Fluorescence recovery after photobleaching (FRAP) assesses lipid mobility, a key factor in membrane fusion. We investigated how different glass coverslip types and surface treatments influence lipid mobility in supported lipid bilayers (SLBs). Tested coverslips included borosilicate, soda-lime, and Schott D263 glass, with or without silicon dioxide (SiO 2 ) coating. SLBs were formed by incubating fluorescent liposomes onto a Langmuir-Blodgett lipid monolayer. The monolayer lipid mix—cholesterol: brainPC:DSPE-PEG—in a 30:20:3 molar ratio, which mimics the extracellular plasma membrane leaflet, was spread on the water surface, compressed to 32 mN/m, and deposited onto the coverslip by slow vertical withdrawal, maintaining constant surface pressure. Liposomes were prepared by mixing cholesterol, brainPC, brainPE, liverPI, brainPS, and brainPI(4,5)P2 in a 30:25:25:4:15:1 molar ratio, which mimics the cytoplasmic leaflet composition. After 1 h of incubation and subsequent detergent dialysis, the liposomes were incubated on the Langmuir-deposited monolayer for one hour at room temperature, forming a supported lipid bilayer. Excess vesicles were then removed by washing. FRAP was performed using Rhodamine-PE or NBD-PE fluorescent labels at a 1% molar ratio, replacing an equal amount of brainPC. Bilayers containing only neutral lipids (brainPC, brainPE, and cholesterol) showed recovery after photobleaching on all glass types. When negatively charged lipids (brainPS and brainPIP2) were present, recovery occurred only on coverslips with a SiO 2 surface, independent of monolayer presence. Final goal is to elucidate the role of membrane fluidity in regulating membrane fusion efficiency and fusion event properties. The effect of incorporating a 1:1 mixture of syntaxin-1 and dSNAP25 into liposomes and supported lipid bilayers on membrane fluidity is currently being investigated. Our initial findings identify which membrane supports and lipid mixes best mimic natural membrane fluidity, which is important for vesicle fusion experiments. NIH grant R35GM139608.
Castellani et al. (Sun,) studied this question.