Characterization of Membrane Domains by FRAP Experiments at Variable Observation Areas
Overview
Authors
Affiliations
In this paper we show that FRAP experiments at variable beam radii provide an experimental approach for investigating membrane organization and dynamics, with great potential for identifying micrometer-sized domains and determining their size and the diffusion coefficient of the lipid and protein molecules they contain. Monte Carlo simulations of FRAP experiments at variable beam radii R on models of compartmentalized membranes have allowed us to establish the relationships (i) between the mobile fraction M of a diffusing particle and the size r of the domains, and (ii) between the apparent diffusion coefficient Dapp and the real diffusion coefficient DO of this particle inside the domains. Furthermore, in its present stage of development, this approach allows us to specify whether these domains are strictly closed or not. This approach was first validated on an experimental model of a strictly compartmentalized membrane consisting of a monolayer of apposed spherical phospholipid bilayers supported by silica beads of known radius (0.83 micron). To prevent fusion between the spherical bilayers 5 mol% of a polymer-grafted phospholipid was added to the lipids. Analysis of the M versus R data yielded a radius r of 0.92 +/- 0.09 microns for the spherical bilayers, close to that of the supporting silica beads. When applied 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 microns for the lipids and 0.25 micron for the proteins. These domains are not strictly closed and they are believed to be delineated by fluctuating barriers which are more or less permeable to lipid and protein molecules.
The Utility of Fluorescence Recovery after Photobleaching (FRAP) to Study the Plasma Membrane.
Day C, Kang M Membranes (Basel). 2023; 13(5).
PMID: 37233553 PMC: 10221041. DOI: 10.3390/membranes13050492.
Statin-induced increase in actin polymerization modulates GPCR dynamics and compartmentalization.
Sarkar P, Chattopadhyay A Biophys J. 2022; 122(11):1938-1955.
PMID: 36045572 PMC: 10257020. DOI: 10.1016/j.bpj.2022.08.039.
Tracking Single Molecules in Biomembranes: Is Seeing Always Believing?.
Yu Y, Li M, Yu Y ACS Nano. 2019; 13(10):10860-10868.
PMID: 31589406 PMC: 7179047. DOI: 10.1021/acsnano.9b07445.
Favard C Biomolecules. 2018; 8(3).
PMID: 30189682 PMC: 6164371. DOI: 10.3390/biom8030090.
Lagerholm B, Andrade D, Clausen M, Eggeling C J Phys D Appl Phys. 2017; 50(6):063001.
PMID: 28458397 PMC: 5390782. DOI: 10.1088/1361-6463/aa519e.