Functional compartmentalization of the T cell plasma membrane in the maturing immunological synapse

Lead Research Organisation: Imperial College London
Department Name: National Heart and Lung Institute

Abstract

When we are infected with a pathogenic organism our immune system mounts a response that eventually leads to the elimination of the organism and recovery from disease. Crucial to this process is the recognition of small pieces of the organism by a specialised immune cell called a T cell. These pieces are presented to the T cell on the surface of a so-called antigen presenting cell (APC). The T cell and the APC form an unique contact region called an immune synapse (IS) which is divided into concentric rings of molecules specialised for different functions such as recognition, binding together and signalling. We will use a well-accepted model of the IS, in combination with sophisticated microscopical methods to visualise the molecules at the cell surface, to understand how it is organised into these different domains and how they play a role in the efficiency and specificity of recognition and activation between the T cell and the APC.

Technical Summary

Our hypothesis is that interactions between ordered membrane microdomains (lipid rafts, LRs), PIP domains and adhesive domains are crucial to the process of formation and signalling at the immunological synapse (IS) between a T cell and an antigen-presenting cell (APC). To study this we will use T cells from antigen-specific TCR transgenic mice (AND strain), where possible crossed to mouse strains expressing fluorescent protein reporters of PIP domains. We plan to use an established supported planar bilayer system of IS formation with advanced fluorescence microscopy techniques (TIRF, FLIM) to examine the distribution of LRs within the IS during the activation of T cells. We will compare this with the distribution of proteins reported to be predominantly localized inside (e.g. Lck, Fyn) or outside (e.g. TfR, CD45) of these microdomains. We will also image the distribution of phosphatidylinositol(4,5)-bisphosphate (PI(4,5)P2) and its derivatives (PI(3,4)P2, PI(3,4,5)P3 and DAG) during the formation of the IS and compare it with LR distribution. We will visualize the distribution of cytoskeletal linker proteins (e.g. vinculin, paxillin, zyxin and FAK in relation to LR and PIPs during the formation of the IS. To examine the role of cytoskeletal linker components in the IS we will silence them with RNAi in order to assess the effect on stabilization of LRs or PIP domains. We will use the generated data, in the context of the known nano-clustering of TCR and proximal signalling molecules, to generate a time scheme of dynamic events ongoing on the surface of T cells during activation by antigen.

Publications

10 25 50
 
Description Pancreatic Cancer Research Fund project grant
Amount £189,000 (GBP)
Organisation Pancreatic Cancer Research Fund 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2011 
End 01/2014
 
Title Novel microscopy approaches 
Description Novel microscopy approaches to studying cell membrane organisation were developed with P. French, M. Neil and C. Dunsby. 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact Publications PMID 16617080; 17728161; 19550853; 19343675 
URL http://europepmc.org/abstract/MED/16617080
 
Description Collaboration on image processing with A. Serge 
Organisation Aix-Marseille University
Country France 
Sector Academic/University 
PI Contribution Provision of live cell movies of molecular rearrangements at the T cell immune synapse
Collaborator Contribution Dr. Serge has provided novel and powerful image processing analysis for our data.
Impact Cebecauer, M.C., Spitaler, M., Sergé, A. and Magee, A.I. Signalling complexes and clusters: functional advantages and methodological hurdles. J. Cell Sci. In press
Start Year 2008
 
Description Collaboration with E. Jury and T. Butters on T cell glycolipids and membrane domains 
Organisation University College London
Department Division of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided extertise on T cell membrane domains
Collaborator Contribution T. Butters - expertise on glycolipids E. Jury - expertise on T cell function and role in SLE
Impact PMID: 21307290
Start Year 2007
 
Description Collaboration with E. Jury and T. Butters on T cell glycolipids and membrane domains 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided extertise on T cell membrane domains
Collaborator Contribution T. Butters - expertise on glycolipids E. Jury - expertise on T cell function and role in SLE
Impact PMID: 21307290
Start Year 2007
 
Description Collaboration with E. Tate on protein lipidation 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution We are analysing the post-translational lipidation of Hedgehog proteins.
Collaborator Contribution Click chemistry and peptide chemistry, development of in vitro assays.
Impact PMID: 21221452 multidisciplinary - cell biology and chemistry
Start Year 2009