ARIA is concentrated in the synaptic basal lamina of the developing chick neuromuscular junction

ARIA is a member of a family of polypeptide growth and differentiation factors that also includes glial growth factor (GGF), neu differentiation factor, and heregulin. ARIA mRNA is expressed in all cholinergic neurons of the central nervous systems of rats and chicks, including spinal cord motor neurons. In vitro, ARIA elevates the rate of acetylcholine receptor incorporation into the plasma membrane of primary cultures of chick myotubes. To study whether ARIA may regulate the synthesis of junctional synaptic acetylcholine receptors in chick embryos, we have developed riboprobes and polyclonal antibody reagents that recognize isoforms of ARIA that include an amino-terminal immunoglobulin C2 domain and examined the expression and distribution of ARIA in motor neurons and at the neuromuscular junction. We detected significant ARIA mRNA expression in motor neurons as early as embryonic day 5, around the time that motor axons are making initial synaptic contacts with their target muscle cells. In older embryos and postnatal animals, we found ARIA protein concentrated in the synaptic cleft at neuromuscular junctions, consistent with transport down motor axons and release at nerve terminals. At high resolution using immunoelectron microscopy, we detected ARIA immunoreactivity exclusively in the synaptic basal lamina in a pattern consistent with binding to synapse specific components on the presynaptic side of the basal lamina. These results support a role for ARIA as a trophic factor released by motor neuron terminals that may regulate the formation of mature neuromuscular synapses.     

Microtubules, microfilaments and the transport of acetylcholine receptors in embryonic myotubes

Both microtubules and microfilaments have been implicated in the exocytotic and endocytotic transport of coated and smooth surfaced membrane vesicles. We have reexamined this question by using specific pharmacological agents to disrupt these filaments and assess the effect on the movement of acetylcholine receptor (AChR) containing membrane vesicles in embryonic chick myotubes. Myotube cultures treated with nocodazole (0.6 microgram/ml) or colcemid (0.5 microgram/ml) (to disrupt microtubules) show only a 20-25% decrease in the number of cell surface AChRs after 48 h. Addition of chick brain extract (CBE) to cultured myotubes causes a significant increase in the total number of cell surface AChRs (measured by [125I]alpha-bungarotoxin (alpha-BGT) binding), thus providing us with a way to manipulate receptor and transport vesicle populations. Cultures treated with CBE plus nocodazole or colcemid show a 1.7-fold increase in AChR number over drug treatment alone, the same increase seen in cultures treated with CBE alone, although the total number remains about 20-25% less than that seen in control cultures. In cultures treated with cytochalasin D (0.2 microgram/ml) or dihydrocytochalasin B (5.0 micrograms/ml) (to disrupt microfilaments), 35 and 65% decreases in cell surface AChR number were seen after 48 h. However, in cultures treated with CBE and cytochalasin D, the same total number of AChRs was found as in cultures treated with CBE alone. No significant effects were seen with any of these drugs on the receptor incorporation rate (the appearance of new alpha-BGT-binding sites) after 6 h. The half-life for AChRs in control cultures was 23.0 h. In cytochalasin D and dihydrocytochalasin B it was 21.9 and 19.0 h, respectively; with colcemid and nocodazole, it increased to 37.1 and 28.1 h. These results suggest that non-myofibrillar microfilament bundles are not involved in the movement of AChR-containing membrane vesicles; further, the small effects seen with microtubule inhibitors tend to rule out a major role for microtubules in this transport.     

Insertion and internalization of acetylcholine receptors at clustered and diffuse domains on cultured myotubes

Two populations of acetylcholine receptors (AChRs) are present in cultured myotubes. One forms large aggregates or clusters and the other has a much lower density of AChRs, which are diffusely distributed. Both clustered and diffuse AChRs are inserted and removed (internalized) from the sarcolemma. To determine the insertion and removal rates of AChRs in these two plasma membrane domains, we used a double label technique to distinguish and quantitate newly inserted and "old" AChRs. Application of our method revealed that the rate of AChR internalization is the same at the clustered and diffuse regions of the plasma membrane, whereas the rate of insertion is threefold greater at the clusters than elsewhere in the plasma membrane. Thus, the increase in AChR number at the clusters is not due to an increase in their half-life, but to an increase in their rate of insertion.     

Microtubules and the formation of acetylcholine receptor clusters in chick embryonic muscle cells

We have used the microtubule-stabilizing drug taxol to examine the relationship between microtubules and the appearance and cell surface distribution of acetylcholine receptors (AChRs) in primary cultures of chick embryonic muscle cells. Taxol at a 5-microM concentration induced the large scale polymerization of tubulin in muscle cells that was most obvious as intermittent bundles of microtubules along the myotube. Prominent bundles of microtubules were also clearly visible in the fibroblasts. This concentration of taxol had no significant effect on the incorporation rate, increased synthesis induced by brain extract or the total cell surface number of AChRs measured over a 24-h period. Thus, excess polymerization of microtubules does not affect the movement of receptors to the cell surface. However, when cell surface AChR distribution was examined using rhodamine-conjugated alpha-bungarotoxin, taxol treatment of myotubes was shown to induce the aggregation of receptors. If receptors were labeled before taxol addition, aggregation of these prelabeled receptors was also seen, a result indicating that taxol can induce the movement of receptors already in the membrane. We believe this evidence further implicates microtubules as being involved in the movement of these cell surface receptors in the plane of the myotube membrane.     

Regulation of agrin-induced acetylcholine receptor aggregation by Ca++ and phorbol ester

Agrin, a protein extracted from the electric organ of Torpedo californica, induces the formation of specializations on cultured chick myotubes that resemble the postsynaptic apparatus at the neuromuscular junction. The aim of the studies reported here was to characterize the effects of agrin on the distribution of acetylcholine receptors (AChRs) and cholinesterase as a step toward determining agrin's mechanism of action. When agrin was added to the medium bathing chick myotubes small (less than 4 micron 2) aggregates of AChRs began to appear within 2 h and increased rapidly in number until 4 h. Over the next 12-20 h the number of aggregates per myotube decreased as the mean size of each aggregate increased to approximately 15 micron 2. The accumulation of AChRs into agrin-induced aggregates occurred primarily by lateral migration of AChRs already in the myotube plasma membrane at the time agrin was added to the cultures. Aggregates of AChRs and cholinesterase remained as long as agrin was present in the medium; if agrin was removed the number of aggregates declined slowly. The formation and maintenance of agrin-induced AChR aggregates required Ca++, Co++ and Mn++ inhibited agrin-induced AChR aggregation and increased the rate of aggregate dispersal. Mg++ and Sr++ could not substitute for Ca++. Agrin-induced receptor aggregation also was inhibited by phorbol 12-myristate 13-acetate, an activator of protein kinase C, and by inhibitors of energy metabolism. The similarities between agrin's effects on cultured myotubes and events that occur during formation of neuromuscular junctions support the hypothesis that axon terminals release molecules similar to agrin that induce the differentiation of the postsynaptic apparatus.     

Induction of acetylcholine receptor synthesis and aggregation: partial purification of low-molecular-weight activity

We have studied the effect of saline and acid extracts of chick brain on the total number of acetylcholine (ACh) receptors and the number of receptor clusters in cultured chick muscle cells. Myotubes in 7-day cultures responded more rapidly to brain extract than did myotubes in 4-day cultures, so the older cells were used in subsequent bioassays. A large percentage of the receptor inducing activity was soluble in 2% trifluoroacetic acid (TFA), and this material appeared by Sephadex G-25 chromatography to be about 1000 daltons in size. Activity was retained on octadecasilyl silica and was further purified by reverse-phase high-pressure liquid chromatography using a TFA-acetonitrile gradient system. Material that eluted between 35 and 40% acetonitrile, termed C4018, was 500- to 1000-fold more potent than saline extract. The receptor accumulation induced by C4018 was associated with an increased rate of receptor incorporation, presumably receptor synthesis, rather than to a decrease in receptor degradation. An increase in incorporation was detected as early as 3 hr after C4018 was added to 7-day cultures and the effect was maximal after 10 hr. C4018 also promoted the aggregation of receptors that were already incorporated in the surface membrane at the time to addition. It is not yet known if aggregation of "old" receptors and increased receptor synthesis are related or if the two phenomena are mediated by the same molecule.     

Agrin released by motor neurons induces the aggregation of acetylcholine receptors at neuromuscular junctions.

To test the hypothesis that agrin mediates motor neuron-induced aggregation of acetylcholine receptors (AChRs) in skeletal muscle fibers and to determine whether the agrin active in this process is released by motor neurons, we raised polyclonal antibodies to purified ray agrin that blocked its receptor aggregating activity. When the antibodies were applied to chick motor neuron--chick myotube cocultures, they inhibited the formation of AChR aggregates at and near neuromuscular contacts, demonstrating that agrin plays a role in the induction of the aggregates. Rat motor neurons, like chick motor neurons, induce AChR aggregates on chick myotubes. This effect was not inhibited by our antibodies, indicating that, although the antibodies inhibited the activity of chick agrin, they did not have a similar effect on rat agrin. We conclude that agrin released by rat motor neurons induced the chick myotubes to aggregate AChRs.     

Phorbol esters inhibit the synthesis of acetylcholine receptors in cultured muscle cells

The acetylcholine receptor (AChR) synthesis, insertion and degradation rates are regulated by numerous intracellular and extracellular agents. Recent studies have shown that Ca2+ and Ca2+ ionophores have a profound regulatory effect on the appearance of AChR clusters and AChR synthesis. These regulatory effects may be mediated through the activation of calcium and phospholipid-dependent protein kinases by agents such as phorbol esters. In this study, we have utilized 4-beta-phorbol-12-myristate-13-acetate (PMA) in order to determine whether the activation of protein kinase C exerts a regulatory effect on the expression of AChRs in cultured chick myotubes. Our results show that 4-beta-phorbol-12-myristate-13-acetate decreased intracellular AChRs and suppressed AChR synthesis without affecting the turnover rate. Control and PMA treated cells labeled with [35S] methionine and immunoprecipitated with a monoclonal antibody to the alpha subunit of AChRs (mAb35) revealed a significant decrease in radioactivity precipitated after exposure to PMA. Polyacrylamide gel electrophoresis revealed no major changes in protein patterns, or in newly synthesized proteins as determined by [35S] methionine incorporation and autoradiography. Other enzymes important in muscle metabolism were not affected by PMA treatment. Our results indicate that activation of protein kinase C results in the suppression of AChRs synthesis and dispersal of AChR clusters.     

Calmodulin and acetylcholine receptor clustering in embryonic chick myotubes

We have used the calmodulin antagonists, trifluoperazine (TFP) and calmidazolium, to study the potential role of this protein in the movement of acetylcholine receptors (AChRs) to and from the myotube membrane, as well as in the formation of clusters of AChRs within the plasma membrane. Neither calmidazolium (up to 10(-6) M) nor TFP (10(-5) M) inhibited receptor degradation or the incorporation of new receptors (12 to 24 h). In addition, neither drug blocked the increased synthesis of receptors induced by chick brain extract, nor significantly affected AChR clusters already in the plane of the membrane at the time of drug addition. However, both drugs blocked new receptor clusters (induced by a basement membrane extract from Torpedo electric organ) from forming. These results indicate that receptors can move to and from the cell membrane in a calmodulin-independent fashion, but movement in the plane of the membrane to form a cluster requires the participation of calmodulin.     

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

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.     

The dynamics of the rapsyn scaffolding protein at individual acetylcholine receptor clusters

Rapsyn, a cytoplasmic receptor-associated protein, is required for the clustering of acetylcholine receptors (AChRs). Although AChR dynamics have been extensively studied, little is known about the dynamics of rapsyn. Here, we used a rapsyn-green fluorescent protein (GFP) fusion protein and quantitative fluorescent imaging to study the dynamics of rapsyn in transfected C2C12 myotubes. First, we found that rapsyn-GFP expression at clusters did not alter AChR aggregation, function, or turnover. Quantification of rapsyn immunofluorescence indicated that the expression of rapsyn-GFP proteins at clusters does not increase the overall rapsyn density compared with untransfected myotube clusters. Using time lapse imaging and fluorescence recovery after photobleaching, we demonstrated that the recovery of rapsyn-GFP fluorescence at clusters was very fast, with a halftime of about approximately 1.5 h (approximately 3 times faster than AChRs). Inhibition of protein kinase C significantly altered receptor insertion, but it had no effect on rapsyn insertion. When cells were treated with the broad spectrum kinase inhibitor staurosporine, receptor insertion was decreased even further. However, inhibition of protein kinase A had no effect on insertion of either rapsyn or receptors. Finally, when cells were treated with neural agrin, rapsyn and AChRs were both directed away from preexisting clusters and accumulated together in new small clusters. These results demonstrate the remarkable dynamism of rapsyn, which may underlie the stability and maintenance of the postsynaptic scaffold and suggest that the insertion of different postsynaptic proteins may be operating independently.     

Involvement of calpains in the destabilization of the acetylcholine receptor clusters in rat myotubes

The effects of calpain inhibitors on the total number of acetylcholine receptors (AChRs) on cultured rat myotubes and on the stability of AChR clusters in these myotubes were investigated. The degradation rate of total AChRs labeled with (125)I-alpha-bungarotoxin was assessed from radioactivity remaining in the myotubes as a function of time. Treatment with calpain inhibitors resulted in a two- to three-fold increase in the half-life of total AChRs. Incubation with these inhibitors produced 40% increases in intracellular AChRs but no major changes in surface AChRs, indicating that the increased AChR half-life is due to intracellular accumulation. The rate loss of AChRs from the clusters was assessed by measuring the loss of fluorescence intensity in rhodaminated-alpha-bungarotoxin-labeled clusters with time. Treatment with calpain inhibitors resulted in twofold increases in cluster half-life. Thus, there was generally no change in total surface receptors with the calpain inhibitors, whereas cluster half-life was substantially increased. Furthermore, with a low dose of calpeptin there was no change in turnover of total cellular AChRs, whereas cluster half-life was doubled. Taken together, these results suggest that the increased half-life of clusters produced by the calpain inhibitors may be due to retardation of the lateral movement from AChRs in the clusters.     

Immobilization of nicotinic acetylcholine receptors in mouse C2 myotubes by agrin-induced protein tyrosine phosphorylation

Agrin induces the formation of highly localized specializations on myotubes at which nicotinic acetylcholine receptors (AChRs) and many other components of the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction accumulate. Agrin also induces AChR tyrosine phosphorylation. Treatments that inhibit tyrosine phosphorylation prevent AChR aggregation. To examine further the relationship between tyrosine phosphorylation and receptor aggregation, we have used the technique of fluorescence recovery after photobleaching to assess the lateral mobility of AChRs and other surface proteins in mouse C2 myotubes treated with agrin or with pervanadate, a protein tyrosine phosphatase inhibitor. Agrin induced the formation of patches in C2 myotubes that stained intensely with anti-phosphotyrosine antibodies and within which AChRs were relatively immobile. Pervanadate, on the other hand, increased protein tyrosine phosphorylation throughout the myotube and caused a reduction in the mobility of diffusely distributed AChRs, without affecting the mobility of other membrane proteins. Pervanadate, like agrin, caused an increase in AChR tyrosine phosphorylation and a decrease in the rate at which AChRs could be extracted from intact myotubes by mild detergent treatment, suggesting that immobilized receptors were phosphorylated and therefore less extractable. Indeed, phosphorylated receptors were extracted from agrin-treated myotubes more slowly than nonphosphorylated receptors. AChR aggregates at developing neuromuscular junctions in embryonic rat muscles also labeled with anti- phosphotyrosine antibodies, suggesting that tyrosine phosphorylation could mediate AChR aggregation in vivo as well. Thus, agrin appears to induce AChR aggregation by creating circumscribed domains of increased protein tyrosine phosphorylation within which receptors become phosphorylated and immobilized.     

Sciatin: a myotrophic protein increases the number of acetylcholine receptors and receptor clusters in cultured skeletal muscle

Factors present in neural extracts or in media conditioned by neurons have been shown by others to increase both the number of acetylcholine receptors (AChRs) and the number of receptor clusters in cultures of embryonic skeletal muscle. We have recently shown that the glycoprotein, sciatin, exerts trophic effects on developing muscle in vitro. In the present study, we investigated the effect of sciatin on AChRs in aneural cultures of chick skeletal muscle. Sciatin caused a significant increase in the number of AChRs/dish as measured by binding of ¹²⁵I-α-bungarotoxin (α-Btx) and in acetylcholinesterase (AChE) activity/dish in differentiating muscle cells. The increase in AChRs elicited by sciatin was due solely to increased receptor synthesis and incorporation. The rate of AChR synthesis in sciatin-treated cultures was as much as five times the control rate and was significantly reduced by cycloheximide (10 μM). AChR degradation was unaffected by the myotrophic protein. Although the number of AChRs/dish was increased by sciatin during myogenesis, AChR specific activity, expressed as picomoles ¹²⁵I-α-Btx bound/mg cell protein, was only transiently increased by the myotrophic protein. This contrasted with AChE specific activity in sciatin-treated cultures which remained elevated throughout differentiation. Autoradiographs of ¹²⁵I-α-Btx-labeled cultures showed that sciatin caused an increase in the number and size of AChR “hot spots” and maintained the integrity of these AChR clusters in aneural muscle cultures for up to 5 weeks. At this time control cultures had completely degenerated. The mechanism by which sciatin enhanced the synthesis of AChRs appeared to be distinct from that of tetrodotoxin (TTX), an agent which abolishes muscle activity. However, like theophylline, sciatin might evoke increased synthesis of AChRs via regulation of cyclic AMP since the myotrophic protein increased cAMP both in cells and in conditioned medium. The results of this study suggest that sciatin may be related to the diffusible factor(s) from motor neurons described by others which has trophic effects on AChRs. Furthermore, we suggest that this myotrophic protein may be responsible for the clustering of AChRs and maintenance of receptor clusters at neuromuscular junctions in developing avian muscle.     

Regulation of the interaction of nicotinic acetylcholine receptors with the cytoskeleton by agrin-activated protein tyrosine kinase

Agrin induces the accumulation of nicotinic acetylcholine receptors (AChRs) in the myofiber membrane at synaptic sites in vertebrate skeletal muscle and causes an increase in tyrosine phosphorylation of the AChR beta subunit. To examine further the mechanism of agrin- induced AChR phosphorylation and the relationship between changes in protein phosphorylation and AChR aggregation, the effect of the protein tyrosine phosphatase inhibitor sodium pervanadate was tested on chick myotubes in culture. Pervanadate caused an increase in the phosphotyrosine content of a variety of proteins, including the AChR. Pervanadate also prevented agrin-induced AChR aggregation and slowed the rate at which AChRs were extracted from intact myotubes by mild detergent treatment. The rate at which phosphorylation of the AChR beta subunit and receptor detergent extractability changed following pervanadate-induced phosphatase inhibition was increased by agrin, indicating that agrin activates a protein tyrosine kinase rather than inhibiting a protein tyrosine phosphatase. The present results, taken together with previous findings on the inhibition of agrin-induced AChR aggregation by protein kinase inhibitors, demonstrate that protein tyrosine phosphorylation regulates the formation and stability of AChR aggregates, apparently by strengthening the interaction between AChRs and the cytoskelton.     

Developmental regulation of 16S acetylcholinesterase and acetylcholine receptors in a mouse muscle cell line

We have studied the appearance, distribution and regulation of acetylcholinesterase (AChE) and acetylcholine receptors (AChRs) in a mouse skeletal muscle cell line (C2), that was originally isolated and described by Yaffe & Saxel [54]. In culture, cells from this line form spontaneously contracting myotubes, with overshooting action potentials that are TTX-sensitive. After fusion of myoblasts into myotubes, there was a dramatic increase in the amount of both AChE and AChR. Three forms of AChE, distinguished by their sedimentation on sucrose gradients, were synthesized: 4-6S, 10S, and 16S. The 4-6S and 10S forms appeared 1 day after the cells began to fuse, whereas the 16S form appeared only 2 days after fusion began. Maximal levels of the 16S AChE form (25-30% of the total) were obtained by reducing the concentration of horse serum in the fusion medium. Prevention of myoblast fusion by reducing the calcium levels in the medium decreased the total AChE by 70%, and only the 4-6S form was synthesized. Blocking spontaneous contractile activity of the myotubes by tetrodotoxin (TTX) led to a 50% reduction in all three esterase forms. Thus, the 16S, or endplate form of AChE is not specifically regulated by electrical or contractile activity in the C2 cell line. After fusion the number of AChRs increased rapidly for 3-4 days and then stabilized. Receptor clusters, ranging from 10-30 micron in length, appeared 1 day after myoblast fusion began. When cells were grown in medium containing reduced Ca2+, the total number of AChRs was decreased by 20-50%. Reduction of Ca2+ after myotubes and AChR clusters had formed resulted in dispersal of AChR clusters. Inhibition of muscle contractions with TTX did not affect the number of AChRs or their distribution.     

Monoclonal antibodies to intermediate filaments in chick muscle cell cultures

Two monoclonal antibodies, FIFI and PHIL, have been prepared using detergent-washed myogenic cells as immunogen. On Western blots of total protein extracts of muscle cells, both antibodies bind to vimentin (52 kD) and its degradation products (major band at 42 kD), but do not bind to mouse proteins or to actin (42 kD). Specificity for a determinant common to vimentin and desmin was confirmed by 2-D gel electrophoresis of muscle cell extracts and purified desmin. Western blots with FIFI reveal particularly well the extreme sensitivity of intermediate filaments (IFs) to proteolysis, which was preventable in brain tissue only by boiling in 1% SDS, although it could be reduced in both brain and muscle by less extreme methods. Western blots suggest a large increase in IF content of differentiating myoblast cell cultures at the time of cell fusion and an increase of at least 4-fold is confirmed by a quantitative immunoassay using a direct ELISA method. Immunofluorescence microscopy shows that this increase is due to the appearance of high concentrations of the intermediate filament antigen at the ends of early myotubes, preceding the appearance of cross-striations in myofibrils. Furthermore, whereas the polar filaments detected by FIFI run right to the ends of the early myotubes and only sparingly penetrate the central area, cross-striated myofibrils (as detected by the monoclonal antibody, SAM) run the length of the myotube but do not reach the ends. Colcemid and colchicine cause the vimentin filaments in fibroblasts to collapse into perinuclear rings or caps, but do not have this effect on the polar fluorescence in early myotubes. Heat shock (2 h at 45 degrees C) has a similar differential effect. The results suggest that early in muscle differentiation intermediate filament proteins accumulate rapidly at myotube ends, where they are organized differently from those in fibroblasts.     

Lithium inhibits a late step in agrin-induced AChR aggregation

Agrin activates an intracellular signaling pathway to induce the formation of postsynaptic specializations on muscle fibers. In myotubes in culture, this pathway has been shown to include autophosphorylation of the muscle-specific kinase MuSK, activation of Src-family kinases, tyrosine phosphorylation of the acetylcholine receptor (AChR) beta subunit, a decrease in receptor detergent extractability, and the accumulation of AChRs into high-density aggregates. Here we report that treating chick myotubes with lithium prevented any detectable agrin-induced change in AChR distribution without affecting the number of AChRs or the agrin-induced change in AChR tyrosine phosphorylation and detergent extractability. Lithium treatment also increased the rate at which AChR aggregates disappeared when agrin was removed. The effects of lithium developed slowly over the course of approximately 12 h. Thus, sensitivity to lithium identifies a late step in the agrin signaling pathway, after agrin-induced MuSK and AChR phosphorylation, that is necessary for the recruitment of AChRs into visible aggregates.     

The putative agrin receptor binds ligand in a calcium-dependent manner and aggregates during agrin-induced acetylcholine receptor clustering.

Agrin derived from Torpedo electric organ induces the clustering of acetylcholine receptors (AChRs) on cultured myotubes. As a first step toward characterizing the plasma membrane receptor for agrin, we have examined agrin binding to cultured myotubes. Agrin binding is saturable as measured by radioimmunoassay and, like agrin-induced AChR clustering, requires extracellular calcium. Immunofluorescence shows that on myotubes incubated with agrin at 4 degrees C, agrin binds in a uniform, finely punctate pattern that correlates poorly with the distribution of AChRs. Myotubes stimulated with agrin at 37 degrees C for greater than or equal to 2 hr show a coclustering of agrin binding sites and AChRs. By contrast, if anti-AChR antibodies are used either to cluster or to internalize AChRs, the distribution and number of agrin binding sites remain unchanged. The aggregation and calcium dependence of the putative agrin receptor may represent important control points in postsynaptic differentiation.     

PKC and PKA Regulate AChR Dynamics at the Neuromuscular Junction of Living Mice

The steady state of the acetylcholine receptor (AChR) density at the neuromuscular junction (NMJ) is critical for efficient and reliable synaptic transmission. However, little is known about signaling molecules involved in regulating the equilibrium between the removal and insertion of AChRs that establishes a stable postsynaptic receptor density over time. In this work, we tested the effect of activities of two serine/threonine kinases, PKC and PKA, on the removal rate of AChRs from and the re-insertion rate of internalized recycled AChRs into synaptic sites of innervated and denervated NMJs of living mice. Using an in vivo time-lapse imaging approach and various pharmacological agents, we showed that PKC and PKA activities have antagonistic effects on the removal and recycling of AChRs. Inhibition of PKC activity or activation of PKA largely prevents the removal of pre-existing AChRs and promotes the recycling of internalized AChRs into the postsynaptic membrane. In contrast, stimulation of PKC or inactivation of PKA significantly accelerates the removal of postsynaptic AChRs and depresses AChR recycling. These results indicate that a balance between PKA and PKC activities may be critical for the maintenance of the postsynaptic receptor density.