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.     

Diffusion, patching, and capping of stearoylated dextrans on 3T3 cell plasma membranes

Fluorescence-labeled trinitrophenylated stearoylated dextrans have been used as controllable analogues of cell membrane proteins on model membranes and on a variety of natural cell membranes. This paper reports their behavior on 3T3 mouse fibroblast plasma membranes. Spatial distribution on the membrane was studied by fluorescence microscopy, and molecular mobility was measured by fluorescence photobleaching recovery. At concentrations from 10(2) to 3 X 10(3) molecules/micron2 essentially homogeneous fluorescence was observed after treatment with these stearoyldextrans in culture. Diffusion coefficients and fractional recovery of fluorescence after photobleaching were cvoncentration independent. For 3 X 10(3) molecules/micron2 we found at 23 degrees C D = (3.0 +/- 1.8) X 10(-10) cm2/s with 65 +/- 17% recovery and at 37 degrees C D = (7.0 +/- 5.0) X 10(-10) cm2/s without a change of the fractional recovery. Cross-linking with antibodies stopped diffusion on a macroscopic scale and sometimes induced patching, mottling (defined as the development of gaps in the fluorescence layer), and capping (defined as the confinement of the fluorescence to less than 50% of the cell). Capping required approximately 3 h at 37 degrees C and was inhibited by metabolic poisons and cytochalasin B. These drugs did not affect stearoyldextran diffusion or fractional recovery. Colchicine, which did not dramatically affect capping, slowed diffusion two- to threefold but did not affect fractional recovery. The antibody inhibition of the diffusion of stearoyldextrans precedent to capping did not affect the diffusion of a lipid probe or fluorescein isothiocyanate labeled membrane proteins. When the trinitrophenylated stearoyldextran was cleared from most of the surface by capping and the surface subsequently relabeled with stearoyldextran, the diffusion coefficient and fractional recovery of the second label were identical with those of the first label prior to capping. Thus, capping does not clear an immobilizing factor from the membrane.     

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.     

Procyclin gene expression and loss of the variant surface glycoprotein during differentiation of Trypanosoma brucei

In the mammalian host, the unicellular flagellate Trypanosoma brucei is covered by a dense surface coat that consists of a single species of macromolecule, the membrane form of the variant surface glycoprotein (mfVSG). After uptake by the insect vector, the tsetse fly, bloodstream-form trypanosomes differentiate to procyclic forms in the fly midgut. Differentiation is characterized by the loss of the mfVSG coat and the acquisition of a new surface glycoprotein, procyclin. In this study, the change in surface glycoprotein composition during differentiation was investigated in vitro. After triggering differentiation, a rapid increase in procyclin-specific mRNA was observed. In contrast, there was a lag of several hours before procyclin could be detected. Procyclin was incorporated and uniformly distributed in the surface coat. The VSG coat was subsequently shed. For a single cell, it took 12-16 h to express a maximum level of procyclin at the surface while the loss of the VSG coat required approximately 4 h. The data are discussed in terms of the possible molecular arrangement of mfVSG and procyclin at the cell surface. Molecular modeling data suggest that a (Asp-Pro)2 (Glu-Pro)22-29 repeat in procyclin assumes a cylindrical shape 14-18 nm in length and 0.9 nm in diameter. This extended shape would enable procyclin to interdigitate between the mfVSG molecules during differentiation, exposing epitopes beyond the 12-15-nm-thick VSG coat.     

Electric field-induced redistribution and postfield relaxation of epidermal growth factor receptors on A431 cells

The lateral mobility of the epidermal growth factor (EGF) receptor in the plane of the plasma membrane of cultured A431 cells was investigated using direct and indirect fluorescent probes to measure the generation and relaxation of electric field-induced receptor asymmetry. A steady electric field of 15 V/cm for 30 min at 23 degrees C induced a redistribution of the unoccupied EGF receptor such that there was approximately a three-fold higher concentration of receptors at the cathode-facing pole. After termination of the field, the unoccupied receptors back diffused at 37 degrees C with a rate corresponding to a diffusion coefficient of 2.6-3.5 X 10(-10) cm2/s. No diffusion was detected at 4 degrees C. Formation of the hormone-receptor complex is known to induce receptor clustering and internalization. By inhibiting internalization with metabolic poisons, we were able to study the cell surface mobility of clusters of the hormone-receptor complex. The same degree of asymmetry was induced when the occupied receptor was exposed to an electric field and the rate of back diffusion of clusters of the hormone-receptor complex corresponded to a diffusion coefficient of 0.68-0.95 X 10(-10) cm2/s. Although the unoccupied receptor is somewhat more mobile than the hormone-receptor complex, it was still far less mobile than one would predict for an unconstrained protein imbedded in a phospholipid bilayer.     

Insulin effect on translational diffusion of lipids and proteins in the plasma membrane of isolated rat hepatocytes

The effects of insulin (10(-10)-10(-8) mol/l) on lateral diffusion of three fluorescent lipid probes, 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)aminocaproyl phosphatidylcholine (NBD-PC), 5-(N-hexadecanoyl)aminofluorescein (F-C16), 5-(N-dodecanoyl)aminofluorescein (F-C12), and of fluorescein isothiocyanate-labeled proteins in the plasma membrane of intact rat hepatocytes were studied by the technique of fluorescence recovery after photobleaching. The absolute lateral diffusion coefficients of the lipid analogues NBD-PC, F-C16 and F-C12 at 21 degrees C were 2.5 X 10(-9) cm2/s, 5.4 X 10(-9) cm2/s and 19 X 10(-9) cm2/s, respectively. The diffusion coefficient mean of proteins labeled with fluorescein isothiocyanate was 6.4 X 10(-10) cm2/s. Insulin at 10(-9) and 10(-8) mol/l reduced the lateral diffusion coefficient for F-C12- and F-C16-labeled cells by 20% and for NBD-PC-labeled cells by 30% (P less than 0.025). The insulin effect was specific as tested by cell incubation with proinsulin and desoctapeptide insulin (10(-8) mol/l) and was detectable after 7 min of insulin preincubation. In contrast to lateral diffusion of lipid probes, lateral mobility of unselected membrane proteins was not altered by insulin. The observed modulation of lipid dynamics in the plasma membrane of intact hepatocytes, by which a variety of membrane functions can be influenced, may be an important step in the mechanism of insulin action.     

Lateral diffusion of gramicidin C in phospholipid multibilayers. Effects of cholesterol and high gramicidin concentration

We have measured the lateral diffusion coefficient (D), of active dansyl-labeled gramicidin C (DGC), using the technique of fluorescence photobleaching recovery, under conditions in which the cylindrical dimer channel of DGC predominates. In pure, hydrated, dimyristoylphosphatidylcholine (DMPC) multibilayers (MBL), D decreases from 6 X 10(-8) cm2/s at 40 degrees C to 3 X 10(-8) cm2/s at 25 degrees C, and drops 100-fold at 23 degrees C, the phase transition temperature (Tm) of DMPC. Above Tm, addition of cholesterol decreases D; a threefold stepwise drop occurs between 10 and 20 mol %. Below Tm, increasing cholesterol increases D; a 10-fold increase occurs between 10 and 20 mol % at 21 degrees C, between 20 and 25 mol % at 15 degrees C, and between 25 and 30 mol % at 5 degrees C. In egg phosphatidylcholine (EPC) MBL, D decreases linearly from 5 X 10(-8) cm2/s at 35 degrees C to 2 X 10(-8) cm2/s at 5 degrees C; addition of equimolar cholesterol reduces D by a factor of 2. Thus this transmembrane polypeptide at low membrane concentrations diffuses quite like a lipid molecule. Its diffusivity in lipid mixtures appears to reflect predicted changes of lateral composition. Increasing gramicidin C (GC) in DMPC/GC MBL broadened the phase transition, and the diffusion coefficient of the lipid probe N-4-nitrobenzo-2-diazole phosphatidylethanolamine (NBD-PE) at 30 degrees C decreases from 8 X 10(-8) cm2/s below 5 mol % GC to 2 X 10(-8) cm2/s at 14 mol % GC; D for DGC similarly decreases from 4 X 10(-8) cm2/s at 2 mol % GC to 1.4 X 10(-8) cm2/s at 14 mol % GC. Hence, above Tm, high concentrations of this polypeptide restrict the lateral mobility of membrane components.     

Electrophoresis and diffusion in the plane of the cell membrane.

Electrophoretic and diffusional movements of concanavalin A (Con A) receptors and acetylcholine (ACh) receptors in the plane of the plasma membrane of mononucleate, spherical Xenopus myoblasts were studied by microfluorimetry and iontophoresis. We found that (a) a uniform electric field of 10 V/cm applied along the cell surface produces a partial accumulation of both types of receptors toward the cathodal pole of the cell within 30 min: (b) post-field relaxation of the culture results in the complete recovery of the uniform distribution of the Con A receptors within 10 min; and (c) in contrast to the Con A receptor in general, accumulation of ACh receptors by the electric field results in the formation of stable, localized receptor aggregates. Theoretical analyses were carried out for the distribution of charged membrane receptors at equilibrium between electrophoresis and diffusion, and for the rate of back diffusion after the removal of the field. These analyses indicated that, at 22 degrees C, the average electrophoretic mobility of the electrophoretically mobile population of the Con A receptors is about 1.9 X 10(-3) micron/s per V/cm, while their average diffusion coefficient is 5.1 X 10(-9) cm2/s.     

Electric field-induced redistribution and postfield relaxation of low density lipoprotein receptors on cultured human fibroblasts

The lateral mobility of unliganded low density lipoprotein-receptor (LDL-R) on the surface of human fibroblasts has been investigated by studying the generation and relaxation of concentration differences induced by exposure of the cultured cells to steady electric fields. The topographic distribution of receptors was determined by fluorescence microscopy of cells labeled with the intensely fluorescent, biologically active LDL derivative dioctadecylindolcarbocyanine LDL (dil(3)-LDL), or with native LDL and anti-LDL indirect immunofluorescence. Exposure of the LDL-receptor-internalization defective J. D. cells (GM2408A) to an electric field of 10 V/cm for 1 h at 22 degrees C causes greater than 80% of the cells to have an asymmetric distribution of LDL-R; receptors accumulate at the more negative pole of the cell. In contrast, only 20% of LDL-internalization normal GM3348 cells exposed to identical conditions have asymmetrical distributions. Phase micrographs taken during electric-field exposure rule out cell movement as the responsible mechanism for the effect. In both cell types, postfield labeling with the F-actin-specific fluorescent probe nitrobenzoxadiazole-phallacidin shows that no topographic alteration of the actin cytoskeleton accompanies the redistribution of cell surface LDL-Rs, and indirect immunofluorescence labeling of the coat protein clathrin shows that coated pits do not redistribute asymmetrically. Measurements of the postfield relaxation in the percentage of GM2408A cells showing an asymmetric distribution allow an estimate of the effective postfield diffusion coefficient of the unliganded LDL-R. At 37 degrees C, D = 2.0 X 10(-9) cm2/s, decreasing to 1.1 X 10(-9) cm2/s at 22 degrees C, and D = 3.5 X 10(-10) cm2/s at 10 degrees C. These values are substantially larger than those measured by photobleaching methods for the LDL-R complexed with dil(3)-LDL on intact cells, but are comparable to those measured on membrane blebs, and are consistent with diffusion coefficients measured for other unliganded integral membrane receptor proteins by postfield-relaxation methods.     

Lateral electromigration and diffusion of Fc epsilon receptors on rat basophilic leukemia cells: effects of IgE binding

We have used in situ electromigration and post-field relaxation (Poo, M.-m., 1981, Annu. Rev. Biophys. Bioeng., 10:245-276) to assess the effect of immunoglobulin E (IgE) binding on the lateral mobility of IgE- Fc receptors in the plasmalemma of rat basophilic leukemia (RBL) cells. Bound IgE sharply increased the receptor's electrokinetic mobility, whereas removal of cell surface neuraminic acids cut it to near zero. In contrast, we found only a small difference between the lateral diffusion coefficients (D) of vacant and IgE-occupied Fc receptors (D: 4 vs. 3 X 10(-10) cm2/s at 24 degrees C). This is true for monomeric rat IgE; with mouse IgE, the difference in apparent diffusion rates was slightly greater (D: 4.5 vs. 2.3 X 10(-10) cm2/s at 24 degrees C). This range of D values is close to that found in previous photobleaching studies of the IgE-Fc epsilon receptor complex in RBL cells and rat mast cells. Moreover, enzymatic depletion of cell coat components did not measurably alter the diffusion rate of IgE-occupied receptors. Thus, binding of fluorescent macromolecular probes to cell surface proteins need not severely impede lateral diffusion of the probed species. If the glycocalyx of RBL cells does limit lateral diffusion of the Fc epsilon receptor, it must act primarily on the receptor itself, rather than on receptor-bound IgE.     

Surface diffusion in human serum lipoproteins

From the viscosity dependence of the 31P NMR signals, the diffusion coefficients DT of phospholipid molecules in the surface monolayer of HDL, LDL and VLDL have been determined. DT for HDL3 and HDL2 are found to be 2.3 X 10(-8) cm2/s and 1.8 X 10(-8) cm2/s, respectively. These values are similar to values reported for diffusion of phospholipid molecules in phospholipid bilayers above the gel to liquid crystalline phase transition temperature. Viscosity dependence of [16,16,16-2H3]phosphatidylcholine incorporated into HDL2 yielded a value similar to that determined by 31P (DT = 1.9 X 10(-8) cm2/s). Slower diffusion coefficients were measured for LDL2 and VLDL. VLDL had a value DT = 9.1 X 10(-9) cm2/s. The diffusion coefficient for LDL2 was 1.4 X 10(-9) cm2/s. Thus, diffusion of phospholipids in LDL2 is a full order of magnitude slower at 25 degrees C than diffusion of phospholipids in the HDLs.     

Lateral diffusion of the PH-20 protein on guinea pig sperm: evidence that barriers to diffusion maintain plasma membrane domains in mammalian sperm

PH-20 protein on the plasma membrane (PH-20PM) is restricted to the posterior head of acrosome-intact guinea pig sperm. During the exocytotic acrosome reaction the inner acrosomal membrane (IAM) becomes continuous with the posterior head plasma membrane, and PH-20PM migrates to the IAM. There it joins a second population of PH-20 protein localized to this region of the acrosomal membrane (PH-20AM) (Cowan, A.E., P. Primakoff, and D.G. Myles, 1986, J. Cell Biol. 103:1289-1297). To investigate how the localized distributions of PH-20 protein are maintained, the lateral mobility of PH-20 protein on these different membrane domains was determined using fluorescence redistribution after photobleaching. PH-20PM on the posterior head of acrosome-intact sperm was found to be mobile, with a diffusion coefficient and percent recovery typical of integral membrane proteins (D = 1.8 X 10(-10) cm2/s; %R = 73). This value of D was some 50-fold lower than that found for the lipid probe 1,1-ditetradecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (C14diI) in the same region (D = 8.9 X 10(-9) cm2/s). After migration to the IAM of acrosome-reacted sperm, this same population of molecules (PH-20PM) exhibited a 30-fold increase in diffusion rate (D = 4.9 X 10(-9) cm2/s; %R = 78). This rate was similar to diffusion of the lipid probe C14diI in the IAM (D = 5.4 X 10(-9) cm2/s). The finding of free diffusion of PH-20PM in the IAM of acrosome-reacted sperm supports the proposal that PH-20 is maintained within the IAM by a barrier to diffusion at the domain boundary. The slower diffusion of PH-20PM on the posterior head of acrosome-intact sperm is also consistent with localization by barriers to diffusion, but does not rule out alternative mechanisms.     

Lateral diffusion of lipid probes in the surface membrane of human platelets. An electron-electron double resonance (ELDOR) study.

Electron-electron double resonance (ELDOR) techniques employing [14N], [15N] 16-Doxylstearate spin-label pairs have been used to measure the lateral diffusion constant, D, of lipids in the surface membrane of intact human blood platelets. For freshly prepared platelets, D is 1.0 X 10(-8) cm2/s at 37 degrees C and for platelets stored for 3 d at room temperature under accepted routine blood bank conditions, D is 2.6 X 10(-8) cm2/s at 37 degrees C. This is the first time that D in the surface membrane of platelets is reported. The marked increase in D for stored platelets may be attributed at least partly to loss of cholesterol during storage, suggesting a correlation between lipid lateral diffusion and cholesterol levels in cell membranes.     

Lateral mobility of a lipid analog in the membrane of irreversible sickle erythrocytes

The major feature of sickle cell anemia is the tendency of erythrocytes to sickle when exposed to decreased oxygen tension and to unsickle when reoxygenated. Irreversible sickle cells (ISCs) are sickle erythrocytes which retain bipolar elongated shapes despite reoxygenation. ISCs are believed to owe their biophysical abnormalities to acquired membrane alterations which decrease membrane deformability. While increased membrane surface viscosity has been measured in ISCs, the lateral dynamics of membrane lipids in these cells have not heretofore been examined. We have measured the lateral diffusion of the lipid analog 3,3'-dioctadecylindocyanine iodide (DiI) in the plasma membrane of intact normal erythrocytes, reversible sickle cells (RSCs), and irreversible sickle cells by fluorescence photobleaching recovery (FPR). The diffusion coefficients +/- standard errors of the mean of DiI in intact normal red blood cells (RBCs), RSCs, and ISCs at 37 degrees C are (8.06 +/- 0.29) X 10(-9) cm2 X s-1, (7.74 +/- 0.22) X 10(-9) cm2 X s-1, and (7.29 +/- 0.24) X 10(-9) cm2 X s-1, respectively. A similar decrease in the diffusion coefficient of DiI in the plasma membranes of the three cell types was observed at 4, 10, 17, 23, and 30 degrees C. ANOVA analysis of the changes in DiI diffusion showed significant differences between the RBC and ISC membranes at all temperatures examined. The characteristic breaks in Arrhenius plots of the diffusion coefficients for the RBCs, RSCs, and ISCs occurred at 20, 19, and 18.6 degrees C, respectively. Photobleaching recovery data were used to estimate (Boullier, J.A., Melnykovich, G. and Barisas, B.G. (1982) Biochim. Biophys. Acta 692, 278-286) the microviscosities of the plasma membranes of the three cell types at 25 degrees C. We find significant differences between our microviscosity values and those obtained in previous fluorescence depolarization studies. However, both methods indicate qualitatively similar differences in membrane microviscosity among the various cell types.     

Oxygen diffusion in biological and artificial membranes determined by the fluorochrome pyrene

Quenching of pyrene fluorescence by oxygen was used to determine oxygen diffusion coefficients in phospholipid dispersions and erythrocyte plasma membranes. The fluorescence intensity and lifetime of pyrene in both artificial and natural membranes decreases about 80% in the presence of 1 atm O2, while the fluorescence excitation and emission spectra and the absorption spectrum are unaltered. Assuming the oxygen partition coefficient between membrane and aqueous phase to be 4.4, the diffusion coefficients for oxygen at 37 degrees C are 1.51 X 10(-5) cm2/s in dimyristoyl lecithin vesicles, 9.32 X 10(-6) cm2/s in dipalmitoyl lecithin vesicles, and 7.27 X 10(-6) cm2/s in erythrocyte plasma membranes. The heats of activation for oxygen diffusion are low (less than 3 kcal/degree-mol). A dramatic increase in the diffusion constant occurs at the phase transition of dimyristoyl and dipalmitoyl lecithin, which may result from an increase in either the oxygen diffusion coefficient, partition coefficient, or both. The significance of the change in oxygen diffusion below and above the phase transition for biological membranes is discussed.     

Regional differences in the lateral mobility of plasma membrane lipids in a molluscan embryo

Regional and temporal differences in plasma membrane lipid mobility have been analyzed during the first three cleavage cycles of the embryo of the polar-lobe-forming mollusc Nassarius reticulatus by the fluorescence photobleaching recovery (FPR) method, using 1,1'-ditetradecyl 3,3,3',3'-tetramethylindocarbocyanine iodide (C14diI) as a fluorescent lipid probe. During this period of development the lateral diffusion coefficient of membrane lipids is consistently greater in the vegetal polar lobe area as compared to the animal plasma membrane area (on average 30%), demonstrating the existence of an animal-vegetal polarity in plasma membrane properties. At third cleavage, the differences between animal and vegetal plasma membrane region become even more pronounced; in the four animal micromeres the diffusion coefficient (D) and mobile fraction (MF) are 2.9 +/- 0.2 X 10(-9) cm2/sec and 51 +/- 2%, respectively, while in the four vegetal macromeres D = 5.0 +/- 0.3 X 10(-9) cm2/sec and MF = 78 +/- 2%. Superimposed upon the observed animal-vegetal polarity, the lateral diffusion in the polar lobe membrane area shows a cell-cycle-dependent modulation. The highest mean values for D are reached during the S phase (ranging from 7.0 to 7.8 X 10(-9) cm2/sec in the three cycles measured), while at the end of G2 phase and during early mitosis mean values for D have decreased significantly (ranging from 5.0 to 5.9 X 10(-9) cm2/sec). Diffusion rates in the animal membranes of the embryo are constant during the three successive cell cycles (D = 4.3-5.0 X 10(-9) cm2/sec), except for a peak at the S phase of the first cell cycle (D = 6.0 X 10(-9) cm2/sec). These results are discussed in relation with previously observed ultrastructural heterogeneities in the Nassarius egg plasma membrane. It is speculated that the observed animal-vegetal polarity in the organization of the egg membrane might play an important role in the process of cell diversification during early development.     

Increased diffusional mobility of CFTR at the plasma membrane after deletion of its C-terminal PDZ binding motif

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated Cl- channel expressed at the apical plasma membrane. It has been proposed that the C-terminal PDZ binding motif of CFTR is required for its apical membrane targeting and that PDZ-domain interactions may tether CFTR to the actin cytoskeleton via soluble proteins including EBP50/NHERF1 and ezrin. We measured the diffusional mobility of human CFTR in the plasma membrane of Madin-Darby canine kidney cells by photobleaching of green fluorescent protein (GFP)-CFTR chimeras. After bleaching by a focused laser beam, GFP-CFTR fluorescence in the bleached membrane region recovered to approximately 90% of its initial level, indicating that nearly all of the CFTR was mobile. The GFP-CFTR diffusion coefficient (D) was 0.99 +/- 0.09 x 10(-10) cm2/s at 37 degrees C, similar to that of other membrane proteins. GFP-CFTR diffusion was not altered by protein kinase A or C activators but was blocked by paraformaldehyde and filipin. CFTR mutants lacking functional PDZ-binding domains (GFPCFTR-DeltaTRL and GFP-CFTR-DeltaTRA) were also mobile with D significantly increased by approximately 60% compared with GFP-CFTR. However, GFP-CFTR, GFP-CFTR-Delta TRL, and GFP-CFTR-DeltaTRA had similar mobilities (D approximately 12 x 10(-10) cm2/s) at the endoplasmic reticulum in brefeldin A-treated cells. Agents that modulate the actin cytoskeleton (cytochalasin D and jasplakinolide) altered the plasma membrane mobility of CFTR but not CFTR- DeltaTRL. EBP50 (NHERF1), a PDZ domain-containing protein that interacts with the C terminus of CFTR, diffused freely in the cytoplasm with a diffusion coefficient of 0.9 +/- 0.1 x 10(-7) cm2/s. EBP50 diffusion increased by approximately 2-fold after deletion of its ezrin-binding domain. These results indicate that wild-type CFTR is not tethered statically at the plasma membrane but that its diffusion is dependent on PDZ-domain interactions and an intact actin skeleton. PDZ-domain interactions of CFTR are thus dynamic and occur on a time scale of seconds or faster.     

Monoclonal antibodies directed against protoplasts of soybean cells: analysis of the lateral mobility of plasma membrane-bound antibody MVS- 1

A monoclonal antibody (MVS-1) was used to monitor the lateral mobility of a defined component (Mr approximately 400,000) of the plasma membrane of soybean protoplasts prepared from suspension cultures of Glycine max (SB-1 cell line). The diffusion coefficient (D) of antibody MVS-1 bound to its target was determined (D = 3.2 X 10(-10) cm2/s) by fluorescence redistribution after photobleaching. Pretreatment of the protoplasts with soybean agglutinin (SBA) resulted in a 10-fold reduction of the lateral mobility of antibody MVS-1 (D = 4.1 X 10(-11) cm2/s). This lectin-induced modulation could be partially reversed by prior treatment of the protoplasts with either colchicine or cytochalasin B. When used together, these drugs completely reversed the modulation effect induced by SBA. These results have refined our previous analysis of the effect of SBA on receptor mobility to the level of a defined receptor and suggest that the binding of SBA to the plasma membrane results in alterations in the plasma membrane such that the lateral diffusion of other receptors is restricted. These effects are most likely mediated by the cytoskeletal components of the plant cell.     

Effects of mutations in three domains of the vesicular stomatitis viral glycoprotein on its lateral diffusion in the plasma membrane [published erratum appers in J Cell Biol 1988 Jan;106(1):325]

The lateral mobility of the vesicular stomatitis virus spike glycoprotein (G protein) and various mutant G proteins produced by site-directed mutagenesis of the G cDNA has been measured. Fluorescence recovery after photobleaching results for the wild type G protein in transfected COS-1 cells yielded a mean diffusion coefficient (D) of 8.5 (+/- 1.3) X 10(-11) cm2/s and a mean mobile fraction of 75% (+/- 3%). Eight mutant proteins were also examined: dTM14, lacking six amino acids from the transmembrane domain; TA2, lacking an oligosaccharide in the extracellular domain; QN2, possessing an extra N-linked oligosaccharide in the extracellular domain; CS2, possessing a serine instead of a cysteine at residue 489 in the cytoplasmic domain, preventing palmitate addition to the glycoprotein; TMR-stop, lacking the entire cytoplasmic domain except an arginine at residue 483; and three chimeric proteins, G mu, G23, and GHA, containing in place of the 29 amino acid wild type cytoplasmic domain the cytoplasmic domains from the surface IgM from the spike protein of the infectious bronchitis virus or from the hemagglutinin protein of the influenza virus, respectively. The mean D for the mutant proteins varied over a relatively small range, with the slowest mutant, G23, exhibiting a value of 11.3 (+/- 1.4) X 10(-11) cm2/s and the fastest mutant, GHA, having a D of 28.6 (+/- 4.5) X 10(-11) cm2/s. The mean mobile fraction similarly varied over a small range, extending from 55 to 68%. None of the mutations resulted in the more rapid diffusion characteristic of membrane proteins embedded in artificial bilayers. Therefore, it appears that the cytoplasmic and transmembrane domains themselves contribute little to restraining the lateral mobility of this integral membrane protein when expressed in transfected cells.     

cAMP regulated membrane diffusion of a green fluorescent protein-aquaporin 2 chimera

To study the membrane mobility of aquaporin water channels, clones of stably transfected LLC-PK1 cells were isolated with plasma membrane expression of GFP-AQP1 and GFP-AQP2, in which the green fluorescent protein (GFP) was fused upstream and in-frame to each aquaporin (AQP). The GFP fusion did not affect AQP tetrameric association or water transport function. GFP-AQP lateral mobility was measured by irreversibly bleaching a spot (diameter 0.8 microm) on the membrane with an Argon laser beam (488 nm) and following the fluorescence recovery into the bleached area resulting from GFP translational diffusion. In cells expressing GFP-AQP1, fluorescence recovered to >96% of its initial level with t(1/2) of 38 +/- 2 s (23 degrees C) and 21 +/- 1 s (37 degrees C), giving diffusion coefficients (D) of 5.3 and 9.3 x 10(-11) cm(2)/s. GFP-AQP1 diffusion was abolished by paraformaldehyde fixation, slowed >50-fold by the cholesterol-binding agent filipin, but not affected by cAMP agonists. In cells expressing GFP-AQP2, fluorescence recovered to >98% with D of 5.7 and 9.0 x 10(-11) cm(2)/s at 23 degrees C and 37 degrees C. In contrast to results for GFP-AQP1, the cAMP agonist forskolin slowed GFP-AQP2 mobility by up to tenfold. The cAMP slowing was blocked by actin filament disruption with cytochalasin D, by K(+)-depletion in combination with hypotonic shock, and by mutation of the protein kinase A phosphorylation consensus site (S256A) at the AQP2 C-terminus. These results indicate unregulated diffusion of AQP1 in membranes, but regulated AQP2 diffusion that was dependent on phosphorylation at serine 256, and an intact actin cytoskeleton and clathrin coated pit. The cAMP-induced immobilization of phosphorylated AQP2 provides evidence for AQP2-protein interactions that may be important for retention of AQP2 in specialized membrane domains for efficient membrane recycling.