CHARACTERIZATION OF MEMBRANE DOMAINS BY FRAP EXPERIMENTS AT VARIABLE OBSERVATION AREAS

In this paper we show that FRAP experiments at variable beam radii pro vide an experimental approach for investigating membrane organization and dynamics, with great potential for identifying micrometer-sized do mains and determining their size and the diffusion coefficient of the lipid and protein molecules they contain. Monte Carlo simulations of F RAP experiments at variable beam radii R on models of compartmentalize d membranes have allowed us to establish the relationships (i) between the mobile fraction M of a diffusing particle and the size r of the d omains, and (ii) between the apparent diffusion coefficient D-app and the real diffusion coefficient D-0 of this particle inside the domains . Furthermore, in its present stage of development, this approach allo ws us to specify whether these domains are strictly closed or not. Thi s approach was first validated on an experimental model of a strictly compartmentalized membrane consisting of a monolayer of apposed spheri cal phospholipid bilayers supported by silica beads of known radius (0 .83 mu m). To prevent fusion between the spherical bilayers 5 mol% of a polymer-grafted phospholipid was added to the lipids. Analysis of th e M versus R data yielded a radius r of 0.92+/-0.09 mu m for the spher ical bilayers, close to that of the supporting silica beads. When appl ied to the experimental data available for lipids and proteins in the plasma membrane of living cells, this approach suggests the existence of domains within these membranes with a radius of about 0.4-0.7 mu m for the lipids and 0.25 mu m for the proteins. These domains are not s trictly closed and they are believed to be delineated by fluctuating b arriers which are more or less permeable to lipid and protein molecule s.