Surface Of Myotubes Research Articles

Laminin induces acetylcholine receptor aggregation on cultured myotubes and enhances the receptor aggregation activity of a neuronal factor

The effect of several basement membrane components on the aggregation of acetylcholine (ACh) receptors on cultured myotubes was studied. Cultures were incubated for 16 to 24 hr with laminin, a heparan sulfate proteoglycan, collagen types IV and V, or fibronectin, alone, or together with medium conditioned by NG108-15 neuroblastoma X glioma hybrid cells (NCM). The number of ACh receptor aggregates per myotube was assayed by fluorescence microscopy of cultures stained with tetramethylrhodamine-labeled alpha-bungarotoxin. Laminin induced ACh receptor aggregation on primary rat myotubes and on myotubes formed by G8-1 clonal rat muscle cells. Laminin enhanced the receptor-aggregating activity of NCM in a concentration-dependent manner (0.6 to 6.0 micrograms/ml) and the number of aggregates formed in the presence of laminin and NCM together was greater than the sum of the aggregates induced by NCM and laminin separately. The aggregation factor in NCM is probably not laminin, since less than 10 ng/ml of laminin-like immunoreactivity was detected in NCM, and antiserum against laminin blocked the effects of laminin but had little effect on NCM aggregation activity. Collagen type V enhanced the receptor aggregation activity of NCM, but less strongly than laminin, and had little or no effect by itself. The other basement membrane components did not induce receptor aggregation or enhance the effect of NCM. Experiments in which ACh receptors were labeled before exposure of cultures to NCM and laminin indicated that laminin enhanced the rearrangement of receptors at the cell surface. Immunofluorescence microscopy indicated that laminin binds to the myotubes within 30 min and forms patches on the cell surface over a period of hours. Laminin bound to the myotube surface enhanced receptor aggregation as well as laminin continuously present in the culture medium. The results suggest the possibility that laminin could enhance the receptor aggregation activity of a neuronal factor(s) released at the developing neuromuscular junction.

Characterization and distribution of acetylcholine receptors and acetylcholinesterase during electric organ development in Torpedo marmorata

The changes that occur in the distribution and properties of the nicotinic acetylcholine receptor and acetylcholinesterase during the development of the electric organ of Torpedo marmorata have been investigated. At early stages of development, both proteins are distributed diffusely over the myotube surface and with differentiation of the myotubes into electrocytes, they become increasingly restricted to the ventral cell surface. This process occurs before axons contact the electrocytes. The concentrations of the acetylcholine receptor and acetylcholinesterase remain at rather low and stable levels during these developmental changes. The acetylcholine receptor concentration begins to increase rapidly as soon as electromotor axons begin to contact the electrocytes. No significant differences in the subunit composition, affinity for d-tubocurarine, isoelectric point or immunochemical properties of the embryonic acetylcholine receptors were detected when they were compared to the receptors of the adult electric organ. The onset of receptor accumulation occurs before the increase in the amount of 17 S acetylcholinesterase, suggesting that increases in acetylcholine receptor and in acetylcholinesterase are not regulated by the same mechanisms. The various molecular forms of acetylcholinesterase undergo characteristic changes during development. Sequential extraction of the esterase forms indicates that their interaction with cellular compartments changes during development. The solubility properties of the esterase forms suggest that most of the 17 S and 13 S acetylcholinesterase become strongly associated with cellular components via ionic interactions and that a hydrophobic 6 S form begins to accumulate at later embryonic stages when the number of mature presynaptic terminals is beginning to show its rapid phase of increase. The results show that events concerning the distribution and some properties of essential components of the electromotor synapse occur during early embryonic development before synaptogenesis, whereas the sequential accumulation of acetylcholine receptor and esterase begins after the axons start contacting the electrocytes. This suggests that the ingrowing nerves exert some regulative influence on the metabolic state of the electrocytes during development.

Differentiation--dependent changes of the nicotinic acetylcholine receptor and other synapse-associated proteins.

Developmentally regulated changes were followed by analyzing the appearance of synapse-associated proteins of the electric organ of Torpedo marmorata. At early stages of development acetylcholine receptor and acetylcholine esterase are distributed diffusely over the myotube surface. With differentiation they become increasingly restricted to the central cell surface. This process occurs before axons contact the electrocytes. As soon as axons begin to contact electrocytes one finds a rapid increase in acetylcholine receptor concentration, which is shortly followed by an increased synthesis of 17 S AChE. The final stages of the synapse formation coincide with increasing amounts of a hydrophobic 6 S AChE and increasing amounts of Mr43000 polypeptides, suggesting that the appearance of these components is linked to the maturation of receptor and synapse function.

Synthesis of type IV collagen and laminin in cultures of skeletal muscle cells and their assembly on the surface of myotubes

The synthesis of two components of the basal lamina, laminin and type IV collagen, and their extracellular deposition on the surface of myotubes was studied in cultures of embryonic mouse and quail skeletal muscle cells and in the rat myoblast cell line L6. Production of type IV collagen and laminin by myoblasts and muscle fibroblasts was demonstrated by incorporation of radioactive amino acids into proteins and by immunoprecipitation with specific antibodies and electrophoretic analysis of labeled proteins. Immunofluorescence staining experiments revealed strong intracellular reactions with antibodies to laminin and type IV collagen in mononucleated myogenic and fibrogenic cells. Cells of fibroblast-like morphology showed a more intense staining than bipolar, spindle-shaped cells which perhaps represented postmitotic myoblasts. Myotubes did not show detectable intracellular staining. The formation of a basal lamina on myotubes was indicated by the deposition of laminin and type IV collagen on the surface of myotubes as viewed by immunofluorescence examination of unfixed cells. Staining for extracellular laminin was stronger in mass cultures than in myogenic clones, suggesting that secretion and deposition of components of the basal lamina on the myotube surface are complex processes which may involve cooperation between myogenic and fibrogenic cells.

Extracts of electric lobe and electric organ from Torpedo californica increase the total number as well as the number of aggregates of chick myotube acetylcholine receptors

Saline extracts prepared from the electric lobe, the electromotor nerves, and the electric organ (the electromotor system) of Torpedo californica increase the number of ACh receptors on uninnervated chick myotubes in culture, while extracts from T. californica liver or skeletal muscle do not. The extracts also increase the ACh sensitivity of treated myotubes, indicating that newly synthesized receptors are functional. The active substance(s) is heat sensitive but not trypsin sensitive. Gel filtration on Bio-Gel P-150 shows that the activity is associated with a peak of low (less than 5,000-dalton) molecular weight activity. Labeling studies with rhodamine-conjugated alpha-bungarotoxin show that, in addition to their effect on receptor number, these extracts also cause aggregation of prelabeled ACh receptors on the myotube surface.

Aggregation of acetylcholine receptors in nerve-muscle cocultures is decreased by inhibitors of collagen production

Coculturing of rat embryonic muscle cells with spinal cord explants resulted in the formation of large numbers of acetylcholine receptor aggregates on the myotube surface, compared to those found on muscle cells grown in the absence of nervous tissue. Remarkably fewer receptor aggregates were formed when, upon addition of nerve explants, these cocultures were treated with either cis-hydroxyproline (a specific inhibitor of collagen production) or collagenase. The possibility is raised that collagen participates in the aggregation of acetylcholine receptors and in synapse formation.

A monoclonal antibody which binds to the surface of chick brain cells and myotubes: Cell selectivity and properties of the antigen

A monoclonal antibody has been obtained which binds to the cell surface of cultured chick myotubes and retinal and tectal neurons but not fibroblasts, myoblasts and embryonic liver cells. Indirect immunocytochemistry reveals that antigen is present in all layers of the chick neural retina. The antibody therefore recognizes an antigen common to most, if not all, chick neurons. The antigen has been identified by staining SDS gels with [ 125I]monoclonal antibody and appears to be a polydisperse collection of polypeptides with molecular weights centered about 250 kdalton.

Neural cell adhesion molecule is on embryonic muscle cells and mediates adhesion to nerve cells in vitro.

The interaction of nerves with muscles, particularly at neuromuscular junctions, is one of the most extensively studied areas of neurophysiology. From the point of view of developmental biology, however, little is known about the cellular and molecular events that lead to formation of these junctions1,2. In previous investigations of cell-cell adhesion in the chick embryo, we identified a molecule on the surface of essentially all nerve cells, called neural cell adhesion molecule (N-CAM)3,4, that appears to be a ligand in the formation of cell-cell bonds5. N-CAM is involved in a variety of developmental processes including neurite fasciculation and the formation of plexiform and cell layers in retinal tissue6–8. We report here that N-CAM antigenic determinants are also present on chick embryo skeletal muscle cells. This observation raised the possibility that N-CAM is involved in adhesion between nerve and muscle cells. In model systems set up to explore this possibility, we observed that myoblasts will adhere in vitro to nerve cells from retina, and that this adhesion can be inhibited by F(ab′) fragments of antibodies against N-CAM. In addition, membrane vesicles obtained from chick embryo brain or artificial vesicles5 reconstituted from lipid and affinity-purified N-CAM9 bound to the surface of myoblasts and myotubes; this binding was also blocked by anti-N-CAM F(ab′). These results suggest that the N-CAM adhesion system can facilitate nerve-muscle as well as nerve-nerve interactions in vitro.

Staining of acetylcholine receptors at the adult mouse and embryonic chick neuromuscular junctions using erabutoxin b and the antibody against acetylcholine receptors.

Acetylcholine receptors (AChRs) at the adult mouse and embryonic chick neuromuscular junctions were visualized by using erabutoxin b (Eb), one of the short-chain neurotoxins from Laticauda semifasciata, and antibody against AChRs of the electric organ from Narke japonica. Visualization of AChRs was performed by the following three methods: (1) staining with rhodaminelabeled Eb (TMR-Eb), (2) staining with horseradish peroxidase-labeled Eb (HRP-Eb), and (3) indirect immunofluorescence microscopy using the antibody against purified AChRs. At the light microscopic level, TMR-Eb offered the most simple and rapid procedure for producing clear images. The HRP-Eb method was suitable for viewing the distribution of AChRs at the ultrastructural level. These procedures revealed that adult neuromuscular junctions had a nearly uniform distribution of AChRs at their postsynaptic membrane, whereas embryonic ones possessed aggregations of small discrete regions of high AChR concentration. Similar regions of AChRs were observed on the nonjunctional myotube surface.

Embryonic growth and innervation of rat skeletal muscles. III. Neural regulation of junctional and extra-junctional acetylcholine receptor clusters.

The number and distribution of acetylcholine (ACh) receptors on muscle cells was studied during development of normal, paralysed and aneural embryonic rat diaphragm muscles. (i) ACh receptors initially are dispersed over the surface of rat embryo myotubes. At day 15| of gestation junctional receptor clusters (‘J-clusters’) form in a well ordered band across the midline of the diaphragm muscle; these also form in denervated and paralysed muscles. At about day 18 of gestation additional ‘EJ-clusters’ develop to either side of the midpoint of treated muscles. (ii) If a nerve terminal is present, J-clusters increase in length with time. The time course of generation of new endplates calculated from frequency distributions of J-cluster lengths accurately predicts the muscle growth curve established from muscle fibre counts. (iii) The mean length of J-clusters in paralysed muscles was greater than in controls, due to small new-formed clusters failing to appear. In muscles allowed to recover from paralysis the mean length was less, due to a preponderance of small, new-formed clusters. These observations show that development of new endplates, which is thought to reflect the development of new muscle cells, is halted in paralysed muscles, and recovery from paralysis is associated with the generation of many new endplates. (iv) J-clusters appeared, but failed to grow, in aneural muscles. In muscles denervated during the later stages of gestation, analysis of the distribution of J-cluster lengths shows that new clusters failed to appear, and existing clusters showed little or no increase in length after the time of removal of the nerve. (v) EJ-clusters form by aggregation of dispersed receptors, and their mean length increases with time. They do not appear to be stable entities, and are removed within 2 d of recovery from paralysis. In paralysed muscles, with both J-clusters and EJclusters present, only J-clusters attract nerve sprouts or become innervated. (vi) A curve is derived showing development of the total number of synaptic terminals in a muscle. This number increases during days 13-18 of gestation, reaching a peak of about 170 % of the adult value during dl8 and d l9 of gestation. There are two episodes of terminal elimination, one during days 19-21 of gestation, and another about 2 weeks postnatally. During the first postnatal week the number of terminals remains constant at about 140% of the adult number, while the average number of inputs per fibre goes down and the number of muscle fibres increases. (vii) Innervation is essential for muscle development. Motoneurons cannot regulate the number of muscle fibres by requiring a simple one-to-one relation between nerve terminal and muscle fibre, and if their role is regulatory as well as supportive of muscle development then some more complex relationship between nerve terminals and developing myotubes must be postulated.

The effect of calcium on acetylcholine receptor synthesis

The level of cytoplasmic calcium has been proposed to act as a regulator of acetylcholine receptor synthesis (Betz, H., and J. P. Changeaux (1979) Nature 278: 749-751). However, there is little known about the effect of altered calcium levels on the metabolism of the acetylcholine receptor. We have investigated the effect of decreased extracellular calcium on the metabolism of acetylcholine receptors in cultured rat myotubes. Our results show that the acetylcholine receptor levels on the surface of myotubes were decreased 25 to 30% following overnight incubation in calcium-deficient medium. In contrast, creatine phosphokinase activity levels and total protein synthesis were unaffected. Calcium depletion did not change the rate of receptor degradation significantly (0.037 hr-1, compared to 0.033 hr-1 for control cells) but dramatically decreased the rate of incorporation of new acetylcholine receptors into the plasma membrane. The time course for incorporation of new acetylcholine receptors into the plasma membrane of calcium-depleted cells was similar to control cells treated with cycloheximide, suggesting that de novo receptor synthesis was inhibited. These results indicate that intracellular calcium levels and acetylcholine receptor synthesis are not related in a simple reciprocal fashion and suggest that the regulation of acetylcholine receptor levels involves more than one intracellular compartment of calcium.

Endogenous lectins in chickens and slime molds: transfer from intracellular to extracellular sites.

Endogenous lectins in both cellular slime molds and chicken tissues have been localized primarily intracellularly, in contrast with the predominantly extracellular localization of the glycoproteins, glycolipids, and glycosaminoglycans with which they might interact. Here we present evidence that lectins in both of these organisms may be externalized and become associated with the cell surface and/or extracellular materials. In chicken intestine, chicken-lactose-lectin-II is shown to be localized in the secretory granules of the goblet cells, along with mucin, and to be secreted onto the intestinal surface. In embryonic muscle, chicken-lactose-lectin-I is shown to be externalized with differentiation, ultimately becoming localized on the surface of myotubes and in the extracellular spaces. In a cellular slime mold, Dictyostelium purpureum, externalization of lectin is elicited by either polyvalent glycoproteins that bind the small amount of endogenous cell surface lectin, or by slime mold or plant lectins that bind unoccupied complementary cell surface oligosaccharides. These results suggest that externalization of endogenous lectin may be a response to specific external signals. We conclude that lectins are frequently held in intracellular reserves awaiting release for specific external functions.

Crosslinkage and visualization of acetylcholine receptors on myotubes with biotinylated alpha-bungarotoxin and fluorescent avidin.

A biotinylated derivative of alpha-bungarotoxin and tetramethylrhodamine-labeled avidin were employed to fluorescence label the acetylcholine receptors (AcChoR) on the surface of rat myotubes in primary culture. Because of the multivalency of both the biotinylated bungarotoxin and the avidin, this treatment extensivey crosslinks the AcChoR. AcChoR crosslinking immobilizes more than 90% of the normally laterally mobile AcChoR as verified by the fluorescence photobleaching recovery technique; it also redistributes the AcChoR into visible micropatches. Biotinylated alpha-bungarotoxin/avidin-induced AcChoR crosslinking greatly accelerates the rate of internalization of surface AcChoR; this rapid internalization affects both the normally immobile AcChoR in areas of diffuse distribution and the normally immobile AcChoR in preexisting patches. The peculiar pattern of fluorescent avidin binding to AcChoR patches previously bound with biotinylated bungarotoxin suggests that almost all AcChoR patches are in very close contact (< 70 A) with the glass substrate. AcChoR immobilization leads to a partial immobilization of concanavalin A receptors in the myotube membrane.

Localization of acetylcholine receptors by means of horseradish peroxidase-alpha-bungarotoxin during formation and development of the neuromuscular junction in the chick embryo.

The localization of acetylcholine receptors (AChR) in the surface of developing myogenic cells of the chick embryo anterior and posterior latissimus dorsi muscles in relation to the process of innervation has been studied at the ultrastructural level utilizing a horseradish peroxidase-alpha-bungarotoxin conjugate. Localized concentrations of AChR were found in small regions 0.1-0.4 micron in width on the surface of myogenic cells of 10- to 14-d-old muscles. Surface specializations consisting of an external coating of extraneous material and an internal accumulation of dense material are associated with the plasma membrane in the regions of AChR concentration. As the muscle fibers are innervated, reactive surface patches are found at the region of contact of the growing nerve fiber and the surface of myotubes or their fusing myoblasts. After the establishment of contact, the patches of reaction product become more numerous and coextensive within the region of the neuromuscular junction and its immediate surroundings forming a dense continuous deposit on the postsynaptic sarcolemma. Activity becomes increasingly restricted to the site of the neuromuscular junction as the embryos approach hatching. At all stages, specializations external and internal to the plasmalemma are found at regions of high density of AChR, suggesting that they play a role in the maintenance of a higher concentration of receptors at these sites. These specializations also occur at the region of initial synaptic contact, indicating that they might be recognized by the nerve and represent preferred sites of innervation. Innervation appears to exert a stabilizing influence on the area of high AChR concentration in contact with the nerve and to induce a further increase in the AChR density of this site while the number of AChR in the remaining portions of the muscle surface declines.

The synthesis and localization of glycosaminoglycans in striated muscle differentiating in cell culture.

AbstractDay 3 mass muscle cultures incorporated 35SO4= and 3H· glucosamine into specific glycosaminoglycans that were identified by a variety of methods (enzyme susceptibility, nitrous acid degradation, electrophoresis, thin layer chromatography). More than 75% of the newly synthesized glycosaminoglycans appeared in the medium. In the medium, the glycosaminoglycans consisted of primarily hyaluronic acid (∼35%) and chondroitin sulfate (∼40%) with smaller amounts of chondroitin (∼15%) and heparan sulfate (∼10%). Among the glycosaminoglycans that remained in the cell layer, chondroitin sulfate (∼45%) and heparan sulfate (20‐40%) predominated and only a small amount of chondroitin (∼15%) and hyaluronic acid (∼5%) were present. In both the medium and cell layer, greater than 80% of the chondroitin sulfate was chondroitin‐6‐sulfate. Although mass cultures contain some non‐muscle cell types which undoubtedly contributed to total glycosaminoglycan synthesis, susceptibility of the autoradiographic grains associated with cells in muscle clones to testicular hyaluronidase digestion suggests that at least some glycosaminoglycans (chondroitin sulfate, in particular) were synthesized by muscle cells.Transmission electron microscopy revealed the presence of ruthenium red staining globular units along the surfaces of myotubes. Destruction of the integrity of surface associated ruthenium red stained material by both Streptomyces hyaluronidase and testicular hyaluronidase suggests that both hyaluronic acid and chondroitin sulfate are present on muscle cell surfaces.

Lateral motion of fluorescently labeled acetylcholine receptors in membranes of developing muscle fibers.

We have made direct, quantitative measurements of the lateral motion and age-dependent distribution of acetylcholine receptors (AChR) on the surface of rat myotubes in primary culture. AChR were fluorescently marked with tetramethylrhodamine-labeled alpha-bungarotoxin and AChR lateral motion was measured by the fluoresence photobleaching recovery technique. We found two coexisting distinct classes of AChR: (i) mobile, uniformly distributed AChR that appear on all myotubes shortly after fusion from myoblasts; and (ii) immobile, dense, highly granular AChR in patches of 10-60 mum size that appear shortly after fusion and disappear after myotubes have become extensively interconnected. In addition, evidence of turnover of AChR labeled with tetramethylrhodamine-alpha-bungarotoxin is seen in the gradual internalization of surface fluorescence within 36 hr after labeling. The relevance of these results to an understanding of the membrane dynamics and localization of muscle AChR is discussed.

Appearance and disappearance of acetycholine receptor during differentiation of chick skeletal muscle in vitro

During differentiation of embryonic chick skeletal muscle in culture, elaboration of acetylcholine receptor (AChR) and acetylcholinesterase occurs shortly after myoblast fusion. During further development, AChR was found to decrease markedly on the myotube surface, while acetylcholinesterase continued to increase. Surface distribution of AChR, as followed by autoradiography using 125I-α-bungarotoxin, was homogeneous in newly fused myotubes. With further differentiation, clusters of AChR appeared on the surface of the myotubes, and their subsequent disappearance paralleled a decrease in overall AChR levels. Quantitative autoradiography showed a reduction of over 75% in the density of AChR on the surface of well differentiated, cross-striated myotubes. Thus the appearance of AChR on the cell surface, its condensation into clusters, and finally its depletion seem to be sequential events in the differentiation of skeletal muscle in culture in the absence of direct neuronal influence.

Ultrastructure of motor end-plates during pharmacologically-induced degeneration and subsequent regeneration of skeletal muscle.

A local anesthetic, methyl-bupivacaine was injected into the planta of adult mice, and the ultrastruct of motor end-plates was studied during the degenerative and regenerative cycle induced in lumbrical muscles. Muscle degeneration took place during the first day after drug administration. The postsynaptic part of the neuromuscular junction completely degenerated as did the whole injured muscle fibre. Nerve terminals, however, remained unaffected. By the second day after muscle injury, axon terminals were enclosed within Schwann cell cytoplasm and thus became separated from the residual sarcolemmal tube. One to three days later, when myotubes were formed by fusion of the surviving myoblasts, the layer of Schwann cell cytoplasm on nerve terminals was discontinuous. Subsequently nerve terminals approached the regenerating muscle cell and the subneural apparatus began to differentiate. Slight depressions and furrows appeared on the myotube surface below the nerve ending and the myotube membrane, covered with basement membrane, became undercoated by dense material in this region. Where the distance between nerve ending and myotube was reduced to that found in the normal neuromuscular junction, i.e. to about 500 A, junctional folds were formed. Fourteen days after drug administration, newly formed end-plates were indistinguishable from those in normal control lumbrical muscles.