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 Agrin regulates CLASP2-mediated capture of microtubules at the neuromuscular junction synaptic membrane

2012 ◽  
Vol 198 (3) ◽  
pp. 421-437 ◽  
Author(s):  
Nadine Schmidt ◽  
Sreya Basu ◽  
Stefan Sladecek ◽  
Sabrina Gatti ◽  
Jeffrey van Haren ◽  
...  
Keyword(s):  
Neuromuscular Junction ◽  
Acetylcholine Receptor ◽  
Three Dimensional ◽  
Postsynaptic Membrane ◽  
Dimensional Structure ◽  
Muscle Membrane ◽  
Synaptic Membrane ◽  
Dense Network ◽  
Phosphatidylinositol 3 Kinase ◽  
Neuronal Regulation

Agrin is the major factor mediating the neuronal regulation of postsynaptic structures at the vertebrate neuromuscular junction, but the details of how it orchestrates this unique three-dimensional structure remain unknown. Here, we show that agrin induces the formation of the dense network of microtubules in the subsynaptic cytoplasm and that this, in turn, regulates acetylcholine receptor insertion into the postsynaptic membrane. Agrin acted in part by locally activating phosphatidylinositol 3-kinase and inactivating GSK3β, which led to the local capturing of dynamic microtubules at agrin-induced acetylcholine receptor (AChR) clusters, mediated to a large extent by the microtubule plus-end tracking proteins CLASP2 and CLIP-170. Indeed, in the absence of CLASP2, microtubule plus ends at the subsynaptic muscle membrane, the density of synaptic AChRs, the size of AChR clusters, and the numbers of subsynaptic muscle nuclei with their selective gene expression programs were all reduced. Thus, the cascade linking agrin to CLASP2-mediated microtubule capturing at the synaptic membrane is essential for the maintenance of a normal neuromuscular phenotype.

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 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.

scholarly journals Absence of filipin-sterol complexes from the membranes of active zones and acetylcholine receptor aggregates at frog neuromuscular junctions.

1981 ◽  
Vol 88 (2) ◽  
pp. 453-458 ◽  
Author(s):  
Y Nakajima ◽  
P C Bridgman
Keyword(s):  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Freeze Fracture ◽  
Cholesterol Content ◽  
Postsynaptic Membrane ◽  
Large Particles ◽  
Glutaraldehyde Solution ◽  
Active Zones ◽  
Low Cholesterol

The polyene antibiotic filipin reacts specifically with membrane cholesterol and produces distinctive membrane lesions. We treated frog cutaneous and sartorius muscles with 0.04% filipin in a glutaraldehyde solution with or without prefixation with glutaraldehyde. Freeze-fracture of these muscles revealed numerous 19 to 38-nm protuberances and depressions (filipin-sterol complexes) in most areas of muscle, axon, and Schwann cell membranes. In the presynaptic membrane, however, these filipin-sterol complexes were absent from active zones consisting of ridges bordered with double rows of particles. In the postsynaptic membrane, filipin-sterol complexes were also virtually absent from the areas occupied by aggregates of large particles representing acetylcholine receptors. These results suggest that the membrane regions of active zones and acetylcholine receptor aggregates have a low cholesterol content.

scholarly journals 300-kD subsynaptic protein copurifies with acetylcholine receptor-rich membranes and is concentrated at neuromuscular synapses

1987 ◽  
Vol 104 (4) ◽  
pp. 939-946 ◽  
Author(s):  
ML Woodruff ◽  
J Theriot ◽  
SJ Burden
Keyword(s):  
Acetylcholine Receptor ◽  
Electric Organ ◽  
Postsynaptic Membrane ◽  
Synaptic Membrane ◽  
Binding Assays ◽  
Western Blots ◽  
Neuromuscular Synapses ◽  
Peripheral Membrane Proteins ◽  
Torpedo Electric Organ ◽  
Skeletal Muscle Antibodies

Acetylcholine receptor-rich membranes from the electric organ of Torpedo californica are enriched in the four different subunits of the acetylcholine receptor and in two peripheral membrane proteins at 43 and 300 kD. We produced monoclonal antibodies against the 300-kD protein and have used these antibodies to determine the location of the protein, both in the electric organ and in skeletal muscle. Antibodies to the 300-kD protein were characterized by Western blots, binding assays to isolated membranes, and immunofluorescence on tissue. In Torpedo electric organ, antibodies to the 300-kD protein stain only the innervated face of the electrocytes. The 300-kD protein is on the intracellular surface of the postsynaptic membrane, since antibodies to the 300-kD protein bind more efficiently to saponin-permeabilized, right side out membranes than to intact membranes. Some antibodies against the Torpedo 300-kD protein cross-react with amphibian and mammalian neuromuscular synapses, and the cross-reacting protein is also highly concentrated on the intracellular surface of the post-synaptic membrane.

Rituximab in AChR subtype of myasthenia gravis: systematic review

2020 ◽  
Vol 91 (4) ◽  
pp. 392-395 ◽  
Author(s):  
Vincenzo Di Stefano ◽  
Antonino Lupica ◽  
Marianna Gabriella Rispoli ◽  
Antonio Di Muzio ◽  
Filippo Brighina ◽  
...  
Keyword(s):  
Systematic Review ◽  
Neuromuscular Junction ◽  
Myasthenia Gravis ◽  
Acetylcholine Receptor ◽  
Autoimmune Disorder ◽  
Postsynaptic Membrane ◽  
Receptor Subtype ◽  
Registration Number ◽  
Randomised Controlled ◽  
Time To Relapse

Myasthenia gravis (MG) is a chronic autoimmune disorder of the neuromuscular junction characterised by an autoantibody against acetylcholine receptor (AChR-Ab), autoantibody against muscle-specific kinase (MuSK-Ab), lipoprotein-related protein 4 or agrin in the postsynaptic membrane at the neuromuscular junction. Many patients are resistant to conventional treatment and effective therapies are needed. Rituximab (RTX) is a monoclonal antibody directed against CD20 antigen on B cells which has been successfully employed in anti-MuSK-Ab+MG, but the efficacy in anti-AChR-Ab+MG is still debated. The purpose of this systematic review was to describe the best evidence for RTX in the acetylcholine receptor subtype. The authors undertook a literature search during the period of 1999–2019 according to the Preferred Reporting Items for Systematic Reviews and Meta-Analys methodology, employing (myasthenia)+(gravis)+(RTX) as search terms. The analysis was confined to studies that include at least five patients with confirmed anti-AChR-Ab+MG. Thirteen studies have been selected, showing a good safety. The data obtained were heterogeneous in terms of posology, administration scheme and patients’ evaluation, ranging from a minimum of two to a maximum of three cycles. RTX led to a sustained clinical improvement with prolonged time to relapse, in parallel to a reduction or discontinuation of other immunosuppressive therapies. Treatment with RTX appears to work in some but not all patients with anti-AChR-Ab+MG, but randomised controlled trials are needed. Future studies should take into account the subtype of MG and employ reliable measures of outcome and severity focusing on how to identify patients who may benefit from the treatment. Trial registration number: NCT02110706.

Disorders of the neuromuscular junction

2010 ◽  
pp. 5177-5184 ◽  
Author(s):  
David Hilton-Jones ◽  
Jacqueline Palace
Keyword(s):  
Ion Channels ◽  
Neuromuscular Junction ◽  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Muscle Receptor ◽  
Male Preponderance ◽  
Female Bias ◽  
Anti Acetylcholine

Two fundamentally different pathological processes are associated with disease at the neuromuscular junction: (1) acquired disorders in which autoantibodies are directed against nerve or muscle receptor or ion channels; (2) rare inherited conditions in which the defect may be pre- or postsynaptic. Aetiology and epidemiology—the fundamental disorder is loss of functional acetylcholine receptors most frequently as a result of binding of anti-acetylcholine receptor (anti-AChR) antibodies. Incidence is about 10 per million population and prevalence about 8 per 100 000, with a marked female bias in cases aged under 40 years and male preponderance in those over 50 years. Thymomas occur in about 10% of cases....

scholarly journals An unusual beta-spectrin associated with clustered acetylcholine receptors.

1989 ◽  
Vol 108 (2) ◽  
pp. 481-493 ◽  
Author(s):  
R J Bloch ◽  
J S Morrow
Keyword(s):  
Neuromuscular Junction ◽  
Acetylcholine Receptors ◽  
Room Temperature ◽  
Peptide Mapping ◽  
Postsynaptic Membrane ◽  
Human Erythrocytes ◽  
Early Event ◽  
Alpha Spectrin

The clustering of acetylcholine receptors (AChR) in the postsynaptic membrane is an early event in the formation of the neuromuscular junction. The mechanism of clustering is still unknown, but is generally believed to be mediated by the postsynaptic cytoskeleton. We have identified an unusual isoform of beta-spectrin which colocalizes with AChR in AChR clusters isolated from rat myotubes in vitro. A related antigen is present postsynaptically at the neuromuscular junction of the rat. Immunoprecipitation, peptide mapping and immunofluorescence show that the beta-spectrin in AChR clusters resembles but is distinct from the beta-spectrin of human erythrocytes. alpha-Spectrin appears to be absent from AChR clusters. Semiquantitative immunofluorescence techniques indicate that there are from two to seven beta-spectrin molecules present for every clustered AChR, the higher values being obtained from rapidly prepared clusters, the lower values from clusters that require several minutes or more for isolation. Upon incubation of isolated AChR clusters for 1 h at room temperature, beta-spectrin is slowly depleted and the AChR redistribute into microaggregates. The beta-spectrin that remains associated with the myotube membrane is concentrated at these microaggregates. beta-Spectrin is quantitatively lost from clusters upon digestion with chymotrypsin, which causes AChR to redistribute in the plane of the membrane. These results suggest that AChR in clusters is closely linked to an unusual isoform of beta-spectrin.

scholarly journals Asynchronous assembly of the acetylcholine receptor and of the 43-kD nu1 protein in the postsynaptic membrane of developing Torpedo marmorata electrocyte.

1989 ◽  
Vol 108 (1) ◽  
pp. 127-139 ◽  
Author(s):  
E Kordeli ◽  
J Cartaud ◽  
H O Nghiêm ◽  
A Devillers-Thiéry ◽  
J P Changeux
Keyword(s):  
Plasma Membrane ◽  
Acetylcholine Receptor ◽  
Postsynaptic Membrane ◽  
Synaptic Membrane ◽  
Asymmetric Distribution ◽  
Subsequent Stage ◽  
Torpedo Marmorata ◽  
Intracellular Compartments ◽  
Membrane Compartments

The assembly of the nicotinic acetylcholine receptor (AchR) and the 43-kD protein (v1), the two major components of the post synaptic membrane of the electromotor synapse, was followed in Torpedo marmorata electrocyte during embryonic development by immunocytochemical methods. At the first developmental stage investigated (45-mm embryos), accumulation of AchR at the ventral pole of the newly formed electrocyte was observed within columns before innervation could be detected. No concomitant accumulation of 43-kD immunoreactivity in AchR-rich membrane domains was observed at this stage, but a transient asymmetric distribution of the extracellular protein, laminin, which paralleled that of the AchR, was noticed. At the subsequent stage studied (80-mm embryos), codistribution of the two proteins was noticed on the ventral face of the cell. Intracellular pools of AchR and 43-kD protein were followed at the EM level in 80-mm electrocytes. AchR immunoreactivity was detected within membrane compartments, which include the perinuclear cisternae of the endoplasmic reticulum and the plasma membrane. On the other hand, 43-kD immunoreactivity was not found associated with the AchR in the intracellular compartments of the cell, but codistributed with the AchR at the level of the plasma membrane. The data reported in this study suggest that AchR clustering in vivo is not initially determined by the association of the AchR with the 43-kD protein, but rather relies on AchR interaction with extracellular components, for instance from the basement membrane, laid down in the tissue before the entry of the electromotor nerve endings.

scholarly journals Degradation rates of acetylcholine receptors can be modified in the postjunctional plasma membrane of the vertebrate neuromuscular junction.

1986 ◽  
Vol 103 (4) ◽  
pp. 1399-1403 ◽  
Author(s):  
M M Salpeter ◽  
D L Cooper ◽  
T Levitt-Gilmour
Keyword(s):  
Neuromuscular Junction ◽  
Acetylcholine Receptors ◽  
Degradation Rate ◽  
Postsynaptic Membrane ◽  
Nerve Crush ◽  
Adult Mice ◽  
Vertebrate Muscle ◽  
Degradation Rates ◽  
Alpha Bungarotoxin ◽  
Two Populations

Denervation of vertebrate muscle causes an acceleration of acetylcholine receptor turnover at the neuromuscular junction. This acceleration reflects the composite behavior of two populations of receptors: "original receptors" present at the junction at the time of denervation, and "new receptors" inserted into the denervated junction to replace the original receptors as they are degraded (Levitt, T. A., and M. M. Salpeter, 1981, Nature (Lond.), 291:239-241). The present study examined the degradation rate of original receptors to determine whether reinnervation could reverse the effect of denervation. Sternomastoid muscles in adult mice were denervated by either cutting or crushing the nerve, and the nerves either allowed to regenerate or ligated to prevent regeneration. The original receptors were labeled with 125I-alpha-bungarotoxin at the time of denervation, and their degradation rate followed by gamma counting. We found that when the nerve was not allowed to regenerate, the degradation decreased from a t1/2 of approximately 8-10 d to one of approximately 3 d (as reported earlier for denervated original receptors) and remained at that half-life throughout the experiment (approximately 36 d). If the axons were allowed to regenerate (which occurred asynchronously between day 14 and day 30 after nerve cut and between day 7 and 13 after nerve crush), the accelerated degradation rate of the original receptors reverted to a t1/2 of approximately 8 d. Our data lead us to conclude that the effect of denervation on the degradation rate of original receptors can be reversed by reinnervating. The nerve can thus slow the degradation rate of receptors previously inserted into the postsynaptic membrane.

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