The effects of lipid fluidity on the rotational diffusion of complex I and complex III in reconstituted NADH-cytochrome c oxidoreductase

NADH-ubiquinone oxidoreductase (Complex I) can be recombined with ubiquinol-cytochrome c oxidoreductase (Complex III) to reconstitute NADH-cytochrome c oxidoreductase. Two modes of interaction have been found. In one, the Complexes interact stoichiometrically in one to one molar ratios to give a binary Complex I-III unit. In the other, the kinetics of NADH-cytochrome c oxidoreductase are characteristic of 'Q-pool' behaviour seen in intact mitochondria and submitochondrial particles in which the Complexes need not interact directly but can do so via a pool of mobile ubiquinone. Stoichiometric behaviour is found when only boundary layer or annular lipid is present or the lipid is in the gel phase. The lipid is immobile on the ESR time scale and protein rotational diffusion, measured by saturation transfer ESR, is very slow. Q-pool behaviour is found when mobile extra-annular lipid phase is also present. Protein rotational diffusion is rapid and characteristic of a fully disaggregated state. We have also used freeze-fracture electron microscopy of reconstituted NADH-cytochrome c oxidoreductase to monitor protein aggregation and lateral phase separation of lipids and proteins under various conditions. We discuss our findings in relation to models for lateral interactions between respiratory chain enzymes.     

Lateral diffusion of proteins in the periplasm of Escherichia coli.

We have introduced biologically active, fluorescently labeled maltose-binding protein into the periplasmic space of Escherichia coli and measured its lateral diffusion coefficient by the fluorescence photobleaching recovery method. Diffusion of this protein in the periplasm was found to be surprisingly low (lateral diffusion coefficient, 0.9 X 10(-10) cm2 s-1), about 1,000-fold lower than would be expected for diffusion in aqueous medium and almost 100-fold lower than for an equivalent-size protein in the cytoplasm. Galactose-binding protein, myoglobin, and cytochrome c were also introduced into the periplasm and had diffusion coefficients identical to that determined for the maltose-binding protein. For all proteins nearly 100% recovery of fluorescence was obtained after photobleaching, indicating that the periplasm is a single contiguous compartment surrounding the cell. These data have considerable implications for periplasmic structure and for the role of periplasmic proteins in transport and chemotaxis.     

Lateral diffusion and fluorescence microscope studies on a monoclonal antibody specifically bound to supported phospholipid bilayers

Supported phospholipid bilayers prepared by Langmuir-Blodgett techniques were introduced recently as a new model membrane system [Tamm, L.K., & McConnell, H.M. (1985) Biophys. J. 47, 105-113]. Here, supported bilayers are applied to study the lateral diffusion and lateral distribution of membrane-bound monoclonal antibodies. A monoclonal anti-trinitrophenol antibody was found to bind strongly and with high specificity to supported phospholipid bilayers containing the lipid hapten (trinitrophenyl)phosphatidylethanolamine at various mole fractions. The lateral distribution of the membrane-bound antibodies was studied by epifluorescence microscopy. The bound antibodies aggregated into patches on a host lipid bilayer of dimyristoylphosphatidylcholine below the lipid chain melting phase transition and redistributed uniformly on fluid-phase supported bilayers. Lateral diffusion coefficients and mobile fractions of fluorescent phospholipid analogues and fluorescein-labeled antibodies were measured by fluorescence recovery after pattern photobleaching. The lateral diffusion coefficients of the membrane-bound antibodies resembled those of the phospholipids but were reduced by a factor of 2 in the fluid phase. The lipid chain melting phase transition was also reflected in the lateral diffusion coefficient of the bound antibody but occurred at a temperature about 3 deg higher than the phase transition in supported bilayers of pure phospholipids. The antibody lateral diffusion coefficients decreased in titration experiments monotonically with increasing antibody surface concentrations by a factor of 2-3. Correspondingly, a relatively small decrease of the antibody lateral diffusion coefficient was observed with increasing mole fractions of lipid haptens in the supported bilayer.     

Lateral diffusion in model membranes is independent of the size of the hydrophobic region of molecules.

We have systematically investigated the probe size and shape dependence of lateral diffusion in model dimyristoyl phosphatidylcholine membranes. Linear hydrophobic polymers, which differ in length by an order of magnitude, were used to explore the effect on the lateral diffusion coefficient of hydrodynamic restrictions in the bilayer interior. The polymers employed are isoprenoid alcohols--citronellol, solanesol, and dolichol. Tracer lateral diffusion coefficients were measured by fluorescence photobleaching recovery. Despite the large difference in lengths, the nitrobenzoxadiazole labelled alcohols all diffuse at the rate of lipid self-diffusion (5.0 x 10(-12) m2 s-1, 29 degrees C) in the liquid crystal phase. Companion measurements in isotropic polymer solution, in gel phase lipid membranes and with nonpolar fluorescent polyaromatic hydrocarbons, show a marked dependence of the lateral diffusion coefficient on the probe molecule size. Our results in the liquid crystal phase are in accord with free area theory which asserts that lateral diffusion in the membrane is restricted by the surface-free area. Probe molecules which are significantly longer than the host phospholipid, seven times longer in the case of dolichol, are still restricted in their lateral motion by the surface properties of the bilayer in the liquid crystal phase. Fluorescence quenching experiments indicate that the nitrobenzoxadiazole label does not reside at the aqueous interface, although it must reside in close proximity according to the diffusion measurements.     

The multicollisional, obstructed, long-range diffusional nature of mitochondrial electron transport

Data are presented which reveal that ubiquinone (Q)-mediated electron transport is a multicollisional, obstructed, long-range diffusion process, where factors that affect the rate of lateral diffusion also affect the rate of electron transport. Based on fluorescence recovery after photobleaching measurements, it was concluded that Q-mediated electron transport occurs by the random collision of redox components which are independent lateral diffusants, each greater than 86% mobile and diffusing in a common pool. The diffusion process of Q-mediated electron transport is 1) multicollisional since the transfers of reducing equivalents between appropriate redox partners occur with less than 100% collision efficiency; 2) obstructed since its maximal rate as well as the rates of diffusion of all redox components involved vary as a function of the membrane protein density; and 3) long-range since the diffusion of all redox components is protein density-dependent, and the diffusion distance required for Q to catalyze the transfer of a reducing equivalent from Complex II to III must be, on average, greater than 37.6 nm. These findings and other theoretical treatments reveal that measurements of short-range diffusion (less than 10 nm), in which collisions between appropriate redox partners do not occur, on average, and which are not affected by membrane protein density, are irrelevant to the collisional process of electron transport. Thus, the data show that the maximum electron transport rate is dependent on both the diffusion rate and the concentration of the redox components. Sucrose was found to inhibit both the mobility of redox components as well as their electron transport rates. Data presented on the relationships between membrane viscosity, rates of lateral and rotational diffusion, and mobile fractions of redox components do not support rotationally immobile aggregates in the functional inner membrane. The high degree of unsaturated phospholipids and the absence of cholesterol in the bilayer of the native inner membrane reflect a requirement for a low resistance to motion of the redox components to compensate for the multicollisional, obstructive nature of their catalytically important collisions in this membrane. These findings support the Random Collision Model of electron transport in which the diffusion and concentration of redox components limit the maximum rate of electron transport.     

Lateral diffusivity of lipid analogue excimeric probes in dimyristoylphosphatidylcholine bilayers.

The lateral mobility of pyrenyl phospholipid probes in dimyristoylphosphatidylcholine (DMPC) vesicles was determined from the dependence of the pyrene monomeric and excimeric fluorescence yields on the molar probe ratio. The analysis of the experimental data makes use of the milling crowd model for two-dimensional diffusivity and the computer simulated random walks of probes in an array of lipids. The fluorescence yields for 1-palmitoyl-2-(1'-pyrenedecanoyl)phosphatidylcholine (py10PC) in DMPC bilayers are well fitted by the model both below and above the fluid-gel phase transition temperature (Tc) and permit the evaluation of the probe diffusion rate (f), which is the frequency with which probes take random steps of length L, the host membrane lipid-lipid spacing. The lateral diffusion coefficient is then obtained from the relationship D = fL2/4. In passing through the fluid-gel phase transition of DMPC (Tc = 24 degrees C), the lateral mobility of py10PC determined in this way decrease only moderately, while D measured by fluorescence photobleaching recovery (FPR) experiments is lowered by two or more orders of magnitude in gel phase. This difference in gel phase diffusivities is discussed and considered to be related either to (a) the diffusion length in FPR experiments being about a micrometer or over 100 times greater than that of excimeric probes (approximately 1 nm), or (b) to nonrandomicity in the distribution of the pyrenyl probes in gel phase DMPC. At 35 degrees C, in fluid DMPC vesicles, the diffusion rate is f = 1.8 x 10(8) s-1, corresponding to D = 29 microns2 s-1, which is about three times larger than the value obtained in FPR experiments. The activation energy for lateral diffusion in fluid DMPC was determined to be 8.0 kcal/mol.     

Protein rotational diffusion and lipid/protein interactions in recombinants of bovine rhodopsin with saturated diacylphosphatidylcholines of different chain lengths studied by conventional and saturation-transfer electron spin resonance.

Bovine rhodopsin has been reconstituted in seven different saturated diacylphosphatidylcholine species of odd and even chain lengths from C-12 to C-18 at a lipid/protein ratio (60:1 mol/mol) comparable to that in the native rod outer segment disk membrane. All recombinants were found to be photochemically active, in that optical bleaching produced a temperature- and lipid chain-length-dependent mixture of species absorbing at 480 and 380 nm. Both the rotational diffusion of rhodopsin and lipid-protein interactions in the various recombinants were studied by saturation transfer and conventional electron spin resonance spectroscopy of spin-labeled rhodopsin and of spin-labeled phosphatidylcholine, respectively. In the fluid lipid phase, the rotational diffusion rate of rhodopsin was found to be dependent on the lipid chain length of the different recombinants in a nonmonotonic manner. The diffusion rate in dilauroylphosphatidylcholine was found to be very slow, indicating extensive protein aggregation, whereas that in dipentadecanoylphosphatidylcholine was rapid (effective correlation time ca. 7 microseconds), consistent with the presence of monomeric protein. For recombinants with longer lipid chain lengths, the rotational diffusion rate again decreased, indicating the presence of di- or oligomeric protein. The fraction of lipid motionally restricted at temperatures in the fluid phase was also dependent on the chain length of the phosphatidylcholine used in the reconstitution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microinjection in combination with microfluorimetry to study proton diffusion along phospholipid membranes

Proton diffusion along the surface of a planar bilayer lipid membrane was measured by means of acid/base injection with a micropipette and recording of the kinetics of fluorescence changes of fluorescein-labelled lipid on the surface. The dimensionality of the process was assayed by fitting the kinetic curves with two-dimensional (2D) or three-dimensional (3D) diffusion equations. In agreement with Serowy et al. (Biophys J 84:1031-1037, 2003), lateral proton diffusion proceeded via bulk phase by means of buffer molecules as proton carriers (D = 600 microm2/s) under the conditions of 1 mM buffer in the solution. Introduction of proton binding sites on the membrane surface led to the appearance of a considerable contribution of two-dimensional proton diffusion on the membrane surface with D = 1,100 mum(2)/s. The system described can be used to study the dependence of the proton diffusion rate on the phospholipid and protein composition of the membrane.     

Lateral diffusion of cholesterol and dimyristoylphosphatidylcholine in a lipid bilayer measured by pulsed field gradient NMR spectroscopy

The pulsed field gradient NMR method for measuring self-diffusion has been used for a direct determination of the lateral diffusion coefficient of cholesterol, fluorine labeled at the 6-position, for an oriented lamellar liquid-crystalline phase of dimyristoylphosphatidylcholine (DMPC)/cholesterol/water. It is found that the diffusion coefficients of DMPC and cholesterol are equal over a large temperature interval. The apparent energy of activation for the diffusion process (58 kJ/mol) is about the same as for a lamellar phase of DMPC/water, whereas the phospholipid lateral diffusion coefficient is approximately four times smaller in the presence of cholesterol.     

Obstructed diffusion in phase-separated supported lipid bilayers: a combined atomic force microscopy and fluorescence recovery after photobleaching approach

Proteins and other macromolecules are believed to hinder molecular lateral diffusion in cellular membranes. We have constructed a well-characterized model system to better understand how obstacles in lipid bilayers obstruct diffusion. Fluorescence recovery after photobleaching was used to measure the lateral diffusion coefficient in single supported bilayers composed of mixtures of 1,2-dilauroylphosphotidylcholine (DLPC) and 1,2-distearoylphosphotidylcholine (DSPC). Because these lipids are immiscible and phase separate at room temperature, a novel quenching technique allowed us to construct fluid DLPC bilayers containing small disk-shaped gel-phase DSPC domains that acted as obstacles to lateral diffusion. Our experimental setup enabled us to analyze the same samples with atomic force microscopy and exactly characterize the size, shape, and number of gel-phase domains before measuring the obstacle-dependent diffusion coefficient. Lateral obstructed diffusion was found to be dependent on obstacle area fraction, size, and geometry. Analysis of our results using a free area diffusion model shows the possibility of unexpected long-range ordering of fluid-phase lipids around the gel-phase obstacles. This lipid ordering has implications for lipid-mediated protein interactions in cellular membranes.     

Lateral diffusion of gramicidin S, M-13 coat protein and glycophorin in bilayers of saturated phospholipids. Mean field and Monte Carlo studies

We have developed a general model that relates the lateral diffusion coefficient of one isolated large intrinsic molecule (mol. wt. greater than or approximately 1000) in a phosphatidylcholine bilayer to the static lipid hydrocarbon chain order. We have studied how protein lateral diffusion can depend upon protein-lipid interactions but have not investigated possible non-specific contributions from gel-state lattice defects. The model has been used in Monte Carlo simulations or in mean-field approximations to study the lateral diffusion coefficients of Gramicidin S, the M-13 coat protein and glycophorin in dimyristoyl- and dipalmitoylphosphatidylcholine (DMPC and DPPC) bilayers as functions of temperature. Our calculated lateral diffusion coefficients for Gramicidin S and the M-13 coat protein are in good agreement with what has been observed and suggest that Gramicidin S is in a dimeric form in DMPC bilayers. In the case of glycophorin we find that the 'ice breaker' effect can be understood as a consequence of perturbation of the lipid polar region around the protein. In order to understand this effect is necessary that the protein hydrophilic section perturb the polar regions of at least approx. 24 lipid molecules, in good agreement with the numbers of 29-30 measured using 31P-NMR. Because of lipid-lipid interactions this effect extends itself out to four or five lipid layers away from the protein so that the hydrocarbon chains of between approx. 74 and approx. 108 lipid molecules are more disordered in the gel phase, so contributing less to the transition enthalpy, in agreement with the numbers of 80-100 deduced from differential scanning calorimetry (DSC). An understanding of the abrupt change in the diffusion coefficient at a temperature below the main bilayer transition temperature requires an additional mechanism. We propose that this change may be a consequence of a 'coupling-uncoupling' transition involving the protein hydrophilic section and the lipid polar regions, which may be triggered by the lipid bilayer pretransition. Our calculation of the average number of gauche bonds per lipid chain as a function of temperature and distance away from an isolated polypeptide or integral protein shows the extent of statically disordered lipid around such molecules. The range of this disorder depends upon temperature, particularly near the main transition.     

Surface-directed structure formation of β-lactoglobulin inside droplets

The morphology of β-lactoglobulin structures inside droplets was studied during aggregation and gelation using confocal laser scanning microscopy (CLSM) equipped with a temperature stage and transmission electron microscopy (TEM). The results showed that there is a strong driving force for the protein to move to the interface between oil and water in the droplet, and the β-lactoglobulin formed a dense shell around the droplet built up from the inside of the droplets. Less protein was found inside the droplets. The longer the β-lactoglobulin was allowed to aggregate prior to gel formation, the larger the part of the protein went to the interface, resulting in a thicker shell and very little material being left inside the droplets. The droplets were easily deformed because no network stabilizes them. When 0.5% emulsifier, polyglycerol polyresinoleat (PGPR), was added to the oil phase, the β-lactoglobulin was situated both inside the droplets and at the interface between the droplets and the oil phase; when 2% PGPR was added, the β-lactoglobulin structure was concentrated to the inside of the droplets. The possibility to use the different morphological structures of β-lactoglobulin in droplets to control the diffusion rate through a β-lactoglobulin network was evaluated by fluorescence recovery after photobleaching (FRAP). The results show differences in the diffusion rate due to heterogeneities in the structure: the diffusion of a large water-soluble molecule, FITC-dextran, in a dense particulate gel was 1/4 of the diffusion rate in a more open particulate β-lactoglobulin gel in which the diffusion rate was similar to that in pure water.     

Domain-formation in DOPC/SM bilayers studied by pfg-NMR: effect of sterol structure

Pulsed field gradient (pfg)-NMR spectroscopy was utilized to determine lipid lateral diffusion coefficients in oriented bilayers composed of 25 mol % sterol and equimolar amounts of dioleoylphosphatidylcholine and sphingomyelin. The occurrence of two lipid diffusion coefficients in a bilayer was used as evidence of lateral phase separation into liquid ordered and liquid disordered domains. It was found that cholesterol, ergosterol, sitosterol, and lathosterol induced domains, whereas lanosterol, stigmasterol, and stigmastanol resided in homogeneous membranes in the temperature interval of 24-70 degrees C. Among the domain-forming sterols, differences in the upper miscibility temperature indicated that the stability of the liquid ordered phase could be modified by small changes in the sterol structure. The domain-forming capacity for the different sterols is discussed in terms of the ordering effect of the sterols on the lipids, and it is proposed that the driving force for the lateral phase separation is the reduced solubility of the unsaturated lipid in the highly ordered phase.     

Modulation of lateral diffusion in the plasma membrane by protein density

The rate of lateral diffusion of proteins over micron-scale distances in the plasma membrane (PM) of mammalian cells is much slower than in artificial membranes [1, 2]. Different models have been advanced to account for this discrepancy. They invoke either effects on the apparent viscosity of cell membranes through, for example, protein crowding [3, 4], or a role for cortical factors such as actin or spectrin filaments [1]. Here, we use photobleaching to test specific predictions of these models [5]. Neither loss of detectable cortical actin nor knockdown of spectrin expression has any effect on diffusion. Disruption of the PM by formation of ventral membrane sheets or permeabilization induces aggregation of membrane proteins, with a concomitant increase in rates of diffusion for the nonaggregated fraction. In addition, procedures that directly increase or decrease the total protein content of the PM in live cells cause reciprocal changes in lateral diffusion rates. Our data imply that slow diffusion over micron-scale distances is an intrinsic property of the membrane itself and that the density of proteins within the membrane is a significant parameter in determining rates of lateral diffusion.     

Two-dimensional diffusion of amphiphiles in phospholipid monolayers at the air-water interface.

Steady-state and time-resolved fluorescence spectroscopy has been used to examine lateral diffusion in dipalmitoyl-L-alpha-phosphatidylcholine (DPPC) and dimyristoyl-L-alpha-phosphatidylcholine (DMPC) monolayers at the air-water interface, by studying the fluorescence quenching of a pyrene-labeled phospholipid (pyrene-DPPE) by two amphiphilic quenchers. Steady-state fluorescence measurements revealed pyrene-DPPE to be homogeneously distributed in the DMPC lipid matrix for all measured surface pressures and only in the liquid-expanded (LE) phase of the DPPC monolayer. Time-resolved fluorescence decays for pyrene-DPPE in DMPC and DPPC (LE phase) in the absence of quencher were best described by a single-exponential function, also suggesting a homogeneous distribution of pyrene-DPPE within the monolayer films. Addition of quencher to the monolayer film produced nonexponential decay behavior, which is adequately described by the continuum theory of diffusion-controlled quenching in a two-dimensional environment. Steady-state fluorescence measurements yielded lateral diffusion coefficients significantly larger than those obtained from time-resolved data. The difference in these values was ascribed to the influence of static quenching in the case of the steady-state measurements. The lateral diffusion coefficients obtained in the DMPC monolayers were found to decrease with increasing surface pressure, reflecting a decrease in monolayer fluidity with compression.     

Lateral diffusion study of excimer-forming lipids in lamellar to inverted hexagonal phase transition of unsaturated phosphatidylethanolamine

Using multi-frequency cross-correlation fluorometry, the monomer fluorescence lifetime of 1-palmitoyl-2-[10-(1-pyrenyl)decanoyl)phosphatidylcholine (Py-PC) was employed to determine the lateral diffusion constant (DT) of dioleoylphosphatidylethanolamine (DOPE) in both the lamellar (L alpha) and the inverted hexagonal (HII) phases. The values of DT increased with temperature in both phases. However, the rate of increase of DT declined abruptly at approximately 10-13 degrees C (L alpha -HII transition temperature), as indicated by the existence of an inflection point in the log (DT/T) vs. 1/T plot. This observation suggests that the translational motion of lipids in the HII phase is lower than that in the L alpha phase upon temperature extrapolation. Lipid perturbants, cholesterol and diacylglycerol, were found to destabilize the L alpha phase of DOPE. This was demonstrated by a down-shift of the inflection point in the log(DT/T) vs. 1/T plot in the presence of the perturbants. Both cholesterol and 1,2-dioleoyl-sn-glycerol (diolein) decreased the lateral diffusion constant in both phases. Diolein promoted the HII phase more effectively than did the cholesterol. This is explained by an intrinsic wedge-shape geometry of diolein which strongly favors the formation of inverted cylindrical packing of the lipids.     

Effects of protein concentration on IgE receptor mobility in rat basophilic leukemia cell plasma membranes.

The ability of variations of membrane protein concentrations to modulate the lateral diffusion rate of an exemplary membrane protein has been studied in healthy and osmotically shocked cultured cells of the rat basophilic leukemia cell line, 2H3 subclone. Cell surface protein was redistributed by the method of in situ electrophoresis; exposure to electric fields of 1.25-5 V/cm results in cathodal migration of the majority of the surface proteins on this cell type (Ryan, T. A., J. Myers, D. Holowka, B. Baird, and W. W. Webb. Science [Wash. DC]. 239:61-64). Even in these small fields, the steady-state distribution becomes "crowded" with more than an 80% protein occupancy of accessible membrane area at the cathodal end of these spheroidal cells, and the anodal end becomes significantly depleted. We have employed fringe pattern fluorescence photobleaching with CCD imaging detection to measure lateral diffusion coefficients of the liganded IgE receptor on both crowded and uncrowded regions of individual rat basophilic leukemia cells. We find no significant difference in lateral diffusion rates in these regions. Cells swollen by hypoosmotic stress exhibit faster diffusion overall, with the uncrowded regions having a significantly greater increase in diffusion coefficient than the crowded regions. These results are consistent with the partial or total release of cytoskeletal constraints to membrane protein diffusion induced by osmotic stress.     

Lipid lateral diffusion measurements in model membranes

The rate of lipid lateral diffusion has been investigated by computer simulation of electron spin resonance (ESR) spectra of spin-labelled dimyristoyl phosphatidylcholine (DMPC) vesicles. An optimization method has been developed to fit the experimental spectra to the theoretical ones calculated from the modified Bloch-equations in order to determine frequencies of probe-probe collisions and the lipid lateral diffusion coefficients. The main results of this study are: (i) Due to the sensitivity of our method to the extent of the overlapping of hyperfine spectral lines it is possible to determine the spin exchange contribution to linebroadening. (ii) It is obvious from these computer analyses that over a wide range of temperatures well above the phase transition both static dipolar interaction and dynamic spin exchange make significant contributions to the linebroadening. (iii) Lipid lateral diffusion coefficient in DMPC bilayers at 36 degrees C was (2.3 +/- 0.2) x 10(-11) m2 s-1.     

Regulation of lipid composition in Acholeplasma laidlawii and Escherichia coli membranes: NMR studies of lipid lateral diffusion at different growth temperatures

Lipid lateral diffusion coefficients have been directly determined by pulsed field gradient NMR spectroscopy on macroscopically aligned, fully hydrated lamellar phases containing dimyristoylphosphatidylcholine and total lipid extracts from Acholeplasma laidlawii and Escherichia coli. The temperature dependence of the diffusion coefficient was of the Arrhenius type in the temperature interval studied. The sharp increase in the diffusion coefficient at the growth temperature of E. coli obtained by FRAP measurements, using a fluorescent probe molecule (Jin, A. J., Edidin, M., Nossal, R., and Gershfeld, N. L. (1999) Biochemistry 38, 13275-13278), was not observed. Thus, we conclude that the lipid structural properties (i.e., those affecting the lipid phase behavior), rather than the lipid dynamics, are involved in the adjustment of the membrane lipid composition. Further support for this conclusion is given by the finding that lipid extracts from A. laidlawii grown at different temperatures have about the same diffusion coefficients. Finally, the lipid lateral diffusion in bilayers of phospholipids was found to be much faster than that in bilayers of mainly glucolipids, which can be understood in terms of a free volume theory for the diffusion process.     

The Lateral Membrane Organization and Dynamics of Myelin Proteins PLP and MBP Are Dictated by Distinct Galactolipids and the Extracellular Matrix

In the central nervous system, lipid-protein interactions are pivotal for myelin maintenance, as these interactions regulate protein transport to the myelin membrane as well as the molecular organization within the sheath. To improve our understanding of the fundamental properties of myelin, we focused here on the lateral membrane organization and dynamics of peripheral membrane protein 18.5-kDa myelin basic protein (MBP) and transmembrane protein proteolipid protein (PLP) as a function of the typical myelin lipids galactosylceramide (GalC), and sulfatide, and exogenous factors such as the extracellular matrix proteins laminin-2 and fibronectin, employing an oligodendrocyte cell line, selectively expressing the desired galactolipids. The dynamics of MBP were monitored by z-scan point fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS), while PLP dynamics in living cells were investigated by circular scanning FCS. The data revealed that on an inert substrate the diffusion rate of 18.5-kDa MBP increased in GalC-expressing cells, while the diffusion coefficient of PLP was decreased in sulfatide-containing cells. Similarly, when cells were grown on myelination-promoting laminin-2, the lateral diffusion coefficient of PLP was decreased in sulfatide-containing cells. In contrast, PLP''s diffusion rate increased substantially when these cells were grown on myelination-inhibiting fibronectin. Additional biochemical analyses revealed that the observed differences in lateral diffusion coefficients of both proteins can be explained by differences in their biophysical, i.e., galactolipid environment, specifically with regard to their association with lipid rafts. Given the persistence of pathological fibronectin aggregates in multiple sclerosis lesions, this fundamental insight into the nature and dynamics of lipid-protein interactions will be instrumental in developing myelin regenerative strategies.