Quantitative analysis of the assembly and disassembly of clathrin cages.

Lead Research Organisation: University of Warwick
Department Name: School of Life Sciences

Abstract

Clathrin is a protein which rapidly and reversibly forms large cage structures of varying sizes. These properties are exploited by cells in order to absorb and transport the substances they need to survive through clathrin-mediated endocytosis. Clathrin-mediated endocytosis plays a central role in multiple cellular functions including nutrient uptake, nerve cell function, communication within the cell and organism development. In addition, such apparatus is used by some viruses (notably HIV) and bacteria to gain entry into cells and there is accumulating evidence that proteins involved in endocytosis are associated with a wide range of diseases including neurodegenerative disease and cancer. Given the importance of clathrin-mediated endocytosis to health and disease, understanding the principles of clathrin coat assembly and disassembly is vital if we are to learn how to tackle disease-causing malfunctions of this system.

The vehicles which are used are formed from cell membranes through the action of a network of many different proteins, including clathrin, which form a specialised coat around transport vesicles to form 'clathrin-coated vesicles'. Assembly and disassembly of clathrin-coated vesicles is essential for their life cycle and yet many details about how this process works are not understood. Disassembly is handled by two relatively small proteins, Hsc70 which is a molecular chaperone or 'helper' protein and auxilin/GAK, which is a cofactor for Hsc70. Their role is to take apart the assembly which has been created as a result of multiple clathrin molecules coming together around the vesicle. The clathrin molecules are much larger than Hsc70 and auxilin and have an intriguing three-legged appearance which gives clathrin assemblies the appearance and geometry of an irregular football, with pentagonal and hexagonal faces. Our aim is to find out how these smaller molecules dismantle the clathrin coat speedily and without mishaps and to understand how association of individual three-legged clathrin molecules leads to formation of cage structures.

We will adopt three strategies for investigating clathrin assembly and disassembly:
First, we have made a tool-kit of Hsc70 and auxilin molecules which can be labelled at known sites. By watching clathrin disassembly using these labels we will be able to see which parts of auxilin and Hsc70 are more important for pulling the clathrin molecules apart.

Second, we will investigate the related mechanism by which clathrin triskelions assemble by analysing the way in which the cages scatter light when they form. This will allow us to measure what factors, particularly other coat components, influence assembly and data analysis will help us piece together an order of events for cage formation.

Our final aim is to obtain detailed measurements of the mechanical properties of the clathrin triskelion. We have been able to image individual clathrin triskelions as they move in real time using the highly novel and state-of-the-art technique of high-speed atomic force microscopy. As a result we will be able to observe cage assembly directly, calculate the rigidity of clathrin triskelions as they associate and understand how the mechanical properties of the clathrin triskelion determine successful assembly into clathrin cages.

Technical Summary

Clathrin rapidly and reversibly forms large cage structures of varying sizes. These properties are exploited by eukaryotic cells to encapsulate endocytic transport vesicles and coordinate their formation, contents selection and delivery to target locations. We have a sensitive assay for clathrin disassembly which has allowed us to monitor clathrin disassembly with excellent time resolution. Kinetic analysis of these results reveals a model for the action of Hsc70 in which three Hsc70 molecules act in sequence to release a clathrin triskelion. We aim to quantitate and define the assembly and disassembly of clathrin cages through the following specific objectives:

1. To obtain molecular level information on the precise way in which domains of Hsc70 and auxilin carry out this mechanism. In order to do this we have created a series of mutants of Hsc70 and auxilin which contain only a single cysteine residue and to which we will attach fluorescent labels. This will provide us with a set of site-specific fluorescent probes with which we can monitor, at the domain level, the binding interactions and protein conformational changes which occur during clathrin disassembly.

2. To investigate the mechanism by which clathrin triskelions assemble both through pH jump and addition of the adaptor protein, AP2. By analysing stopped flow light scattering data of the assembly process we will be able to evaluate potential mechanisms, obtain kinetic rate constants for triskelion interactions during cage assembly and then probe how AP2 influences these rate constants.

3. To investigate how the mechanical properties of the clathrin triskelion determine successful assembly into clathrin cages. We have been successful in imaging clathrin triskelions using the highly novel and state-of-the-art technique of high-speed atomic force microscopy. As a result we will be able to observe cage assembly directly and calculate the rigidity of clathrin triskelions as they associate.

Planned Impact

Clathrin-mediated endocytosis plays a central role in multiple cellular functions including nutrient uptake, synaptic vesicle recycling, signaling, maintenance of cell polarity and development. In addition, the endocytic apparatus is used by some viruses (notably HIV) and bacteria to gain entry into cells and there is accumulating evidence that mutations or differences in expression levels in endocytic proteins are associated with a wide range of diseases including neurodegenerative disease and cancer. Given the importance of clathrin-mediated endocytosis to health and disease, the understanding that we will gain in this project on the principles of clathrin coat assembly and disassembly will enable us to learn how to tackle disease-causing malfunctions of this system. In addition, the knowledge that we will generate concerning the ability of a cell to carry out this precise remodelling of membranes will help synthetic biologists who wish to devise systems for controlling such events outside the cell.

The immediate beneficiaries of this work will include ACADEMIC SCIENTISTS who will use the new knowledge that is gained and the new techniques that are developed and alter their own research and development activities in light of this.

The insights we uncover will be published in journals of the highest possible calibre, thus sustaining the reputation of the UK as a world leader in scientific enquiry. Learned bodies, such as the Biochemical Society and the British Society for Cell Biology will benefit because they will be able to communicate this work to the wider scientific community and the public through their public engagement activities.

The EARLY CAREER RESEARCHER associated with the project will benefit from collaborations across the disciplines of biochemistry, microscopy, biophysics and modelling and from the associated skills to accomplish this that they will gain. These skills will transfer into their future careers in whatever sector they work. 'UK plc.' will benefit from such well-trained cross-disciplinary scientists who will be suited to many avenues of research.

INDUSTRIAL SCIENTISTS who are interested in how proteins control the shape of membranes and those interested in controlling the uptake and release of specific molecules e.g. as part of a drug delivery system, will also benefit from detailed understanding of how this very process is achieved in biology. This impact will take place as soon as the knowledge is disseminated e.g. through attendance of the next BioNano Collaborative International meeting in 2013.

The academics and early stage researcher will continue their programme of SCIENCE COMMUNICATION WITH THE GENERAL PUBLIC, engaging with local schools, local and national media, science fairs, IGGY (International Gateway for Gifted Youth), MoleClues, open days etc. thus benefiting the level of education and of science debate outside academia as well as within. Encouraging scientists to take part in such activities early in their careers will establish habits and expertise for benefitting the general public that will stay with them for their careers. This impact will be immediate as well as over the next few decades.
 
Description We have discovered how clathrin, which is a protein that facilitates endocytosis, forms multiple cage structures. We have also discovered how clathrin's binding partners influence the process of cage disassembly.
Exploitation Route We are in the process of publishing our work and one paper has been published. This will enable others to put to use the methods we have developed. We also anticipate our work on the biochemistry of clathrin disassembly will inform cell biology studies of endocytosis.
Sectors Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Alert14
Amount £1,500,000 (GBP)
Funding ID BB/M01228X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2015 
End 12/2015
 
Description Clathrin assembly regulation of glucose metabolism
Amount £491,534 (GBP)
Funding ID BB/V001434/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 12/2020 
End 11/2023
 
Description High resolution cryo-electron microscopy of clathrin cage complexes
Amount £5,500 (GBP)
Funding ID BB/L018888/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2013 
End 12/2014
 
Description Project grant - Exploiting direct electron detection to resolve protein-protein interactions in clathrin-mediated endocytosis
Amount £550,000 (GBP)
Funding ID BB/N008391/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2016 
End 03/2019
 
Description Strategic research development award
Amount £19,000 (GBP)
Organisation University of Warwick 
Sector Academic/University
Country United Kingdom
Start 10/2015 
End 07/2016
 
Title Method for detecting map differences 
Description Developed method for detecting differences between 2 low resolution 3D maps 
Type Of Material Data analysis technique 
Provided To Others? No  
Impact Anticipiated impacts are that interactions between molecules which are part of low resolution 3D maps can now be seen when this would not previously have been possible. 
 
Title Methods for analysing dynamic light scattering data 
Description Improvements in fitting of light scattering data which exploit knowledge of the biological system. 
Type Of Material Improvements to research infrastructure 
Provided To Others? No  
Impact Once fully developed this will be of use to others interested in measuring the size distribution of particles 
 
Description Improving image analysis of clathrin cage complexes 
Organisation University of California, San Francisco
Country United States 
Sector Academic/University 
PI Contribution Biochemical knowledge of the system and sample preparation
Collaborator Contribution Use of cryo-electron microscope and direct electron detector. Advice on image processing approaches.
Impact As a result of the collaboration we were able to obtain preliminary data for a full project grant application to BBSRC which was successful. We have also now succeeded in publishing our work in Nature Structural and Molecular Biology (Morris et al, 2019)
Start Year 2013
 
Description Hosted outreach event 'Scientist for the day' 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Hosted civil servants from BIS to enable them to gain an insight into life in a working lab. They did hands-on experiments in two research labs and had the opportunity to talk to PhD students and postdocs in my group.
Year(s) Of Engagement Activity 2015
 
Description National Science Week Activity 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Ran a 'murder mystery' outreach activity for all ages in Leamington Spa Museum as part of National Science week which used mass spectrometry and DNA analysis to find the murderer. There was very good engagement from both parents and children.
Year(s) Of Engagement Activity 2015
 
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/