Fluorescence recovery after photobleaching membrane FRAP

Lateral diffusion of phospholipids in model membranes at ambient pressure has been studied over the years by a variety of techniques including fluorescence recovery after photobleaching (FRAP), spin-label ESR, pulse field gradient NMR (PFG-NMR), quasielastic neutron scattering (QENS), excimer fluorescence and others.In general, the values reported for the lateral diffusion coefficient (D) range from 10 to 10 cm /s in the... 

Looks at the role of caveolins in the formation of membrane caveolae Covers the investigation of hop diffusion of membrane lipids using FRAP (fluorescence recovery after photobleaching)... 

A very suitable method for measurement of the lateral diffusion of molecules adsorbed at the foam film surfaces is Fluorescence Recovery after Photobleaching (FRAP) ([491-496], see also Chapter 2). Measurements of the lateral diffusion in phospholipid microscopic foam films, including black foam films, are of particular interest as they provide an alternative model system for the study of molecular mobility in biological membranes in addition to phospholipid monolayers at the air/water interface, BLMs, single unilamellar vesicles, and multilamellar vesicles. 

Ras trafficking to cellular membranes can be measured by fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) (54). Both techniques rely on the expression of fluorescent-labeled Ras proteins to monitor different parameters of Ras movement across and between cellular membranes. FRAP involves photobleaching a membrane subdomain and measuring the kinetics of fluorescence recovery—and hence Ras trafflcking—into the bleached area. With FLIP, a cellular membrane is photobleached repeatedly and the subsequent intercellular movement of the photobleached area is monitored. 

Fluorescence recovery after photobleaching (FRAP) for measuring lateral diffusion in membranes Section 12.6... 

A third technique for studying foam films is the fluorescence recovery after photobleaching (FRAP). This techniques was applied by Clarke et al. [36] for lateral diffusion in foam films, and involves irreversible photobleaching by intense laser light of fluorophore molecules in the sample. The time of redistribution of probe molecules (which are assumed to be randomly distributed within the constitutive membrane lipids in the film) is monitored. The lateral diffusion coefficient, D, is calculated from the rate of recovery of fluorescence in the bleaching region due to the entry of unbleaching fluoroprobes of adjacent parts of the membranes. 

Besides the membrane thickness, the copolymer molecular weight affects also the lateral mobility of polymer chains within the membrane. Using fluorescence recovery after photobleaching (FRAP), Lee et al. [131] discovered that entanglements are responsible for the reduced mobility of copolymers with sufficiently high MW. 

Another technique, referred to as fluorescence recovery after photobleaching (FRAP), is also used to observe lateral diffusion. Cell plasma membranes are uniformly labeled with a fluorescent marker. Using a laser beam, the fluorescence in a small area is destroyed (or bleached ). Using video equipment, the lateral movement of membrane components into and out of the bleached area can be tracked as a function of time. 

A EXPERIMENTAL FIGURE 5-6 Fluorescence recovery after photobleaching (FRAP) experiments can quantify the lateral movement of proteins and lipids within the plasma membrane, (a) Experimental protocol. Step H Cells are first labeled with a fluorescent reagent that binds uniformly to a specific membrane lipid or protein. Step B A laser light is then focused on a small area of the surface, irreversibly bleaching the bound reagent and thus reducing the fluorescence in the illuminated area. Step B In time, the fluorescence of the bleached patch increases as unbleached fluorescent surface molecules diffuse into it and bleached ones diffuse outward. The extent of recovery of fluorescence in the bleached patch is... 

Biological membranes are not rigid, static structures. On the contrary, lipids and many membrane proteins are constantly in lateral motion, a process called lateral diffusion. The rapid lateral movement of membrane proteins has been visualized by means of fluorescence microscopy through the use of the technique of fluorescence recovery after photobleaching (FRAP Figure 12.29). 

Fluorescence recovery after photobleaching (FRAP) is not a novel technique. In fact, FRAP was first developed in the 1970s as a technique to study the mobility of proteins in living cells (8, 9). Experiments were initially aimed at investigating changes in lateral membrane transport as an indicator or consequence of changes in the physiological state of cells. Early success of FRAP... 

The FRAP (fluorescence recovery after photobleaching) technique uses photobleaching to measure molecular diffusion and can be used if the material in question is confined to a specific plane (e.g., a membrane or cytoskeletal filaments adsorbed on a surface). The fluorophores... 

Based on these experiments, studies are in progress to exploit the functionality of transmembrane proteins in S-layer stabilized, solid-supported lipid membranes in which the S-layer is directly attached to the solid support and the lipid layer associated with the S-layer (Fig. 16). Most recently lateral diffusion of fluorescence lipid probes in S-layer supported lipid membranes on solid supports have been investigated with fluorescence recovery after photobleaching FRAP [94]. It was demonstrated that the S-layer cover induced an enhanced mobility of the probe in the lipid layer which also supported the semifluid membrane concept [83,86]. Furthermore it was noticed that the S-layer lattice cover could prevent the formation of cracks and other inhomogeneties in the bilayer. 

Lipids diffuse freely in fluid model membranes. FRAP measurements show full recovery and diffusion coefficients on the order of magnitude of 10 cm /sec. Free diffusion with a similar rate is often observed for lipids in the biomembrane. However, many cell membrane proteins show lower diffusion rates and incomplete recovery after photobleaching. For membrane proteins, dramatically different behavior in model and biological membranes is a common case. In model membranes, membrane proteins also diffuse freely and their diffusion coefficients are often similar to the diffusion coefficients of lipids. On the contrary, in biomembranes, the diffusion of proteins is 2-3 orders of magnitude slower and the fluorescence recovery is often incomplete. This observation points to limitations of the fluid mosaic model as will be discussed below. 

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