Investigating the spatio-temporal dynamics of actin filaments at microclusters during T-cell activation

Project Overview

A pivotal event promoting adaptive immune responses is binding of antigens to antigen receptors and subsequent actin cytoskeleton reorganisations resulting in receptor microcluster (MC) formation, intracellular signalling, and ultimately immunological synapse (IS) formation. The IS is a specialised interface at the cell-cell contact between lymphocytes and antigen presenting cells (APCs), which has been unleashed to enhance T-cell function in cancer immunotherapy or restrained to suppress inappropriate immune responses in autoimmune diseases. T-cell receptor (TCR) mediated activation of T cells by APCs depends on actin assembly, while inhibition of actin dynamics arrests the IS and results in impaired T-cell activation. Specifically, TCR ligation triggers a molecular program that results in activation of phospholipase C-γ1 (PLCγ1) and its recruitment by linker of activated T cells (LAT), resulting in intracellular calcium release and cortical actin remodelling. Recent studies by others and us have reported a complex role for the cortical cytoskeleton in both promoting and inhibiting PLCγ1 activation by the formation actin foci facilitating MC dynamics during IS formation (1,2). Historically, advances in understanding the mechanisms underlying key events in the IS have been driven by innovations in microscopy that provide greater spatial and temporal resolution coupled to appropriate immunochemical, genetic, and biochemical tools (3,4). Until recently, state-of-the-art microscopy has not been informative about the underlying real-time spatio-temporal dynamics of the actin cytoskeleton. Especially, despite our knowledge of the molecular processes underlying actin assembly, the mechanisms controlling actin foci recruitment to MCs and their regulation by PLCγ1 and intracellular calcium release during IS formation remain elusive. To overcome this challenge, we will monitor the dynamics of actin, PLCγ1, and calcium ions at extended spatio-temporal resolutions by applying the novel eTIRF-SIM (extended total-internal-reflection-fluorescence (TIRF) structured-illumination-microscope (SIM)) developed by our close collaborators Professor Dong Li (Biophysics Institute, Beijing) and Dr Eric Betzig (HHMI Janelia FARM, USA). This research project aims to elucidate the dynamic interplay of PLCγ1 recruitment to LAT, intracellular calcium release, and actin recruitment to MCs during the formation of the IS. Further, we make use of a high-speed spinning-disk calcium assay optimised for large cell ensembles and analysed by our software package CalQuo (3). Single molecule based biophysical analysis tools will enable us to study the nanoscale actin filament network structures at the IS (4). We will initially investigate the dynamics of the above detailed players in Jurkat T-cells interacting with protein-functionalised glass surfaces and then extend our studies to primary lymphocyte interacting with mobile supported-lipid-bilayer (SLB) systems.

Training Opportunities

The candidate will be based in the new, purpose built labs at the Kennedy Institute of Rheumatology (KIR), a world-leading centre in the fields of cytokine biology and inflammation, with a strong emphasis on clinical translation. In the first term you will attend 20 core lectures. Throughout the studentship, we will encourage the candidate to attend weekly seminars at the Institute given by global leaders in the fields of immunology and biophysics. The DPhil candidate will benefit from combined supervision by a biophysicist with expertise in advanced fluorescent imaging of actin cytoskeleton dynamics as well as a cell-biologist with long-lasting expertise in the dynamics of the IS formation. Weekly lab meetings, where students and post-doctoral scientists discuss their findings will allow the candidate to gain invaluable research experience, manuscript publication, and conference presentations. There is support available from post-doctoral scientists in our groups and laboratory managers to become proficient in cell and molecular biological techniques, advanced biophysical and microscopy techniques including super-resolution eTIF-SIM, high-speed spinning-disk calcium measurements, tissue culture, and supported lipid-bilayer systems. Overall, the Kennedy Institute provides an ideal environment for intellectual development and translational research. 

Relevant Publications

1. A Babich, S Li, RS O'Connor, MC Milone, BD Freedman, and JK Burkhardt, F-actin polymerization and retrograde flow drive sustained PLC γ 1 signaling during T cell activation, JCB 2012.
2. S Kumari, D Depoil, R Martinelli, E Judokusumo, G Carmona, FB Gertler, LC Kam, CV Carman, JK Burkhardt, DJ Irvine, ML Dustin, Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway, Elife, 2015.
3. M Fritzsche, RA Fernandes, H Colin-York, AM Santos, SF Lee, BC Lagerholm, SJ Davis, and C Eggeling, CalQuo: automated, simultaneous single-cell and population-level quantification of global intracellular Ca2+ responses, Sci Reports, 2015.
4. Marco Fritzsche,1 * Christoph Erlenkämper,2,3 Emad Moeendarbary,4,5 Guillaume Charras, Karsten Kruse, Actin kinetics shapes cortical network structure and mechanics, Science Advances, 2016.

Scientific Themes

Translational Medicine and Medical Technology; Molecular, Cell and Systems Biology.

Further information

Dr Marco Fritzsche, Kennedy Institute, University of Oxford
marco.fritzsche@rdm.ox.ac.uk