Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes

Fluorescence recovery after photobleaching (FRAP) is a widely used tool for estimating mobility parameters of fluorescently tagged molecules in cells. Despite the widespread use of confocal laser scanning microscopes (CLSMs) to perform photobleaching experiments, quantitative data analysis has been limited by lack of appropriate practical models. Here, we present a new approximate FRAP model for use on any standard CLSM. The main novelty of the method is that it takes into account diffusion of highly mobile molecules during the bleach phase. In fact, we show that by the time the first postbleach image is acquired in a CLSM a significant fluorescence recovery of fast-moving molecules has already taken place. The model was tested by generating simulated FRAP recovery curves for a wide range of diffusion coefficients and immobile fractions. The method was further validated by an experimental determination of the diffusion coefficient of fluorescent dextrans and green fluorescent protein. The new FRAP method was used to compare the mobility rates of fluorescent dextrans of 20, 40, 70, and 500 kDa in aqueous solution and in the nucleus of living HeLa cells. Diffusion coefficients were lower in the nucleoplasm, particularly for higher molecular weight dextrans. This is most likely caused by a sterical hindrance effect imposed by nuclear components. Decreasing the temperature from 37 to 22 degrees C reduces the dextran diffusion rates by approximately 30% in aqueous solution but has little effect on mobility in the nucleoplasm. This suggests that spatial constraints to diffusion of dextrans inside the nucleus are insensitive to temperature.     

Effect of microsome-liposome fusion on the rotational mobility of cytochrome P450IIB4 in rabbit liver microsomes

Membrane fusion of microsomes with soybean phospholipid vesicles was performed at pH 6.5 to investigate the effect of lipid-enrichment in the membrane on the rotational mobility of cytochrome P450. Rotational diffusion of cytochrome P450 in the microsomal membrane of phenobarbital-induced rabbit liver was measured by detecting the decay of absorption anisotropy after photolysis of the heme CO complex by a vertically polarized laser flash. The fusion procedures yielded three separate fractions upon sucrose density gradient centrifugation with lipid-to-protein ratio in weight (L/P) as follows: 1.5 in the bottom fraction, 2.2 in the middle fraction, and 3.9 in the top fraction. In each fraction, co-existence of mobile and immobile cytochrome P450 was observed. The percentage of rotationally mobile P450 (with the mean rotational relaxation time of phi=505-828 micros) in each of the different bands was found to be 59% in the bottom fraction, 61% in the middle fraction, and 68% in the top fraction. This increase in mobile population of P450 due to lipid-enrichment indicates that aggregated proteins in microsomal membranes dissociate with increasing L/P which is inversely proportional to the protein concentration in the membrane. With freeze-fracture electron microscopy, it was shown that the average distance increased between intramembrane particles by lipid-enrichment. Thus, the significant immobile population (32%) of P450 in microsomal membranes can be explained by nonspecific protein aggregation which is a consequence of the low L/P of 0.8. The decrease in the mobile population in the bottom fraction compared with intact microsomes was shown to be due to the pH 6.5 incubation used for fusion.     

Dynamic control of Rad51 recombinase by self-association and interaction with BRCA2

Here, we visualize GFP-Rad51 fusion proteins in the nucleus of living cells to demonstrate the dynamic compartmentalization of Rad51 by self-association or by binding to BRCA2. Mutants of Rad51 that fail to oligomerize and/or to bind BRCA2 distinguish three fractions of Rad51 within the nucleoplasm: a relatively mobile fraction, an immobile oligomerized fraction, and an immobile BRCA2-bound fraction. Strikingly, inhibition of replication by hydroxyurea reduces the immobile fraction of nucleoplasmic Rad51. This effect is specific to Rad51 mutants that retain the capacity to bind BRCA2, indicating that the BRCA2-bound fraction is selectively mobilized. We propose that arrested replication triggers a switch between dual functions of BRCA2 in sequestering or mobilizing a small fraction of nucleoplasmic Rad51 and suggest a mechanism for the dynamic control of protein complexes that participate in homologous recombination.     

Depth-of-Focus Correction in Single-Molecule Data Allows Analysis of 3D Diffusion of the Glucocorticoid Receptor in the Nucleus

Single-molecule imaging of proteins in a 2D environment like membranes has been frequently used to extract diffusive properties of multiple fractions of receptors. In a 3D environment the apparent fractions however change with observation time due to the movements of molecules out of the depth-of-field of the microscope. Here we developed a mathematical framework that allowed us to correct for the change in fraction size due to the limited detection volume in 3D single-molecule imaging. We applied our findings on the mobility of activated glucocorticoid receptors in the cell nucleus, and found a freely diffusing fraction of 0.49±0.02. Our analysis further showed that interchange between this mobile fraction and an immobile fraction does not occur on time scales shorter than 150 ms.     

Direct imaging of lateral movements of AMPA receptors inside synapses

Trafficking of AMPA receptors in and out of synapses is crucial for synaptic plasticity. Previous studies have focused on the role of endo/exocytosis processes or that of lateral diffusion of extra-synaptic receptors. We have now directly imaged AMPAR movements inside and outside synapses of live neurons using single-molecule fluorescence microscopy. Inside individual synapses, we found immobile and mobile receptors, which display restricted diffusion. Extra-synaptic receptors display free diffusion. Receptors could also exchange between these membrane compartments through lateral diffusion. Glutamate application increased both receptor mobility inside synapses and the fraction of mobile receptors present in a juxtasynaptic region. Block of inhibitory transmission to favor excitatory synaptic activity induced a transient increase in the fraction of mobile receptors and a decrease in the proportion of juxtasynaptic receptors. Altogether, our data show that rapid exchange of receptors between a synaptic and extra-synaptic localization occurs through regulation of receptor diffusion inside synapses.     

Both lamin A and lamin C mutations cause lamina instability as well as loss of internal nuclear lamin organization

We have applied the fluorescence loss of intensity after photobleaching (FLIP) technique to study the molecular dynamics and organization of nuclear lamin proteins in cell lines stably transfected with green fluorescent protein (GFP)-tagged A-type lamin cDNA. Normal lamin A and C proteins show abundant decoration of the inner layer of the nuclear membrane, the nuclear lamina, and a generally diffuse localization in the nuclear interior. Bleaching studies revealed that, while the GFP-tagged lamins in the lamina were virtually immobile, the intranuclear fraction of these molecules was partially mobile. Intranuclear lamin C was significantly more mobile than intranuclear lamina A. In search of a structural cause for the variety of inherited diseases caused by A-type lamin mutations, we have studied the molecular organization of GFP-tagged lamin A and lamin C mutants R453W and R386K, found in Emery-Dreifuss muscular dystrophy (EDMD), and lamin A and lamin C mutant R482W, found in patients with Dunnigan-type familial partial lipodystrophy (FPLD). In all mutants, a prominent increase in lamin mobility was observed, indicating loss of structural stability of lamin polymers, both at the perinuclear lamina and in the intranuclear lamin organization. While the lamin rod domain mutant showed overall increased mobility, the tail domain mutants showed mainly intranuclear destabilization, possibly as a result of loss of interaction with chromatin. Decreased stability of lamin mutant polymers was confirmed by flow cytometric analyses and immunoblotting of nuclear extracts. Our findings suggest a loss of function of A-type lamin mutant proteins in the organization of intranuclear chromatin and predict the loss of gene regulatory function in laminopathies.     

Protein-protein interactions of cytochrome oxidase in inner mitochondrial membranes. The effect of liposome fusion on protein rotational mobility

Rotational diffusion of cytochrome oxidase in the inner membrane of rat liver mitochondria was measured by detecting the decay of absorption anisotropy after photolysis of the heme a3.CO complex by a vertically polarized laser flash. As in previous experiments with beef heart mitochondria (Kawato, S., Sigel, E., Carafoli, E., and Cherry, R. J. (1980) J. Biol. Chem. 255, 5508-5510), co-existence of rotating cytochrome oxidase (mean rotational relaxation time, phi, of 700 to 1400 microseconds) and immobilized cytochrome oxidase (phi greater than 20 ms) was observed in mitochondria and mitoplasts. The effect of lipid/protein ratio by weight (L/P) on the relative proportions of mobile and immobile cytochrome oxidase was investigated following the fusion of soybean phospholipid vesicles with mitoplasts. The fusion procedure yielded four separate fractions upon sucrose density gradient centrifugation with L/P as follows: 0.3 in Pellet, 0.7 in Band 3, 1.5 in Band 2, and 3.0 in Band 1. The percentage of rotationally mobile cytochrome oxidase (phi = 700 to 1000 microseconds) in each of the different bands was found to be 16% in Pellet, 25% in Band 3, 47% in Band 2, and 76% in Band 1 at 37 degrees C. The dependence of the amount of mobile cytochrome oxidase on L/P indicates that the fraction of aggregated protein progressively decreases with decreasing concentration of proteins in the membrane. Thus, the large immobile fraction of cytochrome oxidase in mitochondrial inner membranes can be explained by nonspecific protein aggregation which is a consequence of the low L/P. The decrease in the mobile fraction in Pellet compared with mitoplasts was shown to be due to the pH 6.5 incubation used for fusion.     

Lateral diffusion in a mixture of mobile and immobile particles. A Monte Carlo study.

The lateral diffusion coefficient for mixtures of mobile and immobile particles is obtained from Monte Carlo calculations of random walks by mobile tracers in the presence of immobile obstacles on a triangular lattice. The diffusion coefficient of the mobile species is obtained as a function of the area fractions of mobile and immobile species. The results are applied to diffusion of band 3 in the erythrocyte membrane, and indicate that obstruction of diffusion of mobile band 3 by band 3 and glycophorin attached to the membrane skeleton is not sufficient to explain the observed diffusion coefficient.     

Effect of hydrodynamic interactions on the diffusion of integral membrane proteins: diffusion in plasma membranes.

Tracer diffusion coefficients of integral membrane proteins (IMPs) in intact plasma membranes are often much lower than those found in blebbed, organelle, and reconstituted membranes. We calculate the contribution of hydrodynamic interactions to the tracer, gradient, and rotational diffusion of IMPs in plasma membranes. Because of the presence of immobile IMPs, Brinkman's equation governs the hydrodynamics in plasma membranes. Solutions of Brinkman's equation enable the calculation of short-time diffusion coefficients of IMPs. There is a large reduction in particle mobilities when a fraction of them is immobile, and as the fraction increases, the mobilities of the mobile particles continue to decrease. Combination of the hydrodynamic mobilities with Monte Carlo simulation results, which incorporate excluded area effects, enable the calculation of long-time diffusion coefficients. We use our calculations to analyze results for tracer diffusivities in several different systems. In erythrocytes, we find that the hydrodynamic theory, when combined with excluded area effects, closes the gap between existing theory and experiment for the mobility of band 3, with the remaining discrepancy likely due to direct obstruction of band 3 lateral mobility by the spectrin network. In lymphocytes, the combined hydrodynamic-excluded area theory provides a plausible explanation for the reduced mobility of sIg molecules induced by binding concanavalin A-coated platelets. However, the theory does not explain all reported cases of "anchorage modulation" in all cell types in which receptor mobilities are reduced after binding by concanavalin A-coated platelets. The hydrodynamic theory provides an explanation of why protein lateral mobilities are restricted in plasma membranes and why, in many systems, deletion of the cytoplasmic tail of a receptor has little effect on diffusion rates. However, much more data are needed to test the theory definitively. We also predict that gradient and tracer diffusivities are the same to leading order. Finally, we have calculated rotational diffusion coefficients in plasma membranes. They decrease less rapidly than translational diffusion coefficients with increasing protein immobilization, and the results agree qualitatively with the limited experimental data available.     

Probe diffusion in cross-linked actin gels

The diffusion of monodisperse polystyrene latex spheres (PLS) in column-purified 0.65 mg/mL actin solutions, polymerized with 100 mM KCl in the absence and presence of a cross-linker, actin binding protein (ABP), has been studied using dynamic light scattering. Measurements over a wide range of scattering angles from 90 degrees to 8 degrees, corresponding to inverse scattering vector probing distances of about 40-400 nm, respectively, give a measure of both the fraction of PLS mobile over the probing distance (from the normalized time autocorrelation function amplitude) and the average diffusion coefficient of the mobile PLS. Both 100- and 500-nm diameter PLS are fully mobile in polymerized actin solutions over distances of less than 100 nm, as reported previously (Newman, J., Schick, K. S. & Gukelberger, G. Biophys. J. 53, 573a, and Newman, J., Mroczka, N. & Schick, K. L. Biopolymers, 28, 655-666). At increasing probing distances, or when ABP is added at molar ratios of 1:750 or 1:150, greater fractions of the PLS are immobilized, up to almost 99% at the conditions of a 400-nm probing distance with 500-nm probes and at a ratio of 1:150 added ABP to actin. The degree of immobilization correlated well with the amount of added ABP, the size of the PLS, and the probing distance. At increasing probe distances, as the degree of immobilization increases, the remaining mobile fraction of PLS has an increasing average diffusion coefficient. These results suggest a range of pore sizes in the actin gels with a mean size of a few hundred nanometers.     

Lateral diffusion of an 80,000-dalton glycoprotein in the plasma membrane of murine fibroblasts: relationships to cell structure and function

The lateral diffusion of an 80,000-dalton major cell surface glycoprotein of murine fibroblasts has been measured. This antigen, identified through the use of monoclonal antibodies, is an integral glycoprotein distributed through the plasma membrane as judged by immunofluorescence and immunoelectron microscopy (see preceding paper). Measurements of fluorescence recovery after photobleaching were performed on the antigen-antibody complex within the plasma membrane of C3H/10T1/2 and NIH/3T3 cells after labeling the monoclonal antibody with fluorescein. Measurements were performed as a function of temperature, for interphase, mitotic, and G0 C3H/10T1/2 cells. The mean lateral diffusion coefficients (D) for the antibody-protein complex in interphase cells were in the range of 0.7-3.5 X 10(-10) cm2/s between 9 degrees and 37 degrees C, while that for the lipid analog probe, dihexadecylindocarbocyanine was about two orders of magnitude greater. This comparison indicates that peripheral interactions other than bilayer fluidity limit the lateral mobility of the antigen. The mobile fraction of mitotic, G0, and interphase cells showed a monotonic increase with temperature with most of the antibody-antigen complexes being free to move about 25 degrees C. Semi-quantitative interpretations of both the slow glycoprotein diffusion and the immobile fraction are offered. Comparison of diffusion coefficients for cells in different phases of the cell cycle does not reveal striking differences. Mobile fractions for G0 cells at 25 degrees C or less are substantially lower than in interphase cells. In all cases, there was a remarkably broad range of the fluorescence recovery data between different cells, resulting in up to a 10-fold variation in diffusion coefficients, which is far greater than the precision limits of the experiment. Diffusion values and mobile fractions were generally well within a factor of two when measured at several arbitrary points on a single cell. The origins of this cellular heterogenity remain to be elucidated. Lateral mobility in cell fragments and specific regions of single cells was also examined. The glycoprotein was mobile in ventral surface cell fragments. Its mobility was not altered in regions of cell- cell underlapping. However, the diffusion coefficient was threefold higher near the leading edge of motile cells compared to the trailing region. This difference may reflect weaker coupling of the glycoprotein to the underlying cytoskeleton in the dynamic leading edge region.     

Fringe pattern photobleaching, a new method for the measurement of transport coefficients of biological macromolecules.

The conventional method of studying mass transport in membranes by spot photobleaching and then following the recovery of fluorescence has disadvantages. Among them, the need for a high density of fluorescent molecules, the measurement of the beam profile, and a knowledge of the photobleaching processes are of a crucial importance. The application of a planar fringe pattern of light both for the bleaching and the monitoring of the fluorescent molecules solves these three major difficulties. Brownian diffusion coefficients and flow velocities can be measured independently and are averaged over the whole fringe pattern volume. These transport coefficients are explored over the wide range of experimentally accessible distances (from an interfringe spacing 0.5-50 micron). The quantification of the mobile and immobile components is further simplified by scanning the fringe pattern and detecting only a modulated fluorescence recovery signal. The fringe pattern photobleaching method is particularly adapted to the measurements of diffusion coefficients and flow velocity of membrane components, as well as of cytoplasmic proteins. The theoretical results and the test experiments with fluorescent bovine serum albumin are described.     

Cellular effects of endotoxin in vitro: mobility of endotoxin in the plasma membrane of hepatocytes and neuroblastoma cells

Lipopolysaccharide labeled with fluorescein isothiocyanate (FITC-LPS) was used to examine interactions between endotoxin and plasma membrane in isolated rat hepatocytes and mouse neuroblastoma NB41A3 cells. At the same endotoxin to cell ratio, hepatocytes bound more toxin than did neuroblastoma cells. At a dose of 12 micrograms/mg dry wt, a bound mobile fraction of between 60 and 75% of FITC-LPS was found on hepatocytes at 25 degrees C with a lateral diffusion coefficient (D) of 4.0 X 10(-9) cm2/s. In neuroblastoma cells, the mobile fraction was larger (85-90%), with D 1.0 X 10(-8) cm2/s. D was temperature-dependent between 10 and 37 degrees C and increased from 1.8 X 10(-9) to 1.0 X 10(-8) cm2/s in hepatocytes and from 9.4 X 10(-9) to 1.9 X 10(-8) cm2/s in neuroblastoma cells. In both types of cell, nonviable (cells which did not exclude Trypan blue) as compared to viable cells showed different recovery patterns and 100% of the probe molecules were mobile. These results suggest that: (1) endotoxin binding to mammalian cells consists of two subpopulations with different mobilities; (2) binding of the immobile fraction is dependent on cellular integrity; and (3) the differences in binding, lateral mobility, and size of the immobile fraction in hepatocytes and neuroblastoma cells may be due to variations in membrane composition and/or number of binding sites.     

Lateral diffusion of wild-type and mutant Ld antigens in L cells

We have compared the lateral diffusion of intact transmembrane proteins, wild-type H-2Ld antigens, with that of mutants truncated in the cytoplasmic domain. Diffusion coefficients and mobile fractions were similar for all molecules examined, from wild-type Ld antigens with 31 residues on the cytoplasmic side of the plasma membrane to mutants with only four residues in the cytoplasmic domain. This result limits ways in which the lateral diffusion of a major histocompatibility antigen, a transmembrane protein, can be constrained by interactions with other molecules.     

Physiological importance of RNA and protein mobility in the cell nucleus

Trafficking of proteins and RNAs is essential for cellular function and homeostasis. While it has long been appreciated that proteins and RNAs move within cells, only recently has it become possible to visualize trafficking events in vivo. Analysis of protein and RNA motion within the cell nucleus have been particularly intriguing as they have revealed an unanticipated degree of dynamics within the organelle. These methods have revealed that the intranuclear trafficking occurs largely by energy-independent mechanisms and is driven by diffusion. RNA molecules and non-DNA binding proteins undergo constrained diffusion, largely limited by the spatial constraint imposed by chromatin, and chromatin binding proteins move by a stop-and-go mechanism where their free diffusion is interrupted by random association with the chromatin fiber. The ability and mode of motion of proteins and RNAs has implications for how they find nuclear targets on chromatin and in nuclear subcompartments and how macromolecular complexes are assembled in vivo. Most importantly, the dynamic nature of proteins and RNAs is emerging as a means to control physiological cellular responses and pathways.     

Sendai virus envelope glycoproteins become laterally mobile on the surface of human erythrocytes following fusion

Fluorescence photobleaching recovery has been employed to study the lateral mobility of the Sendai virus envelope glycoproteins (HN, neuraminidase/hemagglutinin protein (HN) fusion protein (F) on the surface of human erythrocytes. Our results indicate that the two viral glycoproteins are laterally immobile on the cell surface prior to fusion, and become mobile during the fusion process. The two fused glycoproteins are mobilized to the same extent (diffusion coefficients of 3.1-3.3 X 10(-10) cm2/sec with mobile fractions of 0.53-0.57 for both HN and F). Their mobilization is blocked under conditions that allow virus adsorption and hemagglutination, but not virus-cell or cell-cell fusion. These findings suggest a possible role for the lateral diffusion of the viral glycoproteins in the mechanism of cell-cell fusion, enabling them to perturb the membranes of adjacent cells and lead to cell-cell fusion.     

Fluorescence fluctuation spectroscopy in the presence of immobile fluorophores

Fluorescence contributions from immobile sources present a challenge for fluorescence fluctuation spectroscopy (FFS) because the absence of signal fluctuations from stationary fluorophores leads to a biased analysis. This is especially of concern for cellular FFS studies on proteins that interact with immobile structures. Here we present a method that correctly analyzes FFS experiments in the presence of immobile sources by exploiting selective photobleaching of immobile fluorophores. The fluorescence decay due to photobleaching of the immobile species is modeled taking into account the nonuniform illumination volume. The experimentally observed decay curve serves to separate the mobile and immobile fluorescence contribution, which is used to calculate the molecular brightness from the FFS data. We experimentally verify this approach in vitro using the fluorescent protein EGFP as our immobilized species and a diffusing dye of a different color as the mobile one. For this special case, we also use an alternative method of determining the brightness by spectrally resolving the two species. By conducting a dilution study, we show that the correct parameters are obtained using either technique for a wide range of mobile fractions. To demonstrate the application of our technique in living cells, we perform experiments using the histone core protein H2B fused with EGFP expressed in COS-1 cells. We successfully recovered the brightness of the mobile fraction of H2B-EGFP.     

Lateral diffusion in an archipelago. Distance dependence of the diffusion coefficient.

An understanding of the distance dependence of the lateral diffusion coefficient is useful in comparing the results of diffusion measurements made over different length scales, and in analyzing the kinetics of mobile redox carriers in organelles. A distance-dependent, concentration-dependent diffusion coefficient is defined, and it is evaluated by Monte Carlo calculations of a random walk by mobile point tracers in the presence of immobile obstacles on a triangular lattice, representing the diffusion of a lipid or a small protein in the presence of immobile membrane proteins. This work confirms and extends the milling crowd model of Eisinger, J., J. Flores, and W. P. Petersen (1986. Biophys J. 49:987-1001). Similar calculations for diffusion of mobile particles interacting by a hard-core repulsion yield the distance dependence of the self-diffusion coefficient. An expression for the range of short-range diffusion is obtained, and the distance scales for various diffusion measurements are summarized.     

Factors controlling fibroblast growth factor receptor-1's cytoplasmic trafficking and its regulation as revealed by FRAP analysis

Biochemical and microscopic studies have indicated that FGFR1 is a transmembrane and soluble protein present in the cytosol and nucleus. How FGFR1 enters the cytosol and subsequently the nucleus to control cell development and associated gene activities has become a compelling question. Analyses of protein synthesis, cytoplasmic subcompartmental distribution and movement of FGFR1-EGFP and FGFR1 mutants showed that FGFR1 exists as three separate populations (a) a newly synthesized, highly mobile, nonglycosylated, cytosolic receptor that is depleted by brefeldin A and resides outside the ER-Golgi lumen, (b) a slowly diffusing membrane receptor population, and (c) an immobile membrane pool increased by brefeldin A. RSK1 increases the highly mobile cytosolic FGFR1 population and its overall diffusion rate leading to increased FGFR1 nuclear accumulation, which coaccumulates with RSK1. A model is proposed in which newly synthesized FGFR1 can enter the (a) "nuclear pathway," where the nonglycosylated receptor is extruded from the pre-Golgi producing highly mobile cytosolic receptor molecules that rapidly accumulate in the nucleus or (b) "membrane pathway," in which FGFR1 is processed through the Golgi, where its movement is spatially restricted to trans-Golgi membranes with limited lateral mobility. Entrance into the nuclear pathway is favored by FGFR1's interaction with kinase active RSK1.     

Alteration of the lateral organization of the plasma membrane of Chinese hamster ovary cells by synthetic lipopeptide, Pam3Cys-Ser-Lys4.

The cationic lipohexapeptide (S)-[2, 3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-(S)- Lys 4-OH, trihydrochloride (Pam3Cys-Ser-Lys4) is a synthetic analog of the triacylated N-terminal part of bacterial lipoproteins. In this study we addressed the question of whether Pam3Cys-Ser-Lys4 could modify the organization of the plasma membrane of Chinese hamster ovary cells. 1-Acyl-2-[6-(7-nitro-2-1, 3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphocholine (C6-NBD-PC) diffusion was followed by fluorescence recovery after photobleaching experiments carried out on the plasma membrane of Chinese hamster ovary cells. Incubation of cells in the presence of Pam3Cys-Ser-Lys4 induced an increase in the lateral diffusion coefficient and in the immobile fraction of C6-NBD-PC probes. Various control experiments have shown that the increase in the immobile fraction was not due to probe internalization induced by Pam3Cys-Ser-Lys4. Back-exchange experiments showed that a good correlation exists between the fractions of immobilized probes and nonextractable probes in the plasma membrane of Chinese hamster ovary cells. A useful way to analyze the origin of probe immobilization (micrometer-sized domains or aggregated patches of proteins) is to carry out fluorescence recovery after photobleaching experiments at variable observation radii. This type of experiment, carried out on the plasma membrane of Chinese hamster ovary cells incubated with Pam3Cys-Ser-Lys4, confirmed that the lipopeptide induced the aggregation of proteins of Chinese hamster ovary plasma membrane. Lipids which were trapped inside these aggregates were thus prevented from diffusing at long range in the plasma membrane plane and behave as an immobile fraction.