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Integration of the movement of signaling microclusters with cellular motility in immunological synapses

Nature Immunology volume 13, pages 787–795 (2012)Cite this article

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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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Figure 1: T cells remain motile during TCR signaling triggered by a large range of agonist doses.
Figure 2: Microcluster flow aligns with movement in motile synapses.
Figure 3: Synapse motility is independent of cSMAC formation.
Figure 4: TCR centralization and synapse dynamics are myosin II independent.
Figure 5: The polymerization and depolymerization of actin organizes synapses.
Figure 6: Microcluster centralization correlates with F-actin depolymerization.
Figure 7: TCR centralization requires actin depolymerization.

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References

  1. Krummel, M.F., Sjaastad, M.D., Wülfing, C. & Davis, M.M. Differential clustering of CD4 and CD3ζ during T cell recognition. Science 289, 1349–1352 (2000).

    Article  CAS  PubMed  Google Scholar 

  2. Grakoui, A. et al. The immunological synapse: a molecular machine controlling T cell activation. Science 285, 221–227 (1999).

    Article  CAS  PubMed  Google Scholar 

  3. Wülfing, C. & Davis, M.M.A. Receptor/cytoskeletal movement triggered by costimulation during T cell activation. Science 282, 2266–2269 (1998).

    Article  PubMed  Google Scholar 

  4. Monks, C.R.F., Freiberg, B.A., Kupfer, H., Sciaky, N. & Kupfer, A. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395, 82–86 (1998).

    Article  CAS  PubMed  Google Scholar 

  5. Yokosuka, T. et al. Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76. Nat. Immunol. 6, 1253–1262 (2005).

    Article  CAS  PubMed  Google Scholar 

  6. Varma, R., Campi, G., Yokosuka, T., Saito, T. & Dustin, M.L. T cell receptor-proximal signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster. Immunity 25, 117–127 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Mossman, K.D., Campi, G., Groves, J.T. & Dustin, M.L. Altered TCR signaling from geometrically repatterned immunological synapses. Science 310, 1191–1193 (2005).

    Article  CAS  PubMed  Google Scholar 

  8. Kaizuka, Y., Douglass, A.D., Varma, R., Dustin, M.L. & Vale, R.D. Mechanisms for segregating T cell receptor and adhesion molecules during immunological synapse formation in Jurkat T cells. Proc. Natl. Acad. Sci. USA 104, 20296–20301 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Dustin, M.L. & Springer, T.A. T-cell receptor cross-linking transiently stimulates adhesiveness through LFA-1. Nature 341, 619–624 (1989).

    Article  CAS  PubMed  Google Scholar 

  10. Miller, M.J., Safrina, O., Parker, I. & Cahalan, M.D. Imaging the single cell dynamics of CD4+ T cell activation by dendritic cells in lymph nodes. J. Exp. Med. 200, 847–856 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Mempel, T.R., Henrickson, S.E. & von Andrian, U.H. T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases. Nature 427, 154–159 (2004).

    Article  CAS  PubMed  Google Scholar 

  12. Celli, S., Garcia, Z. & Bousso, P. CD4 T cells integrate signals delivered during successive DC encounters in vivo. J. Exp. Med. 202, 1271–1278 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Gunzer, M. et al. Antigen presentation in extracellular matrix: interactions of T cells with dendritic cells are dynamic, shortlived, and sequential. Immunity 13, 323–332 (2000).

    Article  CAS  PubMed  Google Scholar 

  14. Campi, G., Varma, R. & Dustin, M.L. Actin and agonist MHC-peptide complex-dependent T cell receptor microclusters as scaffolds for signaling. J. Exp. Med. 202, 1031–1036 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lee, K.-H. et al. The immunological synapse balances T cell receptor signaling and degradation. Science 302, 1218–1222 (2003).

    Article  CAS  PubMed  Google Scholar 

  16. Friedman, R.S., Beemiller, P., Sorensen, C.M., Jacobelli, J. & Krummel, M.F. Real-time analysis of T cell receptors in naive cells in vitro and in vivo reveals flexibility in synapse and signaling dynamics. J. Exp. Med. 207, 2733–2749 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Skokos, D. et al. Peptide-MHC potency governs dynamic interactions between T cells and dendritic cells in lymph nodes. Nat. Immunol. 8, 835–844 (2007).

    Article  CAS  PubMed  Google Scholar 

  18. Negulescu, P.A., Krasieva, T.B., Khan, A., Kerschbaum, H.H. & Cahalan, M.D. Polarity of T cell shape, motility, and sensitivity to antigen. Immunity 4, 421–430 (1996).

    Article  CAS  PubMed  Google Scholar 

  19. Sims, T.N. et al. Opposing effects of PKCθ and WASp on symmetry breaking and relocation of the immunological synapse. Cell 129, 773–785 (2007).

    Article  CAS  PubMed  Google Scholar 

  20. Weiss, A., Imboden, J., Shoback, D. & Stobo, J. Role of T3 surface molecules in human T-cell activation: T3-dependent activation results in an increase in cytoplasmic free calcium. Proc. Natl. Acad. Sci. USA 81, 4169–4173 (1984).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ehrlich, J.S., Hansen, M.D.H. & Nelson, W.J. Spatio-temporal regulation of Rac1 localization and lamellipodia dynamics during epithelial cell-cell adhesion. Dev. Cell 3, 259–270 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Beemiller, P., Jacobelli, J. & Krummel, M.F. Imaging and analysis of OT1 T cell activation on lipid bilayers. Nat. Protoc. published online, doi:10.1038/protex.2012.028 (1 July 2012).

  23. Jacobelli, J., Chmura, S.A., Buxton, D.B., Davis, M.M. & Krummel, M.F. A single class II myosin modulates T cell motility and stopping, but not synapse formation. Nat. Immunol. 5, 531–538 (2004).

    Article  CAS  PubMed  Google Scholar 

  24. Ilani, T., Vasiliver-Shamis, G., Vardhana, S., Bretscher, A. & Dustin, M.L. T cell antigen receptor signaling and immunological synapse stability require myosin IIA. Nat. Immunol. 10, 531–539 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Yu, Y., Fay, N.C., Smoligovets, A.A., Wu, H.-J. & Groves, J.T. Myosin IIA modulates T cell receptor transport and CasL phosphorylation during early immunological synapse formation. PLoS ONE 7, e30704 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Yi, J., Wu, X.S., Crites, T. & Hammer, J.A. Actin retrograde flow and acto-myosin II arc contraction drive receptor cluster dynamics at the immunological synapse in Jurkat T-cells. Mol. Biol. Cell 23, 834–852 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Straight, A.F. et al. Dissecting temporal and spatial control of cytokinesis with a myosin II inhibitor. Science 299, 1743–1747 (2003).

    Article  CAS  PubMed  Google Scholar 

  28. Kolega, J. Phototoxicity and photoinactivation of blebbistatin in UV and visible light. Biochem. Biophys. Res. Commun. 320, 1020–1025 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. Sakamoto, T., Limouze, J., Combs, C.A., Straight, A.F. & Sellers, J.R. Blebbistatin, a myosin II inhibitor, is photoinactivated by blue light. Biochemistry 44, 584–588 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. Jacobelli, J., Bennett, F.C., Pandurangi, P., Tooley, A.J. & Krummel, M.F. Myosin-IIA and ICAM-1 regulate the interchange between two distinct modes of T cell migration. J. Immunol. 182, 2041–2050 (2009).

    Article  CAS  PubMed  Google Scholar 

  31. Jacobelli, J. et al. Confinement-optimized three-dimensional T cell amoeboid motility is modulated via myosin IIA–regulated adhesions. Nat. Immunol. 11, 953–961 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Riedl, J. et al. Lifeact: a versatile marker to visualize F-actin. Nat. Methods 5, 605–607 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Bubb, M., Senderowicz, A., Sausville, E., Duncan, K. & Korn, E. Jasplakinolide, a cytotoxic natural product, induces actin polymerization and competitively inhibits the binding of phalloidin to F-actin. J. Biol. Chem. 269, 14869–14871 (1994).

    CAS  PubMed  Google Scholar 

  34. Bubb, M., Spector, I., Beyer, B.B. & Fosen, K.M. Effects of Jasplakinolide on the Kinetics of Actin Polymerization. An explanation for certain in vivo observations. J. Biol. Chem. 275, 5163–5170 (2000).

    Article  CAS  PubMed  Google Scholar 

  35. Bunnell, S.C., Kapoor, V., Trible, R.P., Zhang, W. & Samelson, L.E. Dynamic actin polymerization drives T cell receptor–Induced spreading: a role for the signal transduction adaptor LAT. Immunity 14, 315–329 (2001).

    Article  CAS  PubMed  Google Scholar 

  36. Vallotton, P., Gupton, S.L., Waterman-Storer, C.M. & Danuser, G. Simultaneous mapping of filamentous actin flow and turnover in migrating cells by quantitative fluorescent speckle microscopy. Proc. Natl. Acad. Sci. USA 101, 9660–9665 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Delorme, V. et al. Cofilin activity downstream of Pak1 regulates cell protrusion efficiency by organizing lamellipodium and lamella actin networks. Dev. Cell 13, 646–662 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Eibert, S.M. et al. Cofilin peptide homologs interfere with immunological synapse formation and T cell activation. Proc. Natl. Acad. Sci. USA 101, 1957–1962 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Hogquist, K.A. et al. T cell receptor antagonist peptides induce positive selection. Cell 76, 17–27 (1994).

    Article  CAS  PubMed  Google Scholar 

  40. Friedman, R.S., Jacobelli, J. & Krummel, M.F. Surface-bound chemokines capture and prime T cells for synapse formation. Nat. Immunol. 7, 1101–1108 (2006).

    Article  CAS  PubMed  Google Scholar 

  41. Rogers, S.S., Waigh, T.A., Zhao, X. & Lu, J.R. Precise particle tracking against a complicated background: polynomial fitting with Gaussian weight. Phys. Biol. 4, 220–227 (2007).

    Article  CAS  PubMed  Google Scholar 

  42. Grynkiewicz, G., Poenie, M. & Tsien, R. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450 (1985).

    CAS  PubMed  Google Scholar 

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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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  1. Jordan Jacobelli

    Present address: Present address: National Jewish Health, Denver, Colorado, USA.,

Authors and Affiliations

  1. Department of Pathology, University of California, San Francisco, San Francisco, California, USA.,

    Peter Beemiller, Jordan Jacobelli & Matthew F Krummel

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Contributions

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.

Corresponding author

Correspondence to Matthew F Krummel.

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The authors declare no competing financial interests.

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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