Lateral Membrane Mobility Research Articles

Spectral Characterization of Stem Cell-Derived Myelination within the Injured Adult PNS Using the Solvatochromic Dye Nile Red.

Background: Myelin is an essential component of the peripheral and central nervous system, enabling fast axonal conduction and supporting axonal integrity; limited tools exist for analysis of myelin composition in-vivo. Objective: To demonstrate that the photophysical properties of myelin-incorporated solvatochromic dyes can be exploited to probe the biochemical composition of living peripheral nerve myelin at high spatial resolution. Methods: Using the myelin-incorporated fluorescent dye Nile Red we sequentially analyzed the spectral characteristics of remyelinating myelin membranes both in-vitro and in-vivo, including in living rats. Results: We demonstrated a consistent bi-phasic evolution of emission spectra during early remyelination, and visually report the reliable biochemical flux of myelin membrane composition in-vitro and in-vivo. Conclusions: Solvatochromic spectroscopy enables the analysis of myelin membrane maturity during remyelination, and can be performed in-vivo. As the formation of myelin during early-to-late remyelination likely incorporates fluctuating fractions of lipophilic components and changes in lateral membrane mobility, we propose that our spectrochemical data reflects the observation of these biochemical processes.

Endocytosis sustains release at photoreceptor ribbon synapses by restoring fusion competence.

Endocytosis is an essential process at sites of synaptic release. Not only are synaptic vesicles recycled by endocytosis, but the removal of proteins and lipids by endocytosis is needed to restore release site function at active zones after vesicle fusion. Synaptic exocytosis from vertebrate photoreceptors involves synaptic ribbons that serve to cluster vesicles near the presynaptic membrane. In this study, we hypothesize that this clustering increases the likelihood that exocytosis at one ribbon release site may disrupt release at an adjacent site and therefore that endocytosis may be particularly important for restoring release site competence at photoreceptor ribbon synapses. To test this, we combined optical and electrophysiological techniques in salamander rods. Pharmacological inhibition of dynamin-dependent endocytosis rapidly inhibits release from synaptic ribbons and slows recovery of ribbon-mediated release from paired pulse synaptic depression. Inhibiting endocytosis impairs the ability of second-order horizontal cells to follow rod light responses at frequencies as low as 2 Hz. Inhibition of endocytosis also increases lateral membrane mobility of individual Ca2+ channels, showing that it changes release site structure. Visualization of single synaptic vesicles by total internal reflection fluorescence microscopy reveals that inhibition of endocytosis reduces the likelihood of fusion among vesicles docked near ribbons and increases the likelihood that they will retreat from the membrane without fusion. Vesicle advance toward the membrane is also reduced, but the number of membrane-associated vesicles is not. Endocytosis therefore appears to be more important for restoring later steps in vesicle fusion than for restoring docking. Unlike conventional synapses in which endocytic restoration of release sites is evident only at high frequencies, endocytosis is needed to maintain release from rod ribbon synapses even at modest frequencies.

The Ca2+-activated Cl- channel Ano1 controls microvilli length and membrane surface area in the oocyte.

Ca(2+)-activated Cl(-) channels (CaCCs) play important physiological functions in epithelia and other tissues. In frog oocytes the CaCC Ano1 regulates resting membrane potential and the block to polyspermy. Here, we show that Ano1 expression increases the oocyte surface, revealing a novel function for Ano1 in regulating cell morphology. Confocal imaging shows that Ano1 increases microvilli length, which requires ERM-protein-dependent linkage to the cytoskeleton. A dominant-negative form of the ERM protein moesin precludes the Ano1-dependent increase in membrane area. Furthermore, both full-length and the truncated dominant-negative forms of moesin co-localize with Ano1 to the microvilli, and the two proteins co-immunoprecipitate. The Ano1-moesin interaction limits Ano1 lateral membrane mobility and contributes to microvilli scaffolding, therefore stabilizing larger membrane structures. Collectively, these results reveal a newly identified role for Ano1 in shaping the plasma membrane during oogenesis, with broad implications for the regulation of microvilli in epithelia.

Regulation of Basal Lateral Membrane Mobility and Permeability to Divalent Cations by Membrane Associated-Protein Kinase C

Biological membrane stabilization is essential for maintenance of cellular homeostasis, functionality and appropriate response to various stimuli. Previous studies have showed that accumulation of PKCs in the cell membrane significantly downregulates the membrane fluidity and Ca2+ influxes through the membranes in activated cells. In addition, membrane-inserted form of PKCs has been found in a variety of resting mammalian cells and tissues. This study is aimed to investigate possible role of the endogenous membrane-associated PKCs in the modulation of basal membrane fluidity. Here, we showed that interfering PKC expression by chronic activation of PKC with phorbol myristate acetate (PMA) or shRNA targeting at PKCα lowered the levels of PKCα in cytosol, peripheral membrane and integral membrane pools, while short-term activation of PKC with PMA induced accumulation of PKCα in the membrane pool accompanied by a dramatic decrease in the cytosol fraction. The lateral membrane mobility increased or decreased in accordance with the abundance alterations in the membrane-associated PKCα by these treatments. In addition, membrane permeability to divalent cations including Ca2+, Mn2+ and Ba2+ were also potentiated or abrogated along with the changes in PKC expression on the plasma membrane. Membrane stabilizer ursodeoxycholate abolished both of the enhanced lateral membrane mobility and permeability to divalent cations due to PKCα deficiency, whereas Gö6983, a PKC antagonist, or Gd3+ and 2-aminoethyoxydipheyl borne, two Ca2+ channels blockers, showed no effect, suggesting that this PKC-related regulation is independent of PKC activation or a modulation of specific divalent cation channel. Thus, these data demonstrate that the native membrane-associated PKCα is involved in the maintenance of basal membrane stabilization in resting cells.

E-cadherin Polarity Is Determined by a Multifunction Motif Mediating Lateral Membrane Retention through Ankyrin-G and Apical-lateral Transcytosis through Clathrin

We report a highly conserved motif in the E-cadherin juxtamembrane domain that determines apical-lateral polarity by conferring both restricted mobility at the lateral membrane and transcytosis of apically mis-sorted protein to the lateral membrane. Mutations causing either increased lateral membrane mobility or loss of apical-lateral transcytosis result in partial mis-sorting of E-cadherin in Madin-Darby canine kidney cells. However, loss of both activities results in complete loss of polarity. We present evidence that residues required for restricted mobility mediate retention at the lateral membrane through interaction with ankyrin-G, whereas dileucine residues conferring apical-lateral transcytosis act through a clathrin-dependent process and function in an editing pathway. Ankyrin-G interaction with E-cadherin is abolished by the same mutations resulting in increased E-cadherin mobility. Clathrin heavy chain knockdown and dileucine mutation of E-cadherin both cause the same partial loss of polarity of E-cadherin. Moreover, clathrin knockdown causes no further change in polarity of E-cadherin with dileucine mutation but does completely randomize E-cadherin mutants lacking ankyrin-binding. Dileucine mutation, but not loss of ankyrin binding, prevented transcytosis of apically mis-sorted E-cadherin to the lateral membrane. Finally, neurofascin, which binds ankyrin but lacks dileucine residues, exhibited partial apical-lateral polarity that was abolished by mutation of its ankyrin-binding site but was not affected by clathrin knockdown. The polarity motif thus integrates complementary activities of lateral membrane retention through ankyrin-G and apical-lateral transcytosis of mis-localized protein through clathrin. Together, the combination of retention and editing function to ensure a high fidelity steady state localization of E-cadherin at the lateral membrane.

Cortactin and the actin cytoskeleton control the lateral membrane mobility of Kv1.3 channels

The proper Kv1.3 membrane motility is necessary for T cell polarization and activation. The exact mechanisms regulating Kv1.3 membrane movement, however, remain unclear. Increased activity of actin has been found during T cell activation. Additionally, multiple protein complexes including cortactin and PDZ proteins like Dlg1 have been found to bind both Kv1 channels and actin. The purpose of the present investigation was to determine if actin and cortactin/PDZ proteins modulate Kv1.3 membrane motility. HEK293 cells were transfected with EGFP‐tagged Kv1.3 wild‐type or C‐terminal mutants lacking either cortactin or the PDZ binding sites, and their membrane diffusion (D) and mobile fraction (Mf) was determined by FRAP. Wild‐type and mutant channels exhibited similar baseline D and Mf. Upon induction of F‐actin polymerization by jasplakinolide wild‐type and PDZ mutants showed a 40% reduction in Mf and no change in D, while cortactin mutants exhibited no change in Mf and an increase in D. Kv1.3 wild‐type and mutants displayed similar biophysical and electrophysiological properties. Furthermore, the ability of Kv1.3 to bind to cortactin and Dlg1 was confirmed by immunoprecipitation. These data indicate that Kv1.3 lateral membrane motility is regulated by the actin cytoskeleton and cortactin as only the deletion of the cortactin binding site inhibited the actin‐mediated Kv1.3 mobility. (NIH 2R01CA095286)

Epitope‐tagged dopamine transporter knock‐in mice reveal rapid endocytic trafficking and filopodia targeting of the transporter in dopaminergic axons

The plasma membrane dopamine (DA) transporter (DAT) is essential for reuptake of extracellular DA. DAT function in heterologous cells is regulated by subcellular targeting, endocytosis, and intracellular trafficking, but the mechanisms regulating neuronal DAT remain poorly understood. Hence, we generated a knock-in mouse expressing a hemagglutinin (HA)-epitope-tagged DAT to study endogenous transporter trafficking. Introduction of the HA tag into the second extracellular loop of mouse DAT did not perturb its expression level, distribution pattern, or substrate uptake kinetics. Live-cell fluorescence microscopy imaging using fluorescently labeled HA-specific antibody and a quantitative HA-antibody endocytosis assay demonstrated that in axons HA-DAT was primarily located in the plasma membrane and internalized mostly in growth cones and varicosities, where synaptic vesicle markers were also concentrated. Formation of varicosities was frequently preceded or accompanied by highly dynamic filopodia-like membrane protrusions. Remarkably, HA-DAT often concentrated at the tips of these filopodia. This pool of HA-DATs exhibited low lateral membrane mobility. Thus, DAT-containing filopodia may be involved in synaptogenesis in developing DA neurons. Treatment of neurons with amphetamine increased mobility of filopodial HA-DAT and accelerated HA-DAT endocytosis in axons, suggesting that chronic amphetamine may interfere with DA synapse development. Interestingly, phorbol esters did not accelerate endocytosis of axonal DAT.

Lateral Mobility of Presynaptic L-Type Calcium Channels at Photoreceptor Ribbon Synapses

At most synapses, presynaptic Ca(2+) channels are positioned near vesicle release sites, and increasing this distance reduces synaptic strength. We examined the lateral membrane mobility of presynaptic L-type Ca(2+) channels at photoreceptor ribbon synapses of the tiger salamander (Ambystoma tigrinum) retina. Movements of individual Ca(2+) channels were tracked by coupling quantum dots to an antibody against the extracellular α(2)δ(4) Ca(2+) channel subunit. α(2)δ(4) antibodies labeled photoreceptor terminals and colocalized with antibodies to synaptic vesicle glycoprotein 2 and voltage-gated Ca(2+) channel 1.4 (Ca(V)1.4) α(1) subunits. The results show that Ca(2+) channels are dynamic and move within a confined region beneath the synaptic ribbon. The size of this confinement area is regulated by actin and membrane cholesterol. Fusion of nearby synaptic vesicles caused jumps in Ca(2+) channel position, propelling them toward the outer edge of the confinement domain. Channels rebounded rapidly toward the center. Thus, although Ca(V) channels are mobile, molecular scaffolds confine them beneath the ribbon to maintain neurotransmission even at high release rates.

Palmitoylation Controls Dopamine Transporter Kinetics, Degradation, and Protein Kinase C-dependent Regulation

Palmitoylation is a lipid modification that confers diverse functions to target proteins and is a contributing factor for many neuronal diseases. In this study, we demonstrate using [(3)H]palmitic acid labeling and acyl-biotinyl exchange that native and expressed dopamine transporters (DATs) are palmitoylated, and using the palmitoyl acyltransferase inhibitor 2-bromopalmitate (2BP), we identify several associated functions. Treatment of rat striatal synaptosomes with 2BP using lower doses or shorter times caused robust inhibition of transport V(max) that occurred with no losses of DAT protein or changes in DAT surface levels, indicating that acute loss of palmitoylation leads to reduction of transport kinetics. Treatment of synaptosomes or cells with 2BP using higher doses or longer times resulted in DAT protein losses and production of transporter fragments, implicating palmitoylation in regulation of transporter degradation. Site-directed mutagenesis indicated that palmitoylation of rat DAT occurs at Cys-580 at the intracellular end of transmembrane domain 12 and at one or more additional unidentified site(s). Cys-580 mutation also led to production of transporter degradation fragments and to increased phorbol ester-induced down-regulation, further supporting palmitoylation in opposing DAT turnover and in opposing protein kinase C-mediated regulation. These results identify S-palmitoylation as a major regulator of DAT properties that could significantly impact acute and long term dopamine transport capacity.

PSD-95 mediates membrane clustering of the human plasma membrane Ca 2+ pump isoform 4b

Besides the control of global calcium changes, specific plasma membrane calcium ATPase (PMCA) isoforms are involved in the regulation of local calcium signals. Although local calcium signaling requires the confinement of signaling molecules into microdomains, little is known about the specific organization of PMCA molecules within the plasma membrane. Here we show that co-expression with the postsynaptic density-95 (PSD-95) scaffolding protein increased the plasma membrane expression of PMCA4b and redistributed the pump into clusters. The clustering of PMCA4b was fully dependent on the presence of its PDZ-binding sequence. Using the fluorescence recovery after photobleaching (FRAP) technique, we show that the lateral membrane mobility of the clustered PMCA4b is significantly lower than that of the non-clustered molecules. Disruption of the actin-based cytoskeleton by cytochalasin D resulted in increased cluster size. Our results suggest that PSD-95 promotes the formation of high-density PMCA4b microdomains in the plasma membrane and that the membrane cytoskeleton plays an important role in the regulation of this process.

Glutamate Receptor Subunit 3 Is Modified by Site-specific Limited Proteolysis Including Cleavage by γ-Secretase

Ionotropic glutamate receptor (GluR) expression and function is regulated through multiple pre- and post-translational mechanisms. We find that limited proteolytic cleavage of GluR3 at two distinct sites generates stable GluR3 short forms that are glycosylated and found in association with other full-length GluRs in the mouse brain and cultured primary neurons. A combination of mutagenesis and transfection into HEK293 cells revealed cleavage by a gamma-secretase-like activity within the membrane-localized re-entry loop at or near the leucine-glycine pair (amino acids 585-586, GluR3sbeta) and a second site within a proline-rich PEST-like sequence in the first cytoplasmic loop (Asp570-Pro571, GluR3salpha). Generation of the prominent GluR3salpha form was effectively abolished in the mutant, GluR3D570A, but inhibitors of lysosomes, the proteasome, caspases, or calpains had no effect. The possible impact of cleavage on receptor function was suggested when the co-expression of the GluR3P571Stop mutant (creating GluR3salpha) co-assembled with other GluR subunits and decreased receptor function in Xenopus oocytes. In transiently transfected HEK293 cells, co-expression of GluR3salpha alters the relative association between GluR1 and GluR3 during assembly, and the presence of the novel C-terminal proline-rich domain of GluR3salpha imparts lateral membrane mobility to GluR complexes. These results suggest that limited proteolysis is another post-translational mechanism through which functional diversity and specialization between closely related GluR subunits is accomplished.

L-FABP and I-FABP expression increase NBD-stearate uptake and cytoplasmic diffusion in L cells.

The effects of intestinal and liver fatty acid binding protein (I- and L-FABP, respectively) expression on single-cell fatty acid uptake, internalization, and cytoplasmic diffusion were determined in transfected L cell fibroblasts. These parameters were measured using the nonesterifiable fluorescent fatty acid probe 12-N-methyl-(7-nitrobenz-2-oxa-1,3-diazol)aminostearate (NBD-stearate) and fluorescence digital imaging. In single-cell fluorescence imaging experiments, L-FABP-expressing cells, but not I-FABP-expressing cells, increased NBD-stearate uptake 1.7-fold compared with control cells. Both I- and L-FABP increased the cytoplasmic diffusion rate of the internalized NBD-stearate 2.6- and 1.9-fold, respectively, compared with control cells. However, increased NBD-stearate lateral membrane mobility was observed only in L-FABP-expressing cells. After incubation of the cells with 4 microM NBD-stearate at 37 degrees C for 30 min, fluorescence deconvolution imaging indicated that NBD-stearate was localized primarily into lipid droplets in all cell lines. The differential effect of these proteins on fatty acid uptake and intracellular trafficking in single cells illustrates a possible difference in the physiological function of I- and L-FABP in intact cells.

Pyrene Fluorescence: A Potential Tool for Estimation of Short-Range Lateral Mobility in Membranes of Living Renal Epithelial Cells

The excimer-to-monomer (E/M) fluorescence ration of pyrene (p) and its derivatives has been found to correlate with membrane fluidity. We explored the possible use of this method in living renal epithelial cells. In Modin-Darby canine kidney cells, successful fluorescent labeling was obtained with p-cholesterol, p-trimethylammonium (TMA), p-sulfonamidethyl-TMA, p-butyrate, p-propanoate or p-dodecanoate. Among them, the labeling with p-cholesterol was most stable. The whole cell cholesterol content did not significantly increase after loading p-cholesterol and the probe incorporated was not degraded. The E/M ratio changed reproducibly and significantly by temperature change, a standard method to alter membrane fluidity. Thus, the E/M ratio of p-cholesterol fluorescence is suggested to be a potential parameter of membrane fluidity in living renal epithelial cells and might be useful in investigating the relationship between the physical state of the membrane and epithelial function.

Receptor patching and capping of platelet membranes induced by monoclonal antibodies

Redistribution of glycoproteins (GP) Ib, glycocalicin, IIb, and IIIa on the surface of human platelets in response to stimulation with corresponding monoclonal antibodies (MoAb) and a polyclonal antiglycocalicin antibody was studied by immunofluorescence, immunoelectron microscopy, and a quantitative radioimmune assay. Immobilization of the antigens by prefixation with formaldehyde showed a uniform distribution over the surface of the platelet. Incubation of unfixed platelets with specific MoAb against various epitopes on GPIIb and/or IIIa resulted in a time-dependent patching, subsequent capping, and after prolonged exposure to the antibody/label complex, internalization of the complex, possibly by endocytosis. In contrast, GPIb could not be shown to cap. From these results we conclude that platelet GPIIb and/or IIIa undergo spatial rearrangement in a manner analogous to that observed in lymphocytes, whereas GPIb does not. Since both GPIb and GPIIb and/or IIIa seem to be transmembraneous GP associated with the cytoskeleton, a special, though unidentified, role of GPIIb/IIIa in the induction of lateral membrane mobility is postulated.

Receptor Patching and Capping of Platelet Membranes Induced by Monoclonal Antibodies

Redistribution of glycoproteins (GP) Ib, glycocalicin, IIb, and IIIa on the surface of human platelets in response to stimulation with corresponding monoclonal antibodies (MoAb) and a polyclonal antiglycocalicin antibody was studied by immunofluorescence, immunoelectron microscopy, and a quantitative radioimmune assay. Immobilization of the antigens by prefixation with formaldehyde showed a uniform distribution over the surface of the platelet. Incubation of unfixed platelets with specific MoAb against various epitopes on GPIIb and/or IIIa resulted in a time-dependent patching, subsequent capping, and after prolonged exposure to the antibody/label complex, internalization of the complex, possibly by endocytosis. In contrast, GPIb could not be shown to cap. From these results we conclude that platelet GPIIb and/or IIIa undergo spatial rearrangement in a manner analogous to that observed in lymphocytes, whereas GPIb does not. Since both GPIb and GPIIb and/or IIIa seem to be transmembraneous GP associated with the cytoskeleton, a special, though unidentified, role of GPIIb/IIIa in the induction of lateral membrane mobility is postulated.

Fluorometry of absorbant and turbid samples and the lateral mobility in membranes of intact erythrocytes

Intrinsic and extrinsic fluorophores attached to large biological assemblies (e.g. cells) find many useful applications in biophysics and medical science. Since such samples frequently absorb and/or scatter light strongly, special techniques have been developed for dealing with the artifacts which result. The use of excimerization probes as indicators of the local lateral mobility in the membranes of intact red blood cells provides an illustration.

Lateral mobility in membranes as detected by fluorescence recovery after photobleaching

The evaluation of lateral diffusion coefficients of membrane components by the technique of fluorescence recovery after photobleaching (FRAP) is often complicated by uncertainties in the values of the intensities F(O), immediately after bleaching, and F(infinity), after full recovery. These uncertainties arise from instrumental settling time immediately after bleaching and from cell, tissue, microscope, or laser beam movements at the long times required to measure F(infinity). We have developed a method for precise analysis of FRAP data that minimizes these problems. The method is based on the observation that a plot of the reciprocal function R(tau) = F(infinity)/[F(infinity)-F(tau)] is linear over a large time range when (a) the laser beam has a Gaussian profile, (b) recovery involves a single diffusion coefficient, and (c) there is no membrane flow. Moreover, the ratio of intercept to slope of the linear plot is equal to tau 1/2, the time required for the bleached fluorescence to rise to 50% of the full recovery value, F(infinity). The lateral diffusion coefficient D is related to tau 1/2 by tau 1/2 = beta w2/4D where beta is a defined parameter and w is the effective radius of the focused laser beam. These results are shown to indicate that the recovery of fluorescence F(tau) can be represented over a large range of percent bleach, and recovery time tau by the relatively simple expression F(tau) = [ F(o) + F(infinity) (tau/tau 1/2)]/[1 + tau/tau 1/2)]. FRAP data can therefore be easily evaluated by a nonlinear regression analysis with this equation or by a linear fit to the reciprocal function R(tau). It is shown that any error in F(infinity) can be easily detected in a plot of R(tau) vs. tau which deviates significantly from a straight line when F(infinity) is in error by as little as 5%. A scheme for evaluating D by linear analysis is presented. It is also shown that the linear reciprocal plot provides a simple method for detecting flow or multiple diffusion coefficients and for establishing conditions (data precision, differences in multiple diffusion coefficients, magnitude of flow rate compared to lateral diffusion) under which flow or multiple diffusion coefficients can be detected. These aspects are discussed in some detail.

33] Pyrenedecanoic acid and pyrene lecithin

Publisher Summary This chapter describes the synthesis and biophysical properties of pyrenedecanoic acid and pyrene lecithin. The excimer-forming lipids pyrenedecanoic acid and pyrene lecithin are used to investigate the dynamic properties and the structural organization of both artificial and biological membranes. Pyrenedecanoic acid and pyrene lecithin have been used to investigate lipid lateral diffusion and lipid exchange processes, as well as phase separation phenomena in artificial and natural membranes. The lipids to be studied and a given amount of excimer-forming probe were dissolved in chloroform and evaporated under a stream of nitrogen to yield a film on the wall of the glass vessel. The excimer formation technique is not restricted to the measurement of lateral mobility in membranes. It can also be used to determine the transverse mobility— that is, the lipid exchange between the lipid layers of one bilayer or between bilayers of different vesicles. It is found that because of the changes in fluidity and a modified solubility of the pyrene probes within different membrane regions, this method can also be applied to the investigation of phase separation phenomena and to lipid-protein interactions.

Excimer-forming lipids in membrane research

Pyrenedecanoic acid and pyrene lecithin are optical probes well suited to investigate lipid bilayer membranes. The method is based on the determination of the formation of excited dimers or excimers. The rate of excimer formation yields information on the dynamic molecular properties of artificial as well as of natural membranes. This article will review applications of the excimer-forming probes. Pyrene lipid probes are used to determine the coefficient of the lateral diffusion in fluid lipid membranes. Results in artificial membranes are comparable to the values obtained in erythrocyte membranes. Moreover, the excimer formation rate is a very sensitive measure of changes in membrane fluidity. Membrane fluidity is an important regulator of membrane functional proteins. For example, there is a correlation between membrane fluidity and enzyme activities of the adenylate cyclase system. The excimer formation technique is not restricted to the measurement of lateral mobility in membranes. It can also be used to determine the transversal mobility, that is, the lipid exchange between the lipid layers of one bilayer or between bilayers of different vesicles. Again, artificial as well as natural membranes can be investigated by this technique. Another important area of investigation in membrane research is the interaction between lipids and proteins. Lipids, in the presence of a protein, show a different dynamic behavior from free lipids. Because of changes in fluidity and a modified solubility of the pyrene probes within different membrane regions, our methods could also be applied to the examination of phase separation phenomena and to lipid-protein interactions.