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D M Fambrough

Publications and source records attributed to D M Fambrough.

At least 73 records · Page 4Linked to original sources
PubMed1984

Extracellular matrix organization in developing muscle: correlation with acetylcholine receptor aggregates.

Monoclonal antibodies recognizing laminin, heparan sulfate proteoglycan, fibronectin, and two apparently novel connective tissue components have been used to examine the organization of extracellular matrix of skeletal muscle in vivo and in vitro. Four of the five monoclonal antibodies are described for the first time here. Immunocytochemical experiments with frozen-sectioned muscle demonstrated that both the heparan sulfate proteoglycan and laminin exhibited staining patterns identical to that expected for components of the basal lamina. In contrast, the remaining matrix constituents were detected in all regions of muscle connective tissue: the endomysium, perimysium, and epimysium. Embryonic muscle cells developing in culture elaborated an extracellular matrix, each antigen exhibiting a unique distribution. Of particular interest was the organization of extracellular matrix on myotubes: the build-up of matrix components was most apparent in plaques overlying clusters of an integral membrane protein, the acetylcholine receptor (AChR). The heparan sulfate proteoglycan was concentrated at virtually all AChR clusters and showed a remarkable level of congruence with receptor organization; laminin was detected at 70-95% of AChR clusters but often was not completely co-distributed with AChR within the cluster; fibronectin and the two other extracellular matrix antigens occurred at approximately 20, 8, and 2% of the AChR clusters, respectively, and showed little or no congruence with AChR. From observations on the distribution of extracellular matrix components in tissue cultured fibroblasts and myogenic cells, several ideas about the organization of extracellular matrix are suggested. (a) Congruence between AChR clusters and heparan sulfate proteoglycan suggests the existence of some linkage between the two molecules, possibly important for regulation of AChR distribution within the muscle membrane. (b) The qualitatively different patterns of extracellular matrix organization over myotubes and fibroblasts suggest that each of these cell types uses somewhat different means to regulate the assembly of extracellular matrix components within its domain. (c) The limited co-distribution of different components within the extracellular matrix in vitro and the selective immune precipitation of each antigen from conditioned medium suggest that each extracellular matrix component is secreted in a form that is not complexed with other matrix constituents.

Animals↗
PubMed1983

Multiple forms of (Na+ + K+)-ATPase in the chicken. Selective detection of the major nerve, skeletal muscle, and kidney form by a monoclonal antibody.

Evidence is presented that monoclonal gamma-immunoglobulin secreted by hybridoma-24 recognizes the sodium and potassium ion-stimulated ATPase of neurons, muscle fibers, and kidney tubule cells in the chicken. The antigen consists of alpha (Mr = 105,000) and beta (Mr = 47,000) subunits. Only the beta subunit bears major oligosaccharide additions (including binding sites for wheat germ agglutinin) to its polypeptide core (Mr = 32,000). The detergent-solubilized antigen sediments as a 7 S complex of alpha beta or alpha beta 2. The antigen has a basolateral distribution in the plasma membrane of renal tubule cells, and in myogenic cell cultures there is up-regulation of the antigen in low potassium medium. Monoclonal antibody-24 specifically recognizes purified gull salt gland (Na+ + K+)-ATPase as well as the major candidate molecule for the (Na+ + K+)-ATPase in enriched preparations from chicken kidney. On cultured myotubes, the number of ouabain-binding sites (4.8 X 10(5)/nucleus) and antigenic sites (4.4 X 10(5)) are approximately equal. However, the antigenic sites on fibroblasts (1.1 X 10(4)) account for only about 4% of the ouabain-binding sites, and there is little or no antibody binding to capillary endothelial cells, Schwann cells, or erythrocytes. Evidence is presented that the antigenic determinant is proteinaceous. It is concluded that at least two antigenically distinct (Na+ + K+)-ATPases exist in the chicken.

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PubMed1983

Characterization of a plasma membrane glycoprotein common to myoblasts, skeletal muscle satellite cells, and glia.

A plasma membrane glycoprotein common to embryonic chick myoblasts and adult chicken skeletal muscle satellite cells is the antigen recognized by monoclonal antibody C3/1. Although traces of the same antigen are present on some muscle-derived fibroblasts, the density of antigenic sites on myoblasts and satellite cells is so high that these cell types can be identified in tissues by immunocytochemical techniques. The antigen is exposed on the surfaces of myogenic cells growing in tissue culture and can be solubilized with detergent. This and other criteria establish that the antigen is a plasma membrane protein. The antigen, purified by affinity techniques, consists of a single type of polypeptide chain which migrates as a relatively broad band of apparent molecular weight 38,000 Da in SDS-polyacrylamide gel electrophoresis. It has a very small sedimentation constant, suggesting that the solubilized form is either monomeric or dimeric. The concentration of antigenic sites increases during myogenesis in vitro; but during maturation the antigenic sites are lost from muscle fibers. Electron microscopic autoradiographic study of adult muscle labeled with iodinated monoclonal antibody demonstrated unequivocally that the antigenic sites in adult muscle are concentrated in the satellite cells. Although selective for myoblasts, immature myotubes and satellite cells in the myogenic lineage, the monoclonal antibody also binds at rather high levels to peripheral Schwann cells and teloglia, to some nonneuronal cells in cultures derived from embryonic spinal cord, to some glial elements of adult chicken brain, and to several cell types in the early embryo.

Animals↗
PubMed1983

Fibronectin expression during myogenesis.

The biosynthesis and localization of fibronectin during chick muscle differentiation are described. This study employed two monoclonal antibodies, one that selectively killed mononucleated cells and one specific for avian fibronectin. These antibodies allowed precise analyses of fibronectin expression in well-defined cultures of myoblasts or myotubes and avoided the complications of exogenous fibronectin and contamination by fibroblasts or unfused myoblasts. Fibronectin synthesis, as a fraction of total protein synthesis, remains constant at 0.3-0.4% before and after myoblast fusion, suggesting that the absolute rate of fibronectin synthesis may increase somewhat when myotubes synthesize and accumulate myofibrillar proteins. The pattern of fibronectin arrangement does change during myogenesis. In myotube cultures, the appearance of pulse-labeled fibronectin at the cell surface and its secretion into the medium begin after a 2-3-h lag period, in contrast to the 30-min lag period observed in fibroblast cultures. This lag between polypeptide biosynthesis and the exteriorization of the new protein is thus a characteristic of each cell type rather than the protein. All of the major secretory proteins of myogenic cells, including fibronectin and collagenous components, share this 2-3-h intracellular transit time.

Animals↗
PubMed1983

(Na+ + K+)-ATPase correlated with a major group of intramembrane particles in freeze-fracture replicas of cultured chick myotubes.

Immunofluorescence microscopy with a fluorescein-labeled monoclonal antibody was used to map the distribution of sodium- and potassium-ion stimulated ATPase [( Na,K]-ATPase) on the surface of tissue-cultured chick skeletal muscle. At this level of resolution it appeared that the (Na,K)-ATPase molecules were distributed nearly uniformly over the plasma membrane. These molecules could be cross-linked by use of the monoclonal antibody followed by a second antibody directed against the monoclonal antibody; the resulting fluorescent pattern was a set of small dots (patches) on the muscle surface. This pattern was stable over several hours, and there was little evidence of interiorization or of coalescence of the patches. Myotubes labeled with immunofluorescence were fixed in glutaraldehyde, cryoprotected with glycerin, then fractured and replicated by standard methods. Replicas of the immunofluorescence-labeled myotubes revealed clusters of intramembrane particles (IMP) only when the immunofluorescent images indicated a patching of the (Na,K)-ATPase molecules. Double antibody cross-linking of antigenic sites on myotubes with each of three other monoclonal antibodies to plasma membrane antigens likewise resulted in patched patterns of immunofluorescence, but in none of these cases were clusters of intramembrane particles found in freeze-fracture replicas. In each case it was shown that the (Na,K)-ATPase molecules were not patched. Other control experiments showed that patching of (Na,K)-ATPase molecules did not cause co-patching of one of the other plasma membrane proteins defined by a monoclonal antibody and did not cause detectable co-clustering of acetylcholine receptors. Detailed mapping showed that there was a one-to-one correspondence between immunofluorescent patches related to the (Na,K)-ATPase and clusters of IMP in a freeze-fracture replica of the same cell. We conclude that the intramembrane particles patched by double antibody cross-linkage of the (Na,K)-ATPase are caused by (Na,K)-ATPase molecules in the fracture plane. Quantification of the IMP indicated that the (Na,K)-ATPase-related particles account for up to 50% of particles evident in the replicas, or up to about 400 particles/micrometers2 of plasma membrane. Particles related to the (Na,K)-ATPase were similar to the average particle size and were as heterodisperse in size as the total population of IMP.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Aggregates of acetylcholine receptors are associated with plaques of a basal lamina heparan sulfate proteoglycan on the surface of skeletal muscle fibers.

Hybridoma techniques have been used to generate monoclonal antibodies to an antigen concentrated in the basal lamina at the Xenopus laevis neuromuscular junction. The antibodies selectively precipitate a high molecular weight heparan sulfate proteoglycan from conditioned medium of muscle cultures grown in the presence of [35S]methionine or [35S]sulfate. Electron microscope autoradiography of adult X. laevis muscle fibers exposed to 125I-labeled antibody confirms that the antigen is localized within the basal lamina of skeletal muscle fibers and is concentrated at least fivefold within the specialized basal lamina at the neuromuscular junction. Fluorescence immunocytochemical experiments suggest that a similar proteoglycan is also present in other basement membranes, including those associated with blood vessels, myelinated axons, nerve sheath, and notochord. During development in culture, the surface of embryonic muscle cells displays a conspicuously non-uniform distribution of this basal lamina proteoglycan, consisting of large areas with a low antigen site-density and a variety of discrete plaques and fibrils. Clusters of acetylcholine receptors that form on muscle cells cultured without nerve are invariably associated with adjacent, congruent plaques containing basal lamina proteoglycan. This is also true for clusters of junctional receptors formed during synaptogenesis in vitro. This correlation indicates that the spatial organization of receptor and proteoglycan is coordinately regulated, and suggests that interactions between these two species may contribute to the localization of acetylcholine receptors at the neuromuscular junction.

Animals↗
PubMed1983

Studies on the Na+-K+ ATPase of skeletal muscle and nerve.

We have used monoclonal antibodies directed against skeletal muscle and neuronal plasma membranes to define surface antigens and have identified one of these antigens as the Na+-K+ ATPase. The monoclonal antibody to the Na+-K+ ATPase has facilitated an exploration of the biosynthesis and assembly of the Na+-K+ ATPase, the distribution of molecules on nerve and muscle, the regulation of expression of Na+-K+ ATPase, and the relation of the Na+-K+ ATPase to the intramembrane particles seen in freeze-fracture replicas. We have obtained new information on the glycosylation sites on the beta-subunit and evidence for antigenically different forms of Na+-K+ ATPase related to differences in proteinaceous aspects of the molecule. Finally, we are attempting to move from the protein level to the nucleic acid level of analysis by using antiserum prepared against the Na+-K+ ATPase to search for encoding cDNAs in an expression library.

Animals↗
PubMed1982

Acetylcholinesterase of human erythrocytes and neuromuscular junctions: homologies revealed by monoclonal antibodies.

Human erythrocyte acetylcholinesterase was used to immunize mice, and hybridomas were generated by fusion of mouse spleen cells with cells of the Sp 2/0 myeloma cell line. Five independently derived hybridoma clones produced antibodies that bound to purified erythrocyte acetylcholinesterase. All of these antibodies crossreacted with human and monkey neuromuscular junctions; immunocytochemical staining patterns corresponded to the distribution of junctional acetylcholinesterase. The monoclonal antibodies fell into at least four categories based on differences in crossreactivity with neuromuscular acetylcholinesterase of rabbit, dog, calf, and guinea pig, and competition tests indicated that the antibodies defined five different antigenic sites on the acetylcholinesterase molecule. It is concluded that there is a high level of homology between the acetylcholinesterases of erythrocytes and neuromuscular junctions.

Acetylcholinesterase↗
PubMed1980

Synthesis, transport and fate of acetylcholinesterase in cultured chick embryos muscle cells.

We have found that approximately one third of the total cell-associated acetylcholinesterase (AChE) is located on the plasma membrane of cultured chick embryo muscle, the remaining two thirds being found within the cells. This cell surface AChE appears to be an integral membrane protein. The surface enzyme is synthesized by the muscle cells in culture and is transported over a 2-3 hr period to the plasma membrane, where it accumulates at the rate of 2-3% of total surface AChE per hour. Once on the plasma membrane the AChE molecules are degraded by a process that exhibits first-order decay kinetics with a half-life of about 50 hr. Under the same experimental conditions, the acetylcholine receptor, a well described muscle cell integral membrane protein, has a half-life of approximately 19 hr. These studies provide the first direct evidence that the numbers of different muscle plasma membrane glycoprotein molecules are determined not only by differential rates of biosynthesis but also by differential rates of degradation. The intracellular AChE constitutes a rapidly turning-over pool of molecules. The rate of synthesis of AChE in culture is approximately 20% of the total cell-associated enzyme per hour, most of which is destined for secretion into the medium. Only a small portion of the newly synthesized AChE is retained on the plasma membrane. The time from synthesis to release of the enzyme is 2-3 hr. Using 3H-DFP to label the newly synthesized AChE, we can also show a quantitative transfer of AChE molecules from the intracellular to the extracellular compartments without any detectable residence time on the plasma membrane. By studying the synthesis transport and externalization of AChE we have defined the intracellular transport pathway and metabolic requirements for secretion in cultured muscle cells. These studies form the basis for a comparison of the metabolism of membrane-bound and secreted glycoproteins from this cell type.

Acetylcholinesterase↗
PubMed1980

Secretion of acetylcholinesterase: relation to acetylcholine receptor metabolism.

Acetylcholinesterase (AChE) and acetylcholine receptors (AChR) are muscle-specific glycoproteins present (AChR) are muscle-specific glycoproteins present in cultured chick embryo muscle cells. The first is found as both a secreted and a membrane-bound enzyme whereas the ACh receptor is strictly an integral membrane protein. We have studied the transport and externalization of these two proteins in the same cells using several compounds known to affect secretory processes: colchicine, tunicamycin and the ionophores X-537A, Nigericin and Monensin. Under all experimental conditions, any change in the rate of AChE secretion was accompanied by an identical change in the rate of ACh receptor incorporation into the plasma membrane. These studies were designed to test directly the hypothesis that secreted and integral membrane proteins are transported together to the plasma membrane. Our results are consistent with a single transport pathway in muscle cells for the externalization of membrane and secreted proteins.

Acetylcholinesterase↗
PubMed1979

Molecular forms of chicken embryo acetylcholinesterase in vitro and in vivo. Isolation and characterization.

The four molecular forms of chick embryo leg muscle acetylcholinesterase have been isolated by velocity sedimentation; their apparent sedimentation coefficients are 19.5 S, 11.5 S, 7.1 S, and 5.4 S. All four forms are glycoproteins, exhibit the same Km for acetylcholine, and are inhibited to the same extent by specific inhibitors of acetyl- and buryrylcholinesterase. Treatment of the 19.5 S form of acetylcholinesterase with trypsin generates an array of molecular forms, several of which have sedimentation coefficients identical with the naturally occurring forms. Collagenase treatment of the 19.5 S acetylcholinesterase results in a somewhat different pattern of acetylcholinesterase forms including a novel 20.6 S form. Only the 19.5 S acetylcholinesterase is sensitive to collagenase treatment. Our results indicate that the several acetylcholinesterase forms share a common catalytic subunit, and suggest that the molecular forms of acetylcholinesterase in the chick represent different ensembles of a common monomer. In culture, the muscle cells contain only the 11.5 and 7.1 S acetylcholinesterase forms; however, they also secrete substantial amounts of enzyme into the medium. These secreted acetylcholinesterases have sedimentation coefficients of 9 S and 15 S. The relative abundance of the different acetylcholinesterase molecular forms changes during muscle development, both in vivo and in vitro, suggesting that the assembly and distribution of this family of membrane glycoproteins is developmentally regulated.

Acetylcholinesterase↗
PubMed1979

Acetylcholine receptor degradation measured by density labeling: effects of cholinergic ligands and evidence against recycling.

The methodology of density labeling of proteins by biosynthetic incorporation of 2H, 13C, 15N-amino acids into newly synthesized polypeptide chains allows the direct measurement of the turnover rate of the acetylcholine receptor in cultured chick skeletal muscle. In this study, receptors synthesized in medium containing 2H, 13C, 15N-amino acids were resolved from 1H, 12C, 14N-receptors by velocity sedimentation in sucrose-deuterium oxide gradients, and their proportions were determined by computer analysis of the gradient profiles. The kinetics of turnover of acetylcholine receptors are identical for developing chick muscle fibers grown in medium containing 2H, 13C, 15N-amino acids or 1H, 12C, 14N-amino acids, and the high degree of substitution of normal aminoacyl residues by 2H, 13C, 15N-residues does not affect the turnover rate of the denser receptor. Comparison of the turnover rates in continuous and pulse-labeling experiments gave independent confirmation of these results. The application of a potent, essentially irreversible blocking agent, alpha-bungarotoxin, increases the median lifetime of receptors from 17 hr for the native unbound receptor to 22 hr for the alpha-bungarotoxin-receptor complex. As predicted, the total number of alpha-bungarotoxin binding sites increased in the continued presence of alpha-bungarotoxin due to extension of receptor lifetime. To determine whether other cholinergic agents affect the turnover rate of the receptor, measurements were performed on cultures grown in the presence of 10(-4) M d-tubocurare or 10(-4) M carbachol, a reversible antagonist and a reversible agonist, respectively, of the nicotinic acetylcholine receptor. The receptor degradation rates of the drug-treated cells were identical to control values. The total number of alpha-bungarotoxin binding sites was reduced by 30% in the presence of carbachol, indicating that this agent affects the rate of synthesis of the acetylcholine receptor. Data formerly interpreted as suggesting a cycling of receptor-containing plasma membrane out of and back into the sarcolemma are now understood to reflect the alteration in receptor lifetime upon complexing with alpha-bungarotoxin. The intracellular "hidden" receptor sites were found to remain inside the myotubes and thus do not signify an intracellular pool of recycling plasma membrane.

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