A comparison of lipid diffusive dynamics in monolayers and bilayers in the context of interleaflet coupling

Cellular membranes are composed of lipids typically organized in a double-leaflet structure. Interactions between these two leaflets – often referred to as interleaflet coupling – play a crucial role in various cellular processes. Despite extensive study, the mechanisms governing such interactions remain incompletely understood. Here, we investigate the effects of interleaflet coupling from a specific point of view, i.e. by comparing diffusive dynamics in bilayers and monolayers, focusing on potential lipid-specific interactions between opposing leaflets. Through quantitative fluorescence microscopy techniques, we characterize lipid diffusion and mean molecular area in monolayers and bilayers composed of different lipids. Our results suggest that the observed decrease in bilayer lipid diffusion compared to monolayers depends on lipid identity. Furthermore, our analysis suggests that lipid acyl chain structure and spatial configuration at the bilayer may strongly influence interleaflet interactions and dynamics in bilayers. These findings provide insights into the role of lipid structure in mediating interleaflet coupling and underscore the need for further experimental investigations to elucidate the underlying mechanisms. ### Competing Interest Statement The authors have declared no competing interest. * OMPC : 1-oleoyl-2-myristoyl-sn-glycero-3-phosphocholine DOPC : 1,2-dioleoyl-sn-glycero-3-phosphocholine POPC : 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine SOPC : 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine mSM : Milk Sphingomyelin Rh-PE : Rhodamine PE TF-PC : TopFluor PC BSA : Bovine serum albumin PBS : Phosphate buffer saline NaP : Sodium phosphate D : Diffusion coefficient MMA : Mean molecular area lsFCS : line-scanning fluorescence correlation spectroscopy RICS : Raster image correlation spectroscopy PTFE : Polytetrafluoroethylene LD : Lipid droplet MD : Molecular dynamics SDHPW : Saffman-Delbrück-Hughes-Pailthorpe-White SEM : Standard error of the mean SD : Standard deviation MMAexpected : Expected Mean molecular area NAv : Avogadro’s number r : radius of the monolayer chamber C : Concentration of lipid solution in molar units V : Volume w0 : Excitation beam waist τD : Diffusion time Aeff : Effective illuminated area S : Structural parameter MMAb : Mean molecular area of lipid in the bilayer Db : Diffusion coefficient of lipids in the bilayer N : Number of fluorescent particles Ntotal : Total number of all lipid particles MMAm-oil : Mean molecular area of lipid in oil-water interface Dm : Diffusion coefficient of lipids in the air-water interface Dm-oil : Diffusion coefficient of lipids in oil-water interface MMAm : Mean molecular area of lipid in the air-water interface C%TF-PC : Concentration of TF-PC molecules a : Radius of diffusing lipid probe head hm : Monolayer membrane thickness µm : Air-water monolayer bulk viscosity µm-oil : Oil-water monolayer bulk viscosity µw : Viscosity of water µs : Viscosity of sucrose µa : Viscosity of air ηm : Monolayer surface viscosity ε : Reduced radius hb : Bilayer membrane thickness hl : Thickness of a leaflet in a bilayer membrane µl : Bulk viscosity of a leaflet in a bilayer membrane µmidplane : Bilayer midplane viscosity µb : Bilayer bulk viscosity.