scholarly journals Characterisation of alpha-dystrobrevin in muscle

1998 ◽  
Vol 111 (17) ◽  
pp. 2595-2605 ◽  
Author(s):  
R. Nawrotzki ◽  
N.Y. Loh ◽  
M.A. Ruegg ◽  
K.E. Davies ◽  
D.J. Blake
Keyword(s):  
Skeletal Muscle ◽  
Neuromuscular Junction ◽  
Cell Surface ◽  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Synapse Formation ◽  
Electric Organ ◽  
Postsynaptic Membrane ◽  
Related Protein ◽  
Receptor Clustering

Dystrophin-related and associated proteins are important for the formation and maintenance of the mammalian neuromuscular junction. Initial studies in the electric organ of Torpedo californica showed that the dystrophin-related protein dystrobrevin (87K) co-purifies with the acetylcholine receptors and other postsynaptic proteins. Dystrobrevin is also a major phosphotyrosine-containing protein in the postsynaptic membrane. Since inhibitors of tyrosine protein phosphorylation block acetylcholine receptor clustering in cultured muscle cells, we examined the role of alpha-dystrobrevin during synapse formation and in response to agrin. Using specific antibodies, we show that C2 myoblasts and early myotubes only produce alpha-dystrobrevin-1, the mammalian orthologue of Torpedo dystrobrevin, whereas mature skeletal muscle expresses three distinct alpha-dystrobrevin isoforms. In myotubes, alpha-dystrobrevin-1 is found on the cell surface and also in acetylcholine receptor-rich domains. Following agrin stimulation, alpha-dystrobrevin-1 becomes re-localised beneath the cell surface into macroclusters that contain acetylcholine receptors and another dystrophin-related protein, utrophin. This redistribution is not associated with tyrosine phosphorylation of alpha-dystrobrevin-1 by agrin. Furthermore, we show that alpha-dystrobrevin-1 is associated with both utrophin in C2 cells and dystrophin in mature skeletal muscle. Thus alpha-dystrobrevin-1 is a component of two protein complexes in muscle, one with utrophin at the neuromuscular junction and the other with dystrophin at the sarcolemma. These results indicate that alpha-dystrobrevin-1 is not involved in the phosphorylation-dependent, early stages of receptor clustering, but rather in the stabilisation and maturation of clusters, possibly via an interaction with utrophin.

scholarly journals Subtle Neuromuscular Defects in Utrophin-deficient Mice

1997 ◽  
Vol 136 (4) ◽  
pp. 871-882 ◽  
Author(s):  
R. Mark Grady ◽  
John P. Merlie ◽  
Joshua R. Sanes
Keyword(s):  
Skeletal Muscle ◽  
Neuromuscular Junction ◽  
Acetylcholine Receptors ◽  
Cultured Cells ◽  
Neuromuscular Junctions ◽  
Becker Muscular Dystrophy ◽  
Postsynaptic Membrane ◽  
Mutant Mice ◽  
Nonmuscle Cells ◽  
And Behavior

Utrophin is a large cytoskeletal protein that is homologous to dystrophin, the protein mutated in Duchenne and Becker muscular dystrophy. In skeletal muscle, dystrophin is broadly distributed along the sarcolemma whereas utrophin is concentrated at the neuromuscular junction. This differential localization, along with studies on cultured cells, led to the suggestion that utrophin is required for synaptic differentiation. In addition, utrophin is present in numerous nonmuscle cells, suggesting that it may have a more generalized role in the maintenance of cellular integrity. To test these hypotheses we generated and characterized utrophin-deficient mutant mice. These mutant mice were normal in appearance and behavior and showed no obvious defects in muscle or nonmuscle tissue. Detailed analysis, however, revealed that the density of acetylcholine receptors and the number of junctional folds were reduced at the neuromuscular junctions in utrophin-deficient skeletal muscle. Despite these subtle derangements, the overall structure of the mutant synapse was qualitatively normal, and the specialized characteristics of the dystrophin-associated protein complex were preserved at the mutant neuromuscular junction. These results point to a predominant role for other molecules in the differentiation and maintenance of the postsynaptic membrane.

scholarly journals A novel 87,000-Mr protein associated with acetylcholine receptors in Torpedo electric organ and vertebrate skeletal muscle.

1989 ◽  
Vol 109 (4) ◽  
pp. 1753-1764 ◽  
Author(s):  
C Carr ◽  
G D Fischbach ◽  
J B Cohen
Keyword(s):  
Skeletal Muscle ◽  
Acetylcholine Receptors ◽  
Large Fraction ◽  
Surface Membrane ◽  
Electric Organ ◽  
Postsynaptic Membrane ◽  
Velocity Sedimentation ◽  
Synaptic Membranes ◽  
Torpedo Electric Organ

To identify proteins associated with nicotinic postsynaptic membranes, mAbs have been prepared to proteins extracted by alkaline pH or lithium diiodosalicylate from acetylcholine receptor-rich (AChR) membranes of Torpedo electric organ. Antibodies were obtained that recognized two novel proteins of 87,000 Mr and a 210,000:220,000 doublet as well as previously described proteins of 43,000 Mr, 58,000 (51,000 in our gel system), 270,000, and 37,000 (calelectrin). The 87-kD protein copurified with acetylcholine receptors and with 43- and 51-kD proteins during equilibrium centrifugation on continuous sucrose gradients, whereas a large fraction of the 210/220-kD protein was separated from AChRs. The 87-kD protein remained associated with receptors and 43-kD protein during velocity sedimentation through shallow sucrose gradients, a procedure that separated a significant amount of 51-kD protein from AChRs. The 87- and 270-kD proteins were cleaved by Ca++-activated proteases present in crude preparations and also in highly purified postsynaptic membranes. With the exception of anti-37-kD antibodies, some of the monoclonals raised against Torpedo proteins also recognized determinants in frozen sections of chick and/or rat skeletal muscle fibers and in permeabilized chick myotubes grown in vitro. Anti-87-kD sites were concentrated at chick and rat endplates, but the antibodies also recognized determinants present at lower site density in the extrasynaptic membrane. Anti-210:220-kD labeled chick endplates, but studies of neuron-myotube cocultures showed that this antigen was located on neurites rather than the postsynaptic membrane. As reported in other species, 43-kD determinants were restricted to chick endplates and anti-51-kD and anti-270-kD labeled extrasynaptic as well as synaptic membranes. None of the cross reacting antibodies recognized determinants on intact (unpermeabilized) myotubes, so the antigens must be located on the cytoplasmic aspect of the surface membrane. The role that each intracellular determinant plays in AChR immobilization at developing and mature endplates remains to be investigated.

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.

Distribution of acetylcholine receptors in the vicinity of nerve terminals on skeletal muscle of the frog

1972 ◽  
Vol 181 (1065) ◽  
pp. 431-440 ◽  
Keyword(s):  
Skeletal Muscle ◽  
Connective Tissue ◽  
Neuromuscular Junction ◽  
Muscle Fibre ◽  
Nerve Terminal ◽  
Acetylcholine Receptors ◽  
Postsynaptic Membrane ◽  
Muscle Membrane ◽  
Muscle Fibres ◽  
Nerve Terminals

1. The acetylcholine (ACh) sensitivity of muscle fibres at the neuromuscular junction of the frog was investigated in preparations in which the nerve terminals could be clearly seen. 2. ACh released iontophoretically from a micropipette that was precisely positioned at various points along the muscle fibre in the vicinity of the synapse showed that the peak chemosensitivity (up to 1900 mV/nC) is confined to an area of postsynaptic membrane within a few micra of the nerve terminal; a tenfold decline in sensitivity was obtained when the ACh was released only 5 to 10 μm from the terminal’s edge. It is estimated that most of the response obtained when ACh is released within 40 μm from the terminal (the area covered in this study) is due to diffusion to the immediate postsynaptic area. The extrasynaptic chemosensitivity of the muscle membrane was too low to be measured with the present methods. 3. The accuracy with which micropipettes could be positioned in synaptic areas and the clarity of viewing nerve terminals were improved by bathing the tissue in collagenase, which reduced the amount of connective tissue. The distribution of chemosensitivity remained unchanged by such treatment. The ACh response was not detectably altered when nerve terminals were lifted off the muscle, exposing the subsynaptic muscle surface.

scholarly journals Localization of dystrophin relative to acetylcholine receptor domains in electric tissue and adult and cultured skeletal muscle.

1991 ◽  
Vol 113 (5) ◽  
pp. 1133-1144 ◽  
Author(s):  
R Sealock ◽  
M H Butler ◽  
N R Kramarcy ◽  
K X Gao ◽  
A A Murnane ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Duchenne Muscular Dystrophy ◽  
Neuromuscular Junction ◽  
Acetylcholine Receptors ◽  
Electric Organ ◽  
Membrane Domains ◽  
High Affinity ◽  
Postsynaptic Protein ◽  
Human Dystrophin ◽  
Surrounding Membrane

Two high-affinity mAbs were prepared against Torpedo dystrophin, an electric organ protein that is closely similar to human dystrophin, the gene product of the Duchenne muscular dystrophy locus. The antibodies were used to localize dystrophin relative to acetylcholine receptors (AChR) in electric organ and in skeletal muscle, and to show identity between Torpedo dystrophin and the previously described 270/300-kD Torpedo postsynaptic protein. Dystrophin was found in both AChR-rich and AChR-poor regions of the innervated face of the electroplaque. Immunogold experiments showed that AChR and dystrophin were closely intermingled in the AChR domains. In contrast, dystrophin appeared to be absent from many or all AChR-rich domains of the rat neuromuscular junction and of AChR clusters in cultured muscle (Xenopus laevis). It was present, however, in the immediately surrounding membrane (deep regions of the junctional folds, membrane domains interdigitating with and surrounding AChR domains within clusters). These results suggest that dystrophin may have a role in organization of AChR in electric tissue. Dystrophin is not, however, an obligatory component of AChR domains in muscle and, at the neuromuscular junction, its roles may be more related to organization of the junctional folds.

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 CLASP2-dependent microtubule capture at the neuromuscular junction membrane requires LL5β and actin for focal delivery of acetylcholine receptor vesicles

2015 ◽  
Vol 26 (5) ◽  
pp. 938-951 ◽  
Author(s):  
Sreya Basu ◽  
Stefan Sladecek ◽  
Isabel Martinez de la Peña y Valenzuela ◽  
Mohammed Akaaboune ◽  
Ihor Smal ◽  
...  
Keyword(s):  
Neuromuscular Junction ◽  
Actin Cytoskeleton ◽  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Postsynaptic Membrane ◽  
Interaction Partner ◽  
Synaptic Membrane ◽  
Microtubule Capture

A novel mechanism is described for the agrin-mediated focal delivery of acetylcholine receptors (AChRs) to the postsynaptic membrane of the neuromuscular junction. Microtubule capture mediated by CLASP2 and its interaction partner, LL5β, and an intact subsynaptic actin cytoskeleton are both required for focal AChR transport to the synaptic membrane.

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 Processing of ARIA and release from isolated nerve terminals

1999 ◽  
Vol 354 (1381) ◽  
pp. 411-416 ◽  
Author(s):  
Bomie Han ◽  
Gerald D. Fischbach
Keyword(s):  
Neuromuscular Junction ◽  
Action Potential ◽  
Acetylcholine Receptors ◽  
Molecular Layer ◽  
Postsynaptic Membrane ◽  
High Density ◽  
Small Contribution ◽  
Presynaptic Nerve Terminal ◽  
Voltage Gated

The neuromuscular junction is a specialized synapse in that every action potential in the presynaptic nerve terminal results in an action potential in the postsynaptic membrane, unlike most interneuronal synapses where a single presynaptic input makes only a small contribution to the population postsynaptic response. The postsynaptic membrane at the neuromuscular junction contains a high density of neurotransmitter (acetylcholine) receptors and a high density of voltage–gated Na + channels. Thus, the large acetylcholine activated current occurs at the same site where the threshold for action potential generation is low. Acetylcholine receptor inducing activity (ARIA), a 42 kD protein, that stimulates synthesis of acetylcholine receptors and voltage–gated Na + channels in cultured myotubes, probably plays the same roles at developing and mature motor endplates in vivo . ARIA is synthesized as part of a larger, transmembrane, precursor protein called proARIA. Delivery of ARIA from motor neuron cell bodies in the spinal cord to the target endplates involves several steps, including proteolytic cleavage of proARIA. ARIA is also expressed in the central nervous system and it is abundant in the molecular layer of the cerebellum. In this paper we describe our first experiments on the processing and release of ARIA from subcellular fractions containing synaptosomes from the chick cerebellum as a model system.

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