Polydimethylsiloxane (PDMS) substrate preparation

PDMS curing agent and PDMS elastomer base (Sylgard 184, Sigma-Aldrich) were mixed at a 1:50 weight ratio. The substrates were prepared following the steps depicted in Fig. 6a. A drop (~5 µL) of PDMS was deposited on a freshly cleaved mica sheet to ensure the flatness of the substrate and a clean HS-AFM sample stage glass rod (height: 2 mm; diameter: 1.5 mm) was placed on top. Next, the PDMS was cured in an oven at 80 °C for 4 h. Once the PDMS was cured, the glass rod was peeled from the mica. The PDMS has higher adherence to the rough glass rod surface than to the atomically flat mica surface. The PDMS films display a thickness of a few (~10) micrometers. O₂ plasma treatment was used to increase the number of hydroxyl groups on the PDMS surface to render the surface hydrophilic, which was found essential for subsequent lipid bilayer spreading, as previously reported^49,50. A JPK-Bruker Nanowizard was used to assess the Young’s modulus of the surface (Supplementary Fig. ⁴). Force curves were acquired with a 2 µm SiO₂ sphere attached at the end of the cantilever (Sqube, NanoAndMore, USA). After PDMS substrate preparation, 2ul of liposomes (~1 mg/ml) were deposited onto the PDMS substrate. After 20 min, the sample was rinsed with imaging buffer (10 mM Tris at pH 7.4, 150 mM KCl). Alternatively, to observe how the SLB spreads on PDMS, 10 uL of liposomes (~1 mg/ml) were added directly to the imaging chamber and SLB spreading was observed until a confluent bilayer was obtained; the imaging solution was subsequently exchanged to remove excess unburst liposomes. After assessment of the surface and the supported lipid bilayer homogeneity, Snf7 was added to the fluid chamber to a final concentration of 3 μM.