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.     

Diffusion of phycobilisomes on the thylakoid membranes of the cyanobacterium Synechococcus 7942. Effects of phycobilisome size, temperature, and membrane lipid composition

A variant of fluorescence recovery after photobleaching allows us to observe the diffusion of photosynthetic complexes in cyanobacterial thylakoid membranes in vivo. The unicellular cyanobacterium Synechococcus sp. PCC7942 is a wonderful model organism for fluorescence recovery after photobleaching, because it has a favorable membrane geometry and is well characterized and transformable. In Synechococcus 7942 (as in other cyanobacteria) we find that photosystem II is immobile, but phycobilisomes diffuse rapidly on the membrane surface. The diffusion coefficient is 3 x 10(-10) cm(2) s(-1) at 30 degrees C. This shows that the association of phycobilisomes with reaction centers is dynamic; there are no stable phycobilisome-reaction center complexes in vivo. We report the effects of mutations that change the phycobilisome size and membrane lipid composition. 1) In a mutant with no phycobilisome rods, the phycobilisomes remain mobile with a slightly faster diffusion coefficient. This confirms that the diffusion we observe is of intact phycobilisomes rather than detached rod elements. The faster diffusion coefficient in the mutant indicates that the rate of diffusion is partly determined by the phycobilisome size. 2) The temperature dependence of the phycobilisome diffusion coefficient indicates that the phycobilisomes have no integral membrane domain. It is likely that association with the membrane is mediated by multiple weak interactions with lipid head groups. 3) Changing the lipid composition of the thylakoid membrane has a dramatic effect on phycobilisome mobility. The results cannot be explained in terms of changes in the fluidity of the membrane; they suggest that lipids play a role in controlling phycobilisome-reaction center interaction.     

Lateral diffusion in an archipelago. The effect of mobile obstacles.

Lateral diffusion of mobile proteins and lipids (tracers) in a membrane is hindered by the presence of proteins (obstacles) in the membrane. If the obstacles are immobile, their effect may be described by percolation theory, which states that the long-range diffusion constant of the tracers goes to zero when the area fraction of obstacles is greater than the percolation threshold. If the obstacles are themselves mobile, the diffusion constant of the tracers depends on the area fraction of obstacles and the relative jump rate of tracers and obstacles. This paper presents Monte Carlo calculations of diffusion constants on square and triangular lattices as a function of the concentration of obstacles and the relative jump rate. The diffusion constant for particles of various sizes is also obtained. Calculated values of the concentration-dependent diffusion constant are compared with observed values for gramicidin and bacteriorhodopsin. The effect of the proteins as inert obstacles is significant, but other factors, such as protein-protein interactions and perturbation of lipid viscosity by proteins, are of comparable importance. Potential applications include the diffusion of proteins at high concentrations (such as rhodopsin in rod outer segments), the modulation of diffusion by release of membrane proteins from cytoskeletal attachment, and the diffusion of mobile redox carriers in mitochondria, chloroplasts, and endoplasmic reticulum.     

Flexibility of the cytoplasmic domain of the anion exchange protein, band 3, in human erythrocytes.

The rotational flexibility of the cytoplasmic domain of band 3, in the region that is proximal to the inner membrane surface, has been investigated using a combination of time-resolved optical anisotropy (TOA) and saturation-transfer electron paramagnetic resonance (ST-EPR) spectroscopies. TOA studies of rotational diffusion of the transmembrane domain of band 3 show a dramatic decrease in residual anisotropy following cleavage of the link with the cytoplasmic domain by trypsin (E. A. Nigg and R. J. Cherry, 1980, Proc. Natl. Acad. Sci. U.S.A. 77:4702-4706). This result is compatible with two independent hypotheses: 1) trypsin cleavage leads to dissociation of large clusters of band 3 that are immobile on the millisecond time scale, or 2) trypsin cleavage leads to release of a constraint to uniaxial rotational diffusion of the transmembrane domain. ST-EPR studies at X- and Q-band microwave frequencies detect rotational diffusion of the transmembrane domain of band 3 about the membrane normal axis of reasonably large amplitude that does not change upon cleavage with trypsin. These ST-EPR results are not consistent with dissociation of clusters of band 3 as a result of cleavage with trypsin. Global analyses of the ST-EPR data using a newly developed algorithm indicate that any constraint to rotational diffusion of the transmembrane domain of band 3 via interactions of the cytoplasmic domain with the membrane skeleton must be sufficiently weak to allow rotational excursions in excess of 32 degrees full-width for a square-well potential. In support of this result, analyses of the TOA data in terms of restricted amplitude uniaxial rotational diffusion models suggest that the membrane-spanning domain of that population of band 3 that is linked to the membrane skeleton is constrained to diffuse in a square-well of approximately 73 degrees full-width. This degree of flexibility may be necessary for providing the unique mechanical properties of the erythrocyte membrane.     

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.     

Mobility of the IsiA chlorophyll-binding protein in cyanobacterial thylakoid membranes

We are using fluorescence recovery after photobleaching (FRAP) to probe the dynamics of thylakoid membranes in vivo in cells of the cyanobacterium Synechococcus sp. PCC7942. We have shown previously that the light-harvesting phycobilisomes diffuse quite rapidly on the thylakoid membrane surface. However, the photosystem II core complexes appear completely immobile. This raises the possibility that all of the membrane integral protein complexes in the thylakoid membrane are locked into a rather rigid array. Alternatively, it is possible that photosystem II is specifically anchored in the membrane, with other membrane proteins able to diffuse around it. We have now resolved this question by studying the diffusion of a second integral membrane protein, the IsiA chlorophyll-binding protein. IsiA is induced under iron starvation and some other stress conditions. In iron-stressed cyanobacterial cells, a high proportion of chlorophyll fluorescence comes from IsiA. This makes it straightforward to examine the diffusion of IsiA by FRAP. We find that the complex is mobile with a mean diffusion coefficient of approximately 3 x 10(-11) cm(2) s(-1). Thus it is clear that some thylakoid membrane proteins are mobile and that there must be a specific anchor that prevents photosystem II diffusion. We discuss the implications for the structure and function of the cyanobacterial thylakoid membrane.     

Cytoplasmic hydrogen ion diffusion coefficient.

The apparent cytoplasmic proton diffusion coefficient was measured using pH electrodes and samples of cytoplasm extracted from the giant neuron of a marine invertebrate. By suddenly changing the pH at one surface of the sample and recording the relaxation of pH within the sample, an apparent diffusion coefficient of 1.4 +/- 0.5 x 10(-6) cm2/s (N = 7) was measured in the acidic or neutral range of pH (6.0-7.2). This value is approximately 5x lower than the diffusion coefficient of the mobile pH buffers (approximately 8 x 10(-6) cm2/s) and approximately 68x lower than the diffusion coefficient of the hydronium ion (93 x 10(-6) cm2/s). A mobile pH buffer (approximately 15% of the buffering power) and an immobile buffer (approximately 85% of the buffering power) could quantitatively account for the results at acidic or neutral pH. At alkaline pH (8.2-8.6), the apparent proton diffusion coefficient increased to 4.1 +/- 0.8 x 10(-6) cm2/s (N = 7). This larger diffusion coefficient at alkaline pH could be explained quantitatively by the enhanced buffering power of the mobile amino acids. Under the conditions of these experiments, it is unlikely that hydroxide movement influences the apparent hydrogen ion diffusion coefficient.     

Tethered polymer-supported planar lipid bilayers for reconstitution of integral membrane proteins: silane-polyethyleneglycol-lipid as a cushion and covalent linker

There is increasing interest in supported membranes as models of biological membranes and as a physiological matrix for studying the structure and function of membrane proteins and receptors. A common problem of protein-lipid bilayers that are directly supported on a hydrophilic substrate is nonphysiological interactions of integral membrane proteins with the solid support to the extent that they will not diffuse in the plane of the membrane. To alleviate some of these problems we have developed a new tethered polymer-supported planar lipid bilayer system, which permitted us to reconstitute integral membrane proteins in a laterally mobile form. We have supported lipid bilayers on a newly designed polyethyleneglycol cushion, which provided a soft support and, for increased stability, covalent linkage of the membranes to the supporting quartz or glass substrates. The formation and morphology of the bilayers were followed by total internal reflection and epifluorescence microscopy, and the lateral diffusion of the lipids and proteins in the bilayer was monitored by fluorescence recovery after photobleaching. Uniform bilayers with high lateral lipid diffusion coefficients (0.8-1.2 x 10(-8) cm(2)/s) were observed when the polymer concentration was kept slightly below the mushroom-to-brush transition. Cytochrome b(5) and annexin V were used as first test proteins in this system. When reconstituted in supported bilayers that were directly supported on quartz, both proteins were largely immobile with mobile fractions < 25%. However, two populations of laterally mobile proteins were observed in the polymer-supported bilayers. Approximately 25% of cytochrome b(5) diffused with a diffusion coefficient of approximately 1 x 10(-8) cm(2)/s, and 50-60% diffused with a diffusion coefficient of approximately 2 x 10(-10) cm(2)/s. Similarly, one-third of annexin V diffused with a diffusion coefficient of approximately 3 x 10(-9) cm(2)/s, and two-thirds diffused with a diffusion coefficient of approximately 4 x 10(-10) cm(2)/s. A model for the interaction of these proteins with the underlying polymer is discussed.     

Rotational dynamics of the integral membrane protein, band 3, as a probe of the membrane events associated with Plasmodium falciparum infections of human erythrocytes

Time-resolved phosphorescence anisotropy was used to study the molecular organisation of band 3 in the erythrocyte membrane. Three different rotational relaxation regimes of mobile band 3 were resolved. These populations may represent different aggregation states of band 3 within the membrane, or they may result from association of band 3 with other proteins at the cytoplasmic surface. The polycation spermine decreases the apparent mobility of band 3 by a mechanism that does not involve the underlying cytoskeleton. A monoclonal antibody directed against the cytoplasmic portion of band 3 can also cause an increase in the immobile fraction of band 3 molecules. This monoclonal antibody will inhibit invasion of erythrocytes by malaria parasites. Membranes prepared from erythrocytes infected with mature stages of the malaria parasite, Plasmodium falciparum, show altered dynamic properties corresponding to a marked restriction of band 3 mobility.     

Aldolase exists in both the fluid and solid phases of cytoplasm [published erratum appears in J Cell Biol 1988 Dec;107(6 Pt 1):following 2463]

We have prepared a functional fluorescent analogue of the glycolytic enzyme aldolase (rhodamine [Rh]-aldolase), using the succinimidyl ester of carboxytetramethyl-rhodamine. Fluorescence redistribution after photobleaching measurements of the diffusion coefficient of Rh-aldolase in aqueous solutions gave a value of 4.7 x 10(-7) cm2/S, and no immobile fraction. In the presence of filamentous actin, there was a 4.5-fold reduction in diffusion coefficient, as well as a 36% immobile fraction, demonstrating binding of Rh-aldolase to actin. However, in the presence of a 100-fold molar excess of its substrate, fructose 1,6-diphosphate, both the mobile fraction and diffusion coefficient of Rh-aldolase returned to control levels, indicating competition between substrate binding and actin cross-linking. When Rh-aldolase was microinjected into Swiss 3T3 cells, a relatively uniform intracellular distribution of fluorescence was observed. However, there were significant spatial differences in the in vivo diffusion coefficient and mobile fraction of Rh-aldolase measured with fluorescence redistribution after photobleaching. In the perinuclear region, we measured an apparent cytoplasmic diffusion coefficient of 1.1 x 10(-7) cm2/s with a 23% immobile fraction; while measurements in the cell periphery gave a value of 5.7 x 10(-8) cm2/s, with no immobile fraction. Ratio imaging of Rh-aldolase and FITC-dextran indicated that FITC-dextran was relatively excluded excluded from stress fiber domains. We interpret these data as evidence for the partitioning of aldolase between a soluble fraction in the fluid phase and a fraction associated with the solid phase of cytoplasm. The partitioning of aldolase and other glycolytic enzymes between the fluid and solid phases of cytoplasm could play a fundamental role in the control of glycolysis, the organization of cytoplasm, and cell motility. The concepts and experimental approaches described in this study can be applied to other cellular biochemical processes.     

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.     

Development of a Model for Analyzing the Swelling Rate of Ionic Gels on the Basis of the Diffusion of Mobile Ions: Application to the pH-Sensitive Swelling of a Polyelectrolyte Complex Gel Prepared from Xanthan and Chitosan

A model for analyzing the swelling rate of ionic gels was developed on the basis of the diffusion of a species of mobile ion. This model was applied to the analysis of pH-sensitive swelling of a xanthan/chitosan complex gel in NaOH solutions of pH 9–12, using the sodium ion as the reference mobile ion. The time–course for swelling of gel beads with a pH change from 11 to 10 was successfully described by the developed model. The values for the diffusion coefficient obtained by fitting the model to the data were of the same order as those for the diffusion coefficient of the sodium ion measured for a membrane of the complex gel. Thus, it was confirmed that the swelling rate of the gel due to pH change was mainly controlled by the diffusion of mobile ions. However, the time-course for swelling of the gel at pH values below 10 was not satisfactorily explained by the model developed, suggesting that the change in the degree of ionization during swelling also affected the swelling rate of the xanthan/chitosan complex gel.     

Diffusion in a Fluid Membrane with a Flexible Cortical Cytoskeleton

We calculate the influence of a flexible network of long-chain proteins, which is anchored to a fluid membrane, on protein diffusion in this membrane. This is a model for the cortical cytoskeleton and the lipid bilayer of the red blood cell, which we apply to predict the influence of the cytoskeleton on the diffusion coefficient of a mobile band 3 protein. Using the pressure field that the cytoskeleton exerts on the membrane, from the steric repulsion between the diffusing protein and the cytoskeletal filaments, we define a potential landscape for the diffusion within the bilayer. We study the changes to the diffusion coefficient on removal of one type of anchor proteins, e.g., in several hemolytic anemias, as well as for isotropic and anisotropic stretching of the cytoskeleton. We predict an overall increase of the diffusion for a smaller number of anchor proteins and increased diffusion for anisotropic stretching in the direction of the stretch, because of the decrease in the spatial frequency as well as in the height of the potential barriers.     

The Proapoptotic Protein tBid Forms Both Superficially Bound and Membrane-Inserted Oligomers

Bid is a proapopotic activator protein of the Bcl-2 family that plays a pivotal role in controlling mitochondrial outer membrane permeabilization during apoptosis. Here, we characterized the interaction of fluorescently labeled truncated Bid (tBid) with a mitochondria-like supported lipid bilayer at the single-molecule level. The proteins observed at the membrane exhibited a very wide range of mobility. Confocal images of the membrane displayed both diffraction-limited Gaussian spots and horizontal streaks, corresponding to immobile and mobile tBid species, respectively. We observed 1), fast-diffusing proteins corresponding to a loosely, probably electrostatically bound state; 2), slowly diffusing proteins, likely corresponding to a superficially inserted state; and 3), fully immobilized proteins, suggesting a fully inserted state. The stoichiometry of these proteins was determined by normalizing their fluorescence intensity by the brightness of a tBid monomer, measured separately using fluorescence fluctuation techniques. Strikingly, the immobile species were found to be mainly tetramers and higher, whereas the mobile species had on average a significantly lower stoichiometry. Taken together, these results show that as soluble Bid progresses toward a membrane-inserted state, it undergoes an oligomerization process similar to that observed for Bax.     

The Proapoptotic Protein tBid Forms Both Superficially Bound and Membrane-Inserted Oligomers

Bid is a proapopotic activator protein of the Bcl-2 family that plays a pivotal role in controlling mitochondrial outer membrane permeabilization during apoptosis. Here, we characterized the interaction of fluorescently labeled truncated Bid (tBid) with a mitochondria-like supported lipid bilayer at the single-molecule level. The proteins observed at the membrane exhibited a very wide range of mobility. Confocal images of the membrane displayed both diffraction-limited Gaussian spots and horizontal streaks, corresponding to immobile and mobile tBid species, respectively. We observed 1), fast-diffusing proteins corresponding to a loosely, probably electrostatically bound state; 2), slowly diffusing proteins, likely corresponding to a superficially inserted state; and 3), fully immobilized proteins, suggesting a fully inserted state. The stoichiometry of these proteins was determined by normalizing their fluorescence intensity by the brightness of a tBid monomer, measured separately using fluorescence fluctuation techniques. Strikingly, the immobile species were found to be mainly tetramers and higher, whereas the mobile species had on average a significantly lower stoichiometry. Taken together, these results show that as soluble Bid progresses toward a membrane-inserted state, it undergoes an oligomerization process similar to that observed for Bax.     

Surface diffusion of interacting proteins. Effect of concentration on the lateral mobility of adsorbed bovine serum albumin.

Surface diffusion of bovine serum albumin absorbed from aqueous solution to poly(methylmethacrylate) surfaces is significantly hindered by protein-protein lateral interactions. The long-time self diffusion coefficient measured by fluorescence recovery after pattern photobleaching decreases by approximately one order of magnitude as the surface area fraction occupied by protein increases from 0.10 to 0.69. Qualitative features of the surface concentration dependence of the self diffusion coefficient can be described by several recent models for lateral diffusion of interacting species. The mobile fraction is independent of the surface concentration, and both the self diffusion coefficient and the mobile fraction are constant between 15 min and 7 h of adsorption.     

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.     

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.     

Constrained diffusion or immobile fraction on cell surfaces: a new interpretation.

Protein lateral mobility in cell membranes is generally measured using fluorescence photobleaching recovery (FPR). Since the development of this technique, the data have been interpreted by assuming free Brownian diffusion of cell surface receptors in two dimensions, an interpretation that requires that a subset of the diffusing species remains immobile. The origin of this so-called immobile fraction remains a mystery. In FPR, the motions of thousands of particles are inherently averaged, inevitably masking the details of individual motions. Recently, tracking of individual cell surface receptors has identified several distinct types of motion (Gross and Webb, 1988; Ghosh and Webb, 1988, 1990, 1994; Kusumi et al. 1993; Qian et al. 1991; Slattery, 1995), thereby calling into question the classical interpretation of FPR data as free Brownian motion of a limited mobile fraction. We have measured the motion of fluorescently labeled immunoglobulin E complexed to high affinity receptors (Fc epsilon RI) on rat basophilic leukemia cells using both single particle tracking and FPR. As in previous studies, our tracking results show that individual receptors may diffuse freely, or may exhibit restricted, time-dependent (anomalous) diffusion. Accordingly, we have analyzed FPR data by a new model to take this varied motion into account, and we show that the immobile fraction may be due to particles moving with the anomalous subdiffusion associated with restricted lateral mobility. Anomalous subdiffusion denotes random molecular motion in which the mean square displacements grow as a power law in time with a fractional positive exponent less than one. These findings call for a new model of cell membrane structure.     

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.