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.
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