Induce Acetylcholine Receptor Research Articles

Transport and Secretion of the Wnt3 Ligand by Motor Neuron-like Cells and Developing Motor Neurons.

The vertebrate neuromuscular junction (NMJ) is formed by a presynaptic motor nerve terminal and a postsynaptic muscle specialization. Cumulative evidence reveals that Wnt ligands secreted by the nerve terminal control crucial steps of NMJ synaptogenesis. For instance, the Wnt3 ligand is expressed by motor neurons at the time of NMJ formation and induces postsynaptic differentiation in recently formed muscle fibers. However, the behavior of presynaptic-derived Wnt ligands at the vertebrate NMJ has not been deeply analyzed. Here, we conducted overexpression experiments to study the expression, distribution, secretion, and function of Wnt3 by transfection of the motor neuron-like NSC-34 cell line and by in ovo electroporation of chick motor neurons. Our findings reveal that Wnt3 is transported along motor axons in vivo following a vesicular-like pattern and reaches the NMJ area. In vitro, we found that endogenous Wnt3 expression increases as the differentiation of NSC-34 cells proceeds. Although NSC-34 cells overexpressing Wnt3 do not modify their morphological differentiation towards a neuronal phenotype, they effectively induce acetylcholine receptor clustering on co-cultured myotubes. These findings support the notion that presynaptic Wnt3 is transported and secreted by motor neurons to induce postsynaptic differentiation in nascent NMJs.

Rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells.

BackgroundHuman pluripotent stem cells (hPSCs) are being applied in regenerative medicine and for the in vitro modeling of human intractable disorders. In particular, neural cells derived from disease-specific human induced pluripotent stem cells (hiPSCs) established from patients with neurological disorders have been used as in vitro disease models to recapitulate in vivo pathogenesis because neural cells cannot be usually obtained from patients themselves.ResultsIn this study, we established a rapid, efficient, and simple method for efficiently deriving motor neurons from hPSCs that is useful for pathophysiological analysis and the development of drugs to treat motor neuron diseases. Treatment with GSK3β inhibitors during the initial phase of differentiation in combination with dual SMAD inhibition was sufficient to induce PAX6+ and SOX1+ neural progenitors within 1 week, and subsequent treatment with retinoic acid (RA) and purmorphamine, which activates sonic hedgehog (SHH) signaling, resulted in the highly efficient induction of HB9+ and ISL-1+ motor neurons within 2 weeks. After 4 weeks of monolayer differentiation in motor neuron maturation medium, hPSC-derived motor neurons were shown to mature, displaying larger somas and clearer staining for the mature motor neuron marker choline acetyltransferase (ChAT). Moreover, hPSC-derived motor neurons were able to form neuromuscular junctions with human myotubes in vitro and induced acetylcholine receptor (AChR) clustering, as detected by Alexa 555-conjugated α-Bungarotoxin (α-BTX), suggesting that these hPSC-derived motor neurons formed functional contacts with skeletal muscles. This differentiation system is simple and is reproducible in several hiPSC clones, thereby minimizing clonal variation among hPSC clones. We also established a system for visualizing motor neurons with a lentiviral reporter for HB9 (HB9e438::Venus). The specificity of this reporter was confirmed through immunocytochemistry and quantitative RT-PCR analysis of high-positive fractions obtained via fluorescence-activated cell sorting (FACS), suggesting its applicability for motor neuron-specific analysis.ConclusionsOur motor neuron differentiation system and lentivirus-based reporter system for motor neurons facilitate the analysis of disease-specific hiPSCs for motor neuron diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-015-0172-4) contains supplementary material, which is available to authorized users.

Identification of an Agrin Mutation that Causes Congenital Myasthenia and Affects Synapse Function

We report the case of a congenital myasthenic syndrome due to a mutation in AGRN, the gene encoding agrin, an extracellular matrix molecule released by the nerve and critical for formation of the neuromuscular junction. Gene analysis identified a homozygous missense mutation, c.5125G>C, leading to the p.Gly1709Arg variant. The muscle-biopsy specimen showed a major disorganization of the neuromuscular junction, including changes in the nerve-terminal cytoskeleton and fragmentation of the synaptic gutters. Experiments performed in nonmuscle cells or in cultured C2C12 myotubes and using recombinant mini-agrin for the mutated and the wild-type forms showed that the mutated form did not impair the activation of MuSK or change the total number of induced acetylcholine receptor aggregates. A solid-phase assay using the dystrophin glycoprotein complex showed that the mutation did not affect the binding of agrin to alpha-dystroglycan. Injection of wild-type or mutated agrin into rat soleus muscle induced the formation of nonsynaptic acetylcholine receptor clusters, but the mutant protein specifically destabilized the endogenous neuromuscular junctions. Importantly, the changes observed in rat muscle injected with mutant agrin recapitulated the pre- and post-synaptic modifications observed in the patient. These results indicate that the mutation does not interfere with the ability of agrin to induce postsynaptic structures but that it dramatically perturbs the maintenance of the neuromuscular junction.

Caffeine and nicotine decrease acetylcholine receptor clustering in C2C12 myotube culture

As motor neurons approach skeletal muscle during development, agrin is released and induces acetylcholine receptor (AChR) clustering. Our laboratory investigates the effect of environmental agents on skeletal muscle development by using C2C12 cell culture. For the current project, we investigated both short-term and long-term exposure to caffeine, nicotine, or both, at physiologically relevant concentrations. Short-term exposure was limited to the last 48 h of myotube formation, whereas a long-term exposure of 2 weeks allowed for several generations of myoblast proliferation followed by myotube formation. Both agrin-induced and spontaneous AChR clustering frequencies were assessed. For agrin-induced AChR clustering, agrin was added for the last 16 h of myotube formation. Caffeine, nicotine, or both significantly decreased agrin-induced AChR clustering during short-term and long-term exposure. Furthermore, caffeine, nicotine, or both significantly decreased spontaneous AChR clustering during long-term, but not short-term exposure. Surprisingly, caffeine and nicotine in combination did not decrease AChR clustering beyond the effect of either treatment alone. We conclude that physiologically relevant concentrations of caffeine or nicotine decrease AChR clustering. Moreover, we predict that fetuses exposed to caffeine or nicotine may be less likely to form appropriate neuromuscular synapses.

Caffeine and Nicotine Decrease Acetylcholine Receptor Clustering in C2C12 Myotube Culture

As motor neurons approach skeletal muscle during development, agrin is released, which induces acetylcholine receptor (AChR) clustering. Our lab investigates the effect of environmental agents on skeletal muscle development using C2C12 myotube cell culture. For the current project, we investigated both long term and short term exposure to caffeine, nicotine, or both, using physiologically relevant concentrations. Long term exposure was two weeks allowing for several generations of myoblast proliferation followed by myotube formation, while short term exposure was limited to the last 48 hours of myotube formation. For both lines of experimental manipulation, agrin was added for the last 16 hours of myotube formation, and AChR cluster frequency assessed. We found that caffeine, nicotine, or both, decreased agrin‐induced AChR clustering by a similar amount for long term or short term exposure. We conclude that physiologically relevant concentrations of caffeine or nicotine may inhibit AChR clustering. Furthermore, we predict that fetuses exposed to caffeine or nicotine may be less likely to form appropriate neuromuscular synapses.Kaia Kordosky‐Herrera was supported by the Midwestern University Physician Assistant Program. Wade A. Grow was supported by Midwestern University intramural funds.

Neuregulin‐1 induces acetylcholine receptor transcription in the absence of GABPα phosphorylation

Localization of acetylcholine receptors (AChRs) to the postsynaptic region of muscle is mediated in part by transcriptional mechanisms, because the genes encoding AChR subunits are transcribed selectively in synaptic myofiber nuclei. Neuregulin-1 (NRG-1) is a synaptic signal and induces transcription of AChRs in muscle cells. Signaling by NRG-1 is thought to involve the transcription factor GA-binding protein (GABP), a heterodimer of GABPalpha, which is a member of the Ets family, and GABPbeta. Phosphorylation of certain other Ets proteins outside the Ets DNA-binding domain serves to stimulate transcriptional activation in response to extracellular signals. According to previous studies, NRG-1 stimulates phosphorylation of GABPalpha at threonine 280 in the N-terminal region adjacent to the Ets domain, suggesting that GABPalpha phosphorylation might contribute to NRG-1 responsiveness. To determine the functional importance of the N-terminal region of GABPalpha and whether its function is regulated by phosphorylation, we generated muscle cell lines in which the endogenous GABPalpha gene was deleted and replaced by variants of GABPalpha mutated in the N-terminal region. We found that NRG-1 can induce transcription in cells with mutated T280 phosphorylation site, indicating that T280 phosphorylation does not contribute to NRG-1 responsiveness. We also found that NRG-1-induced transcription occurs in cells missing the entire N-terminal region of GABPalpha. Because NRG-1 signaling is not expected to alter the function of the C-terminal region, which remains in these cells, these results suggest that GABPbeta, or other interacting components, rather than GABPalpha directly, is targeted by NRG-1 signaling.

Agrin and laminin induce acetylcholine receptor clustering by convergent, Rho GTPase-dependent signaling pathways

During neuromuscular junction formation, extracellular matrix-mediated signals cause muscle surface acetylcholine receptors (AChRs) to aggregate at synaptic sites. Two extracellular matrix proteins, agrin and laminin, have each been shown to initiate signaling pathways that culminate in AChR clustering in cultured muscle cells. Here we present evidence that laminin-induced AChR clustering is mediated by the activation of the Rho GTPases Cdc42, Rac and Rho. Clustering in response to laminin is blocked by the dominant negative mutants Cdc42N17, RacN17 and RhoN19, as well as by the Rho inhibitor C3 transferase. Moreover, laminin-induced AChR clustering is impaired by the Rho kinase inhibitor Y-27632. Agrin-induced AChR clustering has previously been shown to require activation of Cdc42, Rac and Rho. Therefore, although agrin and laminin use distinct transmembrane receptors to initiate AChR clustering, their signaling pathways converge at the level of Rho GTPase activation.

Tyrosine phosphatase regulation of MuSK-dependent acetylcholine receptor clustering

During vertebrate neuromuscular junction (NMJ) development, nerve-secreted agrin induces acetylcholine receptor (AChR) clustering in muscle by activating the muscle-specific tyrosine kinase MuSK. Recently, it has been recognized that MuSK activation-dependent AChR clustering occurs in embryonic muscle even in the absence of agrin, but how this process is regulated is poorly understood. We report that inhibition of tyrosine phosphatases in cultured C2 mouse myotubes using pervanadate enhanced MuSK auto-activation and agrin-independent AChR clustering. Moreover, phosphatase inhibition also enlarged the AChR clusters induced by agrin in these cells. Conversely, in situ activation of MuSK in cultured Xenopus embryonic muscle cells, either focally by anti-MuSK antibody-coated beads or globally by agrin, stimulated downstream tyrosine phosphatases, which could be blocked by pervanadate treatment. Immunoscreening identified Shp2 as a major tyrosine phosphatase in C2 myotubes and down-regulation of its expression by RNA interference alleviated tyrosine phosphatase suppression of MuSK activation. Significantly, depletion of Shp2 increased both agrin-independent and agrin-dependent AChR clustering in myotubes. Our results suggest that muscle tyrosine phosphatases tightly regulate MuSK activation and signaling and support a novel role of Shp2 in MuSK-dependent AChR clustering.

What controls the position, number, size, and distribution of neuromuscular junctions on rat muscle fibers?

This review focuses on mechanisms that determine the position, number, size, and distribution of neuromuscular junctions (NMJs) on skeletal muscle fibers. Most of the data reviewed derive from studies of ectopic NMJ formation on soleus (SOL) muscle fibers in adult rats, which recapitulates essential aspects of NMJ formation in normal development. Transplanted axons induce acetylcholine receptor (AChR) aggregates, which are multiple and irregularly distributed initially but subsequently undergo massive reorganization such that one or a few winners survive and reach a certain size while the rest are eliminated (the losers). Results obtained by blocking nerve activity early and stimulating the SOL electrically show that evoked muscle impulse activity is responsible for the growth of winners to a given size and the creation of refractory zones, about 0.75 long, on each side of the winners, in which the elimination of losers occurs. Consequently, when two or more aggregates or NMJs survive on one fiber, they are, on average, at least 1.5 mm apart. Locally applied neural agrin induces comparable aggregation of AChRs and other postsynaptic proteins on denervated SOL fibers and such aggregates undergo similar activity-dependent selection for survival or elimination in refractory zones. In a dose-dependent way, neural agrin alone also induces expression of epsilon-AChR subunits and stabilizes AChRs to a half-life of 10 days, as found at normal NMJs. It is argued that signs of prepatterning of innervation sites by intrinsic muscle mechanisms may refer to epiphenomena that play no important role in NMJ formation. The conclusion is that neural agrin initiates and then maintains NMJs where motor axons happen to contact receptive muscle fibers and that evoked muscle impulse activity then ensures that the NMJs reach their appropriate size, efficiency and spatial distribution along each fiber.

Neuregulin-Heparan-sulfate Proteoglycan Interactions Produce Sustained erbB Receptor Activation Required for the Induction of Acetylcholine Receptors in Muscle

Neuregulins bind to and activate members of the EGF receptor family of tyrosine kinases that initiate a signaling cascade that induces acetylcholine receptor synthesis in the postsynaptic membrane of neuromuscular synapses. In addition to an EGF-like domain, sufficient for receptor binding and tyrosine auto-phosphorylation, many spliced forms also have an IG-like domain that binds HSPGs and maintains a high concentration of neuregulin at synapses. Here, we show that the IG-like domain functions to keep the EGF-like domain at sufficiently high concentrations for a sufficiently long period of time necessary to induce acetylcholine receptor gene expression in primary chick myotubes. Using recombinant neuregulins with and without the IG-like domain, we found that IG-like domain binding to endogenous HSPGs produces a 4-fold increase in receptor phosphorylation. This enhancement of activity was blocked by soluble heparin or by pretreatment of muscle cells with heparitinase. We show that at least 12-24 h of neuregulin exposure was required to turn on substantial acetylcholine receptor gene expression and that the erbB receptors need to be kept phosphorylated during this time. The need for sustained erbB receptor activation may be the reason why neuregulins are so highly concentrated in the extracellular matrix of synapses.

Muscle activity and muscle agrin regulate the organization of cytoskeletal proteins and attached acetylcholine receptor (AchR) aggregates in skeletal muscle fibers.

In innervated skeletal muscle fibers, dystrophin and beta-dystroglycan form rib-like structures (costameres) that appear as predominantly transverse stripes over Z and M lines. Here, we show that the orientation of these stripes becomes longitudinal in denervated muscles and transverse again in denervated electrically stimulated muscles. Skeletal muscle fibers express nonneural (muscle) agrin whose function is not well understood. In this work, a single application of > or = 10 nM purified recombinant muscle agrin into denervated muscles preserved the transverse orientation of costameric proteins that is typical for innervated muscles, as did a single application of > or = 1 microM neural agrin. At lower concentration, neural agrin induced acetylcholine receptor aggregates, which colocalized with longitudinally oriented beta-dystroglycan, dystrophin, utrophin, syntrophin, rapsyn, and beta 2-laminin in denervated unstimulated fibers and with the same but transversely oriented proteins in innervated or denervated stimulated fibers. The results indicate that costameres are plastic structures whose organization depends on electrical muscle activity and/or muscle agrin.

In vivo acetylcholine receptor expression induced by calcitonin gene-related peptide in rat soleus muscle.

We applied calcitonin gene-related peptide (CGRP) by continuous perfusion of the extrajunctional surface of the adult rat soleus muscle in vivo. We obtained this through a fine polyethylene catheter connected to an Alzet pump implanted in the animal. The perfusion induced a local acetylcholine receptor accumulation in the membrane of the muscle fibres starting with a delay of one to two days, provided a chronic conduction block of soleus innervation was concomitantly present. The effect was prominent, being higher than that following denervation. The lack of acetylcholine receptor accumulation observed in sham perfused animals and the co-administration of CGRP and its competitive antagonist peptide, hCGRP(8-37), eliminates the possibility that the response to CGRP application represents an inflammatory reaction to foreign bodies instead of a specific effect of the peptide. We suggest that CGRP may act on the extrajunctional membrane of muscle fibres to help induce acetylcholine receptor accumulation after appropriate receptors for the peptide are re-expressed due to muscle paralysis. Whilst this is compatible with a role of CGRP in synaptogenesis, a recent study showed that alpha-CGRP(-/-) mutant mice have normal neuromuscular junction development. However, given the redundancy of factors involved in acetylcholine receptor accumulation, further experiments on multiple knock-outs need to be performed before a final conclusion is reached about the physiological significance of CGRP.

The actin-driven movement and formation of acetylcholine receptor clusters.

A new method was devised to visualize actin polymerization induced by postsynaptic differentiation signals in cultured muscle cells. This entails masking myofibrillar filamentous (F)-actin with jasplakinolide, a cell-permeant F-actin-binding toxin, before synaptogenic stimulation, and then probing new actin assembly with fluorescent phalloidin. With this procedure, actin polymerization associated with newly induced acetylcholine receptor (AChR) clustering by heparin-binding growth-associated molecule-coated beads and by agrin was observed. The beads induced local F-actin assembly that colocalized with AChR clusters at bead-muscle contacts, whereas both the actin cytoskeleton and AChR clusters induced by bath agrin application were diffuse. By expressing a green fluorescent protein-coupled version of cortactin, a protein that binds to active F-actin, the dynamic nature of the actin cytoskeleton associated with new AChR clusters was revealed. In fact, the motive force generated by actin polymerization propelled the entire bead-induced AChR cluster with its attached bead to move in the plane of the membrane. In addition, actin polymerization is also necessary for the formation of both bead and agrin-induced AChR clusters as well as phosphotyrosine accumulation, as shown by their blockage by latrunculin A, a toxin that sequesters globular (G)-actin and prevents F-actin assembly. These results show that actin polymerization induced by synaptogenic signals is necessary for the movement and formation of AChR clusters and implicate a role of F-actin as a postsynaptic scaffold for the assembly of structural and signaling molecules in neuromuscular junction formation.

Globular domains of agrin are functional units that collaborate to induce acetylcholine receptor clustering.

Agrin, an extracellular matrix protein involved in neuromuscular junction formation, directs clustering of postsynaptic molecules, including acetylcholine receptors (AChRs). This activity resides entirely in the C-terminal portion of the protein, which consists of three laminin-like globular domains (G-domains: G1, G2 and G3) and four EGF-like repeats. Additionally, alternate mRNA splicing yields G-domain variants G2(0,4) with 0- or 4-amino-acid inserts, and G3(0, 8,11,19) with 0-, 8-, 11- or 19-amino-acid inserts. In order to better understand the contributions of individual domains and alternate splicing to agrin activity, single G-domains and covalently linked pairs of G-domains were expressed as soluble proteins and their AChR clustering activity measured on cultured C2 myotubes. These analyses reveal the following: (1) While only G3(8) exhibits detectable activity by itself, all G-domains studied (G1, G2(0), G2(4), G3(0) and G3(8)) enhance G3(8) activity when physically linked to G3(8). This effect is most pronounced when G2(4) is linked to G3(8) and is independent of the order of the G-domains. (2) The deletion of EGF-like repeats enhances activity. (3) Increasing the physical separation between linked G1 and G3(8) domains produces a significant increase in activity; similar alterations to linked G2 and G3(8) domains are without effect. (4) Clusters induced by two concatenated G3(8) domains are significantly smaller than all other agrin forms studied. These data suggest that agrin G-domains are the functional units which interact independently of their specific organization to yield AChR clustering. G-domain synergism resulting in biological output could be due to physical interactions between G-domains or, alternatively, independent interactions of G-domains with cell surface receptors which require spatially localized coactivation for optimal signal transduction.

Biochemical and functional characterization of basal lamina-bound agrin in the chick central nervous system.

Agrin is a high-molecular weight extracellular matrix molecule, initially purified from the electric organ of the marine ray Torpedo californica, which induces on the surface of cultured myotubes the formation of postsynaptic specializations similar to those found at the neuromuscular junction. Agrin immunoreactivity is highly concentrated in the basal lamina of the synaptic cleft but is also found in a number of other tissues where its function is not known. We characterized agrin associated with two basal laminae from the central nervous system, the inner limiting membrane of the retina and the mesencephalic external limiting membrane. A major broad band with an apparent molecular weight of > 300 kDa was identified in immunoblots of isolated basal laminae from retina, mesencephalon, kidney and muscle, showing that basal lamina-bound agrin from the central nervous system and that from non-neural tissues have similar molecular sizes. Agrin is stably but not covalently bound to the inner limiting membrane and could be completely removed only with strong detergents. Agrin could be partially extracted with buffers that are also able to partially release acetylcholine receptor aggregation activity from the neuromuscular junction or from the electric organ. Despite these immunological and biochemical similarities, agrin from both central nervous system-derived basal laminae was not able to induce acetylcholine receptor aggregation on cultured myotubes. This shows that functionally different agrin isoforms are associated with basal laminae in the central nervous system compared to the neuromuscular junction or the electric organ.

Local Induction of Acetylcholine Receptor Aggregates on Cultured Rat Myotubes by a Partially Purified Protein from Fetal Pig Brain

It has been suggested that at the time of innervation, developing neurites release one or more soluble factors that locally induce acetylcholine receptor (AChR) aggregate formation at the synaptic site. To test this hypothesis, we developed a model system that mimics the local, neural induction of AChR aggregation at developing synapses. Partially purified protein derived from fetal pig brain was applied locally to the surface of cultured myotubes via a micropipet. We found that local application of this factor for as little as 30 min induced the formation of AChR aggregates that were restricted to a region within 30 μm around the release site. In addition, the locally applied factor induced a local AChR aggregation response, but did not cause a detectable change in the myotube resting membrane potential at the release site. Our data support a soluble factor hypothesis and suggest that neither cell-cell contact nor local electric fields are necessary for the initial induction of AChR aggregation.

The ability of agrin to cluster AChRs depends on alternative splicing and on cell surface proteoglycans

Agrin, which induces acetylcholine receptor (AChR) clustering at the developing neuromuscular synapse, occurs in multiple forms generated by alternative splicing. Some of these isoforms are specific to the nervous system; others are expressed in both neural and nonneural tissues, including muscle. We have compared the AChR clustering activity of agrin forms varying at each of the three identified splicing sites, denoted x, y, and z. Agrin isoforms were assayed by applying either transfected COS cells, with agrin bound to their surfaces, or soluble agrin to myotubes of the C2 muscle line, or of two variant lines having defective proteoglycans. Dramatic differences in activity were seen between z site isoforms and lesser differences between y site isoforms. The most active agrin forms contained splicing inserts of 4 amino acids at the γ site and 8 amino acids at the z site. These forms are found exclusively in neural tissue. All forms were active on C2 myotubes in cell-attached assays, but muscle forms were less active than neural forms. AChR clustering activity of all agrin forms was decreased when assayed on the proteoglycan-deficient lines, suggesting that proteoglycans may help mediate the action of agrin. As neural agrin forms are more active than muscle forms, they are likely to play a primary role in synaptogenesis.

Neuroblastoma x spinal cord (NSC) hybrid cell lines resemble developing motor neurons.

We have developed a series of mouse-mouse neural hybrid cell lines by fusing the aminopterin-sensitive neuroblastoma N18TG2 with motor neuron-enriched embryonic day 12-14 spinal cord cells. Of 30 neuroblastoma-spinal cord (NSC) hybrids displaying a multipolar neuron-like phenotype, 10 express choline acetyltransferase, and 4 induce twitching in cocultured mouse myotubules. NSC-19, NSC-34, and their subclones express additional properties expected of motor neurons, including generation of action potentials, expression of neurofilament triplet proteins, and acetylcholine synthesis, storage, and release. In addition, NSC-34 cells induce acetylcholine receptor clusters on cocultured myotubes, and undergo a vimentin-neurofilament switch with maturation in culture, similar to that occurring in neuronal development. NSC cell lines appear to model selected aspects of motor neuron development in an immortalized clonal system.

Muscle-derived agrin in cultured myotubes: Expression in the basal lamina and at induced acetylcholine receptor clusters

The synaptic basal lamina (SBL) directs key aspects of the differentiation of regenerating neuromuscular junctions. A range of experiments indicate that agrin or a closely related molecule is stably associated with the SBL and participates in inducing the formation of the postsynaptic apparatus after damage to adult muscle. The selective concentration of agrin-related molecules in the SBL suggests that agrin is secreted locally by cellular components of the nerve-muscle synapse. In vivo studies on aneural embryonic muscle indicate that the muscle cell is one source of the agrin-like molecules in the SBL. Here we have used cultured chick muscle cells to study the expression of agrin-related molecules in the absence of innervation. Immunofluorescence and immunoelectron microscopy show that myogenic cells in culture express agrin-related molecules on their surfaces, and that at least a subset of these molecules are associated with the basal lamina. Moreover, in short term cultures agrin-like molecules accumulate on the surfaces of myogenic cells grown in unsupplemented basal media. We quantified the expression of agrin-like molecules on the cell surface using a solid-phase radioimmune assay. The expression of these molecules is relatively low during the first 6 days of culture and increases fourfold during the second week. The stimulation of the expression of agrin-related molecules in these long-term cultures requires the presence of chick embryo extract or fetal calf serum. We also characterized the expression of muscle-derived agrin-like molecules at clusters of AChR. These agrin-related molecules are not consistently colocalized at spontaneous AChR aggregates; however, they are selectively concentrated at ⩾90% of the AChR clusters that are induced by Torpedo agrin. These data, together with previous results from in vivo developmental experiments indicate that the agrin-like molecules in the synaptic basal lamina are derived at least in part from the muscle cell. In addition, the expression of agrin-like molecules can be regulated by soluble factors present in CEE and FBS. Finally, the selective localization of these molecules at induced AChR clusters, taken together with their localization in the basal lamina, suggests that agrin-like molecules secreted by the muscle cell play an important role in the formation and/or the stabilization of the postsynaptic apparatus.

Rapid induction of acetylcholine receptor aggregates by a neural factor and extracellular Ca 2+

A soluble fetal brain extract (EBX) induces acetylcholine receptor (AChR) aggregation in cultured rat myotubes within 4 hr at 36°C in a defined medium containing 1.8 m M (normal) extracellular Ca 2+ (Olek et al., 1983). The activity of EBX was Ca 2+ dependent; reducing extracellular Ca 2+ significantly inhibited EBX-induced AChR aggregation and a 15–50% increase in extracellular Ca 2+ synergistically enhanced the activity of EBX. Synergism was specific for Ca 2+ as increases in other divalent cations (Ba 2+, Co 2+, Mg 2+, Mn 2+, Sr 2+) had no effect. A large increase (300–500%) in extracellular Ca 2+ alone also induced AChR aggregation within 4 hr at 36°C. An equivalent increase in other cations (Ba 2+, Co 2+, Mg 2+, Mn 2+, Sr 2+) did not promote AChR aggregation. An initial 15-min pulse of increased extracellular Ca 2+ alone or with EBX was adequate to induce AChR aggregation. Aggregates induced by EBX, Ca 2+ alone, or EBX Ca 2+ were found predominantly on the top surface of the myotube. These treatments did not detectably alter preexisting aggregates present at substrate contact sites on the bottom surface of myotubes. AChR aggregation induced by any treatment was not inhibited by cycloheximide, Ca 2+ channel blockers, or protease inhibitors but was blocked by Co 2+ and sodium azide.