Lateral mobility and capping of rat lymphocyte membrane proteins

Surface immunoglobulin (SIg), RT1 (rat histocompatibility proteins) and thy-1 present on rat lymphocytes cap with similar kinetics and to the same extent, the only difference being the antibody requirement. SIg requires one cross-linking antibody to induce capping, while RT1 and thy-1 require two antibodies. Fluorescence recovery after photobleaching experiments show that there is heterogeneity in the diffusion characteristics of lymphocyte membrane proteins. SIg is relatively immobile when labeled by Fab' and is immobilized by F(ab')2. RT1 diffuses 2.7 times as fast as SIg and the mobile fraction of RT1 is greater. When RT1 is cross-linked by relatively low concentrations of F(ab')2 its mobility is about the same as that of SIg. When the concentration of F(ab')2 is increased, RT1 is immobilized. Thy-1 diffuses faster than SIg and has a higher mobile fraction. When thy-1 is labeled with F(ab')2 it remains mobile and is immobilized when a second antibody layer is added. The relative order of mobility is thy-1 much greater than RT1 greater than SIg.     

Methods to measure the lateral diffusion of membrane lipids and proteins

In this chapter, we discuss methods to measure lateral mobility of membrane lipids and proteins using techniques based on the light microscope. These methods typically sample lateral mobility in very small, micron-sized regions of the membrane so that they can be used to measure diffusion in regions of single cells. The methods are based on fluorescence from the molecules of interest or from light scattered from particles attached to single or small groups of membrane lipids or proteins. Fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and Single particle tracking (SPT) are presented in that order. FRAP and FCS methodologies are described for a dedicated wide field microscope although many confocal microscopes now have software permitting these measurement to be made; nevertheless, the principles of the measurement are the same for a wide field or confocal microscope. SPT can be applied to trace the movements of single fluorescent molecules in membranes but this aspect will not be treated in detail.     

Lateral diffusion of proteins and lipids in the plasma membrane of rose protoplast

Summary Lateral diffusion measurements have been made on lipids and proteins in the plasma membrane of live protoplasts derived from rose (Rosa sp. Paul's Scarlet) suspension-cultured cells. Two different fluorescent lipid probes exhibited markedly different diffusion rates, indicating possible heterogeneity in the lipid domain of the membrane. Membrane proteins were labeled directly with covalently-reactive fluorophores, and factors that might perturb the lateral diffusion of these labeled proteins were investigated. Treatment of the protoplasts with various cytoskeleton-disrupting drugs generally had little effect on protein diffusion, although treatment with oryzalin, a microtubule-disrupting drug, did slightly reduce the mobile fraction of membrane proteins. Elevation of the CaCl₂ concentration in the medium from 1 mM to 10 mM significantly reduced the mobile fraction of membrane proteins and also increased the fraction of protoplasts that were able to regenerate cell walls and divide in culture. These results are discussed in relation to reported evidence of lipid domains in the plasma membranes of other cells and protoplasts. The relative importance of lipid domains and membrane-cytoskeleton interaction in governing protein diffusion is considered.Abbreviations D lateral diffusion coefficient - RCA Ricinus communis agglutinin - BPA Bauhinia purpurea agglutinin - DTAF dichlorotriazinylaminofluorescein - FTSC fluorescein-5-thiosemicarbazide - C₁₈-Fl 5-(N-octadecanoyl)aminofluorescein - LY-Chol Lucifer yellow conjugate of cholesterol, i.e., dilithium 4-amino-N-[(-(carbo(5-cho-lesten-3-yl)oxy)hydrazinocarbonyl)amino]-1,8-naphthalimide-3,6-disulfonate - APM amiprophosmethyl - DMSO dimethylsulfoxide - FPR fluorescence photobleaching recovery - sd standard deviation - FRAF fluorescence redistribution after fusion - M mobile fraction     

The effect of local anesthetics on the lateral mobility of lymphocyte membrane proteins

In this study, we have measured by fluorescence photobleach recovery (FPR) the relative mobilities of two rat lymphocyte membrane proteins, surface immunoglobulin (SIg) and AgB (histocompatability antigens). The results of the FPR experiments showed that SIg was relatively restricted in its mobility as compared with AgB. Treatment of cells with local anesthetics (LA), agents which are assumed to act, in part, by severing the linkage between the cytoskeleton and integral membrane proteins increased the mobility of SIg to a mobility similar to that of AgB. This finding suggests that SIg is relatively restricted in its mobility due to a tethering by cytoskeletal elements. LA also had a small, but significant effect on the mobility of AgB.     

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.     

Effect of integral membrane proteins on the lateral mobility of plastoquinone in phosphatidylcholine proteoliposomes

Pyrene fluorescence quenching by plastoquinone was used to estimate the rate of plastoquinone lateral diffusion in soybean phosphatidylcholine proteoliposomes containing the following integral membrane proteins: gramicidin D, spinach cytochrome bf complex, spinach cytochrome f, reaction centers from Rhodobacter sphaeroides, beef heart mitochondrial cytochrome bc₁, and beef heart mitochondrial cytochrome oxidase. The measured plastoquinone lateral diffusion coefficient varied between 1 and 3 · 10^-7 cm² s^-1 in control liposomes that lacked protein. When proteins were added, these values decreased: a 10-fold decrease was observed when 16-26% of the membrane surface area was occupied by protein for all the proteins but gramicidin. The larger protein complexes (cytochrome bf, Rhodobacter sphaeroides reaction centers, cytochrome bc₁, and cytochrome oxidase), whose hydrophobic volumes were 15-20 times as large as that of cytochrome f and the gramicidin transmembrane dimer, were 15-20 times as effective in decreasing the lateral-diffusion coefficient over the range of concentrations studied. These proteins had a much stronger effect than that observed for bacteriorhodopsin in fluorescence photobleaching recovery measurements. The effect of high-protein concentrations in gramicidin proteoliposomes was in close agreement with fluorescence photobleaching measurements. The results are compared with the predictions of several theoretical models of lateral mobility as a function of integral membrane concentration.     

Regionalization and lateral diffusion of membrane proteins in unfertilized and fertilized mouse eggs

The unfertilized mouse egg has a round and highly villated main body and a "nipple" that is unvillated and buds off on fertilization to form the second polar body. Fluorescent markers stain the body more intensely than the nipple, which has been assumed to result from surface amplification due to microvilli. Using fluorescence recovery after photobleaching and microfluorescence photometry, we have measured the membrane protein diffusion and concentration on the main body and nipple region of unfertilized and on fertilized CD-1 mouse eggs. Two general membrane protein labels were used: rhodamine-labeled succinylated concanavalin A and trinitrobenzene sulfonate visualized with a rhodamine Fab fragment of a sheep anti-trinitrophenyl. We found that while the diffusion coefficient was the same on the nipple and main body, considerably higher recovery was observed on the nipple for both probes. The ratio of intensity of fluorescence on the nipple to main body was significantly lower for the concanavalin A stain than for the trinitrophenyl stain, indicating that true concentration gradients exist beyond those that result from surface amplification. The effect of fertilization was not general. No effect was observed for the concanavalin A stain for either diffusion coefficient or percent recovery. For the trinitrophenyl stain, percent recovery decreased approximately twofold while diffusion coefficient increased approximately threefold.     

Lateral diffusion of membrane proteins in the presence of static and dynamic corrals: suggestions for appropriate observables

We consider the possibility of inferring the nature of cytoskeletal interaction with transmembrane proteins via optical experiments such as single-particle tracking (SPT) and near-field scanning optical microscopy (NSOM). In particular, we demonstrate that it may be possible to differentiate between static and dynamic barriers to diffusion by examining the time-dependent variance and higher moments of protein population inside cytoskeletal "corrals." Simulations modeling Band 3 diffusion on the surface of erythrocytes provide a concrete demonstration that these statistical tools might prove useful in the study of biological systems.     

Distribution, lateral mobility and function of membrane proteins incorporated into giant unilamellar vesicles

GUVs have been widely used for studies on lipid mobility, membrane dynamics and lipid domain (raft) formation, using single molecule techniques like fluorescence correlation spectroscopy. Reports on membrane protein dynamics in these types of model membranes are by far less advanced due to the difficulty of incorporating proteins into GUVs in a functional state. We have used sucrose to prevent four distinct membrane protein(s) (complexes) from inactivating during the dehydration step of the GUV-formation process. The amount of sucrose was optimized such that the proteins retained 100% biological activity, and many proteo-GUVs were obtained. Although GUVs could be formed by hydration of lipid mixtures composed of neutral and anionic lipids, an alternate current electric field was required for GUV formation from neutral lipids. Distribution, lateral mobility, and function of an ATP-binding cassette transport system, an ion-linked transporter, and a mechanosensitive channel in GUVs were determined by confocal imaging, fluorescence correlation spectroscopy, patch-clamp measurements, and biochemical techniques. In addition, we show that sucrose slows down the lateral mobility of fluorescent lipid analogs, possibly due to hydrogen-bonding with the lipid headgroups, leading to larger complexes with reduced mobility.     

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 of membrane proteins in protein-rich membranes. A simple hard particle model for concentration dependence of the two-dimensional diffusion coefficient.

A model for the effect of protein concentration on the rate of lateral diffusion of integral membrane proteins is presented, in which the proteins are represented by equivalent hard circular particles on a surface. As the density of particles increases, the probability of finding a vacancy immediately adjacent to a tracer particle into which it may diffuse decreases, resulting in a concomitant reduction of the tracer diffusion coefficient. Using scaled particle theory to calculate the concentration-dependent probabilities, a simple approximate result is obtained in closed form, that is compared with the results of previously published Monte Carlo lattice simulations and experimental observations.     

Activation of human neutrophil NADPH oxidase and lateral mobility of membrane proteins: A study with crosslinkers

Fluorescence photobleaching recovery was employed to investigate the relationship between the activation of neutrophil NADPH oxidase and lateral mobility of membrane proteins. Treatment of neutrophils with the crosslinking reagent disuccinimidyl suberate (DSS) blocked activation of the respiratory burst without affecting the lateral motion of concanavalin A receptors. Neutrophils treated with DSS after prestimulation with concanavalin A generated superoxide in response to another stimulator, phorbol myristate acetate, in spite of the lateral immobilization of concanavalin A receptors. The apparent lack of correlation between the activation of NADPH oxidase and the lateral motion of membrane proteins is discussed.     

Lateral mobility of integral proteins in red blood cell tethers.

The red blood cell membrane is a complex material that exhibits both solid- and liquidlike behavior. It is distinguished from a simple lipid bilayer capsule by its mechanical properties, particularly its shear viscoelastic behavior and by the long-range mobility of integral proteins on the membrane surface. Subject to sufficiently large extension, the membrane loses its shear rigidity and flows as a two-dimensional fluid. These experiments examine the change in integral protein mobility that accompanies the mechanical phenomenon of extensional failure and liquidlike flow. A flow channel apparatus is used to create red cell tethers, hollow cylinders of greatly deformed membrane, up to 36-microns long. The diffusion of proteins within the surface of the membrane is measured by the technique of fluorescence redistribution after photobleaching (FRAP). Integral membrane proteins are labeled directly with a fluorescein dye (DTAF). Mobility in normal membrane is measured by photobleaching half of the cell and measuring the rate of fluorescence recovery. Protein mobility in tether membrane is calculated from the fluorescence recovery rate after the entire tether has been bleached. Fluorescence recovery rates for normal membrane indicate that more than half the labeled proteins are mobile with a diffusion coefficient of approximately 4 x 10(-11) cm2/s, in agreement with results from other studies. The diffusion coefficient for proteins in tether membrane is greater than 1.5 x 10(-9) cm2/s. This dramatic increase in diffusion coefficient indicates that extensional failure involves the uncoupling of the lipid bilayer from the membrane skeleton.     

Association of spectrin with its membrane attachment site restricts lateral mobility of human erythrocyte integral membrane proteins

Interactions between spectrin and the inner surface of the human erythrocyte membrane have been implicated in the control of lateral mobility of the integral membrane proteins. We report here that incubation of “leaky” erythrocytes with a water-soluble proteolytic fragment containing the membrane attachment site for spectrin achieves a selective and controlled dissociation of spectrin from the membrane, and increases the rate of lateral mobility of fluorescein isothiocyanate-labeled integral membrane proteins (> 70% of label in band 3 and PAS-1). Mobility of membrane proteins is measured as an increase in the percentage of uniformly fluorescent cells with time after fusion of fluorescent with nonfluorescent erythrocytes by Sendai virus. The cells are permeable to macromolecules since virus-fused erythrocytes lose most of their hemoglobin. The membrane attachment site for spectrin has been solubilized by limited proteolysis of inside-out erythrocyte vesicles and has been purified (V). Bennett, J Biol Chem 253:2292 (1978). This 72,000-dalton fragment binds to spectrin in solution, competitively inhibits association of ³²P-spectrin with inside-out vesicles with a K_i of 10^?7M, and causes rapid dissociation of ³²P-spectrin from vesicles. Both acid-treated 72,000-dalton fragment and the 45,000 dalton-cytoplasmic portion of band 3, which also was isolated from the proteolytic digest, have no effect on spectrin binding, release, or membrane protein mobility. The enhancement of membrane protein lateral mobility by the same polypeptide that inhibits binding of spectrin to inverted vesicles and displaces spectrin from these vesicles provides direct evidence that the interaction of spectrin with protein components in the membrane restricts the lateral mobility of integral membrane proteins in the erythrocyte.     

Cell-specific constraints to the lateral diffusion of a membrane glycoprotein.

We have previously shown that the lateral diffusion, D, of the class I Major Histocompatibility Complex (MHC) glycoprotein H-2Ld is constrained by its glycosylation, when expressed in mouse L-cells. Removal of one or more of the 3 N-linked oligosaccharides of H-2Ld glycoproteins results in an increase in D. In order to further examine the influence of glycosylation on D, we compared lateral diffusion of H-2Ld expressed in wild-type CHO cells with lateral diffusion of the same molecule expressed in mutant CHO cells with aberrant surface glycosylation. In addition, we compared lateral diffusion of wild-type and unglycosylated H-2Ld antigens in these cells. In contrast to the large effect of glycosylation state on lateral diffusion of H-2Ld in mouse L-cells, there was little effect of glycosylation on lateral diffusion of H-2Ld in any of the CHO cells. This, together with similar results on hamster class I antigens, indicates that the constraints to D of H-2Ld and other class I MHC molecules are different in CHO cells than in L-cells. Measurements of lateral diffusion after treatment of cells with cytochalasin D make it clear that interactions between MHC class I molecules and a cytoskeleton are important in reducing the mobile fraction of diffusing molecules, R, though they cannot be shown to directly affect the diffusion coefficient, D.     

Lateral diffusion of membrane lipids and proteins is increased specifically in neurites of differentiating neuroblastoma cells

Lateral diffusion of membrane lipids and proteins was determined in differentiating C1300 mouse neuroblastoma cells by fluorescence photo-bleaching recovery measurements. It is demonstrated that upon differentiation the lateral diffusion of membrane lipids and proteins is increased specifically in the extending neurites. This indicates the appearance of a topographical heterogeneity in the cell membrane, whereby more fluid domains become located in the membrane of the neurites.