Similar diffusibility of membrane proteins across the axon-soma and dendrite-soma boundaries revealed by a novel FRAP technique

In polarized mature neurons, the asymmetrical distribution of proteins between axonal and somatodendritic plasma membrane (PM) domains may be maintained by a diffusion barrier at the axon-soma boundary. At the boundary, a complex containing membrane-associated and cytoskeletal proteins is formed, anchoring axonal membrane proteins and indirectly hindering the diffusion of other membrane proteins. We examined the latter case, i.e., secondary diffusion impedance by comparing the mobility of fluorescently labeled membrane proteins within the axon-soma and dendrite-soma boundaries. We performed fluorescence recovery after photobleaching (FRAP) experiments using mature cultured hippocampal neurons that had been labeled specifically at their PMs with fluorescent proteins (FPs). The maturation of these neurons was confirmed by immunolocalization with Ankyrin-G, which is thought to participate in the creation of the diffusion barrier at the axon-soma boundary. We developed a wide-field microscope equipped with a device (digital micromirror device) composed of 1024 x 768 binary mirrors at the field-stop, allowing free control of the illumination area and intensity. After the FPs in peripheral processes were photobleached, nonbleached FPs diffused into all the processes at equivalent speeds. These results indicate that the secondary diffusion barrier to exogenously overexpressed membrane proteins is not specific to the axon-soma boundary.     

Compartmentalized structure of the plasma membrane for receptor movements as revealed by a nanometer-level motion analysis

Movements of transferrin and alpha 2-macroglobulin receptor molecules in the plasma membrane of cultured normal rat kidney (NRK) fibroblastic cells were investigated by video-enhanced contrast optical microscopy with 1.8 nm spatial precision and 33 ms temporal resolution by labeling the receptors with the ligand-coated nanometer-sized colloidal gold particles. For both receptor species, most of the movement trajectories are of the confined diffusion type, within domains of approximately 0.25 microns2 (500-700 nm in diagonal length). Movement within the domains is random with a diffusion coefficient approximately 10(-9) cm2/s, which is consistent with that expected for free Brownian diffusion of proteins in the plasma membrane. The receptor molecules move from one domain to one of the adjacent domains at an average frequency of 0.034 s-1 (the residence time within a domain approximately 29 s), indicating that the plasma membrane is compartmentalized for diffusion of membrane receptors and that long- range diffusion is the result of successive intercompartmental jumps. The macroscopic diffusion coefficients for these two receptor molecules calculated on the basis of the compartment size and the intercompartmental jump rate are approximately 2.4 x 10(-11) cm2/s, which is consistent with those determined by averaging the long-term movements of many particles. Partial destruction of the cytoskeleton decreased the confined diffusion mode, increased the simple diffusion mode, and induced the directed diffusion (transport) mode. These results suggest that the boundaries between compartments are made of dynamically fluctuating membrane skeletons (membrane-skeleton fence model).     

On a set of data for the membrane potential in a neuron

We consider a set of data where the membrane potential in a pyramidal neuron is measured almost continuously in time, under varying experimental conditions. We use nonparametric estimates for the diffusion coefficient and the drift in view to contribute to the discussion which type of diffusion process is suitable to model the membrane potential in a neuron (more exactly: in a particular type of neuron under particular experimental conditions).     

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.     

Some considerations on a carrier mechanism as a model for ion accumulation

A mechanism is described which accounts for the active transport of Na⁺ ions through a membrane. It is assumed that at one side of the membrane the ion combines with a carrier ion, the resulting carrier compound then diffuses through the membrane and decomposes at the other side of the membrane. The free diffusion of the ions is also taken into account. The time rate of accumulation of the ion in question at the latter side of the membrane is calculated in terms of the concentrations of the ion at both sides of the membrane.     

Diffusion in a Fluid Membrane with a Flexible Cortical Cytoskeleton

We calculate the influence of a flexible network of long-chain proteins, which is anchored to a fluid membrane, on protein diffusion in this membrane. This is a model for the cortical cytoskeleton and the lipid bilayer of the red blood cell, which we apply to predict the influence of the cytoskeleton on the diffusion coefficient of a mobile band 3 protein. Using the pressure field that the cytoskeleton exerts on the membrane, from the steric repulsion between the diffusing protein and the cytoskeletal filaments, we define a potential landscape for the diffusion within the bilayer. We study the changes to the diffusion coefficient on removal of one type of anchor proteins, e.g., in several hemolytic anemias, as well as for isotropic and anisotropic stretching of the cytoskeleton. We predict an overall increase of the diffusion for a smaller number of anchor proteins and increased diffusion for anisotropic stretching in the direction of the stretch, because of the decrease in the spatial frequency as well as in the height of the potential barriers.     

Septin-dependent compartmentalization of the endoplasmic reticulum during yeast polarized growth

Polarized cells frequently use diffusion barriers to separate plasma membrane domains. It is unknown whether diffusion barriers also compartmentalize intracellular organelles. We used photobleaching techniques to characterize protein diffusion in the yeast endoplasmic reticulum (ER). Although a soluble protein diffused rapidly throughout the ER lumen, diffusion of ER membrane proteins was restricted at the bud neck. Ultrastructural studies and fluorescence microscopy revealed the presence of a ring of smooth ER at the bud neck. This ER domain and the restriction of diffusion for ER membrane proteins through the bud neck depended on septin function. The membrane-associated protein Bud6 localized to the bud neck in a septin-dependent manner and was required to restrict the diffusion of ER membrane proteins. Our results indicate that Bud6 acts downstream of septins to assemble a fence in the ER membrane at the bud neck. Thus, in polarized yeast cells, diffusion barriers compartmentalize the ER and the plasma membrane along parallel lines.     

On simultaneous determinations of the permeability of a membrane and of the diffusion coefficient in an adjacent medium

This paper deals with diffusion into a medium of finite thickness through a flat structure which can be considered either as a slice of tissue or as a membrane. Formulae are given to determine the diffusion coefficients in both the flat structure and the adjacent medium from the knowledge of the amount of substance penetrating the medium. The meaning of the formula: (permeability coefficient) = (diffusion coefficient)/(membrane thickness) and the experimentally observed variability of the permeability coefficient in the non-steady state are interpreted on the basis of the mathematical theory of diffusion.     

Anomalous diffusion due to binding: a Monte Carlo study.

In classical diffusion, the mean-square displacement increases linearly with time. But in the presence of obstacles or binding sites, anomalous diffusion may occur, in which the mean-square displacement is proportional to a nonintegral power of time for some or all times. Anomalous diffusion is discussed for various models of binding, including an obstruction/binding model in which immobile membrane proteins are represented by obstacles that bind diffusing particles in nearest-neighbor sites. The classification of binding models is considered, including the distinction between valley and mountain models and the distinction between singular and nonsingular distributions of binding energies. Anomalous diffusion is sensitive to the initial conditions of the measurement. In valley models, diffusion is anomalous if the diffusing particles start at random positions but normal if the particles start at thermal equilibrium positions. Thermal equilibration leads to normal diffusion, or to diffusion as normal as the obstacles allow.     

Microfluidity mapping using fluorescence correlation spectroscopy: a new way to investigate plasma membrane microorganization of living cells

Diffusion time distribution analysis has been employed to highlight the microfluidity fingerprint of plasma membrane of living cells. Diffusion time measurements were obtained through fluorescence correlation spectroscopy performed at the single cell level, over various eukaryotic cell lines (MCF7, LR73, KB3.1, MESSA and MDCKII). The nonsymmetric profile of the diffusion time distributions established experimentally, is discussed according to Monte Carlo simulations, which reproduce the diffusion of the fluorescent probe in heterogeneous membrane.     

Confined diffusion without fences of a g-protein-coupled receptor as revealed by single particle tracking

Single particle tracking is a powerful tool for probing the organization and dynamics of the plasma membrane constituents. We used this technique to study the micro -opioid receptor belonging to the large family of the G-protein-coupled receptors involved with other partners in a signal transduction pathway. The specific labeling of the receptor coupled to a T7-tag at its N-terminus, stably expressed in fibroblastic cells, was achieved by colloidal gold coupled to a monoclonal anti T7-tag antibody. The lateral movements of the particles were followed by nanovideomicroscopy at 40 ms time resolution during 2 min with a spatial precision of 15 nm. The receptors were found to have either a slow or directed diffusion mode (10%) or a walking confined diffusion mode (90%) composed of a long-term random diffusion and a short-term confined diffusion, and corresponding to a diffusion confined within a domain that itself diffuses. The results indicate that the confinement is due to an effective harmonic potential generated by long-range attraction between the membrane proteins. A simple model for interacting membrane proteins diffusion is proposed that explains the variations with the domain size of the short-term and long-term diffusion coefficients.     

Maltose-binding protein does not modulate the activity of maltoporin as a general porin in Escherichia coli.

Maltoporin (lambda receptor) is part of the maltose transport system in Escherichia coli and is necessary for the facilitated diffusion of maltose and maltodextrins across the outer membrane. Maltoporin also allows the diffusion of nonmaltodextrin substrates, albeit with less efficiency. The preference of maltoporin for maltodextrins in vivo is thought to be the result of an interaction of maltoporin with the maltose-binding protein, the malE gene product. In a recent report Heuzenroeder and Reeves (J. Bacteriol. 144:431-435, 1980) suggested that this interaction establishes a gating mechanism which inhibits the diffusion of nonmaltodextrin substrates, such as lactose. To reinvestigate this important conclusion, we constructed ompR malTc strains carrying either the malE+ gene, the nonpolar malE444 deletion, or the malE254 allele, which specifies an interaction-deficient maltose-binding protein. Lactose uptake was measured at different concentrations below the Km of this transport system and under conditions where transport was limited by the diffusion through maltoporin. We found no difference in the kinetics of lactose uptake irrespective of the malE allele. We conclude that the maltose-binding protein does not modulate the activity of maltoporin as a general outer membrane porin.     

The membrane-bound domain of the phosphotransferase enzyme IImtl of Escherichia coli constitutes a mannitol translocating unit

The orientation of the mannitol binding site on the Escherichia coli phosphotransferase enzyme IImtl in the unphosphorylated state has been investigated by measuring mannitol binding to cytoplasmic membrane vesicles with a right-side-out and inside-out orientation. Enzyme IImtl is shown to catalyze facilitated diffusion of mannitol at a low rate. At equilibrium, bound mannitol is situated at the periplasmic side of the membrane. The apparent binding constant is 40 nM for the intact membranes. Solubilization of the membranes in detergent decreases the affinity by about a factor of 2. Inside-out membrane vesicles, treated with trypsin to remove the C-terminal cytoplasmic domain of enzyme IImtl, showed identical activities. These experiments indicate that the translocation of mannitol is catalyzed by the membrane-bound N-terminal half of enzyme IImtl which is a structurally stable domain.     

Ankyrin G restricts ion channel diffusion at the axonal initial segment before the establishment of the diffusion barrier

In mammalian neurons, the precise accumulation of sodium channels at the axonal initial segment (AIS) ensures action potential initiation. This accumulation precedes the immobilization of membrane proteins and lipids by a diffusion barrier at the AIS. Using single-particle tracking, we measured the mobility of a chimeric ion channel bearing the ankyrin-binding motif of the Nav1.2 sodium channel. We found that ankyrin G (ankG) limits membrane diffusion of ion channels when coexpressed in neuroblastoma cells. Site-directed mutants with decreased affinity for ankG exhibit increased diffusion speeds. In immature hippocampal neurons, we demonstrated that ion channel immobilization by ankG is regulated by protein kinase CK2 and occurs as soon as ankG accumulates at the AIS of elongating axons. Once the diffusion barrier is formed, ankG is still required to stabilize ion channels. In conclusion, our findings indicate that specific binding to ankG constitutes the initial step for Nav channel immobilization at the AIS membrane and precedes the establishment of the diffusion barrier.     

A mathematical model relating diffusion of hydrophobic ions to their adsorption on biological membranes as detected with a microdialyzer

We analyze the diffusion of hydrophobic molecules in a dialysis apparatus with respect to their adsorption on biological membrane vesicles confined to one dialysis chamber. The process is described with a kinetic model, which shows that, depending on the pattern of the adsorption isotherm, the kinetic parameter of the diffusion process through the dialysis membrane is up to two-fold increased by the presence of the adsorbing vesicle surface. The model successfully describes the diffusion of tetraphenylborate and 9-aminoacridine in the presence of chromatophores from photosynthetic membrane, with which they interact with hyperbolic and S-shaped isotherms, respectively.     

Modeling the signaling endosome hypothesis: Why a drive to the nucleus is better than a (random) walk

Background  

Information transfer from the plasma membrane to the nucleus is a universal cell biological property. Such information is generally encoded in the form of post-translationally modified protein messengers. Textbook signaling models typically depend upon the diffusion of molecular signals from the site of initiation at the plasma membrane to the site of effector function within the nucleus. However, such models fail to consider several critical constraints placed upon diffusion by the cellular milieu, including the likelihood of signal termination by dephosphorylation. In contrast, signaling associated with retrogradely transported membrane-bounded organelles such as endosomes provides a dephosphorylation-resistant mechanism for the vectorial transmission of molecular signals. We explore the relative efficiencies of signal diffusion versus retrograde transport of signaling endosomes.     

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.     

Role of t-tubules in the control of trans-sarcolemmal ion flux and intracellular Ca2+ in a model of the rat cardiac ventricular myocyte

The t-tubules of mammalian ventricular myocytes are invaginations of the surface membrane that form a complex network within the cell, with restricted diffusion to the bulk extracellular space. The trans-sarcolemmal flux of many ions, including Ca(2+), occurs predominantly across the t-tubule membrane and thus into and out of this restricted diffusion space. It seems possible, therefore, that ion concentration changes may occur in the t-tubule lumen, which would alter ion flux across the t-tubule membrane. We have used a computer model of the ventricular myocyte, incorporating a t-tubule compartment and experimentally determined values for diffusion between the t-tubule lumen and bulk extracellular space, and ion fluxes across the t-tubule membrane, to investigate this possibility. The results show that influx and efflux of different ion species across the t-tubule membrane are similar, but not equal. Changes of ion concentration can therefore occur close to the t-tubular membrane, thereby altering trans-sarcolemmal ion flux and thus cell function, although such changes are reduced by diffusion to the bulk extracellular space. Slowing diffusion results in larger changes in luminal ion concentrations. These results provide a deeper understanding of the role of the t-tubules in normal cell function, and are a basis for understanding the changes that occur in heart failure as a result of changes in t-tubule structure and ion fluxes.     

The Validity of the Ussing Flux Ratio Equation in a Three-Dimensionally Inhomogeneous Membrane

Derivations of the Ussing flux ratio equation have, until now, required the membrane to be both bounded by parallel planes and homogeneous, except in the transmembrane direction. These constraints have been necessary for the theoretical demonstration that the equation is independent of membrane parameters in the absence of carriers, coupling, solvent drag, or “single-file” diffusion. In a new derivation, the flux ratio equation is shown to be valid in this kind of diffusion regime without regard to the three-dimensional structure of the membrane. Thus the constraints on both membrane homogeneity and membrane geometry are shown to be unnecessary. The general use of this equation to differentiate between simple, uncoupled diffusion and other membrane transport phenomena is thus placed on a firmer base. However, as in earlier derivations, it is necessary that isopotential, isobaric, constant concentration surfaces exist sufficiently close to the membrane on both of its sides.     

NMR structural analysis of a membrane protein: bacteriorhodopsin peptide backbone orientation and motion

In reconstituted vesicles above the lipid phase transition temperature, bacteriorhodopsin (BR) undergoes rotational diffusion about an axis perpendicular to the plane of the bilayer [Cherry, R. J., Muller, U., & Schneider, G. (1977) FEBS Lett. 80, 465]. This diffusion narrows the 13C NMR powder line shape of the BR peptide carbonyls. In contrast, BR in native purple membrane is relatively immobile and exhibits a rigid-lattice powder line shape. By use of the principal values of the rigid-lattice chemical shift tensor and the motionally narrowed line shape from the reconstituted system, the range of Euler angles of the leucine peptide groups relative to the diffusion axis has been calculated. The experimentally observed line shape is inconsistent with those expected for structures which consist entirely of either alpha helix or beta sheet perpendicular to the membrane or beta sheet tilted at angles up to about 60 degrees from the membrane normal. However, for two more complex structural models, the predicted line shapes agree well with the experimental one. These are, first, a structure consisting entirely of alpha1 helices tilted at 20 degrees from the membrane normal and, second, a combination of 60% alpha II helix perpendicular to the membrane plane and 40% antiparallel beta sheet tilted at 10-20 degrees from the membrane normal. The results also indicate that the peptide backbone of bacteriorhodopsin in native purple membrane is extremely rigid even at 40 degrees. The experiments presented here demonstrate a new approach, using solid-state nuclear magnetic resonance (NMR) methods, for structural studies of transmembrane proteins in fluid membrane environments, either natural or reconstituted.(ABSTRACT TRUNCATED AT 250 WORDS)