Illuminating approaches to cellular physiology.
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Animals↗
Biomedical subjects
R J Bloch
Publications and source records attributed to R J Bloch.
Synapse development. Up the junction.
alpha-Dystroglycan has been suggested to be the receptor for agrin, an extracellular glycoprotein that signals postsynaptic differentiation at the neuromuscular junction, but it may not have the necessary specificity.
Agrin↗
Clustered acetylcholine receptors have two levels of organization in Xenopus muscle cells.
We studied the organization of acetylcholine receptor (AChR) clusters by shearing cultured Xenopus muscle cells with a stream of buffer, and preparing rotary replicas of the exposed cytoplasmic surface of the sarcolemma. AChR clusters contained numerous particles that protruded from the sarcolemma and formed an irregular array composed of discrete aggregates. AChR were located within these particle aggregates, as shown by comparison of the replicas to labeling by fluorescent alpha-bungarotoxin, and by immunogold cytochemistry with antibodies specific for the receptor. The aggregates were cross-linked by a dense network of 7 nm filaments that replicated with the banded pattern characteristic of actin microfilaments. The organization of receptors into the small aggregates was independent of the organization of these aggregates into clusters, as alkaline extraction removed the microfilament network and disrupted the irregular array of particle aggregates, but did not disperse individual receptors from the aggregates. We conclude that two levels of interactions organize AChR clusters in Xenopus muscle cells: short-range interactions that assemble individual AChR into small aggregates, and long-range interactions, perhaps mediated by actin microfilaments, that anchor the aggregates into larger clusters.
Alkalies↗
Association of acetylcholine receptors with peripheral membrane proteins: evidence from antibody-induced coaggregation.
Acetylcholine receptors (AChR) are associated with several peripheral membrane proteins that are concentrated on the cytoplasmic face of the plasma membrane at the neuromuscular junction, and at aggregates of AChR that form in vitro. We tested the linkage among these proteins by inducing microaggregation of AChR, then determining if a given peripheral membrane protein accumulated with the receptors in microaggregates. In most experiments, we used isolated membrane fragments that are rich in AChR and accessible to antibodies against intracellular antigens. We showed that the 43 kD receptor-associated protein always aggregated with AChR, whether microaggregation was driven by antibodies to the 43 kD protein, or to the receptor itself. Antibodies to the 58 kD receptor-associated protein also always aggregated the 58 kD protein with the receptor. Our results are consistent with a model for AChR-rich membrane in which the 43 kD and 58 kD proteins are both closely associated with the AChR. When we induced microaggregation in intact muscle cells with anti-AChR antibodies, our results were less definitive. The 43 kD receptor-associated protein microaggregated with AChR, but the 58 kD protein was not especially enriched at AChR microaggregates. We discuss the advantages of using isolated AChR-rich membrane fragments to study the association of AChR with peripheral membrane proteins.
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Na(+)-Ca2+ exchanger in arteries: identification by immunoblotting and immunofluorescence microscopy.
Antibodies raised against dog cardiac Na(+)-Ca2+ exchanger were employed to determine the presence and distribution of the exchanger in arterial smooth muscle (ASM) cells. The antiserum cross-reacted with protein bands of approximately 70, 120, and 150-160 kDa from the membranes of ASM cells, as well as heart sarcolemma. A cardiac Na(+)-Ca2+ exchanger cDNA probe hybridized to 7-kilobase (kb) mRNA from myocytes of the mesenteric artery. Thus ASM cells possess a "cardiac type" Na(+)-Ca2+ exchanger. The relative amounts of 7-kb mRNA and antigen detected on Northern and Western blots, respectively, however, indicate that vascular myocytes contain much less of this transporter than do cardiac myocytes. Immunofluorescence studies on cultured arterial myocytes suggest that the exchanger molecules are organized in reticular patterns over the cell surfaces. A similar pattern is observed when cells are stained for sarcoplasmic reticulum (SR) Ca(2+)-ATPase. This raises the possibility that the exchanger in the plasmalemma of arterial myocytes may be associated, perhaps functionally as well as structurally, with the underlying SR. The antiserum also cross-reacted with endothelial cell membranes, but labeling was lighter and more diffuse than in the myocytes.
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Evidence for transmembrane anchoring of extracellular matrix at acetylcholine receptor clusters.
Clusters of nicotinic acetylcholine receptor (AChR) in cultured rat myotubes are organized into rectilinear arrays of receptor-rich and receptor-poor domains. Extracellular matrix (ECM) molecules, including fibronectin, heparan sulfate proteoglycan, laminin, and type IV collagen, codistribute with AChR in these clusters. We have examined the stability of this association. We disrupted the AChR clusters in intact myotubes with sodium azide, an energy metabolism inhibitor, and with culture medium free of Ca2+. We also altered or extracted proteins from detergent-isolated AChR clusters by treating with buffers of low ionic strength or alkaline pH or with insoluble chymotrypsin. Each of these treatments dispersed AChR clusters and, simultaneously, caused fibronectin, heparan sulfate proteoglycan, laminin, and type IV collagen to disperse from AChR-rich strips of membrane. Control experiments indicated that insoluble chymotrypsin had no direct effect on the ECM at AChR clusters. It did, however, remove spectrin and the receptor-associated 58-kDa protein from the cytoplasmic surface of receptor clusters. Thus, the ECM at AChR clusters is disrupted by an agent acting at the cytoplasmic surface of the membrane. We discuss the possibility that both AChR and ECM are bound to a common membrane skeleton and the implications this may have for synaptogenesis.
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Phospholipid asymmetry in acetylcholine receptor clusters.
We modified the lipids of rat myotubes in tissue culture to determine the transmembrane orientation of aminophospholipids in clusters of acetylcholine receptors (AChR). Trinitrobenzenesulfonic acid and N-hydroxysuccinimidobiotin were used to modify the amino groups of phospholipids. Reaction conditions were selected to prevent penetration of the chemical probes into the cell interior. Fluorescence microscopy was used to confirm that the probes remained impermeant. Analysis of aminophospholipids associated with clusters isolated from chemically modified cells and comparisons to results of chemically modifying isolated acetylcholine receptor clusters indicated that at least 77% of plasma membrane aminophospholipids was located in the interior leaflet of the lipid bilayer. We address the possibility that aminophospholipids on the inner lipid leaflet may contribute to the association between the cytoskeleton and the membrane at AChR clusters.
Animals↗
Structures linking microfilament bundles to the membrane at focal contacts.
We used quick-freeze, deep-etch, rotary replication and immunogold cytochemistry to identify a new structure at focal contacts. In Xenopus fibroblasts, elongated aggregates of particles project from the membrane to contact bundles of actin microfilaments. Before terminating, a single bundle of microfilaments interacts with several aggregates that appear intermittently over a distance of several microns. Aggregates are enriched in proteins believed to mediate actin-membrane interactions at focal contacts, including beta 1-integrin, vinculin, and talin, but they appear to contain less alpha-actinin and filamin. We also identified a second, smaller class of aggregates of membrane particles that contained beta 1-integrin but not vinculin or talin and that were not associated with actin microfilaments. Our results indicate that vinculin, talin, and beta 1-integrin are assembled into distinctive structures that mediate multiple lateral interactions between microfilaments and the membrane at focal contacts.
Actin Cytoskeleton↗
Dystrophin in a membrane skeletal network: localization and comparison to other proteins.
We studied the location, relative abundance, and stability of dystrophin in clusters of ACh receptors (AChRs) isolated from primary cultures of neonatal rat myotubes. Although variable amounts of dystrophin were found at receptor clusters, dystrophin was always associated with organized, receptor-rich domains (AChR domains). Dystrophin was occasionally seen in focal contact domains, but never in clathrin-coated domains. Dystrophin was also present in a diffuse, punctate distribution in regions of myotube membrane that did not contain AChR clusters. Immunogold labeling at the ultrastructural level localized dystrophin in a spectrin-rich filamentous network closely applied to the cytoplasmic surface of the cell membrane at AChR domains. Dystrophin was not associated with overlying actin filaments. Semiquantitative immunofluorescence studies indicated that dystrophin was present in relatively small amounts in these preparations, with only one molecule of dystrophin for every approximately 5 AChR, 43 kDa and 58 kDa molecules, and for every approximately 20-35 beta-spectrin molecules. Clusters were disrupted, but the total amount of dystrophin was not significantly reduced, when myotubes were incubated with sodium azide or in Ca(2+)-free medium, and when isolated AChR clusters were extracted at low ionic strength, at high pH, or in 6 M urea. These treatments extract other peripheral membrane proteins from AChR clusters. Labeling for dystrophin was completely eliminated when clusters were incubated with chymotrypsin, however. Thus, dystrophin forms part of a membrane skeleton at AChR clusters, but it is more difficult to remove than other proteins in the network. This suggests that dystrophin attaches to cluster membrane in a unique way.
Animals↗
Treatments that extract the 43K protein from acetylcholine receptor clusters modify the conformation of cytoplasmic domains of all subunits of the receptor.
The nicotinic acetylcholine receptor (AChR) of Torpedo electric organ and vertebrate skeletal muscle is closely associated with a Mr 43,000 protein (43K). In this study, we have examined the effects on the AChR of treatments which remove the 43K protein. We used semiquantitative fluorescence techniques to measure the binding of antibodies to clustered AChR in cultured rat myotubes. We found that labeling by antibodies to the cytoplasmic portions of each of the four receptor polypeptides increased significantly upon extraction of the 43K protein. Labeling by an antibody to an extracellular epitope of the alpha subunits was not affected by removal of the 43K protein, suggesting that changes were restricted to the cytoplasmic domains of the AChR. Increases in labeling by antibodies were more limited following protease treatment, which removes most cytoskeletal structures but leaves the 43K protein bound to the membrane. Competition between an antibody to the beta subunit and an antibody to the gamma and delta subunits suggests that the cytoplasmic portion of the AChR still retains a degree of native structure in the absence of the 43K protein. Our results suggest that, although some of these changes may be due to simply exposing additional epitopes on the AChR, the cytoplasmic portions of all the subunits of the AChR undergo significant conformational changes upon extraction of the 43K protein.
Animals↗
Clusters of neural cell adhesion molecule at sites of cell-cell contact.
I have examined the distribution of neural cell adhesion molecule (N-CAM) in cultured C2 myogenic cells and other cell lines to determine if N-CAM accumulates at sites of cell-cell contact. C2 cells growing in log phase display large clusters of neural cell adhesion molecule where they contact each other. These clusters are remarkably stable, do not form at cell-substrate contacts, and appear not to be enriched in a number of other cytoskeletal, membrane, or extracellular proteins. Thus, N-CAM clusters form preferentially in response to cell-cell contact and are specifically enriched in N-CAM. As C2 cultures mature and differentiate, clusters persist at contacts between aligning myoblasts and between myotubes, consistent with a role in myogenesis. N-CAM is also enriched at cell-cell contacts in cultures of PC12, NRK, and CHO cells. These cells have significant amounts of N-CAM as detected on immunoblots. Clusters are not seen in L929 cells, which do not have detectable amounts of N-CAM. Coculture of these cells with C2 cells results in the clustering of N-CAM at heterologous contacts between C2 cells and NRK, CHO, or PC12 cells, but not between C2 cells and L929 cells. These results suggest that N-CAM specifically accumulates where N-CAM-bearing cells contact one another. Clustering of N-CAM may be an important step in strengthening intercellular adhesion.
Animals↗
Dystrophin colocalizes with beta-spectrin in distinct subsarcolemmal domains in mammalian skeletal muscle.
Duchenne's muscular dystrophy (DMD) is caused by the absence or drastic decrease of the structural protein, dystrophin, and is characterized by sarcolemmal lesions in skeletal muscle due to the stress of contraction. Dystrophin has been localized to the sarcolemma, but its organization there is not known. We report immunofluorescence studies which show that dystrophin is concentrated, along with the major muscle isoform of beta-spectrin, in three distinct domains at the sarcolemma: in elements overlying both I bands and M lines, and in occasional strands running along the longitudinal axis of the myofiber. Vinculin, which has previously been found at the sarcolemma overlying the I bands and in longitudinal strands, was present in the same three structures as spectrin and dystrophin. Controls demonstrated that the labeling was intracellular. Comparison to labeling of the lipid bilayer and of the extracellular matrix showed that the labeling for spectrin and dystrophin is associated with the intact sarcolemma and is not a result of processing artifacts. Dystrophin is not required for this lattice-like organization, as similar domains containing spectrin but not dystrophin are present in muscle from the mdx mouse and from humans with Duchenne's muscular dystrophy. We discuss the possibility that dystrophin and spectrin, along with vinculin, may function to link the contractile apparatus to the sarcolemma of normal skeletal muscle.
Amino Acid Sequence↗
Immunofluorescence localization of the Na-Ca exchanger in heart cells.
We investigated the localization of the Na-Ca exchanger in fixed, isolated heart cells from rat and guinea pig using immunocytochemical methods with epifluorescence and confocal microscopy. We found that the Na-Ca exchanger is distributed throughout all membranes in contact with the extracellular space, including the sarcolemma, the transverse tubules (T-tubules), and the intercalated disks. Microscopic nonuniformities in the fluorescent labeling appear to reflect varying views of the membranes containing Na-Ca exchanger protein. Confocal thin-section imaging reveals a regular grid of discrete foci of fluorescence, which represent Na-Ca exchanger in T-tubules viewed en face. These foci are 1.80 +/- 0.01 microns apart from sarcomere to sarcomere and are aligned with the Z-line. Along each Z-line, these foci are spaced at 1.22 +/- 0.11-microns intervals. Longitudinal sections of the sarcolemma-T-tubule junction show a comblike appearance, with T-tubules extending inward from the heavily labeled sarcolemma. Our finding that the Na-Ca exchanger is widely distributed over the cell surface may provide further insight into the role of Na-Ca exchange in the heart.
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Presynaptic localization of sodium/calcium exchangers in neuromuscular preparations.
Calcium ions play a critical role in neurotransmitter release. The cytosolic Ca2+ concentration ([Ca2+]cyt) at nerve terminals must therefore be carefully controlled. Several different mechanisms, including a plasmalemmal Na/Ca exchanger, are involved in regulating [Ca2+]cyt. We employed immunofluorescence microscopy with polyclonal antiserum raised against dog cardiac sarcolemmal Na/Ca exchanger to determine the distribution of the exchanger in vertebrate neuromuscular preparations. Our data indicate that the Na/Ca exchanger is concentrated at the neuromuscular junctions of the rat diaphragm. The exchanger is also present in the nonjunctional sarcolemma, but at a much lower concentration than in the junctional regions. Denervation markedly lowers the concentration of the exchanger in the junctional regions; this implies that the Na/Ca exchanger is concentrated in the presynaptic nerve terminals. In Xenopus laevis nerve and muscle cell cocultures, high concentrations of the exchanger are observed along the neurites as well as at the nerve terminals. The high concentrations of Na/Ca exchanger at presynaptic nerve terminals in vertebrate neuromuscular preparations suggest that the exchanger may participate in the Ca-dependent regulation of neurotransmitter release. The Na/Ca exchanger is also abundant in developing neurites and growth cones, where it may also be important for Ca2+ homeostasis.
Animals↗
Regulation of vascular smooth muscle contractility: roles of the sarcoplasmic reticulum (SR) and the sodium/calcium exchanger.
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Animals↗
Ultrastructure of the human erythrocyte cytoskeleton and its attachment to the membrane.
We attached paraformaldehyde-fixed human erythrocyte ghosts to coated coverslips and sheared them to expose the cytoskeleton. Quick-freeze, deep-etch, rotary-replication, or tannic acid/osmium fixation and plastic embedding revealed the cytoskeleton as a dense network of intersecting straight filaments. Previous negative stain studies on spread skeletons found 5-6 spectrin tetramers intersecting at each actin oligomer, with an estimated 250 such intersections/microns 2 of membrane. In contrast, we found 3-4 filaments at each intersection and approximately 400 intersections/microns 2 of membrane. Immunogold labeling verified that the filaments were spectrin, but their lengths (29-37 nm) were approximately one-third that of extended spectrin dimers. The length and diameter of the filaments were sufficient to accommodate spectrin dimers, but not spectrin tetramers. Our results suggest that, in situ, spectrin dimers may associate as hexamers and octamers, rather than tetramers. We present several explanations that can reconcile our observations on intact cytoskeletons with previous reports on spread material. Extracting sheared ghosts with solutions of low ionic strength removed the cytoskeleton to reveal projections from the cytoplasmic surface of the membrane. These projections contained band 3, as shown by immunogold labeling, and they aggregated to a similar extent as intramembrane particles (IMP) when the cytoskeleton was removed, suggesting a direct relationship between these structures. Quantification indicated a stoichiometry of 2 IMP for each cytoplasmic projection. Cytoplasmic projections presumably contain other proteins besides band 3 since further treatment with high ionic strength solutions extracts peripheral proteins and reduces the diameter of projections by approximately 3 nm.
Actin Cytoskeleton↗
The lipid bilayer of acetylcholine receptor clusters of cultured rat myotubes is organized into morphologically distinct domains.
We have studied the composition and organization of the lipid bilayer at the large, substrate-associated clusters of acetylcholine receptors (AChR) that form in cultured rat myotubes. These clusters have a characteristic morphology consisting of alternating linear domains of AChR-rich and AChR-poor membrane, the latter involved in attaching the myotube to the substrate. We partially purified AChR clusters by extracting cultured rat myotubes with the cholesterol-specific detergent, saponin. The lipid bilayer of the cluster preparation was analyzed biochemically and the substructure of the bilayers was studied morphologically using the fluorescent probes, dansyl polymyxin B, and 3,3'-di(C12H25 and C18H37) indocarbocyanine iodide (C12- and C18-diI). Our results demonstrate that preparations of AChR clusters have a lipid composition biochemically similar to that of the surrounding plasma membrane. Morphologically, however, the lipid bilayer appears to be arranged into domains that resemble the interdigitating pattern seen for the AChR. This distinctive lipid organization is not due to the use of saponin to purify clusters, as we obtained similar results with clusters isolated by physically shearing myotube cultures. The domain-like organization of the bilayer at clusters is disrupted by treatments that disperse AChR clusters in intact myotubes or that remove peripheral membrane proteins from isolated clusters. This suggests that such proteins may contribute to the organization of the bilayer. Two additional factors may also contribute to the organization of the bilayer: physical constraints imposed by sites of substrate attachment and, to a lesser extent, "boundary" lipid associated with AChR.
Actins↗
Cytoplasmic components of acetylcholine receptor clusters of cultured rat myotubes: the 58-kD protein.
A 58-kD protein, identified in extracts of postsynaptic membrane from Torpedo electric organ, is enriched at sites where acetylcholine receptors (AChR) are concentrated in vertebrate muscle (Froehner, S. C., A. A. Murnane, M. Tobler, H. B. Peng, and R. Sealock. 1987. J. Cell Biol. 104:1633-1646). We have studied the 58-kD protein in AChR clusters isolated from cultured rat myotubes. Using immunofluorescence microscopy we show that the 58-kD protein is highly enriched at AChR clusters, but is also present in regions of the myotube membrane lacking AChR. Within clusters, the 58-kD protein codistributes with AChR, and is absent from adjacent membrane domains involved in myotube-substrate contact. Semiquantitative fluorescence measurements suggest that molecules of the 58-kD protein and AChR are present in approximately equal numbers. Differential extraction of peripheral membrane proteins from isolated AChR clusters suggests that the 58-kD protein is more tightly bound to cluster membrane than is actin or spectrin, but less tightly bound than the receptor-associated 43-kD protein. When AChR clusters are disrupted either in intact cells or after isolation, the 58-kD protein still codistributes with AChR. Clusters visualized by electron microscopy after immunogold labeling and quick-freeze, deep-etch replication show that, within AChR clusters, the 58-kD protein is sharply confined to AChR-rich domains, where it is present in a network of filaments lying on the cytoplasmic surface of the membrane. Additional actin filaments overlie, and are attached to, this network. Our results suggest that within AChR domains of clusters, the 58-kD protein lies between AChR and the receptor-associated 43-kD protein, and the membrane-skeletal proteins, beta-spectrin, and actin.
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