scholarly journals Agrin-like molecules at synaptic sites in normal, denervated, and damaged skeletal muscles.

1987 ◽  
Vol 105 (6) ◽  
pp. 2457-2469 ◽  
Author(s):  
N E Reist ◽  
C Magill ◽  
U J McMahan
Keyword(s):  
Monoclonal Antibodies ◽  
Neuromuscular Junction ◽  
Basal Lamina ◽  
Skeletal Muscles ◽  
Neuromuscular Junctions ◽  
Electric Organ ◽  
Synaptic Cleft ◽  
Cellular Components ◽  
Term Maintenance

Several lines of evidence have led to the hypothesis that agrin, a protein extracted from the electric organ of Torpedo, is similar to the molecules in the synaptic cleft basal lamina at the neuromuscular junction that direct the formation of acetylcholine receptor and acetylcholinesterase aggregates on regenerating myofibers. One such finding is that monoclonal antibodies against agrin stain molecules concentrated in the synaptic cleft of neuromuscular junctions in rays. In the studies described here we made additional monoclonal antibodies against agrin and used them to extend our knowledge of agrin-like molecules at the neuromuscular junction. We found that anti-agrin antibodies intensely stained the synaptic cleft of frog and chicken as well as that of rays, that denervation of frog muscle resulted in a reduction in staining at the neuromuscular junction, and that the synaptic basal lamina in frog could be stained weeks after degeneration of all cellular components of the neuromuscular junction. We also describe anti-agrin staining in nonjunctional regions of muscle. We conclude the following: (a) agrin-like molecules are likely to be common to all vertebrate neuromuscular junctions; (b) the long-term maintenance of such molecules at the junction is nerve dependent; (c) the molecules are, indeed, a component of the synaptic basal lamina; and (d) they, like the molecules that direct the formation of receptor and esterase aggregates on regenerating myofibers, remain associated with the synaptic basal lamina after muscle damage.

scholarly journals Basal lamina directs acetylcholinesterase accumulation at synaptic sites in regenerating muscle.

1985 ◽  
Vol 101 (3) ◽  
pp. 735-743 ◽  
Author(s):  
L Anglister ◽  
U J McMahan
Keyword(s):  
Plasma Membrane ◽  
Neuromuscular Junction ◽  
Basal Lamina ◽  
Acetylcholine Receptors ◽  
Skeletal Muscles ◽  
Ache Activity ◽  
Neuromuscular Junctions ◽  
Muscle Fibers ◽  
Synaptic Cleft

In skeletal muscles that have been damaged in ways which spare the basal lamina sheaths of the muscle fibers, new myofibers develop within the sheaths and neuromuscular junctions form at the original synaptic sites on them. At the regenerated neuromuscular junctions, as at the original ones, the muscle fibers are characterized by junctional folds and accumulations of acetylcholine receptors and acetylcholinesterase (AChE). The formation of junctional folds and the accumulation of acetylcholine receptors is known to be directed by components of the synaptic portion of the myofiber basal lamina. The aim of this study was to determine whether or not the synaptic basal lamina contains molecules that direct the accumulation of AChE. We crushed frog muscles in a way that caused disintegration and phagocytosis of all cells at the neuromuscular junction, and at the same time, we irreversibly blocked AChE activity. New muscle fibers were allowed to regenerate within the basal lamina sheaths of the original muscle fibers but reinnervation of the muscles was deliberately prevented. We then stained for AChE activity and searched the surface of the new muscle fibers for deposits of enzyme they had produced. Despite the absence of innervation, AChE preferentially accumulated at points where the plasma membrane of the new muscle fibers was apposed to the regions of the basal lamina that had occupied the synaptic cleft at the neuromuscular junctions. We therefore conclude that molecules stably attached to the synaptic portion of myofiber basal lamina direct the accumulation of AChE at the original synaptic sites in regenerating muscle. Additional studies revealed that the AChE was solubilized by collagenase and that it remained adherent to basal lamina sheaths after degeneration of the new myofibers, indicating that it had become incorporated into the basal lamina, as at normal neuromuscular junctions.

scholarly journals Acetylcholine receptor-aggregating proteins are associated with the extracellular matrix of many tissues in Torpedo.

1988 ◽  
Vol 106 (4) ◽  
pp. 1263-1272 ◽  
Author(s):  
E W Godfrey ◽  
M E Dietz ◽  
A L Morstad ◽  
P A Wallskog ◽  
D E Yorde
Keyword(s):  
Extracellular Matrix ◽  
Monoclonal Antibodies ◽  
Basal Lamina ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Electric Organ ◽  
Synaptic Cleft ◽  
Basement Membranes ◽  
Cardiac Muscle Cells ◽  
Western Blots

The synaptic basal lamina, a component of extracellular matrix (ECM) in the synaptic cleft at the neuromuscular junction, directs the formation of new postsynaptic specializations, including the aggregation of acetylcholine receptors (AChRs), during muscle regeneration in adult animals. Although the molecular basis of this phenomenon is unknown, it is mimicked by AChR-aggregating proteins in ECM-enriched fractions from muscle and the synapse-rich electric organ of the ray Torpedo californica. Molecules immunologically similar to these proteins are concentrated in the synaptic basal lamina at neuromuscular junctions of the ray and frog. Here we demonstrate that immunologically, chemically, and functionally similar AChR-aggregating proteins are also associated with the ECM of several other tissues in Torpedo. Monoclonal antibodies against the AChR-aggregating proteins from electric organ intensely stained neuromuscular junctions and the ventral surfaces of electrocytes, structures with a high density of AChRs. However, they also labeled many other structures which have basal laminae, including the extrajunctional perimeters of skeletal muscle fibers, smooth and cardiac muscle cells, Schwann cell sheaths in peripheral nerves, walls of some blood vessels, and epithelial basement membranes in the gut, skin, and heart. Some structures with basal laminae did not stain with the antibodies; e.g., the dorsal surfaces of electrocytes. Bands of similar molecular weight were detected by the antibodies on Western blots of extracts of ECM-enriched fractions from electric organ and several other tissues. Proteins from all tissues examined, enriched from these extracts by affinity chromatography with the monoclonal antibodies, aggregated AChRs on cultured myotubes. Thus, similar AChR-aggregating proteins are associated with the extracellular matrix of many Torpedo tissues. The broad distribution of these proteins suggests they have functions in addition to AChR aggregation.

scholarly journals ARIA is concentrated in the synaptic basal lamina of the developing chick neuromuscular junction.

1995 ◽  
Vol 130 (6) ◽  
pp. 1423-1434 ◽  
Author(s):  
A D Goodearl ◽  
A G Yee ◽  
A W Sandrock ◽  
G Corfas ◽  
G D Fischbach
Keyword(s):  
Neuromuscular Junction ◽  
Basal Lamina ◽  
Motor Neurons ◽  
Neuromuscular Junctions ◽  
Primary Cultures ◽  
Cholinergic Neurons ◽  
Synaptic Cleft ◽  
Motor Axons ◽  
Amino Terminal

ARIA is a member of a family of polypeptide growth and differentiation factors that also includes glial growth factor (GGF), neu differentiation factor, and heregulin. ARIA mRNA is expressed in all cholinergic neurons of the central nervous systems of rats and chicks, including spinal cord motor neurons. In vitro, ARIA elevates the rate of acetylcholine receptor incorporation into the plasma membrane of primary cultures of chick myotubes. To study whether ARIA may regulate the synthesis of junctional synaptic acetylcholine receptors in chick embryos, we have developed riboprobes and polyclonal antibody reagents that recognize isoforms of ARIA that include an amino-terminal immunoglobulin C2 domain and examined the expression and distribution of ARIA in motor neurons and at the neuromuscular junction. We detected significant ARIA mRNA expression in motor neurons as early as embryonic day 5, around the time that motor axons are making initial synaptic contacts with their target muscle cells. In older embryos and postnatal animals, we found ARIA protein concentrated in the synaptic cleft at neuromuscular junctions, consistent with transport down motor axons and release at nerve terminals. At high resolution using immunoelectron microscopy, we detected ARIA immunoreactivity exclusively in the synaptic basal lamina in a pattern consistent with binding to synapse specific components on the presynaptic side of the basal lamina. These results support a role for ARIA as a trophic factor released by motor neuron terminals that may regulate the formation of mature neuromuscular synapses.

scholarly journals Identification of agrin in electric organ extracts and localization of agrin-like molecules in muscle and central nervous system

1987 ◽  
Vol 132 (1) ◽  
pp. 223-230 ◽  
Author(s):  
M. A. Smith ◽  
Y. M. Yao ◽  
N. E. Reist ◽  
C. Magill ◽  
B. G. Wallace ◽  
...  
Keyword(s):  
Neuromuscular Junction ◽  
Acetylcholine Receptor ◽  
Basal Lamina ◽  
Acetylcholine Receptors ◽  
Electric Organ ◽  
Synaptic Cleft ◽  
Muscle Fibres ◽  
Motor Neurones ◽  
Cultured Myotubes ◽  
Torpedo Electric Organ

The portion of the muscle fibre's basal lamina that occupies the synaptic cleft at the neuromuscular junction contains molecules that cause the aggregation of acetylcholine receptors and acetylcholinesterase on regenerating muscle fibres. Agrin, which is extracted from basal lamina-containing fractions of the Torpedo electric organ and causes the formation of acetylcholine receptor and acetylcholinesterase aggregates on cultured myotubes, may be similar, if not identical, to the acetylcholine receptor- and acetylcholinesterase-aggregating molecules at the neuro-muscular junction. Here we summarize experiments which led to the identification of agrin and established that the basal lamina at the neuromuscular junction contains molecules antigenically similar to agrin. We also discuss results which raise the possibility that agrin-like molecules at the neuromuscular junction are produced by motor neurones.

scholarly journals Acetylcholinesterase from the motor nerve terminal accumulates on the synaptic basal lamina of the myofiber.

1991 ◽  
Vol 115 (3) ◽  
pp. 755-764 ◽  
Author(s):  
L Anglister
Keyword(s):  
Basal Lamina ◽  
Ache Activity ◽  
Neuromuscular Junctions ◽  
Motor Nerve ◽  
Synaptic Cleft ◽  
Motor Nerve Terminal ◽  
Nerve Terminals ◽  
Axon Terminals ◽  
Muscle Nerve ◽  
Almost All

Acetylcholinesterase (AChE) in skeletal muscle is concentrated at neuromuscular junctions, where it is found in the synaptic cleft between muscle and nerve, associated with the synaptic portion of the myofiber basal lamina. This raises the question of whether the synaptic enzyme is produced by muscle, nerve, or both. Studies on denervated and regenerating muscles have shown that myofibers can produce synaptic AChE, and that the motor nerve may play an indirect role, inducing myofibers to produce synaptic AChE. The aim of this study was to determine whether some of the AChE which is known to be made and transported by the motor nerve contributes directly to AChE in the synaptic cleft. Frog muscles were surgically damaged in a way that caused degeneration and permanent removal of all myofibers from their basal lamina sheaths. Concomitantly, AChE activity was irreversibly blocked. Motor axons remained intact, and their terminals persisted at almost all the synaptic sites on the basal lamina in the absence of myofibers. 1 mo after the operation, the innervated sheaths were stained for AChE activity. Despite the absence of myofibers, new AChE appeared in an arborized pattern, characteristic of neuromuscular junctions, and its reaction product was concentrated adjacent to the nerve terminals, obscuring synaptic basal lamina. AChE activity did not appear in the absence of nerve terminals. We concluded therefore, that the newly formed AChE at the synaptic sites had been produced by the persisting axon terminals, indicating that the motor nerve is capable of producing some of the synaptic AChE at neuromuscular junctions. The newly formed AChE remained adherent to basal lamina sheaths after degeneration of the terminals, and was solubilized by collagenase, indicating that the AChE provided by nerve had become incorporated into the basal lamina as at normal neuromuscular junctions.

scholarly journals The influence of basal lamina on the accumulation of acetylcholine receptors at synaptic sites in regenerating muscle.

1984 ◽  
Vol 98 (4) ◽  
pp. 1453-1473 ◽  
Author(s):  
U J McMahan ◽  
C R Slater
Keyword(s):  
Plasma Membrane ◽  
Basal Lamina ◽  
Muscle Fiber ◽  
Acetylcholine Receptors ◽  
Skeletal Muscles ◽  
Neuromuscular Junctions ◽  
High Concentration ◽  
Direct Role ◽  
Synaptic Region ◽  
Membrane Infoldings

If skeletal muscles are damaged in ways that spare the basal lamina sheaths of the muscle fibers, new myofibers develop within the sheaths and neuromuscular junctions form at the original synaptic sites on them. At the regenerated neuromuscular junctions, as at the original ones, the muscle fiber plasma membrane is characterized by infoldings and a high concentration of acetylcholine receptors (AChRs). The aim of this study was to determine whether or not the synaptic portion of the myofiber basal lamina sheath plays a direct role in the formation of the subsynaptic apparatus on regenerating myofibers, a question raised by the results of earlier experiments. The junctional region of the frog cutaneous pectoris muscle was crushed or frozen, which resulted in disintegration and phagocytosis of all cells at the synapse but left intact much of the myofiber basal lamina. Reinnervation was prevented. When new myofibers developed within the basal lamina sheaths, patches of AChRs and infoldings formed preferentially at sites where the myofiber membrane was apposed to the synaptic region of the sheaths. Processes from unidentified cells gradually came to lie on the presynaptic side of the basal lamina at a small fraction of the synaptic sites, but there was no discernible correlation between their presence and the effectiveness of synaptic sites in accumulating AChRs. We therefore conclude that molecules stably attached to the myofiber basal lamina at synaptic sites direct the formation of subsynaptic apparatus in regenerating myofibers. An analysis of the distribution of AChR clusters at synaptic sites indicated that they formed as a result of myofiber-basal lamina interactions that occurred at numerous places along the synaptic basal lamina, that their presence was not dependent on the formation of plasma membrane infoldings, and that the concentration of receptors within clusters could be as great as the AChR concentration at normal neuromuscular junctions.

scholarly journals Peripheral proteins of postsynaptic membranes from Torpedo electric organ identified with monoclonal antibodies.

1984 ◽  
Vol 99 (1) ◽  
pp. 88-96 ◽  
Author(s):  
S C Froehner
Keyword(s):  
Monoclonal Antibodies ◽  
Neuromuscular Junction ◽  
Acetylcholine Receptor ◽  
Electric Organ ◽  
Alkaline Treatment ◽  
Peripheral Proteins ◽  
Torpedo Electric Organ

Highly purified postsynaptic membranes from Torpedo electric organ contain the acetylcholine receptor as well as other proteins. To identify synapse-specific components, we prepared monoclonal antibodies (mabs) to proteins extracted from the membranes with either lithium diiodosalicylate or alkaline treatment. 10 mabs specific for three different proteins were obtained. Seven mabs reacted with a major 43,000-mol-wt protein (43K protein). This protein is composed of isoelectric variants (pl = 7.2-7.8) and each of the mabs reacted with all of the variants. Analysis of these mabs by competition for binding to 43K protein and by reaction with proteolytic fragments of 43K protein in immunoblots showed that they recognize at least five different epitopes. Two mabs reacted with a protein of 90,000 mol wt (90K protein) and one with a protein of 58,000 mol wt composed of isoelectric variants (pl = 6.4-6.7) (58K protein). The 43K and 58K proteins appeared to co-purify with the receptor-containing membranes while the 90K protein did not. Immunofluorescence experiments indicated that the anti-43K mabs bind to the innervated face of Torpedo electrocytes and that a component related to the 43K protein is found at the rat neuromuscular junction. The anti-58K mab stained the innervated face, although rather weakly, while the anti-90K mabs reacted intensely with the non-innervated membrane. Thus, the 43K protein and possibly also the 58K protein are synaptic components while the 90K protein is predominantly nonsynaptic.

scholarly journals The mechanism of agrin-induced acetylcholine receptor aggregation

1991 ◽  
Vol 331 (1261) ◽  
pp. 273-280 ◽  
Keyword(s):  
Neuromuscular Junction ◽  
Tyrosine Phosphorylation ◽  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Electric Organ ◽  
Motor Axon ◽  
Β Subunit ◽  
Axon Terminals ◽  
Receptor Aggregation

Agrin, a protein isolated from the synapse-rich electric organ of Torpedo californica , induces the formation of specializations on myotubes in culture which resemble the post-synaptic apparatus at the vertebrate skeletal neuromuscular junction. For example, the specializations contain aggregates of acetylcholine receptors and acetylcholinesterase. This report summarizes the evidence that the formation of the postsynaptic apparatus at developing and regenerating neuromuscular junctions is triggered by the release of agrin from motor axon terminals and describes results of recent experiments which suggest that agrininduced tyrosine phosphorylation of the β subunit of the acetylcholine receptor may play a role in receptor aggregation.

scholarly journals Aggregating factor from Torpedo electric organ induces patches containing acetylcholine receptors, acetylcholinesterase, and butyrylcholinesterase on cultured myotubes.

1986 ◽  
Vol 102 (3) ◽  
pp. 783-794 ◽  
Author(s):  
B G Wallace
Keyword(s):  
Monoclonal Antibody ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Electric Organ ◽  
Synaptic Cleft ◽  
Dose Dependence ◽  
Defined Medium ◽  
Induced Aggregation ◽  
Three Components

A factor in extracts of the electric organ of Torpedo californica causes the formation of clusters of acetylcholine receptors (AChRs) and aggregates of acetylcholinesterase (AChE) on myotubes in culture. In vivo, AChRs and AChE accumulate at the same locations on myofibers, as components of the postsynaptic apparatus at neuromuscular junctions. The aim of this study was to compare the distribution of AChRs, AChE, and butyrylcholinesterase (BuChE), a third component of the postsynaptic apparatus, on control and extract-treated myotubes. Electric organ extracts induced the formation of patches that contained high concentrations of all three molecules. The extract-induced aggregation of AChRs, AChE, and BuChE occurred in defined medium, and these components accumulated in patches simultaneously. Three lines of evidence indicate that a single factor in the extracts induced the aggregation of all three components: the dose dependence for the formation of patches of AChRs was the same as that for patches of AChE and BuChE; the AChE- and BuChE-aggregating activities co-purified with the AChR-aggregating activity; and all three aggregating activities were immunoprecipitated at the same titer by a monoclonal antibody against the AChR-aggregating factor. We have shown previously that this monoclonal antibody binds to molecules concentrated in the synaptic cleft at neuromuscular junctions. Taken together, these results suggest that during development and regeneration of myofibers in vivo, the accumulation at synaptic sites of at least three components of the postsynaptic apparatus, AChRs, AChE, and BuChE, are all triggered by the same molecule, a molecule similar if not identical to the electric organ aggregating factor.

scholarly journals Globular and asymmetric acetylcholinesterase in the synaptic basal lamina of skeletal muscle.

1994 ◽  
Vol 125 (1) ◽  
pp. 183-196 ◽  
Author(s):  
L Anglister ◽  
B Haesaert ◽  
U J McMahan
Keyword(s):  
Ionic Strength ◽  
Cell Surface ◽  
Basal Lamina ◽  
Ache Activity ◽  
Electrostatic Interactions ◽  
Neuromuscular Junctions ◽  
Hydrophobic Interactions ◽  
High Ionic Strength ◽  
Molecular Forms ◽  
Cellular Components

The aim of this study was to characterize the molecular forms of acetylcholinesterase (AChE) associated with the synaptic basal lamina at the neuromuscular junction. The observations were made on the neuromuscular junctions of cutaneous pectoris muscles of frog, Rana pipiens, which are similar to junctions of most other vertebrates including mammals, but are especially convenient for experimentation. By measuring relative AChE activity in junctional and extrajunctional regions of muscles after selective inactivation of extracellular AChE with echothiophate, or of intracellular AChE with DFP and 2-PAM, we found that > 66% of the total AChE activity in the muscle was junction-specific, and that > 50% of the junction-specific AChE was on the cell surface. More than 80% of the cell surface AChE was solubilized in high ionic strength detergent-free buffer, indicating that most, if not all, was a component of the synaptic basal lamina. Sedimentation analysis of that fraction indicated that while asymmetric forms (A12, A8) were abundant, globular forms sedimenting at 4-6 S (G1 and G2), composed > 50% of the AChE. It was also found that when muscles were damaged in various ways that caused degeneration of axons and muscle fibers but left intact the basal lamina sheaths, the small globular forms persisted at the synaptic site for weeks after phagocytosis of cellular components; under certain damage conditions, the proportion of globular to asymmetric forms in the vacated basal lamina sheaths was as in normal junctions. While the asymmetric forms required high ionic strength for solubilization, the extracellular globular AChE could be extracted from the junctional regions of normal and damaged muscles by isotonic buffer. Some of the globular AChE appeared to be amphiphilic when examined in detergents, suggesting that it may form hydrophobic interactions, but most was non-amphiphilic consistent with the possibility that it forms weak electrostatic interactions. We conclude that the major form of AChE in frog synaptic basal lamina is globular and that its mode of association with the basal lamina differs from that of the asymmetric forms.

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