department Department of Biomedical Science
Country United Kingdom 
Sector Academic/University 
PI Contribution I worked with the collaborator to implement our clathrin disassembly assay using their reagents
Collaborator Contribution Valuable discussions about the project. The opportunity to join forces in developing funding proposals. Shared expertise and knowledge.
Impact A productive visit from my collaborator's postdoc to my laboratory resulted. I was able to contribute a letter of support to aid their funding application. A manuscript is in preparation.
Start Year 2010
 
Description Expression of Hsc70 in insect cells 
Organisation University of Manchester
Department Faculty of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We have been able to help others become established in insect cell use and Hsc70 expression and purification.
Collaborator Contribution The collaboration has brought about useful discussions on our respective research topics which is resulting in further collaborative opportunities elsewhere
Impact This collaboration has increased the capability of researchers, primarily at Warwick University, to use insect cell and baculovirus methodology in their own research. In addition we have provided pure Hsc70 to a number of collaborators for their own studies which has enabled them to test hypotheses on Hsc70 action in other systems.
Start Year 2007
 
Description Improved image processing methods for 3D structural analysis 
Organisation University of Manchester
Department Faculty of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Our project has contributed to our collaborator's repertoire of test systems.
Collaborator Contribution Permitted the extension of 3D structural studies by use of new image processing methods
Impact Publication in the journal 'Traffic' in 2013
 
Description Mathematical Analysis of clathrin disassembly 
Organisation University of Warwick
Department Warwick Mathematics Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Our data and the biological problem we are working on present an interesting scenario for mathemeticians to model.
Collaborator Contribution New insights into the problem from a mathematical perspective. The potential for a PhD student to join the project.
Impact I presented a seminar at a multi-disciplinary research day. The work is being presented to students as a mini project.
Start Year 2010
 
Description Modelling analysis of disassembly data 
Organisation BioKin
Country United States 
Sector Private 
PI Contribution We have provided high quality data and an interesting biological system for analysis.
Collaborator Contribution The collaboration has allowed us to derive new mechanistic information about our system using modelling analysis.
Impact We have just submitted a manuscript to a high profile journal based on the outcome of this work. We have also submitted a joint funding application on this work. The project is multi-disciplinary between biophysics and mathematical moldelling disciplines.
Start Year 2010
 
Description The cell biology of clathrin disassembly 
Organisation University of Birmingham
Department School of Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We are studying clathrin disassembly at a molecular level using biophysical techniques. By working with our collaborators we can provide moleculr insights which can be tested in cells.
Collaborator Contribution Potential to investigate clathrin disassembly in cells
Impact We have now submitted a joint funding proposal to the BBSRC to carry out this work. This is a multi-disciplinary collaboration between the disciplines of biophysics and cell biology.
Start Year 2010
 
Description Correspondence with primary school children - Devon 
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 A number of primary school children wrote to ask me what it was like to be a scientist and I answered their letters.

The feedback was very good and much goodwill was generated with the school. It is to be hoped that this will result in some of these children considering science as a career in the future.
Year(s) Of Engagement Activity 2007
 
Description Pupil visit to laboratory 
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 Hosted a visit from a home-schooled pupil which stimulated a lot of interest in the work we do here and in science in general

Excellent feedback and enthusiasm for pursuing the topic in future lessons
Year(s) Of Engagement Activity 2010
 
Description Royal Society of Biology Blog 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I wrote a short blog for the Royal Society of Biology explaining my research to a general audience. Informal feedback from colleagues and via social media was positive.
Year(s) Of Engagement Activity 2016
URL https://blog.rsb.org.uk/clathrin-maintaining-cell-health-in-geometric-style/
 
Description School Visit 
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 Postdoc visited a school and gave a talk.

Goodwill with school and opportunity for children to learn more about what a scientific career involves.
Year(s) Of Engagement Activity 2008
 
Description University Open Days 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Discussed research, future study and careers with students and parents. Some events included hands-on experience of electron microscopy using everyday examples to highlight the role of this technique in high impact science.

Positive feedback from the audience including wider discussions on the topic and on science in general.
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2014,2015,2016,2017,2018,2019