Tyrosine phosphorylation and acetylcholine receptor cluster formation in cultured Xenopus muscle cells

Aggregation of the nicotinic acetylcholine receptor (AChR) at sites of nerve-muscle contact is one of the earliest events to occur during the development of the neuromuscular junction. The stimulus presented to the muscle by nerve and the mechanisms underlying postsynaptic differentiation are not known. The purpose of this study was to examine the distribution of phosphotyrosine (PY)-containing proteins in cultured Xenopus muscle cells in response to AChR clustering stimuli. Results demonstrated a distinct accumulation of PY at AChR clusters induced by several stimuli, including nerve, the culture substratum, and polystyrene microbeads. AChR microclusters formed by external cross- linking did not show PY colocalization, implying that the accumulation of PY in response to clustering stimuli was not due to the aggregation of basally phosphorylated AChRs. A semi-quantitative determination of the time course for development of PY labeling at bead contacts revealed early PY accumulation within 15 min of contact before significant AChR aggregation. At later stages (within 15 h), the AChR signal came to approximate the PY signal. We have reported the inhibition of bead-induced AChR clustering in response to beads by a tyrphostin tyrosine kinase inhibitor (RG50864) (Peng, H. B., L. P. Baker, and Q. Chen. 1991. Neuron. 6:237-246). RG50864 also inhibited PY accumulation at bead contacts, providing evidence for tyrosine kinase activation in response to the bead stimulus. These results suggest that tyrosine phosphorylation may play an important role in the generative stages of cluster formation, and may involve protein(s) other than or in addition to AChRs.     

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.     

Ultrastructure of acetylcholine receptor clusters on cultured muscle fibers

The structure of regions with a high concentration of ACh receptors (clusters) on cultured skeletal muscle myotubes was examined by immunoperoxidase staining of bound alphaBT. The clusters did not appear to differ from the other regions except in their higher concentration of receptor.     

Agrin-induced acetylcholine receptor clustering in mammalian muscle requires tyrosine phosphorylation

Agrin is thought to be the nerve-derived factor that initiates acetylcholine receptor (AChR) clustering at the developing neuromuscularjunction. We have investigated the signaling pathway in mouse C2 myotubes and report that agrin induces a rapid but transient tyrosine phosphorylation of the AChR beta subunit. As the beta-subunit tyrosine phosphorylation occurs before the formation of AChR clusters, it may serve as a precursor step in the clustering mechanism. Consistent with this, we observed that tyrosine phosphorylation of the beta subunit correlated precisely with the presence or absence of clustering under several experimental conditions. Moreover, two tyrosine kinase inhibitors, herbimycin and staurosporine, that blocked beta-subunit phosphorylation also blocked agrin-induced clustering. Surprisingly, the inhibitors also dispersed preformed AChR clusters, suggesting that the tyrosine phosphorylation of other proteins may be required for the maintenance of receptor clusters. These findings indicate that in mammalian muscle, agrin-induced AChR clustering occurs through a mechanism that requires tyrosine phosphorylation and may involve tyrosine phosphorylation of the AChR itself.     

Membrane-related specializations associated with acetylcholine receptor aggregates induced by electric fields

The localization of membrane-associated specializations (basal lamina and cytoplasmic density) at sites of acetylcholine receptor (AChR) aggregation is consistent with an involvement of these structures in receptor stabilization. We investigated the occurrence of these specializations in association with AChR aggregates that develop at the cathode-facing edge of Xenopus muscle cells during exposure to a DC electric field. The cultures were labeled with a fluorescent conjugate of alpha-bungarotoxin and the receptor distribution on selected cells was determined before and after exposure to the field. In thin sections taken from the same cells, the cathode-facing edge was characterized by plaques of basal lamina and cytoplasmic density co-extensive with sarcolemma of increased density. In sections cut in a plane similar to the fluorescence image, it was possible to demonstrate that the specializations were concentrated at areas of field-induced AChR aggregation, and at receptor clusters existing on control cells. This finding further indicates that these structures participate in AChR stabilization, and that the mechanisms involved in AChR aggregation that result from field exposure and nerve contact may be similar.     

Mechanism of acetylcholine receptor cluster formation induced by DC electric field

Background

The formation of acetylcholine receptor (AChR) cluster is a key event during the development of the neuromuscular junction. It is induced through the activation of muscle-specific kinase (MuSK) by the heparan-sulfate proteoglycan agrin released from the motor axon. On the other hand, DC electric field, a non-neuronal stimulus, is also highly effective in causing AChRs to cluster along the cathode-facing edge of muscle cells.

Methodology/Principal Findings

To understand its molecular mechanism, quantum dots (QDs) were used to follow the movement of AChRs as they became clustered under the influence of electric field. From analyses of trajectories of AChR movement in the membrane, it was concluded that diffuse receptors underwent Brownian motion until they were immobilized at sites of cluster formation. This supports the diffusion-mediated trapping model in explaining AChR clustering under the influence of this stimulus. Disrupting F-actin cytoskeleton assembly and interfering with rapsyn-AChR interaction suppressed this phenomenon, suggesting that these are integral components of the trapping mechanism induced by the electric field. Consistent with the idea that signaling pathways are activated by this stimulus, the localization of tyrosine-phosphorylated forms of AChR β-subunit and Src was observed at cathodal AChR clusters. Furthermore, disrupting MuSK activity through the expression of a kinase-dead form of this enzyme abolished electric field-induced AChR clustering.

Conclusions

These results suggest that DC electric field as a physical stimulus elicits molecular reactions in muscle cells in the form of cathodal MuSK activation in a ligand-free manner to trigger a signaling pathway that leads to cytoskeletal assembly and AChR clustering.     

Sodium transport by the acetylcholine receptor of cultured muscle cells.

Activation of the acetylcholine receptors of cultured muscle cells by carbamylcholine increases the rate of passive 22-Na+ uptake into the muscle cells up to 20-fold. The Na+ transport activity of the receptor desensitizes during exposure to carbamylcholine. The rate and extent of desensitization is reduced by lowering the assay temperature from 36 degrees to 2 degrees, allowing accurate measurements of initial rates of Na+ transport by the receptor. Activation of the receptor by carbamylcholine and acetylcholine is significantly cooperative (Hill coefficients of 1.4 to 2.0). Inhibition by D-tubocurarine is not cooperative. The carbamylcholine-induced Na+ transport activity of the receptor is inhibited 50% by 4 muM D-tubocurarine, 100 muM atropine, or 1.6 nM diiodo-alpha-bungarotoxin but is not affected by tetrodotoxin. The initial rate of Na+ transport by the receptor is temperature-independent between 2 degrees and 36 degrees. Receptor Na+ transport is saturable by Na+ at 2 degrees with an apparent Km of 150 plus and minus 20 mM. Saturation by Na+ not observed at 36 degrees at the concentrations tested. Saturation by Na+ is observed at 2 degrees both under conditions of net Na+ influx and under conditions of isotopic exchange at equilibrium. The receptor does not catalyze obligatory exchange diffusion at a detectable rate. Comparison of binding of [125-I]diiodo-alpha-bungarotoxin with rates of Na+ transport indicates a turnover number of 2 times 10-7 ions per min per receptor. These results are discussed in terms of the mechanism of Na+ transport by the receptor.     

Association of the postsynaptic 43K protein with newly formed acetylcholine receptor clusters in cultured muscle cells

The postsynaptic membrane from Torpedo electric organ contains, in addition to the acetylcholine receptor (AChR), a major peripheral membrane protein of approximately 43,000 mol wt (43K protein). Previous studies have shown that this protein is closely associated with AChR and may be involved in anchoring receptors to the postsynaptic membrane. In this study, binding sites for monoclonal antibodies (mabs) to the 43K protein have been compared to the distribution of AChR in Xenopus laevis muscle cells in culture. In double label immunofluorescence experiments, clusters of AChR that occur spontaneously on these cells were stained with anti-43K mabs. Newly formed receptor clusters induced with positive polypeptide-coated latex beads were also stained with anti-43K mabs as early as 12 h after the application of the beads. Exact correspondence in the distribution of the anti-43K protein binding sites and the AChR was found in both types of clusters. These results suggest that the 43K protein becomes associated with AChR clusters during a period of active postsynaptic membrane differentiation. Thus, this protein may participate in the clustering process.     

Development of acetylcholinesterase induced by basic polypeptide-coated latex beads in cultured Xenopus muscle cells

The formation of acetylcholine receptor (AChR) clusters can be induced by basic polypeptide-coated latex beads in cultured Xenopus muscle cells. Here we investigated the development of acetylcholinesterase (AChE) at the bead-induced AChR clusters. AChE activity began to appear at the clusters after 1 day of bead-muscle coculture and was present at all of the bead-induced clusters within 4-7 days. Electron microscopy revealed that AChE reaction products were discretely localized within the cleft and the membrane invaginations at the bead-muscle contacts. Thus, the beads can mimic the nerve in inducing a local accumulation of both the AChRs and AChE, suggesting that the development of both specializations can be effected by a common stimulus.     

The influence of AChR clustering stimuli on the formation and maintenance of AChR clusters induced by polycation-coated beads in Xenopus muscle cells

Nerve, polycation-coated beads, and electric fields not only induce acetylcholine receptors (AChRs) to cluster, but they also reduce the number of spontaneous AChR patches (hotspots) away from the induced cluster sites on embryonic Xenopus myotomal muscle cells grown in tissue culture (the global effect). In vivo, the ability of an AChR clustering stimulus to depress cluster formation elsewhere on the muscle cell may influence both the site at which the neuromuscular junction develops as well as which axons survive during synapse elimination. Since the causes of hotspot formation may be variable and cannot be controlled, we have further characterized the global effect by using AChR-clustering stimuli that can be controlled by the experimenter. We report that innervation inhibits the formation and maintenance of bead-associated AChR patches (BARPs) by a percentage of polycation-coated beads. We next investigated competition between beads and between beads and electric fields. In competition between beads added to the muscle cells at different times, however, the first set of beads had a competitive advantage over the second set of beads. This advantage was strengthened when the latency between bead applications was extended, or when a relatively large number of BARPs were formed by the first set of beads. Likewise, long-term electric fields were able to prevent the formation of BARPs, but were unable to disperse mature BARPs. Longer electric fields, or electric fields of greater magnitude competed better with the beads than brief or weak field treatments. None of the "winning" stimuli, including nerve, were able to totally block AChR patch formation or maintenance by competing stimuli. Thus, the global effect, at least in the case of competition between nonneuronal stimuli, favors the initial stimulus and appears to be graded.     

Distribution of filipin-sterol complexes on cultured muscle cells: cell- substratum contact areas associated with acetylcholine receptor clusters

Specialized areas within broad, close, cell-substratum contacts seen with reflection interference contrast microscopy in cultures of Xenopus embryonic muscle cells were studied. These areas usually contained a distinct pattern of light and dark spots suggesting that the closeness of apposition between the membrane and the substratum was irregular. They coincided with areas containing acetylcholine receptor clusters identified by fluorescence labeled alpha-bungarotoxin. Freeze-fracture of the cells confirmed these observations. The membrane in these areas was highly convoluted and contained aggregates of large P-face intramembrane particles (probably representing acetylcholine receptors). If cells were fixed and then treated with the sterol- specific antibiotic filipin before fracturing, the pattern of filipin- sterol complex distribution closely followed the pattern of cell- substratum contact. Filipin-sterol complexes were in low density in the regions where the membrane contained clustered intramembrane particles. These membrane regions were away from the substratum (bright white areas in reflection interference contrast; depressions of the P-face in freeze-fracture). Filipin-sterol complexes were also in reduced density where the membrane was very close to the substratum (dark areas in reflection interference contrast; bulges of the P-face in freeze- fracture). These areas were not associated with clustered acetylcholine receptors (aggregated particles). This result suggests that filipin treatment causes little or no artefact in either acetylcholine receptor distribution or membrane topography of fixed cells and that the distribution of filipin-sterol complexes may closely parallel the microheterogeneity of membranes that exist in living cells.     

Formation of acetylcholine receptor clusters at neuromuscular junction in Xenopus cultures

The formation of acetylcholine receptor (AChR) clusters at the neuromuscular junction was investigated by observing the sequential changes in AChR cluster distribution on cultured Xenopus muscle cells. AChRs were labeled with tetramethylrhodamine-conjugated alpha-bungarotoxin (TMR-alpha BT). Before innervation AChRs were distributed over the entire surface of muscle cells with occasional spots of high density (hot spots). When the nerve contacted the muscle cell, the large existing hot spots disappeared and small AChR clusters (less than 1 micron in diameter) initially emerged from the background along the area of nerve contact. They grew in size, increased in number, and fused to form larger clusters over a period of 1 or 2 days. Receptor clusters did not migrate as a whole as observed during "cap" formation in B lymphocytes. The rate of recruitment of AChRs at the nerve-muscle junction varied from less than 50 binding sites to 1000 sites/hr for alpha BT. In this study the diffusion-trap mechanism was tested for the nerve-induced receptor accumulation. The diffusion coefficient of diffusely distributed AChRs was measured using the fluorescence photobleaching recovery method and found to be 2.45 X 10(-10) cm2/sec at 22 degrees C. There was no significant difference in these values among the muscle cells cultured without nerve, the non-nerve-contacted muscle cells in nerve-muscle cultures, and the nerve-contacted muscle cells. It was found that the diffusion of receptors in the membrane is not rate-limiting for AChR accumulation.     

Denervation disperses acetylcholine receptor clusters at the neuromuscular junction in Xenopus cultures

The effect of denervation on acetylcholine receptor (AChR) cluster distribution on cultured Xenopus muscle cells has been examined in order to study the role of intact nerve in the maintenance of clusters at the nerve-muscle junction during development. AChRs on the muscle cell were labeled with tetramethyl rhodamine-conjugated alpha-bungarotoxin and sequential changes in AChR cluster distribution were examined with a fluorescence microscope using an image intensifier. Denervation was carried out by exposing the nerve cell body to a focused laser light of a high intensity. After this procedure the neurites originating from the cell quickly disintegrated and large AChR clusters associated with nerve divided into smaller clusters. Individual clusters subsequently decreased in size and finally disappeared. In about 30% of the cases new AChR clusters appeared at the extrajunctional region after denervation. These observations indicate that intact nerves are necessary for the maintenance of receptor localization at the nerve-muscle junction and that nerve-induced accumulation is seemingly reversible during the early period of synapse formation. We tested the idea that receptor clusters were lost due to diffusion of receptors in the muscle membrane after denervation. However, the rate of receptor cluster dispersal after denervation was much slower than that predicted by the diffusion model, suggesting that diffusion of receptors is not a rate-limiting step. Furthermore, we found that receptor clusters at the junction stabilize during days in culture. Thus, 80-90% of receptor clusters at the nerve-muscle junction disappeared at 7 hr after denervation in 1-day cocultures, while about 50% of receptor clusters remained after denervation in 3-day cocultures.     

Potassium inward rectifier and acetylcholine receptor channels in embryonic Xenopus muscle cells in culture

Embryonic muscle cells of the frog Xenopus laevis were isolated and grown in culture and single-channel recordings of potassium inward rectifier and acetylcholine (ACh) receptor currents were obtained from cell-attached membrane patches. Two classes of inward rectifier channels, which differed in conductance, were apparent. With 140 mM potassium chloride in the electrode, one channel class had a conductance of 28.8 ± 3.4 pS (n = 21), and, much more infrequently, a smaller channel class with a conductance of 8.6 ± 3.6 pS (n = 7) was recorded. Both channel classes had relatively long mean channel open times, which decreased with membrane hyperpolarization. The probability of finding a patch of membrane with an inward rectifier channel was high (66%) and many membrane patches contained more than one inward rectifier channel. The open state probability (with no applied potential) was high for both inward rectifier channel classes so that 70% of the time there was a channel open. Seventy-three percent of the membrane patches with ACh receptor channels (n = 11) also had at least one inward rectifier channel present when the patch electrode contained 0.1 μM ACh. Inward rectifier channels were also found at 71% of the sites of high ACh receptor density (n = 14), which were identified with rhodamine-conjugated α-bungarotoxin. The results indicate that the density of inward rectifier channels in this embryonic skeletal muscle membrane was relatively high and includes sites of membrane that have synaptic specializations. © 1996 John Wiley & Sons, Inc.     

GSH role on platelet-derived growth factor receptor tyrosine phosphorylation induced by H2O2

This study, conducted on NIH3T3 cells, demonstrates that GSH depletion obtained by buthionine sulfoximine (BSO) treatment does not affect platelet-derived growth-factor receptor (PDGFr) autophosphorylation or cell protein phosphorylation induced by exogenous addition of H2O2, while it does decrease tyrosine phosphorylation obtained by PDGF stimulation. This last effect seems due to the lack of H2O2 generation; for the first time a relation between intracellular GSH content and H2O2 production induced by PDGF has been demonstrated. Therefore, changes of GSH levels can affect the early events of the PDGFr signal pathways by redox regulation. It has also demonstrated that in NIH3T3 cells, H2O2 can directly activate tyrosine phosphorylation by a reversible effect with the involvement of SH-group. This H2O2 effect is increased by vanadate and by GSH depleting agent, diethylmaleate, which unlike BSO is able to produce H2O2 as the current study shows.     

Phosphorylation and assembly of nicotinic acetylcholine receptor subunits in cultured chick muscle cells

The assembly of the nicotinic acetylcholine receptor (AChR), an oligomeric cell surface protein, was studied in cultured muscle cells. To measure this process, the incorporation of metabolically labeled alpha-subunit into oligomeric AChR was monitored in pulse-chase experiments, either by the shift of this subunit from the unassembled (5 S) to the assembled (9 S) position in sucrose density gradients, or by its coprecipitation with antisera specific for the delta-subunit. We have found that AChR assembly is initiated 15-30 min after subunit biosynthesis and is completed within the next 60 min. The alpha-subunit is not overproduced, as all detectable pulse-labeled alpha-subunit can be chased into the oligomeric complex, suggesting that AChR assembly in this system is an efficient process. The rate of AChR assembly is decreased by metabolic inhibitors and by monensin, an ionophore that impairs the Golgi apparatus. We have observed that the gamma- and delta-subunits of AChR are phosphorylated in vivo. The delta-subunit is more highly phosphorylated in the unassembled than in the assembled state, indicating that its phosphorylation precedes assembly and that its dephosphorylation is concomitant with AChR assembly. These findings suggest that subunit assembly occurs in the Golgi apparatus and that phosphorylation/dephosphorylation mechanisms play a role in the control of AChR subunit assembly.     

Determination of the tyrosine phosphorylation sites of the nicotinic acetylcholine receptor.

The peripheral nicotinic acetylcholine receptor (nAChR) is phosphorylated on tyrosine residues in vivo and in vitro at a high stoichiometry. We have previously reported that this tyrosine phosphorylation occurs on the beta, gamma, and delta subunits of the receptor and is implicated in both the modulation of the function of the receptor and localization of the receptor at the synapse. The specific tyrosine residue of each subunit which is phosphorylated is now identified. The endogenously phosphorylated nAChR from the electric organ of Torpedo californica was phosphorylated to maximal stoichiometry in vitro exclusively on tyrosine residues as indicated by phosphoamino acid analysis. Two-dimensional phosphopeptide maps of thermolysin limit digests of the isolated phosphorylated subunits indicated that each subunit is phosphorylated at a single site. To determine the site of tyrosine phosphorylation of the beta, gamma, and delta subunits, phosphorylated subunits were isolated and digested with trypsin. A single phosphotyrosine containing peptide from each subunit was purified by antiphosphotyrosine antibody affinity chromatography and reverse phase high performance liquid chromatography. The purified phosphopeptides were subjected to sequential Edman degradation and sequence analysis. Comparison of the phosphopeptide sequence data with the deduced amino acid sequence of each subunit indicated that Tyr-355 of beta, Tyr-364 of gamma, and Tyr-372 of delta are the sites of in vitro and in vivo tyrosine phosphorylation of the nAChR. Identification of these sites should facilitate further studies of the role of tyrosine phosphorylation in the regulation of receptor function.     

Variation among acetylcholine receptor clusters induced by ciliary ganglion neurons in vitro

We have examined the variation in receptor density and area among neurite-associated acetylcholine receptor patches (NARPs) induced by chick ciliary ganglion neurons on nearby myotubes in vitro. Quantitative analysis of rhodamine-alpha-bungarotoxin (RBTX) NARPs revealed that about 15% of the NARPs were "outstanding" in terms of size (greater than 60 micron 2) and fluorescence intensity (greater than 100 units on a 0-255 scale). The total number of receptors at different NARPs ranged over 3 orders of magnitude. It is likely that variation in NARP size and intensity reflects regional variation in the ability of myotubes to respond to the neuronal influence because (1) no gradient in NARP size or intensity with distance from the soma was evident; (2) the intensities and areas of uninnervated receptor clusters (hot spots) were similar to those of NARPs; (3) acetylcholinesterase was present at the same proportion of hot spots and NARPs at all times examined. We found no physiological or morphological evidence that outstanding NARPs were more effective sites of transmitter release. Outstanding NARPs were restricted to the longest neurite of individual neurons, so they may signal trophic interactions of the sort that promote neurite outgrowth and survival.