Effect of temperature on the formation of liquid phase-separating giant unilamellar vesicles (GUV).

Giant unilamellar vesicles (GUVs) are widely used as model systems to study both, lipid and membrane protein behavior. During their preparation by the commonly applied electroformation method, a number of issues must be considered to avoid the production of artifacts due to a poor lipid hydration and protein degradation. Here we focus on the effect of preparation temperature on GUVs composed of the most commonly used domain-forming mixture dioleoylelphospatidylcholine/shingomyelin/cholesterol (DOPC/SM/chol) (2/2/1). Lower production temperatures are generally preferable when aiming at a functional reconstitution of proteins into the membrane. On the other hand, lower growth temperature is suspected to alter the lipid composition and the yield of phase-separating vesicles. By confocal imaging, we find that vesicles prepared significantly above and below the melting temperature T(m) have the same overall morphology, similar size distributions of vesicles and a similar area coverage by liquid-ordered (L(o)) domains. However, a large population analysis indeed reveals a different overall yield of phase-separating vesicles. Two-focus scanning fluorescence correlation spectroscopy measurements did not show any divergence of lipid analog mobility in (L(o)) and (L(d)) phases in vesicles prepared at different temperatures, indicating that the lowered growth temperature did not influence the lipid organization within the two phases. Moreover, the expected advantages of lower preparation temperature for proteo-GUVs could be exemplified by the reconstitution of voltage dependent anion channel (VDAC) into DOPC/SM/chol GUVs, which aggregates at high, but not at low preparation temperatures.