*4.4. Single-Molecule Tracking*

SMT experiments were carried out as previously described [13]. Briefly, cells plated on coverslips were incubated in red phenol-free DMEM at 37 ◦C for 10 min with Atto647Nlabeled Fab fragments of mAbs raised against CD81 (TS81), CD9 (SYB-1) or CD82 (TS82) at concentrations in the range of 1 to 10 ng/mL. For single molecule experiments, ~ one probe per Fab is required. Ensemble labeling was performed with full antibody. Homemade objective-type TIRF setup allowing multicolor single-molecule imaging and equipped with a Plan Fluor 100×/1.45 NA objective (Zeiss, Le Pecq, France Brattleboro, VT) was used. All the experiments were performed with a 100 ms integration time. The localization of each fluorescence peak was determined with subpixel resolution by fitting a two-dimensional elliptical Gaussian function. The accuracy of the position measurement in living cells was estimated to be 50 nm by fitting a 2D Gaussian to the emission intensity distribution of an immobile single molecule conjugated with Atto647N.

Movies were analyzed using homemade software (named 'PaTrack') implemented in visual C++ ([29], freely available using the link). Trajectories were constructed using the individual diffraction limited signal of each molecule. The center of each fluorescence peak was determined with subpixel resolution by fitting a two-dimensional elliptical Gaussian function. The two-dimensional trajectories of single molecules were constructed frame per frame. Only trajectories containing at least 41 points were retained. Diffusion coefficient values were determined from a linear fit to the MSD (mean square displacement)-τ plots

between the first and the fourth points (D1–4) according to the equation MSD(t) = 4Dt. The determination of the motion modes was performed using homemade algorithm based on a neural network that has been trained using synthetic trajectories to detect pure Brownian, confined and directed motion modes. Thanks to a sliding window, the trajectory is analyzed and the different modes can be confidently detected within a trajectory for segments larger than 10 frames. Once the motion mode is identified, the different segments are analyzed by plotting the MSD versus time lag. The MSD curve was linearly fitted (Brownian) or adjusted with a quadratic curve (4Dt + v2t2) (directed diffusion) or exponential curve L2/3(1–exp(–12Dt/L2) (confined diffusion), where L is the side of a square domain, the confinement diameter being related to L by Ø conf = (2/ √L) [51]. The apparent diffusion coefficient values were determined from a linear fit between the first and fourth points (D1–4). The algorithm was tested with simulated trajectories displaying pure Brownian, confined or directed behavior or a combination of these 3 modes and successfully applied to a set of single molecule experiments previously recorded for tetraspanins diffusing into plasma membranes.

#### *4.5. Flow Cytometry Experiments*

Cells were grown in 25 cm<sup>2</sup> flasks until reaching exponential growth. Then, cells were harvested using an enzyme-free cell dissociation reagen<sup>t</sup> (purchased from Gibco) and centrifuged at 100× *g* for 5 min at 4 ◦C. The cell pellet was thoroughly suspended in cold PBS. The cells were then centrifuged again and the pellet was suspended in PBS/1% FBS buffer containing 2 μg/mL of mouse IgG raised against the protein of interest. After 30 min of incubation, cells were centrifuged at 100× *g* for 5 min. The pellet was then suspended in PBS/1% FBS. This washing step was repeated 3 times. The pellet was suspended in PBS/1% FBS containing 1 μg/mL of secondary antibodies raised against mouse IgG and coupled to Alexa568. As a control, one sample was incubated only with the secondary antibody. After one hour of incubation at 4 ◦C in the dark, the washing steps were repeated 3 times. After the last wash, the cell pellet was suspended in 500 μL of cold PBS with 2% PFA. After 10 min, cells were washed to remove PFA and were analyzed using a Bio-Rad flow cytometer. Post-acquisition analyses were performed using the software FlowJo.

#### *4.6. Dual-Color Immunofluorescence Using TIRF Microscopy*

One day prior to the experiment, 2 × 10<sup>5</sup> cells were plated in 6-well culture plates containing 25 mm diameter coverslips that had been previously plasma-cleaned. Cells were then fixed with PBS buffer containing 4% PFA for 10 min. Cells were then washed 3 times with PBS. PBS/1% FBS buffer containing 5 μg/mL of antibodies raised against the proteins of interest and coupled to Cy3b or Atto647N was then added to the coverslips for 30 min. Cells were then washed 3 times with PBS/1% FBS. Coverslips were mounted on a glass slide using Prolong Diamond and incubated overnight at 4 ◦C. Cells were imaged using a TIRF microscope at 10 images/s. One hundred frames were acquired and then stacked. Pearson's correlation coefficients were calculated using the colocalizer studio plugin of the software Icy.

#### *4.7. Mass Spectrometry Analysis of Lipids*

Total lipids were extracted overnight at 4 ◦C from the samples dissolved in 0.5 mL water with 10 volumes (5 mL) of CHCl3/CH3OH (1:1, *v*/*v*). The residual pellet obtained after centrifugation (1500× *g*, 5 min) was extracted twice with 2 mL of the same solvent. The three lipid extracts were pooled, dried under a stream of nitrogen, and solubilized in 3 mL CHCl3/CH3OH (1:1, *v*/*v*). Gangliosides were then separated from other lipids using phase partition by adding 1 mL water. After centrifugation, the upper aqueous phase was collected while the lower organic phase was reextracted twice with 2 mL CH3OH/water (1:1, *v*/*v*). The three upper phases containing gangliosides were combined, dried under a stream of nitrogen, and solubilized in 2 mL CH3OH/PBS 10 mM (1:1, *v*/*v*). This ganglisoside extract was desalted on a C18 silica gel column (Sep-Pak Vac 6 cc, 500 mg; Waters), washed

with 7 mL CH3OH and pre-equilibrated with 7 mL CH3OH/PBS 10 mM (1:1, *v*/*v*) before injection. After washing with 10 mL water, purified gangliosides were eluted with 6 mL CH3OH and 4 mL CHCl3/CH3OH (2:1, *v*/*v*). Liquid chromatography (LC) was performed at 30 ◦C using a Dionex UltiMateTM 3000 LC system from ThermoScientific equipped with an autosampler. Separation of GM3, GM2, GD3, GD1a, GD1b, GT1b, and GQ1b standards was achieved under hydrophilic interaction liquid chromatography (HILIC) conditions using a silica Kinetex column (GM1 measurement was not available when we have performed the experiments). The mobile phase was composed of acetonitrile/water (90:10, *v*/*v*) containing 10 mM ammonium acetate and acetonitrile/water (50:50, *v*/*v*) containing 10 mM ammonium acetate. The solvent-gradient system was as follows: 0–1 min 100%, 4 min 79%, 9 min 78%, 14–18 min 50%, and 19–48 min 100%. The flow rate was 400 μL/min and the injection volume was 10 μL. Eluates from the HPLC system were then analyzed by mass spectrometry (LC-MS/MS).

The cholesterol levels were analyzed using a Cholesterol Quantitation Kit from Sigma following the manufacturer's protocol.

#### *4.8. Immunoprecipitations of Tetraspanins and Partners*

Cells were grown in 75 cm<sup>2</sup> flasks until reaching 80% confluence. Cells were then washed three times with PBS and incubated with 10 mL of biotin at 0.5 mg/mL for 45 min at 4 ◦C. The cells were then washed three times with PBS and lysed in a buffer containing 30 mM Tris pH 7.4, 150 mM NaCl, protease inhibitors and 1% of Brij97. After 30 min of incubation at 4 ◦C, the insoluble material was removed by centrifugation at 10,000× *g* for 15 min. The cell lysate was then incubated with inactivated goa<sup>t</sup> serum and G-sepharose protein beads for 2 h. The beads were removed by centrifugation and the lysate was incubated with 1 μg of primary antibody and 10 μL of G-sepharose protein beads for 400 μL of lysate for 2 h at 4 ◦C on a rotating wheel. The beads were washed four times using the lysis buffer with 0.5% Brij97 and Laemmli buffer was added. Immunoprecipitated proteins were separated by SDS-PAGE under non-reducing conditions and transferred to a low fluorescence PVDF membrane. Immunoprecipitates were analyzed by Western blot using streptavidin coupled to Alexa680 and blots revealed using the Odyssey Infrared Imaging System (LI-COR Biosciences GmbH, Bad Homburg, Germany). The antibody TS82 was coupled to biotin and revealed using streptavidin coupled to Alexa680.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/1422-006 7/22/16/8459/s1.

**Author Contributions:** Conceptualization, E.O., F.B., E.R. and P.-E.M.; methodology, L.F., M.M. and P.-E.M.; software, P.D.; validation, L.F., F.B., E.O., E.R. and P.-E.M.; formal analysis, L.F., P.-E.M.; investigation, L.F., M.M., C.B., E.R., P.D.; resources, P.-E.M.; data curation, P.-E.M.; writing—original draft preparation, L.F., P.-E.M.; writing—review and editing, L.F., E.R., C.B., E.O., F.B., P.-E.M.; supervision, P.-E.M., C.B., E.R., F.B.; project administration, P.-E.M.; funding acquisition, P.-E.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by France BioImaging (FBI, ANR10INSB04) and the GIS IBISA (Infrastructures en Biologie Santé et Agronomie).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article or Supplementary Material.

**Acknowledgments:** L.F. was a recipient of the french ministry of research and a FRM fellow. We thank Tomonori Saotome, Anthony Lozano and Thibaud Dieudonné for technical help. We thank Emmanuel Margeat for his help in single-molecule microscopy experiments. Mass spectrometry analysis was performed by the Chemosens platform in Dijon (France).

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
