Fluroscence recovery after photobleaching (FRAP) experiments:

FRAP experiment on supported bilayer was performed as previously reported⁶⁵. Briefly, supported lipid bilayers were formed onto the glass-bottom part of a petri dish using the Langmuir-Blodgett deposition technique⁶⁶. Glass-bottom petri dishes (35 mm dishes from MatTek with uncoated glass cover slip of 14 mm diameter, thickness number 1.5) were soaked for 1 hour at ^~ 60°C in 2% v/v cleaning detergent (MICRO-90, VWR), thoroughly rinsed with 18.2 MΩ ultra-pure water, and then dried under a stream of nitrogen. A chloroform solution of DOPC constituting the inner monolayer was first spread on degassed water in a NIMA Langmuir trough (model 611 equipped with the PS4 surface pressure sensor) and allowed to dry for 15 min at room temperature. After solvent evaporation, the film was compressed up to 33 mN/m and the monolayer was transferred onto the glass cover slip (with its lipid headgroups facing the glass surface) as the petri dish was slowly (0.5 cm/min) raised out of water. This first monolayer was allowed to dry for at least 30 min at room temperature. Meanwhile, a chloroform solution of the lipid mixture constituting the outer monolayer was spread at the air/water interface. This lipid mixture is a series of increasing cholesterol mol% (0, 15, 25, and 35) with other synaptic vesicle lipids and 1mol% NBD-DOPE. After 15 minutes, this second monolayer was transferred at a constant pressure of 33 mN/m and with a dipping speed of 0.5 cm/min onto the first monolayer (with its lipid headgroups facing the aqueous medium). After this second deposition, the bilayers remained immersed in aqueous medium throughout experiment.

FRAP experiments were performed on a confocal microscope TCS SP8 from Leica equipped with the LCS software, using an HC PL FLUOTAR 10X Dry objective (numerical aperture: 0.3; zoom: 10X) with a pinhole opened at 70.8 μm and an acquisition rate of 1 picture every 635 ms. Fluorescence bleaching and recovery were conducted as follows. For NBD: λ_exc = 488 nm; λ_em = 500–600 nm with 1 scan at 100% laser power for bleaching, and monitoring recovery at 2% of the maximum laser power. For each sample, recovery curves (average over 3 independent experiments, i.e. performed on a different region of the sample using the same bleaching conditions) were fitted with the software Mathematica (FRAP experiments described by modified Bessel functions; code provided upon request).

In order to take into account the contribution of fluorescence recovery that occurs during the photobleaching phase and can thus affect data analysis⁶⁷, the time t = 0 of the recovery phase in all FRAP experiments was set as the time of the last bleaching frame.

When working with photosensitive probes, such as NBD, one has to correct for the intrinsic photobleaching that occurs during the recovery phase and can also affect data analysis^68,69. In FRAP experiments, we measured the intrinsic photobleaching in a region far away from the bleaching zone. The corrected fluorescence recovery signal was then calculated by dividing the raw fluorescence recovery signal by the intrinsic photobleaching signal.

Diffusion coefficient (D) of 18:1 NBD-PE lipids in the outer leaflet of different supported lipid bilayer containing different amount of cholesterol were deduced by varying the area of the bleached region (disks of diameters d= 5, 10, 15 μm). The linear relationship between the bleaching area d² and the recovery time (τ) proves that lipid diffusion is controlled by Brownian motion and then diffusion coefficient (D) is calculated from the slope of these straight lines: D = d²/16τ where D, the diffusion coefficient is in μm²/s, d is bleached regions diameter, and τ is the recovery time in [s]. The diffusion coefficient (D) is then plotted against the mol% of cholesterol in the bilayer. As can be seen from the plot (Extended Data Fig. 5b), with increase in mol% of cholesterol in the bilayer the diffusion coefficient decreases. That means the fluidity of the bilayer decreases with increase in cholesterol in the bilayer.