Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

T cell antigen receptor signaling and immunological synapse stability require myosin IIA

Nature Immunology volume 10, pages 531–539 (2009)Cite this article

Abstract

Immunological synapses are initiated by signaling in discrete T cell antigen receptor microclusters and are important for the differentiation and effector functions of T cells. Synapse formation involves the orchestrated movement of microclusters toward the center of the contact area with the antigen-presenting cell. Microcluster movement is associated with centripetal actin flow, but the function of motor proteins is unknown. Here we show that myosin IIA was necessary for complete assembly and movement of T cell antigen receptor microclusters. In the absence of myosin IIA or its ATPase activity, T cell signaling was interrupted 'downstream' of the kinase Lck and the synapse was destabilized. Thus, T cell antigen receptor signaling and the subsequent formation of immunological synapses are active processes dependent on myosin IIA.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Effect of the inhibition or depletion of myosin IIA on the centripetal motion of TCR microclusters.
Figure 2: Phosphorylation and redistribution of myosin IIA during activation of T cells.
Figure 3: Effect of the inhibition of myosin IIA activity on the formation of the immunological synapse.
Figure 4: Effect of the inhibition of myosin IIA activity on the stability of the immunological synapse.
Figure 5: Effect of the inhibition of myosin IIA activity on intracellular Ca2+ concentrations.
Figure 6: Effect of the inhibition of myosin IIA activity on TCR microclusters.
Figure 7: Effect of the inhibition or depletion of myosin IIA on signaling in T cells.

Similar content being viewed by others

References

  1. Davis, M.M. The αβ T cell repertoire comes into focus. Immunity 27, 179–180 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. DeMond, A.L., Mossman, K.D., Starr, T., Dustin, M.L. & Groves, J.T. T cell receptor microcluster transport through molecular mazes reveals mechanism of translocation. Biophys. J. 94, 3286–3292 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Monks, C.R., 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 

  4. Dustin, M.L. et al. A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts. Cell 94, 667–677 (1998).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  6. 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 

  7. Combs, J. et al. Recruitment of dynein to the Jurkat immunological synapse. Proc. Natl. Acad. Sci. USA 103, 14883–14888 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Bunnell, S.C. et al. T cell receptor ligation induces the formation of dynamically regulated signaling assemblies. J. Cell Biol. 158, 1263–1275 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 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 

  10. Huse, M. et al. Spatial and temporal dynamics of T cell receptor signaling with a photoactivatable agonist. Immunity 27, 76–88 (2007).

    Article  CAS  PubMed  Google Scholar 

  11. 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 

  12. Douglass, A.D. & Vale, R.D. Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in T cells. Cell 121, 937–950 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 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 

  14. 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 

  15. Dustin, M.L. & Cooper, J.A. The immunological synapse and the actin cytoskeleton: molecular hardware for T cell signaling. Nat. Immunol. 1, 23–29 (2000).

    Article  CAS  PubMed  Google Scholar 

  16. Chakraborty, A.K. How and why does the immunological synapse form? Physical chemistry meets cell biology. Sci. STKE 2002, PE10 (2002).

    PubMed  Google Scholar 

  17. 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 

  18. Lin, C.H., Espreafico, E.M., Mooseker, M.S. & Forscher, P. Myosin drives retrograde F-actin flow in neuronal growth cones. Neuron 16, 769–782 (1996).

    Article  CAS  PubMed  Google Scholar 

  19. Conti, M.A. & Adelstein, R.S. Nonmuscle myosin II moves in new directions. J. Cell Sci. 121, 11–18 (2008).

    Article  CAS  PubMed  Google Scholar 

  20. Tan, J.L., Ravid, S. & Spudich, J.A. Control of nonmuscle myosins by phosphorylation. Annu. Rev. Biochem. 61, 721–759 (1992).

    Article  CAS  PubMed  Google Scholar 

  21. Simons, M. et al. Human nonmuscle myosin heavy chains are encoded by two genes located on different chromosomes. Circ. Res. 69, 530–539 (1991).

    Article  CAS  PubMed  Google Scholar 

  22. Golomb, E. et al. Identification and characterization of nonmuscle myosin II–C, a new member of the myosin II family. J. Biol. Chem. 279, 2800–2808 (2004).

    Article  CAS  PubMed  Google Scholar 

  23. Maupin, P., Phillips, C.L., Adelstein, R.S. & Pollard, T.D. Differential localization of myosin-II isozymes in human cultured cells and blood cells. J. Cell Sci. 107, 3077–3090 (1994).

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  25. 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 

  26. Blanchard, N. et al. Strong and durable TCR clustering at the T/dendritic cell immune synapse is not required for NFAT activation and IFN-γ production in human CD4+ T cells. J. Immunol. 173, 3062–3072 (2004).

    Article  CAS  PubMed  Google Scholar 

  27. Ilani, T., Khanna, C., Zhou, M., Veenstra, T.D. & Bretscher, A. Immune synapse formation requires ZAP-70 recruitment by ezrin and CD43 removal by moesin. J. Cell Biol. 179, 733–746 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. 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 

  29. Valitutti, S., Muller, S., Cella, M., Padovan, E. & Lanzavecchia, A. Serial triggering of many T-cell receptors by a few peptide-MHC complexes. Nature 375, 148–151 (1995).

    Article  CAS  PubMed  Google Scholar 

  30. Krogsgaard, M. et al. Agonist/endogenous peptide-MHC heterodimers drive T cell activation and sensitivity. Nature 434, 238–243 (2005).

    Article  CAS  PubMed  Google Scholar 

  31. Janeway, C.A. Jr. & Bottomly, K. Responses of T cells to ligands for the T-cell receptor. Semin. Immunol. 8, 108–115 (1996).

    Article  PubMed  Google Scholar 

  32. 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 

  33. Barda-Saad, M. et al. Dynamic molecular interactions linking the T cell antigen receptor to the actin cytoskeleton. Nat. Immunol. 6, 80–89 (2005).

    Article  CAS  PubMed  Google Scholar 

  34. 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 

  35. Fleire, S.J. et al. B cell ligand discrimination through a spreading and contraction response. Science 312, 738–741 (2006).

    Article  CAS  PubMed  Google Scholar 

  36. Giannone, G. et al. Lamellipodial actin mechanically links myosin activity with adhesion-site formation. Cell 128, 561–575 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Pasternak, C., Spudich, J.A. & Elson, E.L. Capping of surface receptors and concomitant cortical tension are generated by conventional myosin. Nature 341, 549–551 (1989).

    Article  CAS  PubMed  Google Scholar 

  38. Mabuchi, I. & Okuno, M. The effect of myosin antibody on the division of starfish blastomeres. J. Cell Biol. 74, 251–263 (1977).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Matsumura, F. Regulation of myosin II during cytokinesis in higher eukaryotes. Trends Cell Biol. 15, 371–377 (2005).

    Article  CAS  PubMed  Google Scholar 

  40. Munro, E., Nance, J. & Priess, J.R. Cortical flows powered by asymmetrical contraction transport PAR proteins to establish and maintain anterior-posterior polarity in the early C. elegans embryo. Dev. Cell 7, 413–424 (2004).

    Article  CAS  PubMed  Google Scholar 

  41. Mescher, M.F. Surface contact requirements for activation of cytotoxic T lymphocytes. J. Immunol. 149, 2402–2405 (1992).

    CAS  PubMed  Google Scholar 

  42. Galbraith, C.G., Yamada, K.M. & Sheetz, M.P. The relationship between force and focal complex development. J. Cell Biol. 159, 695–705 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Lillemeier, B.F., Pfeiffer, J.R., Surviladze, Z., Wilson, B.S. & Davis, M.M. Plasma membrane-associated proteins are clustered into islands attached to the cytoskeleton. Proc. Natl. Acad. Sci. USA 103, 18992–18997 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Gallagher, P.J., Herring, B.P., Griffin, S.A. & Stull, J.T. Molecular characterization of a mammalian smooth muscle myosin light chain kinase. J. Biol. Chem. 266, 23936–23944 (1991).

    CAS  PubMed  Google Scholar 

  45. Ludowyke, R.I., Peleg, I., Beaven, M.A. & Adelstein, R.S. Antigen-induced secretion of histamine and the phosphorylation of myosin by protein kinase C in rat basophilic leukemia cells. J. Biol. Chem. 264, 12492–12501 (1989).

    CAS  PubMed  Google Scholar 

  46. Koretzky, G.A., Abtahian, F. & Silverman, M.A. SLP76 and SLP65: complex regulation of signalling in lymphocytes and beyond. Nat. Rev. Immunol. 6, 67–78 (2006).

    Article  CAS  PubMed  Google Scholar 

  47. Zeng, R. et al. SLP-76 coordinates Nck-dependent Wiskott-Aldrich syndrome protein recruitment with Vav-1/Cdc42-dependent Wiskott-Aldrich syndrome protein activation at the T cell-APC contact site. J. Immunol. 171, 1360–1368 (2003).

    Article  CAS  PubMed  Google Scholar 

  48. Dustin, M.L. & Springer, T.A. Lymphocyte function-associated antigen-1 (LFA-1) interaction with intercellular adhesion molecule-1 (ICAM-1) is one of at least three mechanisms for lymphocyte adhesion to cultured endothelial cells. J. Cell Biol. 107, 321–331 (1988).

    Article  CAS  PubMed  Google Scholar 

  49. Vasiliver-Shamis, G. et al. HIV-1 envelope gp120 induces a stop signal and virological synapse formation in non-infected CD4+ T cells. J. Virol. 82, 9445–9457 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Bretscher, A. Rapid phosphorylation and reorganization of ezrin and spectrin accompany morphological changes induced in A-431 cells by epidermal growth factor. J. Cell Biol. 108, 921–930 (1989).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank D. Garbett for help with data analysis with Volocity and for comments, and D.W. Pruyne for help in setting up DIC microscopy. Supported by the European Molecular Biology Organization (T.I.) and the US National Institutes of Health (GM36652 to A.B.; and AI44931 and Nanomedicine Development Center EY16586 to M.L.D.).

Author information

Author notes

  1. Anthony Bretscher and Michael L Dustin: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York, USA

    Tal Ilani & Anthony Bretscher

  2. Molecular Pathogenesis Program, Helen L. and Martin S. Kimmel Center for Biology and Medicine of the Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, New York, USA

    Gaia Vasiliver-Shamis, Santosh Vardhana & Michael L Dustin

Authors
  1. Tal Ilani
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gaia Vasiliver-Shamis
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Santosh Vardhana
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anthony Bretscher
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael L Dustin
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

The laboratories of A.B. and M.L.D. did independent work on the involvement of myosin IIA in the formation of immunological synapses and continued the work collaboratively focusing on studies in the human system initiated by T.I. and A.B.; T.I. conceived and did the experiments in Figures 2, 3, 4, 5a,b and 6; T.I., G.V.-S. and S.V. collaborated on Figures 1, 5c and 7; and T.I. and A.B. wrote the first draft of the manuscript, which M.L.D. extensively edited and revised.

Corresponding authors

Correspondence to Anthony Bretscher or Michael L Dustin.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 3739 kb)

Supplementary Movie 1

Jurkat T cells (movies S1–S3, S7) or primary human CD4 cells (movies S4–S6) pre treated with DMSO (movies S1 and S4), blebbistatin (movies S2 and S5) or ML7 (movies S3 and S67) were added to planer a lipid bilayer containing Alexa-568 labeled OKT3 and ICAM1, and imaged during the initial min of synapse formation by TIRF microscopy. In movie S7 blebbistatin was added to the flow cell following initial contact and microcluster formation. (AVI 137 kb)

Supplementary Movie 2

Jurkat T cells (movies S1–S3, S7) or primary human CD4 cells (movies S4–S6) pre treated with DMSO (movies S1 and S4), blebbistatin (movies S2 and S5) or ML7 (movies S3 and S67) were added to planer a lipid bilayer containing Alexa-568 labeled OKT3 and ICAM1, and imaged during the initial min of synapse formation by TIRF microscopy. In movie S7 blebbistatin was added to the flow cell following initial contact and microcluster formation. (AVI 1007 kb)

Supplementary Movie 3

Jurkat T cells (movies S1–S3, S7) or primary human CD4 cells (movies S4–S6) pre treated with DMSO (movies S1 and S4), blebbistatin (movies S2 and S5) or ML7 (movies S3 and S67) were added to planer a lipid bilayer containing Alexa-568 labeled OKT3 and ICAM1, and imaged during the initial min of synapse formation by TIRF microscopy. In movie S7 blebbistatin was added to the flow cell following initial contact and microcluster formation. (AVI 602 kb)

Supplementary Movie 4

Jurkat T cells (movies S1–S3, S7) or primary human CD4 cells (movies S4–S6) pre treated with DMSO (movies S1 and S4), blebbistatin (movies S2 and S5) or ML7 (movies S3 and S67) were added to planer a lipid bilayer containing Alexa-568 labeled OKT3 and ICAM1, and imaged during the initial min of synapse formation by TIRF microscopy. In movie S7 blebbistatin was added to the flow cell following initial contact and microcluster formation. (AVI 429 kb)

Supplementary Movie 5

Jurkat T cells (movies S1–S3, S7) or primary human CD4 cells (movies S4–S6) pre treated with DMSO (movies S1 and S4), blebbistatin (movies S2 and S5) or ML7 (movies S3 and S67) were added to planer a lipid bilayer containing Alexa-568 labeled OKT3 and ICAM1, and imaged during the initial min of synapse formation by TIRF microscopy. In movie S7 blebbistatin was added to the flow cell following initial contact and microcluster formation. (AVI 900 kb)

Supplementary Movie 6

Jurkat T cells (movies S1–S3, S7) or primary human CD4 cells (movies S4–S6) pre treated with DMSO (movies S1 and S4), blebbistatin (movies S2 and S5) or ML7 (movies S3 and S67) were added to planer a lipid bilayer containing Alexa-568 labeled OKT3 and ICAM1, and imaged during the initial min of synapse formation by TIRF microscopy. In movie S7 blebbistatin was added to the flow cell following initial contact and microcluster formation. (AVI 930 kb)

Supplementary Movie 7

Jurkat T cells (movies S1–S3, S7) or primary human CD4 cells (movies S4–S6) pre treated with DMSO (movies S1 and S4), blebbistatin (movies S2 and S5) or ML7 (movies S3 and S67) were added to planer a lipid bilayer containing Alexa-568 labeled OKT3 and ICAM1, and imaged during the initial min of synapse formation by TIRF microscopy. In movie S7 blebbistatin was added to the flow cell following initial contact and microcluster formation. (AVI 261 kb)

Supplementary Movie 8

SEE superantigen loaded B cells were immobilized in dishes with coverslip inserts and ML7 pretreated Jurkat T cells were added and allowed to form immunological synapses. Cells were imaged using DIC microscopy. B cell is depicted in the first frame of the movie prior to T cells addition. (AVI 5942 kb)

Supplementary Movie 9

SEE superantigen loaded B cells were immobilized in dishes with coverslip inserts and primary human CD4 cells were added and allowed to form immunological synapses. Blebbistatin (movie S9) or ML7 (movie S10) were added 1-2 min after synapse formation and cells were imaged using DIC microscopy. T and B cells are depicted by T and B in the first frame of the movies, respectively. (AVI 3917 kb)

Supplementary Movie 10

SEE superantigen loaded B cells were immobilized in dishes with coverslip inserts and primary human CD4 cells were added and allowed to form immunological synapses. Blebbistatin (movie S9) or ML7 (movie S10) were added 1-2 min after synapse formation and cells were imaged using DIC microscopy. T and B cells are depicted by T and B in the first frame of the movies, respectively. (AVI 4983 kb)

Supplementary Movie 11

Jurkat T cells were incubated with the cytoplasmic Ca2+ sensitive dye Fluo-LOJO and then mixed with SEE superantigen loaded B cells that were prestained with CMTPX (red), and allowed to form immunological synapses. Changes in Fluo-LOJO emission intensity were imaged following ML7 addition. The T cell is depicted in the movie while the B cell is depicted in figure S4. (AVI 5027 kb)

Supplementary Movie 12

Primary human CD4 cells were incubated with the cytoplasmic Ca2+ sensitive dye Fluo-LOJO and activated with OKT3. DMSO (movie S12), blebbistatin (movie S13) or ML7 (movie S14) were then added to stimulated cells and Fluo-LOJO emission intensity was imaged. (AVI 454 kb)

Supplementary Movie 13

Primary human CD4 cells were incubated with the cytoplasmic Ca2+ sensitive dye Fluo-LOJO and activated with OKT3. DMSO (movie S12), blebbistatin (movie S13) or ML7 (movie S14) were then added to stimulated cells and Fluo-LOJO emission intensity was imaged. (AVI 480 kb)

Supplementary Movie 14

Primary human CD4 cells were incubated with the cytoplasmic Ca2+ sensitive dye Fluo-LOJO and activated with OKT3. DMSO (movie S12), blebbistatin (movie S13) or ML7 (movie S14) were then added to stimulated cells and Fluo-LOJO emission intensity was imaged. (AVI 651 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ilani, T., Vasiliver-Shamis, G., Vardhana, S. et al. T cell antigen receptor signaling and immunological synapse stability require myosin IIA. Nat Immunol 10, 531–539 (2009). https://doi.org/10.1038/ni.1723

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni.1723

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing