Claire Hivroz

Paola Larghi

Mabel Jouve

Laurence Ardouin

Purification of LAT containing membranes from resting and activated T lymphocytes

Keywords: T lymphocyte, Immune synapse, flotation gradient, Strep-Tag-Strep-Tactin affinity purification, LAT

In T lymphocytes, the immune synapse is an active zone of vesicular traffic. Directional transport of vesicular receptors and signaling molecules from or to the immune synapse has been shown to play an important role in T cell receptor (TCR) signal transduction. However, how vesicular trafficking is regulating the activation of T cells is still a burning question and the characterization of these intracellular compartments remains the first step to understand this process. We describe herein a protocol, which combined a separation of membranes on flotation gradient with an affinity purification of Strep-tagged fusion transmembrane proteins with Strep-Tactin® resins, allowing the purification of membranes containing the Streptagged molecule of interest. By keeping the membranes intact this protocol leads to the purification of molecules physically associated with the Strep-Tagged protein as well as of molecules present in the same membrane compartment: transmembrane proteins, proteins strongly associated with the membranes, luminal proteins. The example shown herein is the purification of membrane compartment prepared from T lymphocytes expressing LAT fused to a Strep-Tag.

Introduction

How the activation of T cells is regulated remains a fundamental question for understanding the adaptive immune response. It has been 17 years now that the immune synapse has been described by both A. Kupfer's and ML. Dustin's groups [START_REF] Monks | Three-dimensional segregation of supramolecular activation clusters in T cells[END_REF][START_REF] Grakoui | The immunological synapse: a molecular machine controlling T cell activation [see comments[END_REF]. Since then a growing number of papers have investigated the spatio-temporal organization of this structure as well as the mechanisms involved in its formation and its role in T cell activation. The application of microscopy techniques, revealed that different receptors expressed by T lymphocytes and involved in T-cell activation localized to defined supramolecular activation clusters (SMACs) in the contact zone between the T lymphocyte and the antigen-presenting cell (APC), with antigen receptors distributing to the center of this region (cSMAC), and adhesion molecules accumulating at the periphery (pSMAC) [START_REF] Monks | Three-dimensional segregation of supramolecular activation clusters in T cells[END_REF][START_REF] Grakoui | The immunological synapse: a molecular machine controlling T cell activation [see comments[END_REF]. Development of high resolution microscopy then allowed for a better description of the dynamic of these contact regions, showing that receptors and signaling molecules aggregate in dynamic microclusters, which form at the periphery of the immune synapse and migrate toward its center [START_REF] Yokosuka | Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76[END_REF][START_REF] Campi | Actin and agonist MHC-peptide complexdependent T cell receptor microclusters as scaffolds for signaling[END_REF]. These microscopy based analysis also allowed the characterization of the polarized trafficking and secretion of vesicular components at the immune synapse [START_REF] Kupfer | Polarized expression of cytokines in cell conjugates of helper T cells and splenic B cells[END_REF][START_REF] Kupfer | On the mechanism of unidirectional killing in mixtures of two cytotoxic T lymphocytes. Unidirectional polarization of cytoplasmic organelles and the membrane-associated cytoskeleton in the effector cell[END_REF][START_REF] Chemin | Cytokine secretion by CD4+ T cells at the immunological synapse requires Cdc42-dependent local actin remodeling but not microtubule organizing center polarity[END_REF]. Since these early studies, it has become evident that the immune synapse is an active zone for directional exocytosis, endocytosis, receptor recycling and more generally vesicular traffic [START_REF] Griffiths | The immunological synapse: a focal point for endocytosis and exocytosis[END_REF][START_REF] Rossy | Super-resolution microscopy of the immunological synapse[END_REF][START_REF] Purbhoo | The function of sub-synaptic vesicles during T-cell activation[END_REF][START_REF] Onnis | Vesicular Trafficking to the Immune Synapse: How to Assemble Receptor-Tailored Pathways from a Basic Building Set[END_REF]. Yet, how vesicular trafficking is involved in T cell activation is still only partially understood.

Several groups including ours have shown that intracellular vesicles containing signaling molecules are actively involved in signal transduction processes in T lymphocytes, providing unique platforms for specific signaling complexes to assemble or be activated [START_REF] Purbhoo | Dynamics of subsynaptic vesicles and surface microclusters at the immunological synapse[END_REF][START_REF] Williamson | Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events[END_REF][START_REF] Soares | Regulated vesicle fusion generates signaling nanoterritories that control T cell activation at the immunological synapse[END_REF][START_REF] Larghi | VAMP7 controls T cell activation by regulating the recruitment and phosphorylation of vesicular Lat at TCR-activation sites[END_REF]. These intracellular vesicles, which localize close to or interact with the membrane at the immune synapse, may regulate T-cell activation by transporting cargo to or from specific sites of the immune synapse. They may also directly control T lymphocyte signaling process by acting as mobile platforms that compartmentalize and organize signaling inside the cell. Along this line, it is worth noting that location and intracellular trafficking regulate the signaling induced by many receptors such as EGFR [START_REF] Vieira | Control of EGF receptor signaling by clathrinmediated endocytosis[END_REF], TGFβR [START_REF] Guglielmo | Distinct endocytic pathways regulate TGF-beta receptor signalling and turnover[END_REF], TLR [START_REF] Mcgettrick | Localisation and trafficking of Toll-like receptors: an important mode of regulation[END_REF] and BCR [START_REF] Chaturvedi | Endocytosed BCRs sequentially regulate MAPK and Akt signaling pathways from intracellular compartments[END_REF]. Intracellular location of signaling also matters in T lymphocytes. Indeed, differential intracellular locations of signaling molecules have been shown to translate to different functional outcomes [START_REF] Mor | The lymphocyte function-associated antigen-1 receptor costimulates plasma membrane Ras via phospholipase D2[END_REF][START_REF] Daniels | Thymic selection threshold defined by compartmentalization of Ras/MAPK signalling[END_REF][START_REF] Perez De Castro | Ras activation in Jurkat T cells following low-grade stimulation of the T-cell receptor is specific to N-Ras and occurs only on the Golgi apparatus[END_REF].

Finally, there is emerging evidence that different pools of sub-synaptic vesicles containing signaling molecules exist [START_REF] Soares | Regulated vesicle fusion generates signaling nanoterritories that control T cell activation at the immunological synapse[END_REF][START_REF] Choudhuri | Polarized release of T-cell-receptor-enriched microvesicles at the immunological synapse[END_REF][START_REF] Finetti | Regulation of vesicular traffic at the T cell immune synapse: lessons from the primary cilium[END_REF]]. Yet, their relative role and content are unknown. The mechanisms controlling the traffic of these different vesicular compartments and their interaction with the immune synapse are still a matter of debate [START_REF] Lillemeier | TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation[END_REF][START_REF] Williamson | Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events[END_REF][START_REF] Larghi | VAMP7 controls T cell activation by regulating the recruitment and phosphorylation of vesicular Lat at TCR-activation sites[END_REF][START_REF] Balagopalan | Cutting edge: cell surface linker for activation of T cells is recruited to microclusters and is active in signaling[END_REF][START_REF] Soares | Regulated vesicle fusion generates signaling nanoterritories that control T cell activation at the immunological synapse[END_REF].

To better understand T cell signaling at the immune synapse, it thus seems important to develop tools that allow the purification of these different intracellular compartments and to analyze their content.

We herein describe a protocol of purification of membrane compartments containing the adaptor molecule LAT. This molecule, which was cloned at the same time as the first characterization of the immune synapse [START_REF] Weber | Molecular cloning of the cDNA encoding pp36, a tyrosinephosphorylated adaptor protein selectively expressed by T cells and natural killer cells[END_REF][START_REF] Zhang | LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation[END_REF], plays a key role in T cell activation. It is present both at the plasma membrane and in vesicles [START_REF] Bonello | Dynamic recruitment of the adaptor protein LAT: LAT exists in two distinct intracellular pools and controls its own recruitment[END_REF]. The traffic of the vesicular pool of LAT to the immune synapse requires the SNARE protein VAMP7 [START_REF] Larghi | VAMP7 controls T cell activation by regulating the recruitment and phosphorylation of vesicular Lat at TCR-activation sites[END_REF]. Our protocol is based on a first step of separation of different intracellular organelles by flotation gradient and a second step of affinity purification of Strep-tagged fusion proteins (Strep-tag® is a nine amino acid peptide with intrinsic streptavidin-binding activity) on resins coated with Strep-Tactin ® (an engineered streptavidin that binds Strep-tag®) [START_REF] Junttila | Single-step Strep-tag purification for the isolation and identification of protein complexes from mammalian cells[END_REF]. Because the purification is done in the absence of detergent, it not only allows the purification of molecules that are physically associated with the tagged-bait, as usually done for interactome studies [START_REF] Roncagalli | Quantitative proteomics analysis of signalosome dynamics in primary T cells identifies the surface receptor CD6 as a Lat adaptor-independent TCR signaling hub[END_REF][START_REF] Paster | A THEMIS:SHP1 complex promotes T-cell survival[END_REF] but also the purification of molecules that are present in the same membranes/vesicles being integral or lumenal proteins of the compartments (see Fig19.1). We think that this protocol, presented here with the example of LAT, can be used to purify membrane compartments containing other tagged transmembrane proteins.

Materials

Prepare all solutions using ultrapure water. Solutions are stored at room temperature unless stated otherwise.

2.1. Solutions and material for cell disruption and gradient preparation.

1. Cells: LAT deficient JCAM2.5 Jurkat T cells [START_REF] Finco | LAT is required for TCR-mediated activation of PLCgamma1 and the Ras pathway[END_REF] expressing the mouse LAT-Strep-tag® protein [START_REF] Roncagalli | Quantitative proteomics analysis of signalosome dynamics in primary T cells identifies the surface receptor CD6 as a Lat adaptor-independent TCR signaling hub[END_REF]. 2. Wash the cells twice in RPMI medium to remove the FCS contained in culture medium: fill a 50 mL tube with RPMI pre-warmed at 37°C and spin for 5 min at 300xg. Remove the supernatant, resuspend the pellet and spin one more time.

3. For the stimulation, resuspend the cells in RPMI at 100 10 6 /mL and then transfer 1 mL of this cell suspension in an Eppendorf tube (2 mL). Do as many tubes as needed. Leave the cells at 37°C for 5 min (in a water bath) without any stimulation. Meanwhile, prepare the appropriate dilutions of anti-CD3 and anti-CD28 antibodies (in RPMI). Anti-CD3 Ab is used at 12.5µg/mL and anti-CD28 Ab at 25 µg/mL. (see Note 2) 4. Add antibodies to the cells directly in Eppendorf tubes and mix by gently pipetting up and down without vortexing the cells. Incubate at 37°C (in water bath) for the requested period of time. 5. To stop the activation, add 1 mL of ice-cold PBS to the cells and spin immediately for 5 min at 250xg at 4°C.

Cells disruption

It is important to keep samples on ice as much as possible. To avoid any contamination between samples, rinse extensively the Dounce homogenizer with sterile water and do the last rinse with homogenization buffer. 7. Wash the resin/membranes pellet 5 times as in 3.5.2. but add phosphatase and protease inhibitors to the IBA washing buffer. 2. For each gradient, use between 100-400 10 6 cells. If more than 500 10 6 cells are used, the mechanical lysis will be less efficient and will reduce both the gradient separation and the protein yield. 4. The total lysate can be immunoblotted for specific phospho-proteins in order to ensure that the stimulation was successful. A proper activation of the cells can also be detected in the different fractions following the flotation gradient. (see Fig19. 8. Carefully equilibrate the tubes between layers to ensure that the centrifuge will be balanced during centrifugation. 9. Without brake, it will take about 1 h for the rotor to stop.

10. The density of each fraction can be measured using a refractometer (Carl Zeiss). After centrifugation, the gradient results in a linear increase in density and can be assessed to ensure the reproducibility of the experiments. 13. Resin binding capacity (from IBA data sheet): 1 mL of sedimented resin (corresponding to 2 mL of a 50 % suspension) is able to purify 50 to 100 nmol recombinant Strep-tag® protein (up to 3 mg in the case of a 30 kDa protein) 14. You can make a mark on the Eppendorf tube to visualize the top of the pellet formed by the resin. Indeed, the resin is whitish and the pellet can be difficult to see. [START_REF] Larghi | VAMP7 controls T cell activation by regulating the recruitment and phosphorylation of vesicular Lat at TCR-activation sites[END_REF]. In our experiments, the volume of fraction sample used for purification is 400 µL, so we resuspend the resin in 400 µL of IBA washing buffer supplemented with proteases and phosphatases inhibitors. 16. In the example described herein, fraction 3 was precipitated because it contains LAT and the vesicular SNARE protein VAMP7 that was shown to control LAT trafficking [START_REF] Larghi | VAMP7 controls T cell activation by regulating the recruitment and phosphorylation of vesicular Lat at TCR-activation sites[END_REF] (see figure19.2A) and thus constitute an enriched pool of the LAT containing vesicles transported to the immune synapse. 17. Elution steps can be repeated to improve the yield of protein bait recovery (see Fig19.4). Each application will require preliminary settings to determine recovery yields of precipitation and of elution. 18. To control for the efficacy of the precipitation and elution steps, run side by side on a gel samples from fraction 3 (before purification, 3.4.1), unbound membranes (3.5.6), elution (membranes containing the Strep-tag® proteins, 3.5.10) and resin after elution (non-eluted proteins, 3.5.11). Use an antibody corresponding to the Strep-tag® protein and quantify its amount in each sample. The comparison of the amount of Strep-tag® protein found in fraction 3 and in "unbound membranes" will give the efficiency of purification, whereas the comparison between the eluted material and material still bound to resin after elution will give the efficiency of elution.

19. As a control for the specificity of the purification, you can run the experiment in the exact same conditions but using a "nude" Sepharose, which does not contain the Strep-Tactin ® (see Fig19.1.C). Proteins that will bind in a non-specific manner on the resin will constitute the background of the experiment. 20. Also, negative control can come from non-activated conditions or using cells that do not express the Strep-tag® protein. Presence of the different intracellular organelles is followed using antibodies for specific markers: mitofilin for mitochondria, LAMP2 for lysosomes, GM130 for cis-Golgi, TGN46 for trans-Golgi, gp 96 for endoplasmic reticulum, VAMP7 for vesicular compartment and CD45, TfR, CD3ζ, LAT for plasma membrane and endocytic compartments. B: Electronmicroscopy images showing immunogold labeling for LAT on the vesicles present in fraction 3. The size of the vesicles is between 50 and 300nm. Two bands are revealed with the anti-CD3ζ, the upper band corresponding to the phosphorylated form of the protein.

2 .

 2 Homogenization buffer: 0.25 M sucrose, 10 mM Tris-HCl pH 7.4, 1 mM EDTA 3. Iodixanol dilution buffer: 0.25 M sucrose, 60 mM Tris-HCl pH 7.4, 6mM EDTA. 4. cOmplete TM , EDTA-free protease inhibitor cocktail from Roche Life Science. Dissolve 1 tablet in 2 mL of water according to the manufacturer instructions to obtain a 25x concentrated stock solution (store at -20°C). 5. Halt TM Phosphatase Inhibitor cocktail from ThermoFisher Scientific. Solution is diluted 100x in appropriate solution. (store at 4°C) 6. OptiPrep TM (Axis-Shield) density gradient medium is a solution of 60% iodixanol in water with a density of 1.32 g/mL 7. Phosphate-buffered saline (PBS) 8. RPMI-1640 9. 2 mL glass Dounce homogenizer 10. Needles (Terumo) 25Gx5/8" and 2 mL Syringes 11. SW 55 Ti Rotor, Swinging Bucket (Beckman-Coulter) 12. Ultra-clear centrifuge tubes 5 mL (Beckman-Coulter) 13. RIPA lysis buffer: 25 mM Tris-HCl pH 7.4 , 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 150 mM NaCl. 2.2. Solutions for the immunoprecipitation and elution steps.

1 . 1 .

 11 Strep-Tactin ® Sepharose ® resin (IBA) 50% suspension (50% suspension in 100mM Tris-HCl pH 8.0 ; 1 mM EDTA; 150 mM NaCl) (Fig19.1B) 2. Sepharose ® 50% suspension (IBA) (Fig19.1C) 3. IBA washing buffer: 100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA.4. Biotin elution buffer: IBA washing buffer supplemented with 2 mM of D-biotin (IBA). Store at 4°C 2.3. Western blot 5. SDS PAGE running buffer: 25 mM Tris-HCl pH 8.3, 0.192 M Glycine, 0.1% SDS 6. Western blot transfer buffer: 25 mM Tris, 0.192 M glycine and 20% methanol 7. Tris buffered saline (TBS; 10x) containing 0.05% Tween-20 (TBST) 8. Blocking solution: 5% BSA in TBST. Filter on 0.45µm and store at 4°C. 9. BIORAD Mini-PROTEAN ® TGX Stain-free TM Precast gels 4%-15% 10. 4x Laemmli sample buffer (277.8 mM Tris-HCl pH 6.8, 44.4% (v/v) glycerol, 0.02% bromophenol blue, 5% SDS, BIORAD) 11. Reducing sample buffer 10x: 500 mM Dithiothreitol (Novex TM ) 12. PVDF transfer membrane (Immuno-Blot ® , BIORAD) 13. ECL Blotting substrate (Pierce) Cells stimulation. It is very important to avoid any difference of temperature. Place the centrifuge at room temperature and pre-warm medium at 37°C. 1. Harvest and count the cells expressing the transmembrane Strep-tag® protein of interest. (see Note 1)

1 . 3 . 4 . 4 . 2 .

 13442 Fig19.1.E).

8 . 10 .

 810 After the last wash, carefully remove the supernatant and add 120 µL of elution buffer containing 2 mM D-biotin to the pellet containing membranes bound Strep-tag® protein coupled to the resin (Fig19.1F). Incubate for 8 min on ice. 9. Centrifuge at 2000xg for 1 min. Remove the supernatant: it corresponds to eluted membranes containing the Strep-tag® proteins (Fig19.1G). (See Note 17) For each elution sample, transfer 20 µL in an Eppendorf tube and mix with 2 µL of 10x reducing sample buffer and 10 µL of 4x Laemmli buffer. Proceed to immunoblotting as in 3.4. 11. Following the last elution step, resuspend the resin in 500 µL of IBA washing buffer and transfer 50 µL in an Eppendorf tube, mix with 5 µL of reducing sample buffer and 18 µL of 4x Laemmli buffer. This will be the control of the efficacy of elution (material staying on Strep-tactin® resin after elution). Proceed to immunoblotting as in 3.4. (See Note 18) 4. Notes 1. This protocol was optimized to purify membranes containing LAT from JCAM2.5 cells (LAT deficient Jurkat cells) expressing the mouse chimeric LAT coupled to aStrep-tag®[START_REF] Junttila | Single-step Strep-tag purification for the isolation and identification of protein complexes from mammalian cells[END_REF][START_REF] Roncagalli | Quantitative proteomics analysis of signalosome dynamics in primary T cells identifies the surface receptor CD6 as a Lat adaptor-independent TCR signaling hub[END_REF][START_REF] Finco | LAT is required for TCR-mediated activation of PLCgamma1 and the Ras pathway[END_REF].

3 .

 3 Use 150-200 10 6 cells/1.5 mL. Increase the volume of homogenization buffer in proportion.

3 ) 5 .

 35 These 2 sequential steps of homogenization should break between 60% and 80% of the cells without damaging nuclei. Cells breakage can be checked by Trypan blue staining under a microscope. 6. Ultracentrifuge settings: acceleration maximum, brake maximum 7. Carefully mix the content of the Optiprep TM bottle before use. Keep the Optiprep TM solution at room temperature, sterile and protected from light.

11 .

 11 Each lane contains 1/40 of total fraction proteins. (see Fig19.2A) 12. The Sepharose suspension has a tendency to stick to plastic pipettes and tips. Moreover 200 µL tips are too tight to let the resin go through so cut off the end of the tip to ensure reproducible pipetting.

Fig19. 1 :

 1 Fig19.1: Experimental approach followed to purify membranes containing a chimeric LAT

Fig19. 2 :

 2 Fig19.2: A. Western blot analysis of the different fractions collected after flotation gradient.

Fig19. 3 :

 3 Fig19.3: Western blot analysis of the phosphorylated signaling molecules in the different fractions. JCAM2.5 T cells expressing a chimeric LAT fused to Strep-Tactin ® were either left

Fig19. 4 :

 4 Fig19.4: Western blot analysis of eluted proteins. Fractions 3 obtained from unstimulated or

Acknowledgments.

We would like to thank R. Roncagalli for providing the LAT-Strep-tag® construct and for helpful discussion. We are grateful to S. Dogniaux, JM. Carpier, M.Saitakis and A.Zucchetti for their kind support. This work was supported by ANR-10-IDEX-0001-02 PSL*, ANR-11-LABX-0043, ANR-13-BSV2-0018 "NeuroImmunoSynapse"), Fondation pour la Recherche Médicale (FRM, FRM DEQ20140329513).

*Dilution is given when antibody concentration is not known.

StrepTactin sepharose

Membrane vesicle

Fraction to be purified "nude" sepharose