scholarly journals Proteolytic disruption of laminin-integrin complexes on muscle cells during synapse formation.

1996 ◽  
Vol 16 (9) ◽  
pp. 4972-4984 ◽  
Author(s):  
M J Anderson ◽  
Z Q Shi ◽  
S L Zackson
Keyword(s):  
Basal Lamina ◽  
Synapse Formation ◽  
Neuromuscular Junctions ◽  
Surrounding Medium ◽  
Muscle Cells ◽  
Cell Contact ◽  
Neural Modulation ◽  
Ligand Interactions ◽  
Nerve Muscle ◽  
Integrin Ligand

To explore whether a neural modulation of muscle integrins' extracellular ligand interactions contributes to synapse induction, we compared the distributions of beta1-integrins and basal lamina proteins on Xenopus myotomal myocytes developing in culture. beta1-Integrins formed numerous organized aggregates scattered over the entire muscle surface, with particularly dense accumulations at specialized sites resembling myotendinous and neuromuscular junctions. Integrin aggregates on muscle cells differed from those on surrounding fibroblasts and epithelial cells, both in their lack of response to cross-linking by multivalent ligands and in their consistent association with the cells' own extracellular matrices. Muscle integrin clusters were usually associated with congruent basal lamina accumulations containing laminin and a heparan sulfate proteoglycan (HSPG), sometimes including fibronectin and vitronectin acquired from the surrounding medium. Immediately prior to synaptic differentiation, any existing laminin and HSPG accumulations along the path of cell contact were eliminated, disrupting otherwise stable laminin-integrin complexes. This apparently proteolytic modulation of integrins' extracellular ligand interactions was soon followed by the accumulation of new congruent accumulations of laminin and HSPG in the developing synaptic basal lamina. Combining these results with earlier findings, we consider the possibility that postsynaptic differentiation is induced, at least in part, by the proteolytic disruption of integrin-ligand complexes at sites of nerve-muscle contact.

scholarly journals Nerve-induced remodeling of muscle basal lamina during synaptogenesis.

1986 ◽  
Vol 102 (3) ◽  
pp. 863-877 ◽  
Author(s):  
M J Anderson
Keyword(s):  
Basal Lamina ◽  
Neuromuscular Junctions ◽  
Muscle Cells ◽  
Cell Contact ◽  
High Background ◽  
Site Density ◽  
Paradoxical Effects ◽  
Muscle Cultures ◽  
Developing Muscle ◽  
Xenopus Laevis Embryos

To identify mechanisms that regulate the deposition of the junctional basal lamina during synaptogenesis, immunocytochemical experiments were carried out on cultured nerve and muscle cells derived from Xenopus laevis embryos. In some experiments successive observations were made on individual muscle cells after pulse-labeling with a fluorescent monoclonal antibody specific for a basal lamina proteoglycan. In others, old and new proteoglycan molecules were differentially labeled with antibody conjugated to contrasting fluorochromes. These observations revealed that surface deposits of antibody-labeled proteoglycan remain morphologically stable for several days on developing muscle cells. Over the same period, however, new sites of proteoglycan accumulation formed that contained primarily those antigenic sites recently exposed at the cell surface. When muscle cells became innervated by cholinergic neurites, new proteoglycan accumulations were induced at the developing neuromuscular junctions, and these too were composed almost exclusively of recently deposited antigen. In older muscle cultures, where many cells possessed relatively high background concentrations of antigen over their surfaces, developing neuromuscular junctions initially showed a markedly reduced proteoglycan site-density compared with the adjacent, extrajunctional muscle surface. Much of this perineural region eventually became filled with dense, nerve induced proteoglycan plaques at later stages of synapse development. Motoneurons thus appear to have two, superficially paradoxical effects on muscle basal lamina organization. They first cause the removal of any existing, extrajunctional proteoglycan from the path of cell contact, and then induce the deposition of dense plaques of recently synthesized proteoglycan within the developing junctional basal lamina. This observation suggests that the proteolytic enzyme systems that have already been implicated in tissue remodeling may also contribute to the inductive interaction between nerve and muscle cells during synaptogenesis.

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.

scholarly journals Purification and characterization of a polypeptide from chick brain that promotes the accumulation of acetylcholine receptors in chick myotubes.

1986 ◽  
Vol 103 (2) ◽  
pp. 493-507 ◽  
Author(s):  
T B Usdin ◽  
G D Fischbach
Keyword(s):  
Acetylcholine Receptors ◽  
Synapse Formation ◽  
Neuromuscular Junctions ◽  
Surface Membrane ◽  
Postsynaptic Membrane ◽  
Size Exclusion ◽  
Response Curves ◽  
Sds Page ◽  
Nerve Muscle ◽  
Receptor Clusters

Acetylcholine receptors (AChRs) are packed in the postsynaptic membrane at neuromuscular junctions at a density of approximately 20,000/micron 2, whereas the density a few micrometers away is less than 20/micron 2. To understand how this remarkable distribution comes about during nerve-muscle synapse formation, we have attempted to isolate factors from neural tissue that can promote the accumulation of AChRs and/or alter their distribution. In this paper we report the purification of a polypeptide from chick brains that can increase the rate of insertion of AChR into membranes of cultured chick myotubes at a concentration of less than 0.5 ng/ml. Based on SDS PAGE and the action of neuraminidase, the acetylcholine receptor-inducing activity (ARIA) appears to be a 42,000-D glycoprotein. ARIA was extracted in a trifluoroacetic acid-containing cocktail and purified to homogeneity by reverse-phase, ion exchange, and size exclusion high pressure liquid chromatography. Dose response curves indicate that the activity has been purified 60,000-fold compared with the starting acid extract and approximately 1,500,000-fold compared with a saline extract prepared from the same batch of brains. Although the ARIA was purified on the basis of its ability to increase receptor incorporation, we found that it increased the number and size of receptor clusters as well. It is not yet clear if the two effects are independent. The 42-kD ARIA is extremely stable: it was not destroyed by exposure to intact myotubes, low pH, organic solvents, or SDS. Its action appears to be selective in that the increase in the rate of receptor insertion was not accompanied by an increase in the rate of protein synthesis. Moreover, there was no change in cellular, surface membrane, or secreted acetylcholinesterase. The effect of ARIA is apparently independent of the state of activity of the target myotubes as its effect on receptor incorporation added to that of maximal concentrations of tetrodotoxin.

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.

Dynamics of nerve-muscle interaction in developing and mature neuromuscular junctions

1995 ◽  
Vol 75 (4) ◽  
pp. 789-834 ◽  
Author(s):  
A. D. Grinnell
Keyword(s):  
Synapse Formation ◽  
Neuromuscular Junctions ◽  
Structure And Function ◽  
Axon Pathfinding ◽  
Control Of Gene Expression ◽  
Synaptic Structure ◽  
Dynamic Cell ◽  
Nerve Muscle ◽  
And Function ◽  
Motoneuron Cell

Neuromuscular connections have long served as models of synaptic structure and function. They also provide illuminating insights into the dynamic cell-cell interactions governing synaptogenesis, neuromuscular differentiation, and the maintenance of effective function. This paper reviews recent advances in our understanding of the regulatory and inductive interactions involved in motor axon pathfinding, target recognition, bidirectional control of gene expression during synapse formation, motoneuron cell death, terminal rearrangement, and the ongoing remodeling of synaptic number, structure, and function to adjust to growth and changes in use.

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 Agrin Binds to the Nerve–Muscle Basal Lamina via Laminin

1997 ◽  
Vol 137 (3) ◽  
pp. 671-683 ◽  
Author(s):  
Alain J. Denzer ◽  
Ralph Brandenberger ◽  
Matthias Gesemann ◽  
Matthias Chiquet ◽  
Markus A. Ruegg
Keyword(s):  
Amino Acids ◽  
Basal Lamina ◽  
Motor Neurons ◽  
Muscle Fiber ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Basement Membranes ◽  
Full Length ◽  
Local Aggregation ◽  
Nerve Muscle

Agrin is a heparan sulfate proteoglycan that is required for the formation and maintenance of neuromuscular junctions. During development, agrin is secreted from motor neurons to trigger the local aggregation of acetylcholine receptors (AChRs) and other proteins in the muscle fiber, which together compose the postsynaptic apparatus. After release from the motor neuron, agrin binds to the developing muscle basal lamina and remains associated with the synaptic portion throughout adulthood. We have recently shown that full-length chick agrin binds to a basement membrane-like preparation called Matrigel™. The first 130 amino acids from the NH2 terminus are necessary for the binding, and they are the reason why, on cultured chick myotubes, AChR clusters induced by full-length agrin are small. In the current report we show that an NH2-terminal fragment of agrin containing these 130 amino acids is sufficient to bind to Matrigel™ and that the binding to this preparation is mediated by laminin-1. The fragment also binds to laminin-2 and -4, the predominant laminin isoforms of the muscle fiber basal lamina. On cultured myotubes, it colocalizes with laminin and is enriched in AChR aggregates. In addition, we show that the effect of full-length agrin on the size of AChR clusters is reversed in the presence of the NH2-terminal agrin fragment. These data strongly suggest that binding of agrin to laminin provides the basis of its localization to synaptic basal lamina and other basement membranes.

scholarly journals Cyclic Mechanical Reinforcement of Integrin–Ligand Interactions

2013 ◽  
Vol 49 (6) ◽  
pp. 1176 ◽  
Author(s):  
Fang Kong ◽  
Zhenhai Li ◽  
William M. Parks ◽  
David W. Dumbauld ◽  
Andrés J. García ◽  
...  
Keyword(s):  
Mechanical Reinforcement ◽  
Ligand Interactions ◽  
Integrin Ligand

scholarly journals Association of beta-cytoplasmic actin with high concentrations of acetylcholine receptor (AChR) in normal and anti-AChR-treated primary rat muscle cultures.

1984 ◽  
Vol 32 (9) ◽  
pp. 973-981 ◽  
Author(s):  
B W Lubit
Keyword(s):  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Morphological Changes ◽  
Muscle Cells ◽  
High Concentration ◽  
Rat Muscle ◽  
Cytoplasmic Actin ◽  
High Concentrations

Previous immunocytochemical studies in which an antibody specific for mammalian cytoplasmic actin was used showed that a high concentration of cytoplasmic actin exists at neuromuscular junctions of rat muscle fibers such that the distribution of actin corresponded exactly to that of the acetylcholine receptors. Although clusters of acetylcholine receptors also are present in noninnervated rat and chick muscle cells grown in vitro, neither the mechanism for the formation and maintenance of these clusters nor the relationship of these clusters to the high density of acetylcholine receptors at the neuromuscular junction in vivo are known. In the present study, a relationship between beta-cytoplasmic actin and acetylcholine receptors in vitro has been demonstrated immunocytochemically using an antibody specific for the beta-form of cytoplasmic actin. Networks of cytoplasmic actin-containing filaments were found in discrete regions of the myotube membrane that also contained high concentrations of acetylcholine receptors; such high concentrations of acetylcholine receptors have been described in regions of membrane-substrate contact. Moreover, when primary rat myotubes were exposed to human myasthenic serum, gross morphological changes, accompanied by an apparent rearrangement of the cytoplasmic actin-containing cytoskeleton, were produced. Although whether the distribution of cytoplasmic actin-containing structures was influenced by the organization of acetylcholine receptor or vice versa cannot be determined from these studies, these findings suggest that in primary rat muscle cells grown in vitro, acetylcholine receptors and beta-cytoplasmic actin-containing structures may be somehow connected.

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