Induction of a specialized muscle basal lamina at chimaeric synapses in culture

Development ◽  
1990 ◽  
Vol 110 (1) ◽  
pp. 51-61 ◽  
Author(s):  
L.E. Swenarchuk ◽  
S. Champaneria ◽  
M.J. Anderson
Keyword(s):  
Monoclonal Antibodies ◽  
Basal Lamina ◽  
Acetylcholine Receptors ◽  
Muscle Cells ◽  
Synaptic Cleft ◽  
Sulfate Proteoglycan ◽  
Cellular Origin ◽  
Initial Deposition ◽  
Species Specific ◽  
Nerve Muscle

To identify mechanisms that regulate the formation of the neuromuscular junction, we examined the cellular origin of a heparan sulfate proteoglycan (HSPG) that becomes highly concentrated within the synaptic cleft during the initial deposition of the junctional basal lamina. Using cultured nerve and muscle cells from anuran and urodele embryos, we prepared species-chimaeric synapses that displayed spontaneous cholinergic potentials, and eventually developed organized accumulations of acetylcholine receptors and HSPG at the sites of nerve-muscle contact. To determine the cellular origin of synaptic HSPG molecules, these chimaeric junctions were stained with both species-specific and cross-reactive monoclonal antibodies, labeled with contrasting fluorochromes. Our results demonstrate that synaptic HSPG is derived almost exclusively from muscle. Since it has already been shown that muscle cells can assemble virtually all of the known constituents of the junctional basal lamina into organized surface accumulations, without any input from nerve cells, we consider the possibility that the specialized synaptic basal lamina may be generated primarily by the myofibre, in response to another ‘inductive’ positional signal at the site of nerve-muscle contact.

scholarly journals Acetylcholinesterase Clustering at the Neuromuscular Junction Involves Perlecan and Dystroglycan

1999 ◽  
Vol 145 (4) ◽  
pp. 911-921 ◽  
Author(s):  
H. Benjamin Peng ◽  
Hongbo Xie ◽  
Susanna G. Rossi ◽  
Richard L. Rotundo
Keyword(s):  
Neuromuscular Junction ◽  
Basal Lamina ◽  
Acetylcholine Receptors ◽  
Muscle Cells ◽  
Cell Types ◽  
Specific Form ◽  
Lateral Migration ◽  
Extracellular Matrix Molecules ◽  
A Cell ◽  
Nerve Muscle

Formation of the synaptic basal lamina at vertebrate neuromuscular junction involves the accumulation of numerous specialized extracellular matrix molecules including a specific form of acetylcholinesterase (AChE), the collagenic-tailed form. The mechanisms responsible for its localization at sites of nerve– muscle contact are not well understood. To understand synaptic AChE localization, we synthesized a fluorescent conjugate of fasciculin 2, a snake α-neurotoxin that tightly binds to the catalytic subunit. Prelabeling AChE on the surface of Xenopus muscle cells revealed that preexisting AChE molecules could be recruited to form clusters that colocalize with acetylcholine receptors at sites of nerve–muscle contact. Likewise, purified avian AChE with collagen-like tail, when transplanted to Xenopus muscle cells before the addition of nerves, also accumulated at sites of nerve–muscle contact. Using exogenous avian AChE as a marker, we show that the collagenic-tailed form of the enzyme binds to the heparan-sulfate proteoglycan perlecan, which in turn binds to the dystroglycan complex through α-dystroglycan. Therefore, the dystroglycan–perlecan complex serves as a cell surface acceptor for AChE, enabling it to be clustered at the synapse by lateral migration within the plane of the membrane. A similar mechanism may underlie the initial formation of all specialized basal lamina interposed between other cell types.

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

1983 ◽  
Vol 97 (5) ◽  
pp. 1396-1411 ◽  
Author(s):  
M J Anderson ◽  
D M Fambrough
Keyword(s):  
Skeletal Muscle ◽  
Neuromuscular Junction ◽  
Heparan Sulfate ◽  
Basal Lamina ◽  
Acetylcholine Receptors ◽  
Muscle Fibers ◽  
Muscle Cells ◽  
Heparan Sulfate Proteoglycan ◽  
Sulfate Proteoglycan ◽  
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.

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 Acetylcholine receptor aggregation parallels the deposition of a basal lamina proteoglycan during development of the neuromuscular junction.

1984 ◽  
Vol 99 (5) ◽  
pp. 1769-1784 ◽  
Author(s):  
M J Anderson ◽  
F G Klier ◽  
K E Tanguay
Keyword(s):  
Neuromuscular Junction ◽  
Basal Lamina ◽  
Acetylcholine Receptors ◽  
Time Course ◽  
Synapse Formation ◽  
Muscle Cells ◽  
Postsynaptic Membrane ◽  
Receptor Aggregation ◽  
Nerve Muscle ◽  
Alpha Bungarotoxin

To determine the time course of synaptic differentiation, we made successive observations on identified, nerve-contacted muscle cells developing in culture. The cultures had either been stained with fluorescent alpha-bungarotoxin, or were maintained in the presence of a fluorescent monoclonal antibody. These probes are directed at acetylcholine receptors (AChR) and a basal lamina proteoglycan, substances that show nearly congruent surface organizations at the adult neuromuscular junction. In other experiments individual muscle cells developing in culture were selected at different stages of AChR accumulation and examined in the electron microscope after serial sectioning along the entire path of nerve-muscle contact. The results indicate that the nerve-induced formation of AChR aggregates and adjacent plaques of proteoglycan is closely coupled throughout early stages of synapse formation. Developing junctional accumulations of AChR and proteoglycan appeared and grew progressively, throughout a perineural zone that extended along the muscle surface for several micrometers on either side of the nerve process. Unlike junctional AChR accumulations, which disappeared within a day of denervation, both junctional and extrajunctional proteoglycan deposits were stable in size and morphology. Junctional proteoglycan deposits appeared to correspond to discrete ultrastructural plaques of basal lamina, which were initially separated by broad expanses of lamina-free muscle surface. The extent of this basal lamina, and a corresponding thickening of the postsynaptic membrane, also increased during the accumulation of AChR and proteoglycan along the path of nerve contact. Presynaptic differentiation of synaptic vesicle clusters became detectable at the developing neuromuscular junction only after the formation of postsynaptic plaques containing both AChR and proteoglycan. It is concluded that motor nerves induce a gradual formation and growth of AChR aggregates and stable basal lamina proteoglycan deposits on the muscle surface during development of the neuromuscular junction.

Effects Of Doxycycline On Mice Neuromuscular Junction, In Situ

2021 ◽  
Vol 21 ◽  
Author(s):  
Natália Tribuiani ◽  
Jocimar de Souza ◽  
Marcos Antônio de Queiroz Junior ◽  
Denicezar Angelo Baldo ◽  
Valéria de Campos Orsi ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Synaptic Cleft ◽  
Diaphragm Muscle ◽  
Na Channel ◽  
Muscle Histology ◽  
Casearia Sylvestris ◽  
Nerve Muscle ◽  
Neuromuscular Preparation

Background: The antibacterial mechanism of doxycycline is known, but on the nerve-muscle apparatus is yet unclear. Objective: To combine molecular targets of the neuromuscular machinery using the neuronal blocker effect doxycycline, a semisynthetic second-generation tetracycline derivative, on mice neuromuscular preparations, in situ. Methods: Doxycycline was assessed at the neurotransmission; presynaptic; synaptic cleft; and postsynaptic, including the muscle fiber, using the traditional myographic technique. Preliminarily, doxycycline showed an "all or nothing" effect, being "all" obtained with 4 µM and "nothing", with 1-3 µM. The rationale of this study was to apply known pharmacological tools against the blocker effect of 4 µM doxycycline such as F55-6 (Casearia sylvestris), CaCl2 (or Ca2+), atropine, neostigmine, polyethylene glycol (PEG 400), and d-Tubocurarine. The evaluation of cholinesterase enzyme activity, the diaphragm muscle histology, and protocols on the neuromuscular preparation submitted to indirect or direct stimuli were complementary. Results: Doxycycline does not affect cholinesterase activity nor cause damage to skeletal muscle diaphragm; acts on ryanodine receptor, sarcolemmal membrane, and on neuronal sodium channel with a postjunctional consequence due to the decreased availability of muscle nicotinic acetylcholine receptors. Conclusions: In conclusion, using the blocker effect we showed that doxycycline acts on multiple targets, among them, is antagonized by F55-6, a neuronal Na+-channel agonist and Ca2+, but not by neostigmine.

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 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 Nerve-Muscle Interaction In Vitro

1973 ◽  
Vol 62 (3) ◽  
pp. 255-270 ◽  
Author(s):  
J. H. Steinbach ◽  
A.J. Harris ◽  
J. Patrick ◽  
D. Schubert ◽  
S. Heinemann
Keyword(s):  
Nerve Cell ◽  
Muscle Cell ◽  
Acetylcholine Receptors ◽  
Muscle Cells ◽  
Acetylcholine Sensitivity ◽  
Release Of Acetylcholine ◽  
Clonal Lines ◽  
Nerve Muscle ◽  
A Site

Nerve and muscle cells from clonal lines interact in vitro, resulting in the association on the muscle surface of an area of increased acetylcholine sensitivity with a site of nerve-muscle contact. This localization of acetylcholine sensitivity on the muscle cell to a site of contact between nerve and muscle was found to occur when acetylcholine receptors on the muscle had been blocked with α-neurotoxin. Localization was also found to occur when the nerve cell had been prevented from releasing acetylcholine. It is concluded that neither the presence of active acetylcholine receptors on the muscle, nor the release of acetylcholine from the nerve, was required for the events leading to the localization of acetylcholine sensitivity in vitro.

scholarly journals Components of Torpedo electric organ and muscle that cause aggregation of acetylcholine receptors on cultured muscle cells.

1984 ◽  
Vol 99 (2) ◽  
pp. 615-627 ◽  
Author(s):  
E W Godfrey ◽  
R M Nitkin ◽  
B G Wallace ◽  
L L Rubin ◽  
U J McMahan
Keyword(s):  
Extracellular Matrix ◽  
Basal Lamina ◽  
Acetylcholine Receptors ◽  
Active Component ◽  
Electric Organ ◽  
Muscle Cells ◽  
Insoluble Fraction ◽  
Lateral Migration ◽  
Cultured Myotubes ◽  
Torpedo Electric Organ

The synaptic portion of a muscle fiber's basal lamina sheath has molecules tightly bound to it that cause aggregation of acetylcholine receptors (AChRs) on regenerating myofibers. Since basal lamina and other extracellular matrix constituents are insoluble in isotonic saline and detergent solutions, insoluble detergent-extracted fractions of tissues receiving cholinergic input may provide an enriched source of the AChR-aggregating molecules for detailed characterization. Here we demonstrate that such an insoluble fraction from Torpedo electric organ, a tissue with a high concentration of cholinergic synapses, causes AChRs on cultured chick muscle cells to aggregate. We have partially characterized the insoluble fraction, examined the response of muscle cells to it, and devised ways of extracting the active components with a view toward purifying them and learning whether they are similar to those in the basal lamina at the neuromuscular junction. The insoluble fraction from the electric organ was rich in extracellular matrix constituents; it contained structures resembling basal lamina sheaths and had a high density of collagen fibrils. It caused a 3- to 20-fold increase in the number of AChR clusters on cultured myotubes without significantly affecting the number or size of the myotubes. The increase was first seen 2-4 h after the fraction was added to cultures and it was maximal by 24 h. The AChR-aggregating effect was dose dependent and was due, at least in part, to lateral migration of AChRs present in the muscle cell plasma membrane at the time the fraction was applied. Activity was destroyed by heat and by trypsin. The active component(s) was extracted from the insoluble fraction with high ionic strength or pH 5.5 buffers. The extracts increased the number of AChR clusters on cultured myotubes without affecting the number or degradation rate of surface AChRs. Antiserum against the solubilized material blocked its effect on AChR distribution and bound to the active component. Insoluble fractions of Torpedo muscle and liver did not cause AChR aggregation on cultured myotubes. However a low level of activity was detected in pH 5.5 extracts from the muscle fraction. The active component(s) in the muscle extract was immunoprecipitated by the antiserum against the material extracted from the electric organ insoluble fraction. This antiserum also bound to extracellular matrix in frog muscles, including the myofiber basal lamina sheath. Thus the insoluble fraction of Torpedo electric organ is rich in AChR-aggregating molecules that are also found in muscle and has components antigenically similar to those in myofiber basal lamina.

scholarly journals Distribution of alpha-dystroglycan during embryonic nerve-muscle synaptogenesis.

1995 ◽  
Vol 129 (4) ◽  
pp. 1093-1101 ◽  
Author(s):  
M W Cohen ◽  
C Jacobson ◽  
E W Godfrey ◽  
K P Campbell ◽  
S Carbonetto
Keyword(s):  
Acetylcholine Receptors ◽  
Culture Medium ◽  
Muscle Cells ◽  
Immunofluorescent Staining ◽  
Western Blots ◽  
Xenopus Embryos ◽  
Alpha Dystroglycan ◽  
Nerve Muscle ◽  
Induced Changes

The distribution of alpha-dystroglycan (alpha DG) relative to acetylcholine receptors (AChRs) and neural agrin was examined by immunofluorescent staining with mAb IIH6 in cultures of nerve and muscle cells derived from Xenopus embryos. In Western blots probed with mAb IIH6, alpha DG was evident in membrane extracts of Xenopus muscle but not brain. alpha DG immunofluorescence was present at virtually all synaptic clusters of AChRs and neural agrin. Even microclusters of AChRs and agrin at synapses no older than 1-2 h (the earliest examined) had alpha DG associated with them. alpha DG was also colocalized at the submicrometer level with AChRs at nonsynaptic clusters that have little or no agrin. The number of large (> 4 microns) nonsynaptic clusters of alpha DG, like the number of large nonsynaptic clusters of AChRs, was much lower on innervated than on noninnervated cells. When mAb IIH6 was included in the culture medium, the large nonsynaptic clusters appeared fragmented and less compact, but the accumulation of agrin and AChRs along nerve-muscle contacts was not prevented. It is concluded that during nerve-muscle synaptogenesis, alpha DG undergoes the same nerve-induced changes in distribution as AChRs. We propose a diffusion trap model in which the alpha DG-transmembrane complex participates in the anchoring and recruitment of AChRs and alpha DG during the formation of synaptic as well as nonsynaptic AChR clusters.

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