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.     

Diffusion rates of cell surface antigens of mouse-human heterokaryons. II. Effect of membrane potential on lateral diffusion

The rate of appearance, in a population of mouse-human heterokaryons, of cells with intermixed mouse and human surface antigens may be used to estimate the rate of lateral diffusion of the antigens in a single cell. Most heterokaryons appear to restrict diffusion of their surface antigens. These restrictions are altered by exposing either heterokaryons or their parent cells to conditions that change cell surface membrane potential. Media containing unphysiological concentrations of potassium ion, drugs, affecting the Na+,K+ ATPase, or a channel-forming antibiotic, gramicidin, all affect lateral mobility of cell surface antigens in a manner consistent with a common effect on membrane potential.     

The lateral diffusion of lipid probes in the surface membrane of Schistosoma mansoni

The technique of fluorescence recovery after photobleaching was used to measure the lateral diffusion of fluorescent lipid analogues in the surface membrane of Schistosoma mansoni. Our data reveal that although some lipids could diffuse freely others exhibited restricted lateral diffusion. Quenching of lipid fluorescence by a non-permeant quencher, trypan blue, showed that there was an asymmetric distribution of lipids across the double bilayer of mature parasites. Those lipids that diffused freely were found to reside mainly in the external monolayer of the outer membrane whereas lipids with restricted lateral diffusion were located mainly in one or more of the monolayers beneath the external monolayer. Formation of surface membrane blebs allowed us to measure the lateral diffusion of lipids in the membrane without the influence of underlying cytoskeletal structures. The restricted diffusion found on the normal surface membrane of mature parasites was found to be released in membrane blebs. Quenching of fluorescent lipids on blebs indicated that all probes were present almost entirely in the external monolayer. Juvenile worms exhibited lower lateral diffusion coefficients than mature parasites: in addition, the lipids partitioned into the external monolayer. The results are discussed in terms of membrane organization, cytoskeletal contacts, and biological significance.     

Lipid lateral diffusion and membrane heterogeneity

The pulsed field gradient (pfg)-NMR method for measurements of translational diffusion of molecules in macroscopically aligned lipid bilayers is described. This technique is proposed to have an appreciable potential for investigations in the field of lipid and membrane biology. Transport of molecules in the plane of the bilayer can be successfully studied, as well as lateral phase separation of lipids and their dynamics within the bilayer organizations. Lateral diffusion coefficients depend on lipid packing and acyl chain ordering and investigations of order parameters of perdeuterated acyl chains, using (2)H NMR quadrupole splittings, are useful complements. In this review we summarize some of our recent achievements obtained on lipid membranes. In particular, bilayers exhibiting two-phase coexistence of liquid disordered (l(d)) and liquid ordered (l(o)) phases are considered in detail. Methods for obtaining good oriented lipid bilayers, necessary for the pfg-NMR method to be efficiently used, are also briefly described. Among our major results, besides determinations of l(d) and l(o) phases, belongs the finding that the lateral diffusion is the same for all components, independent of the molecular structure (including cholesterol (CHOL)), if they reside in the same domain or phase in the membrane. Furthermore, quite unexpectedly CHOL seems to partition into the l(d)and l(o) phases to roughly the same extent, indicating that CHOL has no strong preference for any of these phases, i.e. CHOL seems to have similar interactions with all of the lipids. We propose that the lateral phase separation in bilayers containing one high-T(m) and one low-T(m) lipid together with CHOL is driven by the increasing difficulty of incorporating an unsaturated or prenyl lipid into the highly ordered bilayer formed by a saturated lipid and CHOL, i.e. the phase transition is entropy driven to keep the disorder of the hydrocarbon chains of the unsaturated lipid.     

Lipid lateral diffusion and membrane heterogeneity

The pulsed field gradient (pfg)-NMR method for measurements of translational diffusion of molecules in macroscopically aligned lipid bilayers is described. This technique is proposed to have an appreciable potential for investigations in the field of lipid and membrane biology. Transport of molecules in the plane of the bilayer can be successfully studied, as well as lateral phase separation of lipids and their dynamics within the bilayer organizations. Lateral diffusion coefficients depend on lipid packing and acyl chain ordering and investigations of order parameters of perdeuterated acyl chains, using ²H NMR quadrupole splittings, are useful complements. In this review we summarize some of our recent achievements obtained on lipid membranes. In particular, bilayers exhibiting two-phase coexistence of liquid disordered (l_d) and liquid ordered (l_o) phases are considered in detail. Methods for obtaining good oriented lipid bilayers, necessary for the pfg-NMR method to be efficiently used, are also briefly described. Among our major results, besides determinations of l_d and l_o phases, belongs the finding that the lateral diffusion is the same for all components, independent of the molecular structure (including cholesterol (CHOL)), if they reside in the same domain or phase in the membrane. Furthermore, quite unexpectedly CHOL seems to partition into the l_dand l_o phases to roughly the same extent, indicating that CHOL has no strong preference for any of these phases, i.e. CHOL seems to have similar interactions with all of the lipids. We propose that the lateral phase separation in bilayers containing one high-T_m and one low-T_m lipid together with CHOL is driven by the increasing difficulty of incorporating an unsaturated or prenyl lipid into the highly ordered bilayer formed by a saturated lipid and CHOL, i.e. the phase transition is entropy driven to keep the disorder of the hydrocarbon chains of the unsaturated lipid.     

Lipid lateral diffusion and membrane organization

It is shown that investigating the lateral motion of lipids in biological membranes can provide useful information on membrane lateral organization. After labeling membranes with extrinsic or intrinsic lipophilic fluorescent probes, fluorescence recovery after photobleaching experiments strongly suggests that specialized cells like spermatozoa, eggs and epithelia exhibit surface membrane regionalization or macrocompartmentation and that lateral microheterogeneities or lipid microdomains exist in the plasma membrane of many cellular systems.     

Analysis of lateral diffusion from a spherical cell surface to a tubular projection.

Cell surfaces are often heterogeneous with respect to the lateral distribution and mobility of membrane components. Because lateral mobility is related to membrane structure, measurement of a particular component's local diffusion coefficient within a distinct surface region provides useful information about the formation and maintenance of that region. Many structurally interesting cell surface features can be described as narrow tubular projections from the body of the cell. In a companion paper, we consider the thin "tethers" that can be mechanically drawn from the red blood cell membrane, and we measure the transport of fluorescent integral proteins from the surface of the cell body onto the tether. In this paper we present an analysis to describe the surface diffusion of membrane particles from a spherical shell onto a thin cylindrical process. Provision is made for different rates of diffusion within the two morphologically distinct regions. The relative role of each region in controlling the diffusive flux between regions is determined primarily by a single dimensionless parameter. This parameter incorporates the ratio of the two diffusion coefficients as well as the dimensions of each region. The analysis can be applied to a fluorescence photobleaching experiment in which the extended process is bleached. If the dimensions of the spherical cell body and the cylindrical extension are known, then the diffusion coefficients of both regions can be determined from the experimental fluorescence recovery curve.     

Lateral diffusion in biological membranes. A normal-mode analysis of diffusion on a spherical surface.

A new approach is described for the analysis of lateral diffusion in biological membranes. It is shown that a suitably defined first moment of the concentration distribution on a spherical surface decays as a single exponential with a relaxation rate proportional to the diffusion coefficient and inversely proportional to the square of the radius of the sphere. The approach is illustrated with an example of fluorescence redistribution after photobleaching of membrane proteins in a spectrin-deficient spherocytic mouse erythrocyte membrane.     

Electrostatic field effects on membrane domain segregation and on lateral diffusion

Natural membranes are organized structures of neutral and charged molecules bearing dipole moments which generate local non-homogeneous electric fields. When subjected to such fields, the molecules experience net forces that can modify the lipid and protein organization, thus modulating cell activities and influencing (or even dominating) the biological functions. The energetics of electrostatic interactions in membranes is a long-range effect which can vary over distance within r⁻¹ to r⁻³. In the case of a dipole interacting with a plane of dipoles, e.g. a protein interacting with a lipid domain, the interaction is stronger than two punctual dipoles and depends on the size of the domain. In this article, we review several contributions on how electrostatic interactions in the membrane plane can modulate the phase behavior, surface topography and mechanical properties in monolayers and bilayers.     

Imaging the lateral diffusion of membrane molecules with quantum dots

This protocol describes a sensitive approach to tracking the motion of membrane molecules such as lipids and proteins with molecular resolution in live cells. This technique makes use of fluorescent semiconductor nanocrystals, quantum dots (QDs), as a probe to detect membrane molecules of interest. The photostability and brightness of QDs allow them to be tracked at a single particle level for longer periods than previous fluorophores, such as fluorescent proteins and organic dyes. QDs are bound to the extracellular part of the object to be followed, and their movements can be recorded with a fluorescence microscope equipped with a spectral lamp and a sensitive cooled charge-coupled device camera. The experimental procedure described for neurons below takes about 45 min. This technique is applicable to various cultured cells.     

Catalytic facilitation by diffusion of adsorbed substrate on membrane surface

Membrane-alkaline phosphatase shows greater velocity of reaction than solubilized enzyme at low substrate concentration, whereas at saturation-concentration the opposite is true. The catalytic rate enhancement with the membrane-enzyme, when substrate availability is limiting, is attributed to non-specific adsorption of substrate to the membrane followed by its surface-diffusion to the active site resulting in an enhanced collision rate for the substrate with the enzyme. Experimental evidence for the adsorption-diffusion is provided by the dynamic quenching of 1-anilino-naphthalene-8-sulphonate, a membrane-bound probe's fluorescence by the substrate, 4-nitrophenyl-phosphate.     

Restricted lateral diffusion of luteinizing hormone receptors in membrane microdomains

Single particle tracking was used to evaluate lateral motions of individual FLAG-tagged human luteinizing hormone (LH) receptors expressed on CHO cells and native LH receptors on both KGN human granulosa-derived tumor cells and M17 human neuroblastoma cells before and after exposure to human chorionic gonadotropin (hCG). Compared with LH receptors on untreated cells, LH receptors on cells treated with 100 nm hCG exhibit restricted lateral diffusion and are confined in small, nanometer-scale, membrane compartments. Similar to LH receptors labeled with Au-hCG, LH receptors labeled with gold-deglycosylated hCG, an hCG antagonist, also exhibit restricted lateral diffusion and are confined in nanoscale membrane compartments on KGN cells treated with 100 nm hCG. LH receptor point mutants lacking potential palmitoylation sites remain in large compartments despite treatment with 100 nm hCG as do LH receptors on cells treated with cytochalasin D. Finally, both polarization homotransfer fluorescence resonance energy transfer imaging and photon counting histogram analysis indicate that treatment with hCG induces aggregation of YFP-coupled LH receptors stably expressed on CHO cells. Taken together, our results demonstrate that binding of hCG induces aggregation of LH receptors within nanoscale, cell surface membrane compartments, that hCG binding also affects the lateral motions of antagonist binding LH receptors, and that receptor surface densities must be considered in evaluating the extent of hormone-dependent receptor aggregation.     

The membrane skeleton of erythrocytes: models of its effect on lateral diffusion

The membrane skeleton, a network of structural proteins attached to the cytoplasmic surface of the plasma membrane, hinders lateral diffusion of integral proteins. 2. In some types of cells, such as epithelial cells and nerve cells, the obstruction of lateral diffusion by the membrane skeleton is one of the mechanisms by which proteins are localized to domains on the cell surface. 3. The effect of the membrane skeleton on lateral diffusion may involve steric hindrance, transient binding or both. Three pictures of the effect are reviewed, the discrete barrier model, the continuous barrier model and the transient binding model. 4. Experiments to distinguish the models are discussed.     

Rhodopsin lateral diffusion as a function of rod outer segment disk membrane axial position

Rhodopsin lateral diffusion was measured at three points along the axis of frog outer segments using the method of absorbance recovery after photobleaching. Mean recovery times were slightly longer in distal disk membranes than in proximal disks. A small reduction of pigment mobility with disk age may reflect subtle changes in membrane composition.     

The spectrin network as a barrier to lateral diffusion in erythrocytes. A percolation analysis.

The spectrin network on the cytoplasmic surface of an erythrocyte can be modeled as a triangular lattice of spectrin tetramers (Tsuji, A., and S. Ohnishi, 1986. Biochemistry. 25:6133-6139). The tetramers act as barriers to protein diffusion, while dissociated dimer pairs, single dimers, and missing tetramers do not. Diffusion in the presence of these barriers is shown to be equivalent to bond percolation on the honeycomb lattice. Monte Carlo calculations for this system then yield the relative diffusion constant of a mobile integral protein as a function of the fraction of spectrin tetramers. At high concentrations of spectrin tetramer, long-range diffusion is blocked, but short-range diffusion is still possible. Monte Carlo calculations yield the average distance over which short-range diffusion can occur, as a function of the fraction of spectrin tetramers. Applications to erythrocyte development and hereditary hemolytic anemia are discussed.     

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.     

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.     

Modeling the role of lateral membrane diffusion in AMPA receptor trafficking along a spiny dendrite

AMPA receptor trafficking in dendritic spines is emerging as a major postsynaptic mechanism for the expression of plasticity at glutamatergic synapses. AMPA receptors within a spine are in a continuous state of flux, being exchanged with local intracellular pools via exo/endocytosis and with the surrounding dendrite via lateral membrane diffusion. This suggests that one cannot treat a single spine in isolation. Here we present a model of AMPA receptor trafficking between multiple dendritic spines distributed along the surface of a dendrite. Receptors undergo lateral diffusion within the dendritic membrane, with each spine acting as a spatially localized trap where receptors can bind to scaffolding proteins or be internalized through endocytosis. Exocytosis of receptors occurs either at the soma or at sites local to dendritic spines via constitutive recycling from intracellular pools. We derive a reaction–diffusion equation for receptor trafficking that takes into account these various processes. Solutions of this equation allow us to calculate the distribution of synaptic receptor numbers across the population of spines, and hence determine how lateral diffusion contributes to the strength of a synapse. A number of specific results follow from our modeling and analysis. (1) Lateral membrane diffusion alone is insufficient as a mechanism for delivering AMPA receptors from the soma to distal dendrites. (2) A source of surface receptors at the soma tends to generate an exponential-like distribution of receptors along the dendrite, which has implications for synaptic democracy. (3) Diffusion mediates a heterosynaptic interaction between spines so that local changes in the constitutive recycling of AMPA receptors induce nonlocal changes in synaptic strength. On the other hand, structural changes in a spine following long term potentiation or depression have a purely local effect on synaptic strength. (4) A global change in the rates of AMPA receptor exo/endocytosis is unlikely to be the sole mechanism for homeostatic synaptic scaling. (5) The dynamics of AMPA receptor trafficking occurs on multiple timescales and varies according to spatial location along the dendrite. Understanding such dynamics is important when interpreting data from inactivation experiments that are used to infer the rate of relaxation to steady-state.