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VAMP7 controls T cell activation by regulating the recruitment and phosphorylation of vesicular Lat at TCR-activation sites

Nature Immunology volume 14, pages 723–731 (2013)Cite this article

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Abstract

The mechanisms by which Lat (a key adaptor in the T cell antigen receptor (TCR) signaling pathway) and the TCR come together after TCR triggering are not well understood. We investigate here the role of SNARE proteins, which are part of protein complexes involved in the docking, priming and fusion of vesicles with opposing membranes, in this process. Here we found, by silencing approaches and genetically modified mice, that the vesicular SNARE VAMP7 was required for the recruitment of Lat-containing vesicles to TCR-activation sites. Our results indicated that this did not involve fusion of Lat-containing vesicles with the plasma membrane. VAMP7, which localized together with Lat on the subsynaptic vesicles, controlled the phosphorylation of Lat, formation of the TCR-Lat-signaling complex and, ultimately, activation of T cells. Our findings suggest that the transport and docking of Lat-containing vesicles with target membranes containing TCRs regulates TCR-induced signaling.

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Figure 1: VAMP7 controls the recruitment of Lat to TCR-activation sites.
Figure 2: Recruitment and clustering of Lat in cells depleted of VAMP7.
Figure 3: Subsynaptic vesicles containing Lat do not fuse with the plasma membrane.
Figure 4: Vesicular pools of Lat are decorated with VAMP7 and are recruited to the immunological synapse.
Figure 5: VAMP7 controls Lat phosphorylation after T cell activation.
Figure 6: VAMP7 controls the TCR-induced activation of T cells.
Figure 7: VAMP7 controls the formation of Lat signalosomes.

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Acknowledgements

We thank A.-M. Lennon and S. Amigorena for discussions; and P. Pierobon, V. Fraisier, P. Paul-Gilloteaux, L. Sengmanivong and the Nikon Imaging Centre at Institut Curie (Centre National de la Recherche Scientifique) for technical assistance with microscopy and image analysis. Supported by Fondation pour la Recherche Médicale (P.L. and K.C., and T.G.'s group), la Ligue contre le Cancer (J.-M.C. and A.B.), DC-Biol Labex (C.H.'s group), Institut National de la Santé et de la Recherche Médicale (T.G.'s group) and the Mairie de Paris Medical Research and Health Program (T.G.'s group).

Author information

Author notes

  1. Karine Chemin

    Present address: Present address: Department of Medicine, Rheumatology Unit, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden.,

  2. Thierry Galli and Claire Hivroz: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut Curie, Centre de Recherche, Pavillon Pasteur, Paris, France

    Paola Larghi, Jean-Marie Carpier, Stéphanie Dogniaux, Karine Chemin, Armelle Bohineust & Claire Hivroz

  2. Institut National de la Santé et de la Recherche Médicale, Unité 932, Immunité et Cancer, Paris, France

    Paola Larghi, Jean-Marie Carpier, Stéphanie Dogniaux, Karine Chemin, Armelle Bohineust & Claire Hivroz

  3. Centre for Vascular Research and Australian Centre for Nanomedicine, University of New South Wales, Sydney, Australia

    David J Williamson & Katharina Gaus

  4. Institut Jacques Monod, Unité Mixte de Recherche 7592, Centre National de la Recherche Scientifique, Université Paris Diderot, Sorbonne Paris Cité, Paris, France

    Lydia Danglot & Thierry Galli

  5. Institut National de la Santé et de la Recherche Médicale Equipe de Recherche Labelisée U950, Membrane Traffic in Neuronal and Epithelial Morphogenesis, Paris, France

    Lydia Danglot & Thierry Galli

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Contributions

P.L. designed, did and analyzed three-dimensional microscopy, TIRF microscopy and biochemistry experiments, and prepared the manuscript; D.J.W. designed, did and analyzed PALM experiments and revised the manuscript; J.-M.C. did and analyzed experiments with mouse T cells; S.D. designed vectors for and did mice genotyping; K.C. and A.B. designed and analyzed three-dimensional microscopy experiments; L.D. and T.G. provided VAMP7-deficient mice and tools; K.G. designed PALM experiments and assisted with manuscript preparation; T.G. designed VAMP7-deficient mice, discussed the results and revised the manuscript; and C.H. conceived of the study, did biochemistry experiments and prepared manuscript.

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Correspondence to Claire Hivroz.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 3445 kb)

Supplementary Video 1

Lat subsynaptic vesicles are recruited to the immunological synapse in activated control Jurkat cells. Time lapse TIRF microscopy images of Lat-GFP expressing Jurkat cells infected with a control shRNA encoding lentiviruses put on glass slides coated with anti-CD3 and anti-CD28 mAbs. (AVI 3503 kb)

Supplementary Video 2

VAMP7 silencing inhibits the recruitment of Lat subsynaptic vesicles to the immunological synapse. Time lapse TIRF microscopy images of Lat-GFP expressing Jurkat cells infected with the VAMP7 specific Sh1RNA encoding lentiviruses put on glass slides coated with anti-CD3 and anti-CD28 mAbs. (AVI 4789 kb)

Supplementary Video 3

VAMP7 silencing inhibits the recruitment of Lat subsynaptic vesicles to the immunological synapse. Time lapse TIRF microscopy images of Lat-GFP expressing Jurkat cells infected with the VAMP7 specific Sh5RNA encoding lentiviruses put on glass slides coated with anti-CD3 and anti-CD28 mAbs. (AVI 2722 kb)

Supplementary Video 4

Lat subsynaptic vesicles recruited to the immunological synapse contain VAMP7.Time-lapse TIRF microscopy images of Jurkat cells co-transfected with Lat-mCherry (magenta) and GFP-VAMP7 (green) put on glass slides coated with anti-CD3 and anti-anti-CD28 mAbs. Left: GFP-VAMP7, middle: Lat-mCherry, right: merge. (AVI 6222 kb)

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Larghi, P., Williamson, D., Carpier, JM. et al. VAMP7 controls T cell activation by regulating the recruitment and phosphorylation of vesicular Lat at TCR-activation sites. Nat Immunol 14, 723–731 (2013). https://doi.org/10.1038/ni.2609

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