Abstract
Immune synapses form between T cells and antigen-presenting cells (APCs). Increasing evidence suggests synapses must form flexibly to accommodate ongoing motility and displacement of the synapse. Here, time-lapse total internal reflection fluorescence (TIRF) microscopy showed that signaling via the T cell antigen receptor (TCR) occurred during synapse translation. TCR microclusters in motile synapses did not flow directly into supramolecular activating complexes (SMACs) but were directed, independently of myosin II contractility, toward an F-actin-poor 'sink' region. Inward microcluster flow often followed collapse of the leading edge, which suggested that actin depolymerization regulated microcluster flow and the formation of SMACs. The coordination of TCR movement with the translocation of this 'sink' shows how T cells coordinate TCR signaling and microcluster flow in dynamic physiological synapses.
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Acknowledgements
We thank S. Rogers (University of Manchester) for the polynomial fit Gaussian weight function; R. Wedlich-Soldner (Max Planck Institute of Biochemistry) for Lifeact-GFP; B. Lillemeier (Salk Institute) and M. Davis (Stanford University) for the histidine-tagged ICAM construct; M. Werner and K. Austgen for assistance in preparing histidine-tagged ICAM; and J. Altman (US National Institutes of Health Tetramer Facility at Emory University) for biotinylated pMHC monomers. Supported by the Cancer Research Institute (P.B.) and the US National Institutes of Health (AI52116 to M.F.K.).
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P.B. and M.F.K. designed the experiments for Figures 1,2,3 and 5,6,7 and P.B. did these experiments; P.B., J.J. and M.F.K. designed the experiments for Figure 4 and P.B. and J.J. did these experiments; P.B. wrote the manuscript; J.J. contributed to editing of the manuscript; and M.F.K. edited and revised the manuscript.
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Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–3 (PDF 759 kb)
Supplementary Movie 1
Microcluster flow couples with cell motility. (MP4 1326 kb)
Supplementary Movie 2
cSMAC formation is independent of synapse motility. (MP4 2430 kb)
Supplementary Movie 3
Myosin II inhibition does not impair motile synapse formation under non-detrimental experimental conditions. (MP4 2975 kb)
Supplementary Movie 4
Synapse formation by control, blebbistatin treated and conditional myosin II knockout cells. (MP4 2500 kb)
Supplementary Movie 5
Cell footprint contraction coordinated TCR microcluster flows. (MP4 3098 kb)
Supplementary Movie 6
TCR microclusters flow into zones of actin depolymerization. (MP4 222 kb)
Supplementary Movie 7
TCR microclusters and cSMAC movement are directed to an F-actin-poor sink region in motile synapses. (MP4 3057 kb)
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Beemiller, P., Jacobelli, J. & Krummel, M. Integration of the movement of signaling microclusters with cellular motility in immunological synapses. Nat Immunol 13, 787–795 (2012). https://doi.org/10.1038/ni.2364
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DOI: https://doi.org/10.1038/ni.2364
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