Analysis of SNARE protein spatio-temporal dynamics during exocytosis in living cells

Lead Research Organisation: Heriot-Watt University
Department Name: Sch of Engineering and Physical Science

Abstract

All cells in higher organisms have a requirement to move membranes and proteins around, whilst identifying and maintaining the compartments the various factors are in. A final step in this process may be secretion, or exocytosis, where specialised cells can export hormones (like insulin) or neurotransmitters (like serotonin) to the outside of the cells, thus enabling inter-cell communication. It has been known for some time that all these processes are mediated by specialised proteins in the cell; this was defined in the test-tube, and the protein players identified. Until recently, however, it has not been possible to identify where and when these proteins interact
with one another inside cells. Microscopy has been limited to the simple observational process of seeing where things are. My laboratory, and many others, have helped develop functional imaging , where microscopes can be used not only for seeing things, but looking at their functions. In particular, I have techniques for visualising proteins as they interact with partners inside living cells. These approaches will be used to bridge the gap between our biochemical knowledge from the test-tube, and what actually happens in the cell during secretion.

Technical Summary

Mammalian regulated exocytosis requires an essential set of proteins; the plasma membrane tSNAREs syntaxin and SNAP-25 and the vesicle resident vSNARE synaptobrevin. The interactions between these proteins have been well characterised in vitro. However, the cellular temporal and spatial organisation of these interactions remains unknown.
In order to address this, we will determine the following, using a variety of complimentary techniques, in living neuroendocrine cells in real time:

1. What is the spatio-temporal organisation of the binary tSNARE complex in cells?
2. Do SNARE cluster heterogeneities influence docking site selection?
3. How are the SNARE proteins arranged when an LDCV is present?
4. How are SNARE spatio-temporal interactions influenced by accessory proteins?

In this project we aim to deliver a detailed description of tSNARE protein dynamic interactions in living cells, before and after the moment of vesicle fusion, and examine any effects accessory proteins may have on these interactions. These data are essential to further our understanding of exocytosis.

Publications

10 25 50
 
Description CFI committee
Geographic Reach North America 
Policy Influence Type Participation in advisory committee
 
Title datasets 
Description We have made available large amounts of digital data in an online archive for use by signal processing and mathematical modelling groups 
Type Of Material Biological samples 
Year Produced 2014 
Provided To Others? Yes  
Impact Other groups from diverse backgrounds are now working with exemplar datasets particularly in super-resolution imaging areas, where analysis tools are lacking. 
 
Description 3-D imaging 
Organisation Heriot-Watt University
Department Physics
Country United Kingdom 
Sector Academic/University 
PI Contribution Prof Greenaway, an astronomer, has designed a simple modification to our microscopes which permits faster, higher resolution and less invasive image data acquisition from cells. This has very important implications for our work and for cell biology.
Collaborator Contribution Led to a successful funding award from the STFC.
Impact Successful STFC award (2012-13). Paper to follow (2012)
Start Year 2010
 
Description Mathematical modelling 
Organisation Heriot-Watt University
Department Department of Mathematics
Country United Kingdom 
Sector Academic/University 
PI Contribution Professor Lord has successfully modelled the motions of 100000s of single molecules alongside organelles, based on data from our imaging experiments funded by this MRC award.
Collaborator Contribution Professor Lord has successfully modelled the motions of 100000s of single molecules alongside organelles, based on data from our imaging experiments funded by this MRC award.
Impact We are preparing a manuscript at the moment for submission to Nature. We expect this to lead to funding applications in the near future.
Start Year 2011
 
Description Single Molecule tracking 
Organisation Heriot-Watt University
Department Physics
Country United Kingdom 
Sector Academic/University 
PI Contribution We provide single molecule image data to Dr Lu and colleagues, who write single particle tracking algorithms. They are able to track many 10000s of single molecules inside living cells. This will lead to several high impact publications in the near future.
Impact Multi-discplinary - most definitely. Dr Lu is a theoretical physicist. This will lead to several (3) high impact publications in 2012.
Start Year 2011
 
Description Canadian Foundation for Innovation 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Served on a funding committee

5 large scale imaging facilites were established across Canada.
Year(s) Of Engagement Activity 2012
 
Description IGMM KE workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact 40 academics from the MRC IGMM and Heriot Watt University attended a residential knowledge exchange workshop I organised.

This led to several pilot projects (EPSRC and MRC joint funded) and 3 external funding proposals (to date, OCtober 2012). A strategic alliance has now been formed between our institutes to take advantage of our cross disciplinary skills.
Year(s) Of Engagement Activity 2012
 
Description Keynes Lecture 2012 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Delivered the annual Keynes Lecture at the EMBL 'Advanced Imaging' workshop in the Marine Biological laboratory, Plymouth

The audience was a selection of high achieving postgraduate and postdoctoral researchers. My presentation led directly to an international collaboration and further invitations.
Year(s) Of Engagement Activity 2012
 
Description Keynote lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Invited to deliver the Plenary Lecture at the 40th Anniversary Scottish Microscopy Group Meeting

Reached a wide audience, describing the benefits and developments in super-resolution imaging.
Year(s) Of Engagement Activity 2012
 
Description Nexxus 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Type Of Presentation Paper Presentation
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Nexxus, a science networking organisation, featured the 'Life Science Interface' laboratory in its monthly magazine, as well as on the front cover.

This led to several enquiries from other academics seeking collaborations - notable from the University of Edinburgh
Year(s) Of Engagement Activity 2011
 
Description public exhibitions 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact We have participated in 25 science festivals this year, reaching several 1000 members of the public.

Several new invitations, schools talks, outreach activities
Year(s) Of Engagement Activity 2014