Fig. 3: CEs condense into droplets in model membranes.

A GUVs were made with different concentrations of CE (here, cholesteryl oleate) and imaged under confocal microscopy. Top: images of gradually CE-concentrated GUVs. CE condensates started appearing from the concentration of 15 mol%. Bottom: Schematic representation of gradually increasing CE concentration within GUV bilayer. B Left: Schematic representation of GUV splashing on a bare glass coverslip. Right: microscope image of a splashed GUV, appearing flat on the glass surface. CE condensates were still visible and immobilized. C Signal recovery in a totally bleached CE condensate from splashed GUV as shown in B. The signal was normalized by the initial fluorescence signal. D Quantification of the amount of free CE in the membrane by measuring the CE/PL fluorescence ratio in splashed GUVs as shown in C. Points: Mean ± SD, 5–10 GUVs per experimental point. E Quantification of the occurrences of droplet-containing GUVs according to the [CE/PL] ratio. Above the [CE/PL] ratio of 15%, both the CE enrichment in the membrane and the occurrences of droplet-containing GUVs appear to be almost constant, suggesting that the critical concentration is around 15%. N = 9–37 GUVs per concentration. F Representation of CE distribution in a model ER bilayer at the end (t = 2 μs) of the simulation period. Top-view (left) and side-view (right) representation of the system. CE molecules are shown in orange. Phosphate atoms of the membrane lipids are shown in tan in the side-view representation. Other components of the system (membrane lipids, water, and ions) are not shown for clarity. G Plot showing the fraction of CE molecules clustered in the bilayers during the simulation. The remaining CE molecules that are not bound to clusters are individual monomers. The data have been averaged over three simulation repeats.