Rab6-dependent retrograde traffic of LAT controls immune synapse formation and T cell activation Jean-Marie Carpier, Andres Zucchetti, Laurence Bataille, Stephanie Dogniaux, Massiullah Shafaq-Zadah, Sabine Bardin, Marco Lucchino, Mathieu Maurin, Leonel Joannas, Joao Gamelas Magalhaes, et al. To cite this version: Jean-Marie Carpier, Andres Zucchetti, Laurence Bataille, Stephanie Dogniaux, Massiullah Shafaq- Zadah, et al.. Rab6-dependent retrograde traffic of LAT controls immune synapse formation and T cell activation. Journal of Experimental Medicine, Rockefeller University Press, 2018, 215 (4), pp.1245-1265. 10.1084/jem.20162042. inserm-02446749 HAL Id: inserm-02446749 https://www.hal.inserm.fr/inserm-02446749 Submitted on 21 Jan 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Article Rab6-dependent retrograde traffic of LAT controls immune synapse formation and T cell activation Jean-Marie Carpier,1* Andres E. Zucchetti,1* Laurence Bataille,1** Stéphanie Dogniaux,1** Massiullah Shafaq-Zadah,3** Sabine Bardin,2 Marco Lucchino,3 Mathieu Maurin,1 Leonel D. Joannas,1 Joao Gamelas Magalhaes,1 Ludger Johannes,3 Thierry Galli,4 Bruno Goud,2 and Claire Hivroz1 1Crosstalk between T Cells and Dendritic Cells Group, Institut Curie, Paris Sciences and Lettres Research University, INS ERM U932, Paris, France 2Molecular Mechanisms of Intracellular Transport Group, Institut Curie, Paris Sciences and Lettres Research University, CNRS UMR 144, Paris, France 3Cellular and Chemical Biology of Membranes and Therapeutic Delivery Unit, Institut Curie, Paris Sciences and Lettres Research University, INS ERM U1143, CNRS UMR 3666, Paris, France 4Center of Psychiatry and Neurosciences, Membrane Traffic in Health and Diseased Brain, Université Paris Descartes, Sorbonne Paris Cité, INS ERM ERL U950, Paris, France The adapter molecule linker for activation of T cells (LAT) orchestrates the formation of signalosomes upon T cell receptor Downloaded from (TCR) stimulation. LAT is present in different intracellular pools and is dynamically recruited to the immune synapse upon stimulation. However, the intracellular traffic of LAT and its function in T lymphocyte activation are ill defined. We show herein that LAT, once internalized, transits through the Golgi–trans-Golgi network (TGN), where it is repolarized to the im- mune synapse. This retrograde transport of LAT depends on the small GTPase Rab6 and the target soluble N-ethylmaleim- ide-sensitive factor attachment protein receptor (t-SNA RE) Syntaxin-16, two regulators of the endosome-to-Golgi/TGN retrograde transport. We also show in vitro in Syntaxin-16– or Rab6-silenced human cells and in vivo in CD4+ T lymphocytes jem.rupress.org of the Rab6 knockout mouse that this retrograde traffic controls TCR stimulation. These results establish that the retrograde traffic of LAT from the plasma membrane to the Golgi-TGN controls the polarized delivery of LAT at the immune synapse and T lymphocyte activation. INTRODUCTION on February 13, 2018 T lymphocyte activation relies on the cognate recogni- long-lasting signaling required for T cell activation (Grakoui tion by the TCR of the MHC-associated peptide ligand et al., 1999; Fooksman et al., 2010). (pMHC) presented at the surface of an APC. Engagement The TCR-induced signaling cascade has long been of the TCR induces a cascade of biochemical reactions that presented as a linear cascade of events taking place exclu- involves tyrosine phosphorylation of the CD3-ζ complexes sively at the plasma membrane. However, it is now clear that by the kinase Lck and recruitment of the cytosolic tyrosine TCR-induced signaling is modular and involves distinct pools Journal of Experimental Medicine kinase ZAP70 to these phosphorylated complexes (Weiss of signaling molecules at the plasma membrane and in intra- and Littman, 1994). ZAP70 then phosphorylates multiple cellular compartments (Ehrlich et al., 2002; Das et al., 2004; tyrosines in the cytoplasmic domain of the transmembrane Yudushkin and Vale, 2010; Antón et al., 2011; Finetti et al., protein LAT (for linker of activated T cells; Zhang et al., 2014; Bouchet et al., 2016). Along these lines, we and others 1998). Once phosphorylated, LAT interacts with several have shown that the intracellular vesicular pool of signaling adapter proteins and enzymes near the site of TCR engage- molecules including LAT plays a key role in T cell activation ment and forms nanostructures of multimolecular signaling (Bonello et al., 2004; Purbhoo et al., 2010; Williamson et al., complexes (Sherman et al., 2011; Roncagalli et al., 2014), 2011; Larghi et al., 2013; Soares et al., 2013). Hence, TCR which organize hierarchically (Sherman et al., 2016) and signaling is regulated by the traffic of these vesicles from and segregate from inhibitory molecules (Su et al., 2016). These to the IS (Purbhoo, 2013; Onnis et al., 2016). complexes control downstream signaling and T lymphocyte One of the key questions now relates to the origin development (Zhang et al., 1998) and activation (Samel- of the vesicular pool of signaling molecules. We herein ad- son, 2002). This process is organized in time and space by dress this question for LAT. a structure that forms at the interface between T lympho- LAT is present in two pools at steady-state: one that is cyte and APC: the immune synapse (IS). The IS allows the close or at the plasma membrane and a pericentrosomal one (Bonello et al., 2004). Upon activation, both pools are re- *J.-M. Carpier and A.E. Zucchetti contributed equally to this paper. © 2018 Carpier et al. This article is distributed under the terms of an Attribution–Noncommercial–Share **L. Bataille, S. Dogniaux, and M. Shafaq-Zadah contributed equally to this paper. Alike–No Mirror Sites license for the first six months after the publication date (see http://www .rupress .org /terms /). After six months it is available under a Creative Commons License (Attribution–Noncommercial– Correspondence to Claire Hivroz: [email protected] Share Alike 4.0 International license, as described at https ://creativecommons .org /licenses /by -nc -sa /4 .0 /). Supplemental material can be found at: The Rockefeller University Press http://doi.org/10.1084/jem.20162042 J. Exp. Med. 2018 1 https://doi.org/10.1084/jem.20162042 cruited to the IS, and both have a role in T cell activation (Lil- B) than in cells expressing a control nontargeting shRNA lemeier et al., 2010; Purbhoo et al., 2010; Williamson et al., (shC) (see silencing in Fig. S1 C). We thus analyzed the rela- 2011; Balagopalan et al., 2013; Larghi et al., 2013). As stated, tive distribution of VAMP7 and LAT by confocal microscopy. LAT is a palmitoylated integral membrane protein that traffics In resting Jurkat T cells, LAT was juxtaposed with the VAMP7 from the Golgi apparatus to the plasma membrane (Hundt compartments but was more central (Fig. 1 A). VAMP7, like et al., 2009). Upon TCR activation, microclusters contain- in other cell types (Chaineau et al., 2009) was present in the ing LAT and SLP-76, another adapter protein that binds Golgi of T cells as shown by its proximity with Rab6, a small phosphorylated LAT, transiently associate with the TCR GTPase associated with Golgi-TGN membranes (Goud et and undergo endocytosis (Barr et al., 2006; Balagopalan et al., 1990) and with Syntaxin-16, a t-SNA RE localized to al., 2007). This endocytosis depends on the ubiquitylation of the Golgi stacks (Simonsen et al., 1998; Tang et al., 1998; LAT by c-Cbl (Balagopalan et al., 2011). The pericentrosomal Fig. 1 A). As shown previously for the relative distribution pool of LAT has been shown to colocalize with internalized of VAMP7 and LAT, LAT was juxtaposed to the Golgi com- transferrin (Bonello et al., 2004), suggesting that LAT does partments labeled with Rab6 or Syntaxin-16, but was more recycle. More recently, we showed that the recruitment of central, showing only an inconspicuous colocalization with LAT-containing vesicles to the TCR activation sites depends these markers (Fig. 1 A). Thus, although VAMP7 is involved on the vesicular–soluble N-ethylmaleimide-sensitive factor in LAT trafficking to the immune synapse, at the steady-state attachment protein receptor (SNA RE) VAMP7 (Larghi et al., the central pool of LAT colocalized little with VAMP7, which Downloaded from 2013). We also showed that LAT phosphorylation, formation was mainly present in Golgi–trans-Golgi compartments. We of the LAT signalosome, and T cell activation were controlled then studied the distribution of LAT in VAMP7-silenced Jur- by this VAMP7-dependent traffic of LAT. Thus, although it is kat T cells. In the absence of VAMP7, the intracellular pool clear that LAT follows both an outward flow from centroso- of LAT colocalized more with the t-SNARE Syntaxin-16 mal intracellular compartments to the IS and an inward flow (Fig. 1 B; quantified in Fig. 1 C). from the periphery to recycling endosomes, the exact path- These results suggest that LAT transits through the way followed by this key protein of T lymphocyte activation Golgi–trans-Golgi