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).     

Topography and dynamics of receptors for acetylated and malondialdehyde-modified low-density lipoprotein in the plasma membrane of mouse peritoneal macrophages as visualized by colloidal gold in conjunction with surface replicas

The topography and dynamics of receptors for acetylated (acetyl) and malondialdehyde-modified (MDA) low-density lipoprotein (LDL) in the plasma membrane of cultured mouse peritoneal macrophages were investigated using a new technique. Modified LDL labeled with gold particles was used to visualize LDL receptors in the plane of the plasma membrane in platinum-carbon surface replicas of critical point-dried cells. It was found that the native distribution of unoccupied acetyl-LDL receptors is diffuse, whereas unoccupied MDA-LDL receptors are preclustered in the plasma membrane. Competition and double labeling experiments suggest the existence of two distinct classes of receptor sites for acetyl-LDL and MDA-LDL.     

gamma-Aminobutyric acid-containing terminals can be apposed to glycine receptors at central synapses

The distributions of terminals containing gamma-aminobutyric acid (GABA) and of endings apposed to glycine receptors were investigated cytochemically in the ventral horn of the rat spinal cord. For this purpose, a polyclonal antibody raised to recognize glutamic acid decarboxylase (GAD), a synthetic enzyme for GABA, and three monoclonal antibodies (mAb's) directed against the glycine receptor were used. Double immunofluorescence showed that, surprisingly, GAD-positive terminals are closely associated in this system with glycine receptors at all the investigated cells, most of which were spinal motoneurons. Furthermore, double labeling was performed with immunoenzymatic recognition of GAD and indirect marking of mAb's with colloidal gold. With this combined approach, it was found, at the electron microscopic level, that all GAD-positive terminals are in direct apposition with glycine receptors while, on the other hand, not all glycine receptors are in front of GABA-containing boutons. This result is not due to a cross-reactivity of mAb's with GABA receptors as shown by using as a control synapses known to use GABA as a neurotransmitter in the cerebellar cortex. Indeed, no glycine receptor immunoreactivity was detected on Purkinje cells facing basket axon terminals. However, Purkinje neurons can express glycine receptor immunoreactivity at other synaptic contacts. Assuming that the presence of postsynaptic receptors for glycine indicates that this amino acid is used for neurotransmission at a given synapse, our results strongly support the notion that GABA and glycine, two classical inhibitory transmitters, coexist at some central connections. However, such is not always the case; in the cerebellum, Golgi terminals impinging on the dendrites of granule cells are either GAD-positive or face glycine receptors, in a well-segregated manner.     

Ultrastructural localization of the Mr 43,000 protein and the acetylcholine receptor in Torpedo postsynaptic membranes using monoclonal antibodies

Four mouse monoclonal antibodies (mabs) were shown by immunoblotting procedures to recognize the major, basic, membrane-bound Mr 43,000 protein (43K protein) of acetylcholine receptor-rich postsynaptic membranes from Torpedo nobiliana . These mabs and a mab against an extracellular determinant on the acetylcholine receptor were used to localize the two proteins in electroplax (Torpedo californica) and on unsealed postsynaptic membrane fragments at the ultrastructural level. Bound mabs were revealed with a rabbit anti-mouse Ig serum and protein A-colloidal gold. The anti-43K mabs bound only to the cytoplasmic surface of the postsynaptic membrane. The distributions of the receptor and the 43K protein along the membrane were found to be coextensive. Distances between the membrane center and gold particles were very similar for anti-receptor and anti-43K mabs (29 +/- 7 nm and 26 to 29 +/- 7 to 10 nm, respectively). These results show that the 43K protein is a receptor-specific protein having a restricted spatial relationship to the membrane. They thus support models in which the 43K protein is associated with the cytoplasmic domains of the receptor molecule.     

Subcellular localization of alkaline phosphatase in Bacillus licheniformis 749/C by immunoelectron microscopy with colloidal gold

Subcellular distribution of the alkaline phosphatase of Bacillus licheniformis 749/C was determined by an immunoelectron microscopy method. Anti-alkaline phosphatase antibody labeled with 15- to 18-nm colloidal gold particles (gold-immunoglobulin G [IgG] complex) were used for the study. Both the plasma membrane and cytoplasmic material were labeled with the gold-IgG particles. These particles formed clusters in association with the plasma membrane; in contrast, in the cytoplasm the particles were largely dispersed, and only a few clusters were found. The gold-IgG binding was quantitatively estimated by stereological analysis of labeled, frozen thin sections. This estimation of a variety of control samples showed that the labeling was specific for the alkaline phosphatase. Cluster formation of the gold-IgG particles in association with the plasma membrane suggests that existence of specific alkaline phosphatase binding sites (receptors) in the plasma membrane of B. licheniformis 749/C.     

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.     

Observing FcepsilonRI signaling from the inside of the mast cell membrane

We have determined the membrane topography of the high-affinity IgE receptor, FcstraightepsilonRI, and its associated tyrosine kinases, Lyn and Syk, by immunogold labeling and transmission electron microscopic (TEM) analysis of membrane sheets prepared from RBL-2H3 mast cells. The method of Sanan and Anderson (Sanan, D.A., and R.G.W. Anderson. 1991. J. Histochem. Cytochem. 39:1017-1024) was modified to generate membrane sheets from the dorsal surface of RBL-2H3 cells. Signaling molecules were localized on the cytoplasmic face of these native membranes by immunogold labeling and high-resolution TEM analysis. In unstimulated cells, the majority of gold particles marking both FcepsilonRI and Lyn are distributed as small clusters (2-9 gold particles) that do not associate with clathrin-coated membrane. Approximately 25% of FcepsilonRI clusters contain Lyn. In contrast, there is essentially no FcepsilonRI-Syk colocalization in resting cells. 2 min after FcepsilonRI cross-linking, approximately 10% of Lyn colocalizes with small and medium-sized FcepsilonRI clusters (up to 20 gold particles), whereas approximately 16% of Lyn is found in distinctive strings and clusters at the periphery of large receptor clusters (20-100 gold particles) that form on characteristically osmiophilic membrane patches. While Lyn is excluded, Syk is dramatically recruited into these larger aggregates. The clathrin-coated pits that internalize cross-linked receptors bud from membrane adjacent to the Syk-containing receptor complexes. The sequential association of FcstraightepsilonRI with Lyn, Syk, and coated pits in topographically distinct membrane domains implicates membrane segregation in the regulation of FcstraightepsilonRI signaling.     

Probing functionalized gold colloids for single particle tracking experiments

Functionalized submicroscopic particles are currently used to label proteins or lipids at the surface of living cells for single particle tracking experiments. In many cases, it can be of crucial importance for the particle to be anchored to a single molecule. We have addressed this question for the labeling at the plasma membrane of NRK cells of the mu-opioid receptor bearing a T7 epitope at the N-terminus. Using biophysical methods we were able to prepare quasi-monovalent anti-T7 antibody conjugated gold colloids (40 nm diameter) leading to stable and specific binding to the receptor. The rational method, we report here, can be extended to design customized probes for the labeling of various tagged molecules.     

Internalization and degradation of macrophage Fc receptors during receptor-mediated phagocytosis

Macrophage receptors for the Fc domain of immunoglobulin G (IgG) can mediate the efficient binding and phagocytosis of IgG-coated particles. After internalization, phagocytic vacuoles fuse with lysosomes, initiating the degradation of their contents. Using specific monoclonal and polyclonal antireceptor antibodies, we have now analyzed the internalization and fate of Fc receptors during the uptake of IgG- coated erythrocytes and erythrocyte ghosts by mouse peritoneal macrophages. Receptor-mediated phagocytosis led to the selective and largely irreversible removal of Fc receptors (greater than 50%) from the macrophage plasma membrane. The expression of several other plasma membrane proteins (including a receptor for complement), recognized by a series of antimacrophage monoclonal antibodies, was affected only slightly. Interiorized Fc receptors were rapidly and selectively degraded. This was demonstrated by a series of turnover studies in which Fc receptor was immunoprecipitated from lysates of 125I-labeled macrophages. These experiments were made possible by the development of a polyclonal rabbit antiserum, raised against isolated Fc receptor, which recognized the receptor even in the presence of bound ligand. In control cells, the receptor turned over with a t1/2 of approximately 10 h; after phagocytosis, greater than 50% of the receptors were degraded with a t1/2 of less than 2 h. The turnover of other unrelated plasma membrane proteins was unaffected (t1/2 of 18-23 h) under these conditions.     

Epidermal growth factor receptor in chronic bile duct obstructed rats: implications for maintenance of hepatocellular mass.

Changes of the number and properties of the epidermal growth factor (EGF) receptor occur during liver regeneration and may be of importance in the maintenance of hepatocellular mass in liver cirrhosis. We therefore studied the changes in the number and distribution of EGF receptor in the development of liver cirrhosis induced by bile duct ligation. Receptor binding assays demonstrated a marked decrease in the binding capacity of crude plasma membrane fractions from 45 +/- SD 16 to 19 +/- 10 fmol/mg protein (p < 0.001) in control and bile duct ligated livers, respectively while the Kd increased after 3 days of bile duct ligation from 0.5 +/- 0.2 to 1.4 +/- 0.6 nmol/l. Total receptor concentration in the same membrane fractions, as assessed by Western blot analysis, was not changed. The expression of EGF receptor mRNA was reduced to about one third of control levels after 28 days of bile obstruction. Immunohistochemistry, performed using monoclonal antibodies against EGF receptor, showed a strong labeling of cytoplasm (87 +/- 3% positive) and plasma membranes (84 +/- 24%) but no labeling of nuclei in control livers. In bile duct ligated rats, in contrast, cytoplasmic staining was decreased (15 +/- 12%) already after 3 days of bile obstruction; labeling of canalicular membranes and nuclei appeared after 14 days. The shift of EGF receptor from plasma membranes to nuclei supports the notion that EGF receptor is involved in the maintenance of hepatocellular mass in this model of liver cirrhosis. This concept is supported by the finding of decreased mRNA for EGF receptor presumably representing down-regulation as seen in regenerating rat liver.     

High-resolution immunocytochemical labeling of replicas with ultrasmall gold.

The use of 10-15-nm gold probes in freeze-fracture immunocytochemistry sometimes results in poor immunogold labeling. Replica sites are labeled with only one or two gold particles, making it unlikely that the labeling depicts the true distribution of antigen. In this study, the feasibility of using ultrasmall ( approximately 1.4-nm) gold probes for immunocytochemical labeling of replicas was examined. When HLA Class I in neutrophil membrane replicas was labeled with various sized immunogold particles as the secondary detection system, the apparent distribution density was inversely related to the size of the particles (1.4-nm > 5-nm >10-nm >15-nm). Indeed, the density of the apparent distribution of HLA Class I labeled with 1.4-nm gold particles was about sevenfold greater than when labeling was carried out with the 10-nm gold particles. Similar results were obtained with CD16, another neutrophil membrane protein. Silver enhancement was required to visualize the 1.4-nm gold particles, but this procedure did not adversely affect replica membranes. These results suggest that, when followed by silver enhancement, 1.4-nm gold particles are effective probes for achieving high-resolution immunocytochemical labeling of replicas.(J Histochem Cytochem 47:569-573, 1999)     

Morphological evidence for the association of plasma membrane glycoprotein IIb/IIIa with the membrane skeleton in human platelets

We examined the association between glycoprotein (GP) IIb/IIIa, a receptor for fibrinogen, and membrane skeletons in both unstimulated and thrombin-activated human platelets. After a treatment with dithiobis succinimidyl propionate (DTSP), a cross-linker, unstimulated and activated platelets were simultaneously extracted and fixed with a fixing solution containing Triton X-100. Also, the localization of GPIIb/IIIa on the plasma membrane was observed by a preembedding staining method of unextracted platelets. In unstimulated platelets, 20-40% of the whole plasma membrane remained in the detergent-extracted samples. Amorphous structures with 10-70 nm in diameters are distributed at 20 to 100-nm intervals on the surface of plasma membrane. Similar structures also were identified in the intact platelets by the immunocytochemical method. By careful inspection, we found that most of the amorphous structures that contained gold particles were connected to the submembrane zone just beneath the plasma membrane. The submembrane zone was identified as the membrane skeleton because actin was detected in the zone. After activation, detergent-insoluble granules were surrounded by dense networks of microfilaments in the central part of platelets. The filaments were identified as actin and became associated with myosin. These results demonstrate that GPIIb/IIIa on the plasma membrane is connected to the membrane skeleton and suggest that, during activation, actin filaments which extend into the cytoplasm from the membrane skeleton increase and form dense networks around Triton-insoluble granules.     

Immunochemical characterization of brain synaptic membrane glutamate-binding proteins

Two glutamate-binding proteins (71 and 63 kDa) were previously purified from synaptic plasma membranes (Chen, J.-W., Cunningham, M.D., Galton, V., and Michaelis, E. K. (1988) J. Biol. Chem. 263, 417-426). These proteins may play a role in glutamate neurotransmission in brain. Polyclonal antibodies were raised against the denatured glutamate-binding proteins in rabbits, including sets of antibodies against each of the binding proteins. The antibodies reacted specifically against both 71- and 63-kDa proteins. The antibodies recognized the denatured form of the proteins in Western blots and the native state of the proteins in enzyme-linked immunosorbent assays and in immunoaffinity chromatography and extraction procedures. All antibodies labeled most strongly the 71-kDa protein in Western blots, but extracted both proteins from solubilized synaptic membrane preparations. These findings indicate that the two proteins are closely related immunologically but the reactivity on Western blots differs between these two proteins. Immunoextraction of the 71- and 63-kDa proteins led to a approximately 60% decrease in L-[3H]glutamate-binding activity associated with synaptic membrane proteins. Of the brain subcellular fractions examined, the isolated synaptic plasma membranes had the strongest reaction in enzyme-linked immunosorbent assays toward the antiglutamate-binding protein antisera. Electron microscopy combined with gold particle immunohistochemistry revealed the sites labeled by the antibodies as entities present either on the surface or within the postsynaptic membranes and the associated densities of brain nerve ending particles (synaptosomes). Immunohistochemical procedures of gold labeling with silver enhancement of labeled sites revealed selective neuronal labeling in brain regions enriched in glutamate neurotransmitter pathways such as the hippocampus. Labeling was along dendrites and around cell bodies of pyramidal neurons. Based on the pattern of histochemical labeling, the distribution of immune reactivity in synaptic membranes, and the extractions of a major component of membrane glutamate-recognizing proteins by the antibodies, the glutamate-binding proteins must play a role in glutamate neurotransmission.     

Ultrastructural orientation of laminin 5 in the epidermal basement membrane: an updated model for basement membrane organization.

Laminin 5 is a trimeric glycoprotein involved in cell adhesion in the epidermal basement membrane. To determine the precise orientation of laminin 5 in adult human skin, we used plural epitope-specific monoclonal antibodies, a polyclonal antiserum, and postembedding immunogold electron microscopy (IEM). Immunogold labeling distances from the basal keratinocyte plasma membrane (PM) were measured for each gold particle (>200 particles) and the mean distance (nm) calculated. Antibodies included BM165 (recognizing the alpha 3-chain first globular domain) that was measured at 35.40 +/- 2.20 nm from the keratinocyte PM, K140 (recognizing a region adjacent to the beta 3-chain globular domain IV) that measured 45.20 +/- 3.60 nm from the PM, and an anti-laminin 5 polyclonal antiserum that was 43.43 +/- 6.28 nm from the PM. The laminin 5 gamma 2-chain short arm hinge domain was previously localized to the lower lamina densa (LD) at approximately 56.30 +/- 1.65 nm from the keratinocyte PM. Taken together with previous gamma 2-chain data and the distribution of the polyclonal antisera, we estimate that the long axis of laminin 5 is oriented at an angle of approximately 27 degrees from the horizontal lamina lucida (LL)/LD border and propose that the gamma 2-chain lies farthest from the PM. This novel orientation, with the majority of the laminin 5 molecule lying obliquely along the LL/LD border and not perpendicularly, as was first thought, sheds new light on the organization of the basement membrane and likely molecular interactions.     

The presynaptic particle web: ultrastructure, composition, dissolution, and reconstitution

We report the purification of a presynaptic "particle web" consisting of approximately 50 nm pyramidally shaped particles interconnected by approximately 100 nm spaced fibrils. This is the "presynaptic grid" described in early EM studies. It is completely soluble above pH 8, but reconstitutes after dialysis against pH 6. Interestingly, reconstituted particles orient and bind PSDs asymmetrically. Mass spectrometry of purified web components reveals major proteins involved in the exocytosis of synaptic vesicles and in membrane retrieval. Our data support the idea that the CNS synaptic junction is organized by transmembrane adhesion molecules interlinked in the synaptic cleft, connected via their intracytoplasmic domains to the presynaptic web on one side and to the postsynaptic density on the other. The CNS synaptic junction may therefore be conceptualized as a complicated macromolecular scaffold that isostatically bridges two closely aligned plasma membranes.     

Immunocytochemical identification of the human alpha 2-macroglobulin receptor in monocytes and fibroblasts: monoclonal antibodies define the receptor as a monocyte differentiation antigen

The alpha 2-macroglobulin receptor was recently purified from rat liver and human placenta. Three different monoclonal antibodies have now been raised against the human receptor and expression of the 440-kDa receptor protein is demonstrated in human placenta, fibroblasts, liver, and monocytes by immunoblot analysis. Flow cytometric studies showed that anti-alpha 2-macroglobulin receptor monoclonal antibodies bind to 90-100% of the blood monocyte population and not to other blood cells. This defines the alpha 2-macroglobulin receptor as a monocyte differentiation antigen, different from any of the classified leucocyte cluster determinants. Electron microscopic gold immunocytochemistry revealed the subcellular distribution of the receptor in human cultured monocytes and fibroblasts. In these cells, 18-33% of the gold particles were found on the outside of the plasma membrane, and in fibroblasts, especially, in coated invaginations. The intracellular receptors were mainly distributed in vesicles and tubular structures.     

Receptor-bound thrombin is not internalized through coated pits in mouse embryo cells

The localization of thrombin receptors on mouse embryo (ME) cells was examined using electron microscope (EM) immunocytological techniques. ME cells were fixed with formaldehyde, prior to thrombin binding, and thrombin visualized on cell surfaces using affinity-purified antithrombin rabbit antibody and colloidal gold labeled anti-rabbit IgG. Colloidal gold particles were found in clusters on the surface of cells incubated with thrombin. There were approximately seven particles per cluster observed in thin sections with cluster diameters ranging from 70 to 200 nm. These clusters were not observed on cells incubated without thrombin. The total number of particles present on cells incubated with and without thrombin indicate that the colloidal gold labeling is approximately 98% specific for thrombin. Only four colloidal gold particles out of approximately 1,200 were associated with coated pits. Thus the thrombin receptor clusters do not appear to associate with coated membrane regions. To determine whether receptor-bound thrombin was internalized by receptor-mediated endocytosis, ME cells were incubated with 125I-thrombin and examined using EM autoradiography and the trypsin sensitivity of 125I-thrombin which was associated with the cells. In two types of experiments, where thrombin was incubated with cells at 4 degrees C and the temperature increased to 37 degrees C and where initial incubation was at 37 degrees C, the receptor-directed specific internalization proceeded at approximately the same rate as nonspecific internalization. These studies indicate that thrombin that binds to its receptors on ME cells is not rapidly internalized by receptor-mediated endocytosis.     

Annexin V relocates to the periphery of activated platelets following thrombin activation: an ultrastructural immunohistochemical approach

We have previously shown biochemically that the physiological agonist thrombin can cause translocation of endogenous annexin V to a fraction containing all platelet membranes. This paper reports ultrastructural immunohistochemical data revealing that annexin V molecules localize with plasma membranes of blood platelets following thrombin activation. When ultrathin sections of resting platelets were examined by immunogold staining, annexin V was found to be cytosolic, having a generalized distribution throughout the platelet. After thrombin activation, annexin V became peripheral in location and plasmalemma association increased. Morphometric analysis of gold particles shows that annexin V relocates specifically to the plasma membrane and its underlying cytoskeleton following treatment with thrombin. In control platelets 6.1% +/- 0.78 of annexin V is present at the plasma membrane and 15.0% +/- 0.82 in the region corresponding to the membrane cytoskeleton (10-80 nm); after stimulation with 0.5 unit/ml thrombin for 2 min this increased to 16.7% +/- 0.22 and 40.4% +/- 0.53, respectively.     

Solubilization and characterization of adrenal and uterine angiotensin II receptors after photoaffinity labeling

The physical characteristic of the receptors for angiotensin II in dog adrenal cortex and uterus were determined after affinity labeling. 125I-nitro-5-azido-benzoyl-angiotensin II, a photosensitive angiotensin II analogue which retained aldosterone-stimulating activity, was used to couple the octapeptide specifically and irreversibly to its membrane receptors. After solubilization with Triton X-100, the covalent hormone . receptor complex was analyzed by gel filtration and sucrose density gradient centrifugation. Two radioactive species were consistently observed, with calculated Mr values of 126,000 +/- 10,000 and 64,500 +/- 11,000. the elution profiles of solubilized adrenal and uterine particles were almost identical. When the solubilized complexes were subjected to sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, a single radioactive band was detected upon autoradiography, with Mr - 65,000 +/- 6,000 for adrenal cortex and 68,000 +/- 7,000 for myometrium. These results indicate that the receptors for angiotensin II in adrenal cortex and uterus are composed of two subunits of similar molecular weight, and that the common functional properties of the receptors from both tissues are probably related to their similar physicochemical characteristics.     

Colloidal gold-labeled insulin complex. Characterization and binding to adipocytes

Biologically active insulin gold complex was used as an ultrastructural marker to study insulin binding sites, uptake, and internalization in isolated rat adipocytes. The preparation conditions for monodispersed particles, ca. 16 nm in diameter and loaded with approximately 100 insulin molecules, are reported. The complex is stable for at least six weeks. Single particles or small clusters were scattered across the cell membrane. The distribution of unbound receptors seemed to be independent of the extensive system of pre-existing surface connected vesicles in adipocytes. The uptake of particles took place predominantly via non-coated pinocytotic invaginations; clathrin-coated pits did not seem to be important for this process. Lysosome-like structures contained aggregates of 10-15 particles. These data suggest that insulin gold complex is a useful marker for the specific labeling of insulin binding sites.