Analysis of cell surface interactions by measurements of lateral mobility

Interactions of cell surface components with one another and with structures inside and outside the cell may have important physiological functions in the transmission of signals and the assembly of specialized structures. These interactions may be detected and analyzed through their effects on the lateral mobility of cell surface molecules. Measurements by a fluorescence photobleaching method have shown that in general lipid-like molecules diffuse rapidly and freely through the plasma membrane, whereas proteins move much more slowly or appear to be immobile. This dichotomy has been supposed to result from forces beyond the viscosity of the lipid bilayer, which specifically retard the diffusion of membrane proteins. This general picture should be qualified, however, by noting that the lateral mobility of lipid-like molecules can be influenced in detail by changes in the state of the plasma membrane such as result from mitosis or fertilization. The interactions of cell surface proteins that limit their lateral mobility are unknown. The effects of binding concanavalin A to localized regions of cell surface show that these interactions can vary in subtle and complex ways. It may soon be useful to interpret mobility experiments in terms of simple reaction models that attempt to describe surface interactions in physicochemical terms. More experimental data are needed to carry out this program and to relate interactions that affect mobility to the structural connections between cell surface components and the cytoskeleton, which have been detected by biochemical methods and electron and immunofluorescence microscopy.     

Tracking microdomain dynamics in cell membranes

Studies of the diffusion of proteins and lipids in the plasma membrane of cells have long pointed to the presence of membrane domains. A major challenge in the field of membrane biology has been to characterize the various cellular structures and mechanisms that impede free diffusion in cell membranes and determine the consequences that membrane compartmentalization has on cellular biology. In this review, we will provide a brief summary of the classes of domains that have been characterized to date, focusing on recent efforts to identify the properties of lipid rafts in cells through measurements of protein and lipid diffusion.     

Differently anchored influenza hemagglutinin mutants display distinct interaction dynamics with mutual rafts

Lipid rafts play important roles in cellular functions through concentrating or sequestering membrane proteins. This requires proteins to differ in the stability of their interactions with lipid rafts. However, knowledge of the dynamics of membrane protein-raft interactions is lacking. We employed FRAP to measure in live cells the lateral diffusion of influenza hemagglutinin (HA) proteins that differ in raft association. This approach can detect weak interactions with rafts not detectable by biochemical methods. Wild-type (wt) HA and glycosylphosphatidylinositol (GPI)-anchored HA (BHA-PI) diffused slower than a nonraft HA mutant, but became equal to the latter after cholesterol depletion. When antigenically distinct BHA-PI and wt HA were coexpressed, aggregation of BHA-PI into immobile patches reduced wt HA diffusion rate, suggesting transient interactions with BHA-PI raft patches. Conversely, patching wt HA reduced the mobile fraction of BHA-PI, indicating stable interactions with wt HA patches. Thus, the anchoring mode determines protein-raft interaction dynamics. GPI-anchored and transmembrane proteins can share the same rafts, and different proteins can interact stably or transiently with the same raft domains.     

荧光漂白后恢复技术及其在活细胞分子机制研究中的应用

荧光漂白后恢复（FRAP）是一项利用荧光探针研究活体细胞中各类分子迁移特性的技术。简要介绍了FRAP技术的原理和具体实施要求,列出了动态比和扩散系数的计算公式,并例举了近几年FRAP技术在细胞分子机制研究中的应用。     

Total internal reflection fluorescence microscopy: application to substrate-supported planar membranes

The use of total internal reflection illumination in fluorescence microscopy (TIRFM) is reviewed with emphasis on application to fluorescent macromolecules that specifically and reversibly bind to planar model membranes supported on glass or quartz substrates. Several methods for characterizing macromolecular motion and organization are discussed: the measurement of equilibrium binding curves to obtain values for equilibrium binding constants; the measurement of fluorescence photobleaching recovery curves to obtain values of kinetic rate constants and surface diffusion coefficients; and the measurement of fluorescence intensities as a function of the evanescent field polarization to characterize orientational order. Applications to cell-substrate contact regions are summarized and future directions of TIRFM are outlined. Correspondence to: N. L. Thompson     

Parvalbumin is freely mobile in axons, somata and nuclei of cerebellar Purkinje neurones

The Ca(2+) -binding protein (CaBP) parvalbumin (PV) is strongly expressed in cerebellar Purkinje neurones (PNs). It is considered a pure Ca(2+) buffer, lacking any Ca(2+) sensor function. Consistent with this notion, no PV ligand was found in dendrites of PNs. Recently, however, we observed for a related CaBP that ligand-targeting differs substantially between dendrites and axons. Thus, here we quantified the diffusion of dye-labelled PV in axons, somata and nuclei of PNs by two-photon fluorescence recovery after photobleaching (FRAP). In all three compartments the fluorescence rapidly returned to baseline, indicating that no large or immobile PV ligand was present. In the axon, FRAP was well described by a one-dimensional diffusion equation and a diffusion coefficient (D) of 12 (IQR 6-20) micro m(2)/s. For the soma and nucleus a three-dimensional model yielded similar D values. The diffusional mobility in these compartments was approximately 3 times smaller than in dendrites. Based on control experiments with fluorescein dextrans, we attributed this reduced mobility of PV to different cytoplasmic properties rather than to specific PV interactions in these compartments. Our findings support the notion that PV functions as a pure Ca(2+) buffer and will aid simulations of neuronal Ca(2+) signalling.     

Ion-conformational interaction and charge transport through channels of biological membranes

This work proposes a theory of charge transport through channels in biological membranes, based on ion flow interaction with charged groups of protein macromolecules that form the channel. Displacements of the groups are due to conformational changes of the protein molecule, the relaxation times of which are much larger than the average time of ion ocurrence in the channel. Ion flow is assumed to depend on the conformational changes and vice-versa. The resulting self-organizing ion-conformational system is described by a set of nonlinear differential equations for conformational variables and average occupancy of the channel by ions. The system exhibits multistable behaviour in a certain range of control parameters (potential difference, input ion flow). The stationary states of the system may be identified with the states of discrete conductivity of the ionic channels.     

Activated K-Ras and H-Ras display different interactions with saturable nonraft sites at the surface of live cells

Ras-membrane interactions play important roles in signaling and oncogenesis. H-Ras and K-Ras have nonidentical membrane anchoring moieties that can direct them to different membrane compartments. Ras-lipid raft interactions were reported, but recent studies suggest that activated K-Ras and H-Ras are not raft resident. However, specific interactions of activated Ras proteins with nonraft sites, which may underlie functional differences and phenotypic variation between different Ras isoforms, are unexplored. Here we used lateral mobility studies by FRAP to investigate the membrane interactions of green fluorescent protein-tagged H- and K-Ras in live cells. All Ras isoforms displayed stable membrane association, moving by lateral diffusion and not by exchange with a cytoplasmic pool. The lateral diffusion rates of constitutively active K- and H-Ras increased with their expression levels in a saturable manner, suggesting dynamic association with saturable sites or domains. These sites are distinct from lipid rafts, as the activated Ras mutants are not raft resident. Moreover, they appear to be different for H- and K-Ras. However, wild-type H-Ras, the only isoform preferentially localized in rafts, displayed cholesterol-sensitive interactions with rafts that were independent of its expression level. Our findings provide a mechanism for selective signaling by different Ras isoforms.     

Rapid lateral diffusion of lectin-labelled glycoconjugates in the human colonic adenocarcinoma cell line HT29

The lateral diffusion of lectin-labelled glycoconjugates was studied in the human colon carcinoma cell line HT29 using fluorescence photobleaching techniques. HT29 cells were grown in either Dulbecco's modified Eagle's medium with glucose (25 mM; DMEM-Glu) or with galactose (25 mM; DMEM-Gal). Cell cultivation in the DMEM-Gal medium was assumed to promote a transformation of the cells to become small-intestinal-like with characteristic microvilli and associated enzymes. The diffusion of glycoconjugates labelled with fluoresceinated Triticum vulgaris agglutinin (Wheat germ agglutinin; WGA), Ricinus communis agglutinin-I (RCA-I), Concanavalia ensiformis agglutinin (ConA), Ulex europaeus agglutinin-I (UEA-I) and Arachis hypogaea agglutinin (PNA) was in all cases rapid, with a diffusion constant (D) ranging between 0.4 and 0.8×10^-8 cm² s^-1. As a comparison the diffusion of the fluorescent synthetic lipid analog diI-C₁₄ was characterized by D=0.8 – 1.0 × 10^–8 cm² s^-1. The diffusion of lectin-labelled surface components could not be related to the presence of microvilli on HT29 cells grown in DMEM-Gal, which ought to yield an apparently lower diffusion rate. The results indicate either that surface glycoconjugates in HT29 cells are dominated by glycolipid, or that the labelled glycoproteins are more or less free to diffuse in the plane of the membrane.     

Cholesterol domains in biological membranes

Membrane cholesterol is distributed asymmetrically both within the cell or within cellular membranes. Elaboration of intracellular cholesterol trafficking, targeting and intramembrane distribution has been spurred by both molecular and structural approaches. The expression of recombinant sterol carrier proteins in L-cell fibroblasts has been especially useful in demonstrating for the first time that such proteins actually elicit intracellular and intra-plasma membrane redistribution of sterol. Additional advances in the use of native fluorescent sterols allowed resolution of transbilayer and lateral cholesterol domains in plasma membranes from cultured fibroblasts, brain synaptosomes and erythrocytes. In all three cell surface membranes, cholesterol is enriched in the inner, cytofacial leaflet. Up to three different cholesterol domains have been identified in the lateral plane of the plasma membrane: a fast exchanging domain comprising less than 10% of cholesterol, a slowly exchanging domain comprising about 30% of cholesterol, and a very slowly or non-exchangeable sterol domain comprising 50–60.

Of plasma membrane cholesterol. Factors modulating plasma membrane cholesterol domains include polyunsaturated fatty acids, expression of intracellular sterol carrier proteins, drugs such as ethanol, and several membrane pathologies (systemic lupus erythematosus, sickle cell anaemia and aging). Disturbances in plasma membrane cholesterol domains after transbilayer fluidity gradients in plasma membranes. Such changes are associated with decreased Ca²⁺ -ATPase and Na ⁺, K⁺ -ATPase activity. Thus, the size, dynamics and distribution of cholesterol domains within membranes not only regulate cholesterol efflux/influx but also modulate plasma membrane protein functions and receptor-effector coupled systems.     

The random collision model and a critical assessment of diffusion and collision in mitochondrial electron transport

This review focuses on our studies over the past ten years which reveal that the mitochondrial inner membrane is a fluid-state rather than a solid-state membrane and that all membrane proteins and redox components which catalyze electron transport and ATP synthesis are in constant and independent diffusional motion. The studies reviewed represent the experimental basis for therandom collision model of electron transport. We present five fundamental postulates upon which the random collision model of mitochondrial electron transport is founded: (1) All redox components areindependent lateral diffusants; (2) Cytochromec diffuses primarily inthree dimensions; (3) Electron transport is adiffusion-coupled kinetic process; (4) Electron transport is amulticollisional, obstructed, long-range diffusional process; (5) The rates of diffusion of the redox components have a direct influence on the overall kinetic process of electron transport and can berate limiting, as indiffusion control. The experimental rationales and the results obtained in testing each of the five postulates of the random collision model are presented. In addition, we offer the basic concepts, criteria and experimental strategies that we believe are essential in considering the significance of the relationship between diffusion and electron transport. Finally, we critically explore and assess other contemporary studies on the diffusion of inner membrane components related to electron transport including studies on: rotational diffusion, immobile fractions, complex formation, dynamic aggregates, and rates of diffusion. Review of all available data confirms the random collision model and no data appear to exist that contravene it. It is concluded that mitochondrial electron transport is a diffusion-based random collision process and that diffusion has an integral and controlling affect on electron transport.     

Lateral diffusion of human histocompatibility antigens in isolated plasma membranes

We have prepared large (5–10 μm) plasma membrane fragments by lysis of VA-2, human, cells adherent to Sephadex beads. The membrane fragments may be removed from beads by sonication and stained with fluorescent antibodies to human histocompatibility antigens, HLA antigens. Lateral diffusion of labelled antigens is followed by the method of fluorescence photobleaching recovery (FPR). HLA antigens of isolated membranes diffuse at the same rate, approx. (2–4) · 10^?10 cm² · s^?1 as they do in intact cells. This rate may be modified by incubating membranes in a variety of media. Buffers of slightly acid pH (6.5 or less) enhance lateral diffusion, while the presence of divalent ions slightly reduces diffusion rates. Our major finding is that incubation of 37° in 0.10 M phosphate buffer increases lateral diffusion 3–5-fold.     

A comparison of the translational diffusion of a normal and a membrane-spanning lipid in Lα phase 1-palmitoyl-2-oleoylphosphatidylcholine bilayers

We have used the fluorescence recovery after photobleaching technique to study the translational diffusion, in L phase multibilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), of fluorescent derivatives of 1-palmitoyl-2-oleoylphosphatidylethanolamine (NBD-POPE) and a membrane-spanning phosphatidylethanolamine (NBD-MSPE). The latter derivative was prepared from a membrane-spanning glycerol-dialkyl-glycerol tetraether lipid isolated from the thermophilic and acidophilic archaebacterium Sulfolobus solfataricus. The translational diffusion was examined between about 15° and 45°C. It is shown that over this temperature range the translational diffusion coefficient for NBD-MSPE is 2/3 that for NBD-POPE which spans only one monolayer of the bilayer. The result is interpreted in terms of existing models for translational diffusion in lipid membranes.Abbreviations D _t translational diffusion coefficient - FRAP fluorescence recovery after photobleaching - MSPE a membrane-spanning phosphatidylethanolamine derived from a glycerol-dialkyl-glycerol tetraether lipid isolated from Sulfolobus solfataricus - NBD 4-nitrobenz-2-oxa-1,3-diazolyl - PE phosphatidylethanolamine - POPC 1-palmitoyl-2-oleoylphosphatidylcholine - POPE 1-palmitoyl-2-oleoylphosphatidylethanolamine     

Transport and dynamics of molecules dissolved in maize root cortex membranes

Translational diffusion of a fluorescent sterol probe was measured in the plasma membranes of protoplasts isolated from cortical cells of the primary root of maize seedlings. The apparent lateral diffusion coefficient was typically observed to be nearly insensitive to temperature, while the mobile fraction increased with increasing temperature. These fluorescence photobleaching recovery (FPR) measurements were compared with the electron paramagnetic resonance (EPR) spectra of the methyl ester of 13-doxyl palmitic acid in membranes of corn root tissue in situ. The complex spectra observed with this probe were analyzed as weighted sums of simpler spectra of various order parameters and rotational correlation times. The reconstituted spectra calculated from the model show that EPR also detects a mobile (less ordered, fluid) fraction, distinguished by the order parameter S=0.1 to 0.2, which becomes more abundant as temperature increases and is qualitatively comparable to the mobile fraction determined by the FPR method. The observed results on the mobile fractions and the diffusion rates for translational (FPR) as well as rotational (EPR) motions are interpreted in terms of membrane organization, thus providing information on the population and structural patterns of the coexisting domains with a special emphasis on the response of the membrane to temperature changes.This work was supported in part by grants from the Ministry of Science and Technology of the Republic of Slovenia and the International Research Program of the U.S. Department of Agriculture (USDA-JF 814-51) to M.S., and by grants from the Competitive Grants Program of the U.S. Department of Agriculture (88-37264-3807 and 90-37264-5471) to E.A.N.     

Generalized transition state method and continuous diffusion in multi-dimensional systems with relation to ion transport in channels of biological membranes

An exact expression for the escape rate of a particle in a multi-dimensional system, with respect to an arbitrary reaction coordinate, is derived from first principles according to the transition state method, using a simple geometrical argument. It is shown how the mutual coupling of all degrees of freedom due to the interaction forces leads to the appearance of an effective mass and the potential of the mean force. The same relevant quantities dominate the effective one-particle Fokker-Planck equation, which is derived by a similar projection procedure from the multi-dimensional transport equation. In the limit of a large, position-dependent friction the respective effective Smoluchowski equation is obtained. It allows for the discussion of a diffusing particle which is subject to a temperature bath only through the coupled motion with the constituent lattice particles, or ligands in the case of a molecular ion channel. This treatment is of particular importance for the analysis of ion transport in membrane pores in which the ionic motion is mediated by internal ligand motion.     

Pressure variation of the lateral diffusion in lipid bilayer membranes

A pressure-induced decrease of the lateral diffusion in pure and cholesterol containing phosphatidylcholine bilayer membranes has been determined by the excimer formation technique using pyrene as probe molecule. The experimental results at pressures up to 150 bars are described satisfactorily by the free volume theory of a molecular transport in liquids. A pressure increase of extrapolated 575 bars decreases the lateral diffusion of lipids by a factor of two in pure dipalmitoylphosphatidylcholine membranes. Higher pressures are necessary to induce the same effect in cholesterol containing membranes. This result is interpreted by the condensing effect of cholesterol in fluid bilayer membranes.     

On the decay of lateral phase separations in biological membranes

A theory for the decay of a lateral phase separation in a biological membrane has been developed based upon the edge decay of a population of circular molecular domains of uniform size. The theory has been applied to the case of vesicle fusion at a presynaptic membrane. It is shown that the efficiency of fusion decays exponentially in time with a rate constant $\text{solπ}\text{2τ}\text{N}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ which decreases as the rate at which bonds are broken within each domain (τ^?1) decreases and as the number of molecules within each domain (N) increases. Moreover, it has been speculated that this mechanism may offer in part an explanation for the slow, exponential decay during post-tetanic potentiation where it is known that the efficiency of neurotransmitter release at the presynaptic membrane is rate-controlling and decays exponentially.     

Towards an understanding of complex biological membranes from atomistic molecular dynamics simulations

Computer simulation has emerged as a powerful tool for studying the structural and functional properties of complex biological membranes. In the last few years, the use of recently developed simulation methodologies and current generation force fields has permitted novel applications of molecular dynamics simulations, which have enhanced our understanding of the different physical processes governing biomembrane structure and dynamics. This review focuses on frontier areas of research with important biomedical applications. We have paid special attention to polyunsaturated lipids, membrane proteins and ion channels, surfactant additives in membranes, and lipid–DNA gene transfer complexes.