Tetraspanin 4 stabilizes membrane swellings and facilitates their maturation into migrasomes.

Cell culture HEK293T (ATCC CRL-3216TM) and Normal rat kidney (NRK, ATCC CRL-6509TM) cells were cultured at 37 °C and 5% CO 2 in DMEM (Gibco-Thermo Fisher scientific 11995065) supplemented with 10% Fetal bovine serum (biological industries, 04-001-1A) and 1% penicillin–streptomycin (Gibco-Thermo Fisher scientific 15140122). NRK cells were gifted by Natalie Elia lab in BGU, Israel. MGC803 gastric carcinoma cells (RRID:CVCL 5334, ICLAC-00588) were gifted from Zhijie Chang lab from Tsinghua. MGC803 and NRK cells were cultured at 37 °C and 5% CO 2 in DMEM (Gibco, C11995500BT) supplemented with 10% Fetal bovine serum (Vivicum, 9906-85) and 1% penicillin–streptomycin (Cienry, CR15140). TSPAN4 expression plasmids, cell transfection, and giant plasma-membrane vesicles (GPMVs) isolation Complementary DNAs of mouse TSPAN4 were cloned into pEGFP-N1. HEK293T or NRK cells were plated at 20% confluency in 25 cm 2 flask (Romical) coated with poly- l -lysine (Sigma-Aldrich P6282) to keep the cells attached during the blebbing process and to minimize cell debris in solution. At 50% confluency, cells were transiently transfected with 5 μg DNA using Lipofectamine 2000 (Invitrogen, Thermo Fisher scientific) according to the manufacture’s protocols and then grown 24 h for protein expression. GPMVs were produced according to a published protocol 38 . Briefly, following TSPAN4-GFP expression the cell membrane was stained (except from experiments shown in Figs. S10 and S12 ) with DiI-C12 membrane dye (Invitrogen, Thermo Fisher scientific D383), washed with GPMV buffer (10 mM HEPES, 150 mM NaCl, 2 mM CaCl2, pH 7.4) twice, and incubated with 1 mL of GPMV buffer containing 1.9 mM DTT (Sigma-Aldrich 1019777701) and 27.6 mM formaldehyde (Sigma-Aldrich F8775). Secreted GPMVs were then collected and isolated from the cells and immediately used for optical trapping experiments. Among the GPMVs, about 10% contained TSPAN4-GFP in their membranes. In order to verify the presence of TSPAN4-GFP in the GPMVs’ membrane we scanned the GPMVs using a 488 nm laser before each measurement. For the control experiments GPMVs that did not contain TSPAN4-GFP in their membrane were used.

Cell imaging

Cell sample preparation NRK cells with stable

TSPAN4-GFP expression (NRK-TSPAN4-GFP), WT NRK, WT MGC803 or MGC803-TSPAN4-KO cells were seeded (around 4 × 10 3 cells) into 3.5 cm glass-bottom confocal dish (Cellvis D35C4-20-1.5-N), which was pre-coated with 10 μg/mL fibronectin (Gibco, Thermo Fisher scientific PHE0023) and grew for 12–15 h. The cells were stained with 5 μg/mL FM4-64 (Invitrogen, Thermo Fisher scientific T3166) for 15 min at 37 °C.

Confocal microscopy imaging

Cell imaging shown in Fig. 1a , e and S 1 was conducted under galvanometer scanning mode using a NIKON A1 confocal microscope fitted with a 100× oil objective. The laser power was 0.5% for 488 nm and 3% for 561 nm and each field of 1024 × 1024 pixels was imaged. For time-lapse imaging, the interval was 4 min, and the duration was at least 5 h.

Cell imaging shown in Figure

S4 was conducted by a Nikon HD25 microscope with 60x objective and each field of 1024 × 1024 pixels was imaged for a least 6 h with 5 min interval. The laser power was 3% for 561 nm, 54.35 μW is the maximal laser power.

SIM imaging

The cells shown in Fig. 1b were imaged by structured illumination microscopy (Nikon N-SIM S) with 4 min interval and 1.5 h duration, and then reconstructed by a standard stack-reconstruction process.

Quantitative analysis of migrasomes

For the analysis shown in Fig. 1d , 252 migrasomes of 11 NRK-TSAPN4-GFP cells from 4 movies conducted at three independent experiments were analyzed, each movie 4 h in length, with 60 frames in total. Fully formed migrasomes were chosen in the last frames of the movies, and then tracked back to their initial stage in the earlier frames. Only migrasomes that formed in the time course of the movie were included in the analysis. For the growing stage, several frames were included in the analysis in order to validate the presence of fully formed migrasomes due to possible changes in the focus. An example of initial stage, starting from red puncta, and growing stage is shown in Supplementary Fig. S1 . For the analysis shown in Fig. S4b–d , 1391 pre-migrasomes of 54 WT NRK cells from 16 movies of four independent experiments, and 1899 pre-migrasomes of 55 NRK-TSPAN4-GFP cells from 14 movies of four independent experiments were analyzed. For the analysis shown in Fig. S4f–h , 724 per-migrasomes of 31 WT MGC803 cells from 11 movies of four independent experiments, and 274 pre-migrasomes of 30 MGC803-TSPAN4-KO cells from 13 movies of four independent experiments were analyzed.

Proteolysis and mass spectrometry analysis

In order to confirm TSPAN4 overexpression in the GPMVs, we conducted mass spectrometry (MS) measurements, which showed that TSPAN4 abundance in GPMVs generated by TSPAN4-transfected cells increased 1562-fold compared to control GPMVs (GPMVs that were generated from WT HEK293T cells as described above). The samples were extracted in 10 mM DTT, 100 mM Tris and 5% SDS, boiled in 95 °C for 10 min and sonicated twice for 10 min. The samples were precipitated in 80% acetone overnight and washed three times with 80% acetone. The protein pellets were dissolved in 8.5 M Urea (Sigma-Aldrich U6504) and 400 mM ammonium bicarbonate (Sigma-Aldrich 09830). The protein amount was estimated using Bradford readings. Proteins were reduced with 10 mM DTT (Sigma-Aldrich D9163) at 60 0 C for 30 min, modified with 40 mM iodoacetamide (Sigma-Aldrich I6125) in 100 mM ammonium bicarbonate (room temperature for 30 min in the dark) and digested in 1.5 M Urea, 66 mM ammonium bicarbonate with modified trypsin (Promega), overnight at 37 °C in a 1:50 (M/M) enzyme-to-substrate ratio. An additional trypsin digestion was performed for 4 h at 37 °C in a 1:100 (M/M) enzyme-to-substrate ratio. The resulting tryptic peptides were desalted using C18 stage tips (homemade, 3 M company, USA) dried and resuspended in 0.1% Formic acid (Fluka analytical 94318-250 ML-F). The peptides were resolved by reverse-phase chromatography on 0.075 × 300-mm fused silica capillaries (J&W) packed with Reprosil reversed phase material (Dr. Maisch GmbH, Germany). The peptides mixture was eluted with a 5 to 28% of linear gradient of solvent B (95% acetonitrile (J.T. Baker 9821)) with 0.1% formic acid (Thermo Fisher scientific 85178) in water for 60 min followed by a 28 to 95% linear gradient for 15 min in solvent B at flow rate of 0.15 μL/min. MS was performed by Q Exactive Plus mass spectrometer (Thermo Fisher scientific) in a positive mode using repetitively full MS scan followed by high-collision dissociation (HCD) of the 10 most dominant ions selected from the first MS scan. The MS data was analyzed using Proteome Discoverer 2.4 software with Sequest (Thermo-Fisher scientific) search algorithm against Human Uniprot database with 1% FDR. Semi quantitation was done by calculating the peak area of each peptide based its extracted ion currents (XICs), and the area of the protein is the average of the three most intense peptides from each protein. Tube pulling from aspirated GPMVs The experiments were performed using a C-trap® confocal fluorescence optical tweezers setup (LUMICKS, Amsterdam, the Netherlands) made of an inverted microscope based on a water-immersion objective (NA 1.2) together with a condenser top lens placed above the flow cell. The optical traps are generated by splitting a 10 W 1064-nm laser into two orthogonally polarized, independently steerable optical traps. To steer the two traps, one coarse-positioning piezo stepper mirror and one accurate piezo mirror were used. Optical traps were used to capture polystyrene microbeads. The displacement of the trapped beads from the center of the trap was measured and converted into a force signal by back-focal plane interferometry of the condenser lens using two position-sensitive detectors. The samples were illuminated by a bright-field 850-nm LED and imaged in transmission onto a metal-oxide semiconductor (CMOS) camera. Confocal fluorescence microscopy: The C-Trap uses a 3 color, fiber-coupled laser with wavelengths 488, 561 and 638 nm for fluorescence excitation. Scanning was done using a fast tip/tilt piezo mirror. For confocal detection, the emitted fluorescence was descanned, separated from the excitation by a dichroic mirror, and filtered using emission filters (Blue: 500–550 nm, Green: 575–625 nm and Red: 650–750 nm). Photons were counted using fiber-coupled single-photon counting modules. The multimode fibers serve as pinholes providing background rejection. For confocal imaging the 488 nm and 532 nm lasers were used for GFP and DiI-C12 excitation with 5% and 1% laser power, respectively, 54.34 μW is the maximal laser power, and the emission detected in three channels (Blue, Green, Red).

Experimental chamber Polydimethylsiloxane

(PDMS) walls were placed on the bottom cover slip (Thorlabs CG15KH1) and mounted onto an automated XY-stage. The GPMVs sample was added to the chamber and after about 15 min, a few drops of oil were put on the sample surface to prevent evaporation. A micropipette aspiration setup including micromanipulator (Sensapex) holding a micropipette with diameter of 5 μm (BioMedical instruments) connected to a Fluigent EZ-25 pump was integrated into our optical tweezers instrument. Before each experiment, the zero-suction pressure was found by aspirating a 3.43 μm polystyrene bead (Spherotech) into the pipette and reducing the suction pressure until the bead stopped moving. A membrane tube was pulled from aspirated GPMVs using beads trapped by the optical tweezers. First, a membrane tube was pulled at relatively low suction pressure (0.05–0.1 mbar, correspond to 1.2–2 × 10 −5 N/m membrane tension), then the suction pressure was reduced to zero (corresponds to zero applied membrane tension) for about 15 s. Then, we increased instantaneously the suction pressure to values in the range of 0.2–0.8 mbar (correspond to 2–15 × 10 −5 N/m membrane tension) for HEK293T-GPMVs or 0.5–1.1 mbar (correspond to 10–25 × 10 −5 N/m membrane tension). The control experiments were conducted on HEK293T-GPMVs and NRK-GPMVs (which did not contain TSPAN4-GFP). The same tension jumps used for TSPAN4-GPMVs were done on the GPMVs from the control group (±5 × 10 −6 N/m). Microfluidics: To induce shear forces on TSPAN4-GPMVs, the GPMVs were injected at 1.5 bar into a 5-channel laminar flow cell (LUMICKS, Amsterdam, the Netherlands), which was on the C-trap® confocal fluorescence optical tweezers stage. The pressure was reduced to zero and following the settlement of the GPMVs at the bottom of the flow cell, they were scanned using a 488 nm laser at 5% laser power.

Data analysis

Data acquisition was carried out using Bluelake, a commercial software from Lumicks. This software stores experimental data acquired with the C-trap in HDF5 files, which can be processed using Lumicks’ Pylake python package. Images of the confocal scans were reconstituted from photon count per pixel data in the HDF5 files using Pylake. All data analysis was performed with custom-written Python scripts. Fluorescence intensity profiles were obtained from the images by averaging the photon count of the relevant fluorescent channel (Blue or Green) in the region of interest. Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Experimental chamber Polydimethylsiloxane

(PDMS) walls were placed on the bottom cover slip (Thorlabs CG15KH1) and mounted onto an automated XY-stage. The GPMVs sample was added to the chamber and after about 15 min, a few drops of oil were put on the sample surface to prevent evaporation. A micropipette aspiration setup including micromanipulator (Sensapex) holding a micropipette with diameter of 5 μm (BioMedical instruments) connected to a Fluigent EZ-25 pump was integrated into our optical tweezers instrument. Before each experiment, the zero-suction pressure was found by aspirating a 3.43 μm polystyrene bead (Spherotech) into the pipette and reducing the suction pressure until the bead stopped moving. A membrane tube was pulled from aspirated GPMVs using beads trapped by the optical tweezers. First, a membrane tube was pulled at relatively low suction pressure (0.05–0.1 mbar, correspond to 1.2–2 × 10 −5 N/m membrane tension), then the suction pressure was reduced to zero (corresponds to zero applied membrane tension) for about 15 s. Then, we increased instantaneously the suction pressure to values in the range of 0.2–0.8 mbar (correspond to 2–15 × 10 −5 N/m membrane tension) for HEK293T-GPMVs or 0.5–1.1 mbar (correspond to 10–25 × 10 −5 N/m membrane tension). The control experiments were conducted on HEK293T-GPMVs and NRK-GPMVs (which did not contain TSPAN4-GFP). The same tension jumps used for TSPAN4-GPMVs were done on the GPMVs from the control group (±5 × 10 −6 N/m). Microfluidics: To induce shear forces on TSPAN4-GPMVs, the GPMVs were injected at 1.5 bar into a 5-channel laminar flow cell (LUMICKS, Amsterdam, the Netherlands), which was on the C-trap® confocal fluorescence optical tweezers stage. The pressure was reduced to zero and following the settlement of the GPMVs at the bottom of the flow cell, they were scanned using a 488 nm laser at 5% laser power.

Supplementary information Supplementary Information Description of Additional Supplementary files Supplementary Movie 1 Supplementary Movie 2 Supplementary Movie 3 Supplementary Movie 4 Supplementary Movie 5 Supplementary Movie 6 Reporting Summary Peer Review File