Nir apo 60x wd 2

Patterned Supported Lipid Bilayers (pSLB) were obtained as described above on PDMS membranes, and the ring-containing membranes were mounted under an upright epifluorescence microscope (Nikon Ni, with Hamamatsu Orca Flash 4.0, v2). Images of pSLBs, obtained with either 15 or 30 s plasma cleaning, were acquired with a 60x water dipping objective (NIR Apo 60X/WD 2.8, Nikon) and an Orca R2 camera. A small linear region of the pSLB was frapped by repeatedly scanning and focusing 180 fs pulses generated by a fiber laser (FemtoPower, Fianium) with central wavelength at 1064 nm at 20 MHz. A set of galvo mirrors (Thorlabs) and a telescope before the port of the microscope allowed to position and move (oscillations at 400 Hz) the diffraction-limited spot at a desired place on the bilayer. Once bleached, fluorescence recovery was monitored for 5 min. Time-lapse imaging during the pSLB photobleaching and its recovery after photobleaching was done with a home-made software (Labview 2011). Recovery of the intensity of the bleached lines were plotted either for the full line, or by separating the line into a left and a right area and assess whether the recovery was symmetric (Supplementary Fig. 1).

Cell mechanical stimulation was done as previously described⁷ . Briefly, a 150 μL droplet of a 10 μg/mL fibronectin solution (Sigma) was deposited in the center of the membrane mounted in the ring. After overnight incubation at 4 °C, the fibronectin solution was rinsed, cells were seeded on the fibronectin-coated membranes and allowed to attach during 30–90 min. Then ring-containing membranes were mounted in the stretch system previously described⁷ . Cell images were acquired with a 60x water dipping objective (NIR Apo 60X/WD 2.8, Nikon) and an Orca Flash 4.0 camera (Hamamatsu), in an upright epifluorescence microscope with the Metamorph software. Cells were always imaged in two different channels collecting each fluorophore emission signal, every 3 s. They were imaged for 2 min at rest, 3 min in the 6% stretched state (nominal stretch of the PDMS substrate), and 3 min during the release of the stretch.

Fluorescence live cells images were acquired with Metamorph software using an upright microscope (Nikon eclipse Ni-U) with a 60× water dipping objective (NIR Apo 60X/WD 2.8, Nikon) and an Orca Flash 4.0 camera (Hamamatsu). Fluorophore emission was collected every 3 s. Cells were imaged in a relaxed state and then for 3 min at 5% stretch, and for 3 min during the release of stretch. Fixed cells images were acquired either in the abovementioned acquisition system except for the experiments related to WAVE protein enrichment. In this case, images were acquired in a Zeiss Airyscan microscope (Zeiss LSM880 inverted confocal microscope objective, using Zeiss ZEN2.3 SP1 FP3 [black, version 14.0.24.201] software and a 63×1.46 NA oil immersion objective). Z-stack of single cells were acquired in full Airyscan mode to visualize the PM and the WAVE-C-GFP protein. SEM images were taken using the xTm Microscope Control software in a NOVA NanoSEM 230 microscope (FEI Company) under the high vacuum mode using ET and TL detectors to acquire high and ultra-high resolution images of the cell surface. TEM samples were observed in a Jeol 1010 microscope (Gatan, Japan) equipped with a tungsten cathode in the CCiTUB EM and Cryomicroscopy Units. Images were acquired at 80 kV with a CCD Megaview 1kx1k.