Membrane mechanisms for signal transduction: the coupling of the meso-scale raft domains to membrane-skeleton-induced compartments and dynamic protein complexes

Virtually all biological membranes on earth share the basic structure of a two-dimensional liquid. Such universality and peculiarity are comparable to those of the double helical structure of DNA, strongly suggesting the possibility that the fundamental mechanisms for the various functions of the plasma membrane could essentially be understood by a set of simple organizing principles, developed during the course of evolution. As an initial effort toward the development of such understanding, in this review, we present the concept of the cooperative action of the hierarchical three-tiered meso-scale (2-300 nm) domains in the plasma membrane: (1) actin membrane-skeleton-induced compartments (40-300 nm), (2) raft domains (2-20 nm), and (3) dynamic protein complex domains (3-10nm). Special attention is paid to the concept of meso-scale domains, where both thermal fluctuations and weak cooperativity play critical roles, and the coupling of the raft domains to the membrane-skeleton-induced compartments as well as dynamic protein complexes. The three-tiered meso-domain architecture of the plasma membrane provides an excellent perspective for understanding the membrane mechanisms of signal transduction.     

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).     

Calcium Promotes the Formation of Syntaxin 1 Mesoscale Domains through Phosphatidylinositol 4,5-Bisphosphate

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) is a minor component of total plasma membrane lipids, but it has a substantial role in the regulation of many cellular functions, including exo- and endocytosis. Recently, it was shown that PI(4,5)P₂ and syntaxin 1, a SNARE protein that catalyzes regulated exocytosis, form domains in the plasma membrane that constitute recognition sites for vesicle docking. Also, calcium was shown to promote syntaxin 1 clustering in the plasma membrane, but the molecular mechanism was unknown. Here, using a combination of superresolution stimulated emission depletion microscopy, FRET, and atomic force microscopy, we show that Ca²⁺ acts as a charge bridge that specifically and reversibly connects multiple syntaxin 1/PI(4,5)P₂ complexes into larger mesoscale domains. This transient reorganization of the plasma membrane by physiological Ca²⁺ concentrations is likely to be important for Ca²⁺-regulated secretion.     

Unconfined lateral diffusion and an estimate of pericellular matrix viscosity revealed by measuring the mobility of gold-tagged lipids

Nanovid (video-enhanced) microscopy was used to determine whether lateral diffusion in the plasma membrane of colloidal gold-tagged lipid molecules is confined or is unrestricted. Confinement could be produced by domains within the plane of the plasma membrane or by filamentous barriers within the pericellular matrix. Fluorescein- phosphatidylethanolamine (F1-PE), incorporated into the plasma membranes of cultured fibroblasts, epithelial cells and keratocytes, was labeled with 30-nm colloidal gold conjugated to anti-fluorescein (anti-F1). The trajectories of the gold-labeled lipids were used to compute diffusion coefficients (DG) and to test for restricted motion. On the cell lamella, the gold-labeled lipids diffused freely in the plasma membrane. Since the gold must move through the pericellular matrix as the attached lipid diffuses in the plasma membrane, this result suggests that any extensive filamentous barriers in the pericellular matrix are at least 40 nm from the plasma membrane surface. The average diffusion coefficients ranged from 1.1 to 1.7 x 10(-9) cm2/s. These values were lower than the average diffusion coefficients (DF) (5.4 to 9.5 x 10(-9) cm2/s) obtained by FRAP. The lower DG is partially due to the pericellular matrix as demonstrated by the result that heparinase treatment of keratocytes significantly increased DG to 2.8 x 10(-9) cm2/s, but did not affect DF. Pericellular matrix viscosity was estimated from the frictional coefficients computed from DG and DF and ranged from 0.5 to 0.9 poise for untreated cells. Heparinase treatment of keratocytes decreased the apparent viscosity to approximately 0.1 poise. To evaluate the presence of domains or barriers, the trajectories and corresponding mean square displacement (MSD) plots of gold-labeled lipids were compared to the trajectories and MSD plots resulting from computer simulations of random walks within corrals. Based on these comparisons, we conclude that, if there are domains limiting the diffusion of F1-PE, most are larger than 5 microns in diameter.     

delta-Opioid receptors exhibit high efficiency when activating trimeric G proteins in membrane domains

Low-density membrane fragments (domains) were separated from the bulk of plasma membranes of human embryonic kidney (HEK)293 cells expressing a delta-opioid (DOP) receptor-Gi1alpha fusion protein by drastic homogenization and flotation on equilibrium sucrose density gradients. The functional activity of trimeric G proteins and capacity of the DOP receptor to stimulate both the fusion protein-linked Gi1alpha and endogenous pertussis-toxin sensitive G proteins was measured as d-Ala2, d-Leu5-enkephalin stimulated high-affinity GTPase or guanosine-5'-[gamma-35S]triphosphate ([35S]GTPgammaS) binding. The maximum d-Ala2-d-Leu5 enkephalin (DADLE)-stimulated GTPase was two times higher in low-density membrane fragments than in bulk of plasma membranes; 58 and 27 pmol/mg/min, respectively. The same difference was obtained for [35S]GTPgammaS binding. Contrarily, the low-density domains contained no more than half the DOP receptor binding sites (Bmax = 6.6 pmol/mg versus 13.6 pmol/mg). Thus, when corrected for expression levels of the receptor, low-density domains exhibited four times higher agonist-stimulated GTPase and [35S]GTPgammaS binding than the bulk plasma membranes. The regulator of G protein signaling RGS1, enhanced further the G protein functional activity but did not remove the difference between domain-bound and plasma membrane pools of G protein. The potency of the agonist in functional studies and the affinity of specific [3H]DADLE binding to the receptor were, however, the same in both types of membranes - EC50 = 4.5 +/- 0.1 x 10(-8) and 3.2 +/- 1.4 x 10(-8) m for GTPase; Kd = 1.2 +/- 0.1 and 1.3 +/- 0.1 nm for [3H]DADLE radioligand binding assay. Similar results were obtained when sodium bicarbonate was used for alkaline isolation of membrane domains. By contrast, detergent-insensitive membrane domains isolated following treatment of cells with Triton X100 exhibited no DADLE-stimulated GTPase or GTPgammaS binding. Functional coupling between the DOP receptor and cognate G proteins was also blocked by high-energy ultrasound and repeated freezing-thawing. Our data indicate, for the first time, that membrane domains isolated using 'detergent-free' procedures exhibit higher efficiency of coupling between a G protein-coupled receptor and its corresponding G protein(s) than bulk plasma membranes. Detergent-extraction diminishes these interactions, even when the receptor and G proteins are physically tethered together.     

Detection of lipid domains in model and cell membranes by fluorescence lifetime imaging microscopy of fluorescent lipid analogues

The presence of lipid domains in cellular membranes and their characteristic features are still an issue of dividing discussion. Several recent studies implicate lipid domains in plasma membranes of mammalian cells as short lived and in the submicron range. Measuring the fluorescence lifetime of appropriate lipid analogues is a proper approach to detect domains with such properties. Here, the sensitivity of the fluorescence lifetime of1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]-hexanoyl]-sn-glycero-3-phospholipid (C6-NBD-phospholipid) analogues has been employed to characterize lipid domains in giant unilamellar vesicles (GUVs) and the plasma membrane of mammalian cells by fluorescence lifetime imaging (FLIM). Fluorescence decay of C6-NBD-phosphatidylcholine is characterized by a short and long lifetime. For GUVs forming microscopically visible lipid domains the longer lifetime in the liquid disordered (ld) and the liquid ordered (lo) phase was clearly distinct, being approximately 7 ns and 11 ns, respectively. Lifetimes were not sensitive to variation of cholesterol concentration of domain-forming GUVs indicating that the lipid composition and physical properties of those lipid domains are well defined entities. Even the existence of submicroscopic domains can be detected by FLIM as demonstrated for GUVs of palmitoyloleoyl phosphatidylcholine/N-palmitoyl-d-sphingomyelin/cholesterol mixtures. A broad distribution of the long lifetime was found for C6-NBD-phosphatidylcholine inserted in the plasma membrane of HepG2 and HeLa cells centered around 11 ns. FLIM studies on lipid domains forming giant vesicles derived from the plasma membrane of HeLa cells may suggest that a variety of submicroscopic lipid domains exists in the plasma membrane of intact cells.     

Functional and morphological correlates of connexin50 expressed in Xenopus laevis oocytes

Electrophysiological and morphological methods were used to study connexin50 (Cx50) expressed in Xenopus laevis oocytes. Oocytes expressing Cx50 exhibited a new population of intramembrane particles (9.0 nm in diameter) in the plasma membrane. The particles represented hemichannels (connexin hexamers) because (a) their cross-sectional area could accommodate 24 +/- 3 helices, (b) when their density reached 300-400/microm2, they formed complete channels (dodecamers) in single oocytes, and assembled into plaques, and (c) their appearance in the plasma membrane was associated with a whole-cell current, which was activated at low external Ca2+ concentration ([Ca2+]o), and was blocked by octanol and by intracellular acidification. The Cx50 hemichannel density was directly proportional to the magnitude of the Cx50 Ca2+-sensitive current. Measurements of hemichannel density and the Ca2+-sensitive current in the same oocytes suggested that at physiological [Ca2+]o (1-2 mM), hemichannels rarely open. In the cytoplasm, hemichannels were present in approximately 0.1-microm diameter "coated" and in larger 0.2-0.5-microm diameter vesicles. The smaller coated vesicles contained endogenous plasma membrane proteins of the oocyte intermingled with 5-40 Cx50 hemichannels, and were observed to fuse with the plasma membrane. The larger vesicles, which contained Cx50 hemichannels, gap junction channels, and endogenous membrane proteins, originated from invaginations of the plasma membrane, as their lumen was labeled with the extracellular marker peroxidase. The insertion rate of hemichannels into the plasma membrane (80, 000/s), suggested that an average of 4,000 small coated vesicles were inserted every second. However, insertion of hemichannels occurred at a constant plasma membrane area, indicating that insertion by vesicle exocytosis (60-500 microm2 membranes/s) was balanced by plasma membrane endocytosis. These exocytotic and endocytotic rates suggest that the entire plasma membrane of the oocyte is replaced in approximately 24 h.     

Visualization of protein compartmentation within the plasma membrane of living yeast cells

Different distribution patterns of the arginine/H+ symporter Can1p, the H+ plasma membrane ATPase Pma1p, and the hexose transport facilitator Hxt1p within the plasma membrane of living Saccharomyces cerevisiae cells were visualized using fluorescence protein tagging of these proteins. Although Hxt1p-GFP was evenly distributed through the whole cell surface, Can1p-GFP and Pma1p-GFP were confined to characteristic subregions in the plasma membrane. Pma1p is a well-documented raft protein. Evidence is presented that Can1p, but not Hxt1p, is exclusively associated with lipid rafts, too. Double labeling experiments with Can1p-GFP- and Pma1p-RFP-containing cells demonstrate that these proteins occupy two different nonoverlapping membrane microdomains. The size of Can1p-rich (Pma1p-poor) areas was estimated to 300 nm. These domains were shown to be stable in growing cells for >30 min. To our knowledge, this is the first observation of a cell polarization-independent lateral compartmentation in the plasma membrane of a living cell.     

Sterol carrier protein-2 selectively alters lipid composition and cholesterol dynamics of caveolae/lipid raft vs nonraft domains in L-cell fibroblast plasma membranes

Although the functional significance of caveolae/lipid rafts in cellular signaling and cholesterol transfer is increasingly recognized, almost nothing is known regarding the lipids, cholesterol dynamics, and factors regulating these properties in caveolae/lipid rafts as opposed to nonlipid raft domains of the plasma membrane. The present findings demonstrate the utility of con-A affinity chromatography for simultaneous isolation of caveolae/lipid raft and nonlipid raft domains from plasma membranes of L-cell fibroblasts. These domains differed markedly in both protein and lipid constituents. Although caveolae/lipid rafts were enriched in total lipid, cholesterol, and phospholipid as well as other markers for these domains, the cholesterol/phospholipid ratio of caveolae/lipid rafts did not differ from that of nonlipid rafts. Nevertheless, spontaneous sterol transfer was 7-12-fold faster from caveolae/lipid raft than nonlipid raft domains of the plasma membrane. This was largely due to the near absence of exchangeable sterol in the nonlipid rafts. SCP-2 dramatically and selectively enhanced sterol transfer from caveolae/lipid rafts, but not from nonlipid rafts. Finally, overexpression of SCP-2 significantly altered the sterol dynamics of caveolae/lipid rafts to facilitate retention of cholesterol within the cell. These results established for the first time that (i) caveolae/lipid rafts, rather than the nonlipid raft domains, contain significant levels of rapidly transferable sterol, consistent with their role in spontaneous sterol transfer from and through the plasma membrane, and (ii) SCP-2 selectively regulates how caveolae/lipid rafts, but not nonlipid raft domains, mediate cholesterol trafficking through the plasma membrane.     

Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells.

The movements of E-cadherin, epidermal growth factor receptor, and transferrin receptor in the plasma membrane of a cultured mouse keratinocyte cell line were studied using both single particle tracking (SPT; nanovid microscopy) and fluorescence photobleaching recovery (FPR). In the SPT technique, the receptor molecules are labeled with 40 nm-phi colloidal gold particles, and their movements are followed by video-enhanced differential interference contrast microscopy at a temporal resolution of 33 ms and at a nanometer-level spatial precision. The trajectories of the receptor molecules obtained by SPT were analyzed by developing a method that is based on the plot of the mean-square displacement against time. Four characteristic types of motion were observed: (a) stationary mode, in which the microscopic diffusion coefficient is less than 4.6 x 10(-12) cm2/s; (b) simple Brownian diffusion mode; (c) directed diffusion mode, in which unidirectional movements are superimposed on random motion; and (d) confined diffusion mode, in which particles undergoing Brownian diffusion (microscopic diffusion coefficient between 4.6 x 10(-12) and 1 x 10(-9) cm2/s) are confined within a limited area, probably by the membrane-associated cytoskeleton network. Comparison of these data obtained by SPT with those obtained by FPR suggests that the plasma membrane is compartmentalized into many small domains 300-600 nm in diameter (0.04-0.24 microns2 in area), in which receptor molecules are confined in the time scale of 3-30 s, and that the long-range diffusion observed by FPR can occur by successive movements of the receptors to adjacent compartments. Calcium-induced differentiation decreases the sum of the percentages of molecules in the directed diffusion and the stationary modes outside of the cell-cell contact regions on the cell surface (which is proposed to be the percentage of E-cadherin bound to the cytoskeleton/membrane-skeleton), from approximately 60% to 8% (low- and high-calcium mediums, respectively).     

Pore size of swelling-activated channels for organic osmolytes in Jurkat lymphocytes, probed by differential polymer exclusion

The present study explores the impact of the molecular size on the permeation of low-molecular-weight polyethylene glycols (PEG200-1500) through the plasma membrane of Jurkat cells under iso- and hypotonic conditions. To this end, we analyzed the cell volume responses to PEG-substituted solutions of different osmolalities (100-300 mOsm) using video microscopy. In parallel experiments, the osmotically induced changes in the membrane capacitance and cytosolic conductivity were measured by electrorotation (ROT). Upon moderate swelling in slightly hypotonic solutions (200 mOsm), the lymphocyte membrane remained impermeable to PEG300-1500, which allowed the cells to accomplish regulatory volume decrease (RVD). During RVD, lymphocytes released intracellular electrolytes through the swelling-activated pathways, as proved by a decrease of the cytosolic conductivity measured by electrorotation. RVD also occurred in strongly hypotonic solutions (100 mOsm) of PEG600-1500, whereas 100 mOsm solutions of PEG300-400 inhibited RVD in Jurkat cells. These findings suggest that extensive hypotonic swelling rendered the cell membrane highly permeable to PEG300-400, but not to PEG600-1500. The swelling-activated channels conducting PEG300-400 were inserted into the plasma membrane from cytosolic vesicles via swelling-mediated exocytosis, as suggested by an increase of the whole cell capacitance. Using the hydrodynamic radii R_h of PEGs (determined by viscosimetry), the observed size-selectivity of membrane permeation yielded an estimate of ∼ 0.74 nm for the cut-off radius of the swelling-activated channel for organic osmolytes. Unlike PEG300-1500, the smallest PEG (PEG200, R_h = 0.5 nm) permeated the lymphocyte membrane under isotonic conditions thus leading to a continuous isotonic swelling. The results are of interest for biotechnology and biomedicine, where PEGs are widely used for cryopreservation of cells and tissues.     

Membrane domains in lymphocytes - from lipid rafts to protein scaffolds

Lateral compartmentalization of the plasma membrane into domains is a key feature of immune cell activation and subsequent immune effector functions. Here, we will review the high diversity of membrane domains, ranging from elementary lipid rafts, envisioned as dynamic and small domains (in the tens of nm), to relatively stable μm-scale membrane domains, which form the immunologic synapse of T lymphocytes. We will discuss the relationship between these different types of plasma membrane domains and how raft lipid- and protein-controlled interactions and cell biological processes cooperate to generate functional domains that mediate lymphocyte activity.     

Effect of polylysine on transformations and permeability of negative vesicular membranes

Small (40-60 nm in diameter) and large (300-350 nm) negative vesicles were complexed with a cationic polypeptide, poly-L-lysine (PL). Laser microelectrophoresis experiments showed that in small vesicles rendered anionic with the addition of cardiolipin (CL(2-)), only the CL(2-) in the outer leaflet is involved in the complexation with PL. Calorimetric and other data demonstrate that the binding of PL to the membrane surface causes domains ("rafts") of CL(2-) to form in the outer leaflet, and it is these domains that electrostatically bind the polymer. The kinetics of transmembrane permeation of doxorubicin (Dox, a fluorescent anti-tumor drug) was monitored with and without PL binding to the outer surface of the vesicles. It was found that PL mediates the permeation of Dox into the vesicle interior. In the absence of PL, the Dox molecule (possessing an amino group of pK(a)=8.6) binds to the anionic vesicles in the protonated form and, consequently, suffers an impaired mobility through the membrane. On the other hand, when the PL covers the vesicle surface, Dox passes though the membrane with greater ease. The effects of salt and polyanion on the stability of PL-vesicle complexes and the PL-mediated Dox permeation are also discussed.     

Different subcellular localization and phosphoinositides binding of insulin receptor substrate protein pleckstrin homology domains

Insulin evokes diverse biological effects through receptor-mediated tyrosine phosphorylation of the insulin receptor substrate (IRS) proteins. Here, we show that, in vitro, the IRS-1, -2 and -3 pleckstrin homology (PH) domains bind with different specificities to the 3-phosphorylated phosphoinositides. In fact, the IRS-1 PH domain binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3), the IRS-2 PH domain to phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P2), and the IRS-3 PH domain to phosphatidylinositol 3-phosphate. When expressed in NIH-IR fibroblasts and L6 myocytes, the IRS-1 and -2 PH domains tagged with green fluorescent protein (GFP) are localized exclusively in the cytoplasm. Stimulation with insulin causes a translocation of the GFP-IRS-1 and -2 PH domains to the plasma membrane within 3-5 min. This translocation is blocked by the phosphatidylinositol 3-kinase (PI 3-K) inhibitors, wortmannin and LY294002, suggesting that this event is PI 3-K dependent. Interestingly, platelet-derived growth factor (PDGF) did not induce translocation of the IRS-1 and -2 PH domains to the plasma membrane, indicating the existence of specificity for insulin. In contrast, the GFP-IRS-3 PH domain is constitutively localized to the plasma membrane. These results reveal a differential regulation of the IRS PH domains and a novel positive feedback loop in which PI 3-K functions as both an upstream regulator and a downstream effector of IRS-1 and -2 signaling.     

Three-dimensional structures of the mammalian multidrug resistance P-glycoprotein demonstrate major conformational changes in the transmembrane domains upon nucleotide binding

P-glycoprotein is an ATP-binding cassette transporter that is associated with multidrug resistance and the failure of chemotherapy in human patients. We have previously shown, based on two-dimensional projection maps, that P-glycoprotein undergoes conformational changes upon binding of nucleotide to the intracellular nucleotide binding domains. Here we present the three-dimensional structures of P-glycoprotein in the presence and absence of nucleotide, at a resolution limit of approximately 2 nm, determined by electron crystallography of negatively stained crystals. The data reveal a major reorganization of the transmembrane domains throughout the entire depth of the membrane upon binding of nucleotide. In the absence of nucleotide, the two transmembrane domains form a single barrel 5-6 nm in diameter and about 5 nm deep with a central pore that is open to the extracellular surface and spans much of the membrane depth. Upon binding nucleotide, the transmembrane domains reorganize into three compact domains that are each 2-3 nm in diameter and 5-6 nm deep. This reorganization opens the central pore along its length in a manner that could allow access of hydrophobic drugs (transport substrates) directly from the lipid bilayer to the central pore of the transporter.     

Caveolae,transmembrane signalling and cellular transformation

Caveolae are -50–100 nm membrane micro-invaginations associated with the plasma membrane of a wide variety of cells. Although they were first identified in transmission electron micrographs -40 years ago, their exact function(s) has remained controversial. Two well-established functions include: (1) the transcytosis of both large and small molecules across capillary endothelial cells and (2) the utilization of GPI-linked proteins to concentrate small molecules in caveolae for translocation to the cytoplasm (termed potocytosis). Recently, interest in a ‘third’ proposed caveolar function, namely transmembrane signalling, has been revived by the identification of caveolin — a transformation-dependent v-Src substrate and caveolar marker protein — and the isolation of caveolin-rich membrane domains from cultured cells. Here we will discuss existing evidence that suggests a role for caveolae in signalling events.     

The spatial and temporal dynamics of pleckstrin homology domain binding at the plasma membrane measured by imaging single molecules in live mouse myoblasts

Pleckstrin homology (PH) domains act to target proteins to the plasma membrane and intracellular vesicles by binding to specific phosphoinositol phospholipids. We have investigated the binding kinetics of PH domains found in the tail region of the molecular motor, myosin X. Using total internal reflection fluorescence microscopy, we observed binding and release of individual PH domains fused to green fluorescent protein at the plasma membrane of living cells. Individual spots of light corresponding to single fluorescently tagged molecules were imaged onto a sensitive camera system, and digital image processing was then used to identify each fluorophore and store its trajectory in time and space. The PH domains bound with an apparent on-rate of 0.03 microm(-1) microm(-2) s(-1) and a detachment rate constant of 0.05 s(-1). The average residency time of the domains at the plasma membrane was about 20s. We found very limited movement of the membrane-bound PH domains in the mouse myoblast cells that we studied. This implies that the PH domains must either be attached to the cytoskeleton or corralled in a lipid compartment. Localization of the PH domains together with their rapid detachment rate is probably important in controlling the response of myosin X to signaling events and in regulating its cellular function.     

FM dyes label sterol-rich plasma membrane domains and are internalized independently of the cytoskeleton in characean internodal cells

We applied the endocytic markers FM1-43, FM4-64 and filipin to internodal cells of the green alga Chara corallina. Both FM dyes stained stable, long-living plasma membrane patches with a diameter of up to 1 microm. After 5 min, FM dyes labeled cortical, trembling structures up to 500 nm in size. After 15 min, FM dyes localized to endoplasmic organelles up to 1 microm in diameter, which migrated actively along actin bundles or participated in cytoplasmic mass streaming. After 30-60 min, FM fluorescence appeared in the membrane of small, endoplasmic vacuoles but not in that of the central vacuole. Some of the FM-labeled organelles were also stained by neutral red and lysotracker yellow, indicative of acidic compartments. Filipin, a sterol-specific marker, likewise labeled plasma membrane domains which co-localized with the FM patches. However, internalization of filipin could not be observed. KCN, cytochalasin D, latrunculin B and oryzalin had no effect on size, shape and distribution of FM- and filipin-labeled plasma membrane domains. Internalization of FM dyes was inhibited by KCN but not by drugs which interfere with the actin or microtubule cytoskeleton. Our data indicate that the plasma membrane of characean internodal cells contains discrete domains which are enriched in sterols and probably correspond to clusters of lipid rafts. The inhibitor experiments suggest that FM uptake is active but independent of actin filaments, actin polymerization and microtubules. The possible function of the sterol-rich, FM labeled plasma membrane areas and the significance of actin-independent FM internalization (via endocytosis or energy-dependent flippases) are discussed.     

Molecular Weight and Subunit Composition of the Sensitive to GABA-Ergic Ligands Cl<Superscript>−</Superscript>/HCO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack>-Stimulated Mg<Superscript>2+</Superscript>-ATPase from Plasma Membrane of Rat Brain

The molecular weight and subunit composition of Cl-,HCO3(-)- and picrotoxin-stimulated Mg2+-ATPase from rat brain plasma membrane solubilized in sodium deoxycholate were studied by gel filtration chromatography. The enzyme activity eluted from a Sephacryl S-300 column in a single peak associated with a protein of molecular weight approximately 300 kD and a Stokes radius of 5.4 nm. The enzyme-enriched fraction, concentrated and denatured by SDS, migrated through a Sephacryl S-200 column as three peaks with molecular weights of approximately 57, 53, and 45 kD. SDS-PAGE also showed three major protein bands with molecular weights of about 57, 53, and 48 kD. The molecular weight and subunit composition of the Cl- and HCO3(-)-stimulated Mg2+-ATPase from neuronal membrane of rat brain are similar with the molecular properties of GABA(A)-benzodiazepine receptor complex from mammalian brain but are different from those of P-type transport ATPases.     

Dominant portion of thyrotropin-releasing hormone receptor is excluded from lipid domains. Detergent-resistant and detergent-sensitive pools of TRH receptor and Gqalpha/G11alpha protein

Some G protein-coupled receptors might be spacially targetted to discrete domains within the plasma membrane. Here we assessed the localization in membrane domains of the epitope-tagged, fluorescent version of thyrotropin-releasing hormone receptor (VSV-TRH-R-GFP) expressed in HEK293 cells. Our comparison of three different methods of cell fractionation (detergent extraction, alkaline treatment/sonication and mechanical homogenization) indicated that the dominant portion of plasma membrane pool of the receptor was totally solubilized by Triton X-100 and its distribution was similar to that of transmembrane plasma membrane proteins (glycosylated and non-glycosylated forms of CD147, MHCI, CD29, CD44, transmembrane form of CD58, Tapa1 and Na,K-ATPase). As expected, caveolin and GPI-bound proteins CD55, CD59 and GPI-bound form of CD58 were preferentially localized in detergent-resistant membrane domains (DRMs). Trimeric G proteins G(q)alpha/G(11)alpha, G(i)alpha1/G(i)alpha2, G(s)alphaL/G(s)alphaS and Gbeta were distributed almost equally between detergent-resistant and detergent-solubilized pools. In contrast, VSV-TRH-R-GFP, Galpha, Gbeta and caveolin were localized massively only in low-density membrane fragments of plasma membranes, which were generated by alkaline treatment/sonication or by mechanical homogenization of cells. These data indicate that VSV-TRH-R-GFP as well as other transmembrane markers of plasma membranes are excluded from TX-100-resistant, caveolin-enriched membrane domains. Trimeric G protein G(q)alpha/G(11)alpha occurs in both DRMs and in the bulk of plasma membranes, which is totally solubilized by TX-100.