Small unilamellar vesicles (SUVs) and SLB formation

For the formation of SUVs, DOPC (no. 850375, Avanti Polar Lipids), dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) (no. 840475, Avanti Polar Lipids) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-cap biotinyl (18:1 Biotinyl Cap PE; no. 870273, Avanti Polar Lipids) were dissolved in chloroform (Sigma Aldrich) and mixed in a ratio of 70 mol% DOPC to 30 mol% DOPG or 70 mol% DOPC with 29.99 mol% DOPG and 0.01 mol% Biotinyl Cap PE. After solvent evaporation through nitrogen, residual chloroform was removed for 1 h in a vacuum desiccator. Lipid film hydration was achieved with Min buffer (25 mM Tris/HCl pH 7.5, 150 mM KCl, 5 mM MgCl₂) and SUVs were formed through consecutive freeze–thaw cycles (8–10) using liquid nitrogen and a 90 °C water bath. For monodisperse vesicle distribution, lipid mixtures were extruded across a Whatman nucleopore membrane (no. 110603, Cytiva) with a pore size of 50 nm for 37 passes.

SLBs were formed by fusion of SUVs on cleaned glass cover slides (Paul Marienfeld GmbH & Co. KG) that were assembled into a flow chamber through double-sided sticky tape (Scotch, Conrad Electronic SE). Before their assembly, cover slides (nos. 0102242, 1.5, 24 × 60 mm²; nos. 0102062, 1.5, 24 × 24 mm²) were cleaned by sequential sonication in Milli-Q water, isopropanol and Milli-Q water (15 min each), and subsequently dried under a nitrogen stream. Slides were then activated for 30 s (30% power, 0.3 mbar) in a Zepto plasma cleaner (Diener Electronic GmbH) using oxygen as the process gas. After flow chamber assembly, SUVs were added to each reaction chamber at a final concentration of 0.4 mg ml⁻¹ in Min buffer with additional 2 mM CaCl₂ to promote vesicle rupture. Unfused SUVs were removed through subsequent washing with Min buffer.