Agrin-related molecules are concentrated at acetylcholine receptor clusters in normal and aneural developing muscle

Agrin induces the clustering of acetylcholine receptors (AchRs) and other postsynaptic components on the surface of cultured muscle cells. Molecules closely related if not identical to agrin are highly concentrated in the synaptic basal lamina, a structure known to play a key part in orchestrating synapse regeneration. Agrin or agrin-related molecules are thus likely to play a role in directing the differentiation of the postsynaptic apparatus at the regenerating neuromuscular junction. The present studies are aimed at understanding the role of agrin at developing synapses. We have used anti-agrin monoclonal antibodies combined with alpha-bungarotoxin labeling to establish the localization and time of appearance of agrin-related molecules in muscles of the chick hindlimb. Agrinlike immunoreactivity was observed in premuscle masses from as early as stage 23. AchR clusters were first detected late in stage 25, coincident with the entry of axons into the limb. At this and all subsequent stages examined, greater than 95% of the AchR clusters colocalized with agrin-related molecules. This colocalization was also observed in unpermeabilized whole mount preparations, indicating that the agrin-related molecules were disposed on the external surface of the cells. Agrin-related molecules were also detected in regions of low AchR density on the muscle cell surface. To examine the role of innervation in the expression of agrin-related molecules, aneural limbs were generated by two methods. Examination of these limbs revealed that agrin-related molecules were expressed in the aneural muscle and they colocalized with AchR clusters. Thus, in developing muscle, agrin or a closely related molecule (a) is expressed before AchR clusters are detected; (b) is colocalized with the earliest AchR clusters formed; and (c) can be expressed in muscle and at sites of high AchR density independently of innervation. These results indicate that agrin or a related molecule is likely to play a role in synapse development and suggest that the muscle cell may be at least one source of this molecule.     

Development of the basal lamina and extracellular materials in the early chick embryo

Summary Chick embryos at developmental stages up to primitive streak formation were fixed in a mixture of tannic acid and glutaraldehyde. A basal lamina was present in the unincubated embryo and consisted of a lucent lamina interna and a lamina densa. At the primitive streak stage the lamina densa showed a periodicity of stained elements. Densely stained materials were present on the cell surfaces lining the cavity between the epiblast and endoblast, and on the mesoderm cells within this cavity. Considerable amounts of extracellular material were observed in the cavity. Hyaluronidase treatment removed the cell surface and extracellular material, indicating that hyaluronic acid is a major component. This enzyme disrupted the basal lamina, leaving a fibrillar remnant with no periodic structure. It is therefore suggested that the dense periodicities consist of glycosaminoglycan built on an enzyme-resistant framework which is probably collagen. Enzyme-resistant fibrils, presumably collagen precursors, are present elsewhere within the tissue spaces.     

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 greater than or equal to 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.     

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.     

Localization of surface and internal acetylcholine receptors in developing fast and slow muscles of the chick embryo

The localization of surface and internal acetylcholine (ACh) receptors was investigated in the developing anterior and posterior latissimus dorsi (ALD and PLD) muscles in the chick embryo (11, 15, and 19 days) by autoradiography using ¹²⁵I-α-bungarotoxin (BTX). At 11 days, ACh receptors were already preferentially at neuromuscular junctions. Internal ACh receptors, measured using muscles made permeable to BTX by saponin treatment, were scattered throughout the length of each muscle fiber with or without a slight increase in their number around neuromuscular junctions. Quantitative analysis of grains in montage electron micrographs of muscle fibers from 11-day embryos revealed that intracellular specific BTX binding sites were the Golgi complex and multivesicular bodies. The number of silver grains over the Golgi complex decreased greatly after puromycin treatment of organ-cultured muscles. These findings strongly suggest that the Golgi complex is one of the sites involved in the production of ACh receptors in the skeletal muscle cells in vivo. Multivesicular bodies are assumed to be involved in the degradation of ACh receptors.     

Expression of Eph receptor tyrosine kinases and their ligands in chick embryonic motor neurons and hindlimb muscles

Evidence is accumulating that Eph receptor tyrosine kinases and their ligands regulate cell migration and axonal guidance during development. It was previously found that one of the Eph receptors, EphA4, is transiently expressed in subsets of chick embryonic motor neurons. Here, the expression of EphA and ephrin-A subfamily members was further examined, and the dynamic patterns of expression in chick embryonic motor neurons found. EphA3, EphA4, ephrin-A2, and ephrin-A5 were also expressed in the connective tissues of limb muscles and EphA3 and EphA4 expressing motor neurons innervated EphA3 and EphA4 expressing limb muscles, respectively. These spatiotemporal expression patterns suggest that EphA and ephrin-A proteins play important roles in muscle patterning and motor axonal guidance.     

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.     

Metabolism of acetylcholine receptor in chick embryo muscle cells: effects of RSV and PMA.

We have investigated some aspects of the metabolism of the integral membrane protein acetylcholine receptor (AChR) in normal and transformed cultures of chick embryo muscle cells. Turnover of AChR in control muscle cell cultures was compared with turnover in cultures infected and transformed by a temperature-sensitive mutant of Rous sarcoma virus (RSV) and with cultures treated with the tumor promoter phorbol myristate acetate (PMA). The parameters of AChR metabolism were estimated using 125I-alpha-bungarotoxin as a stoichiometric high affinity ligand for the AChR. We found that both RSV transformation and PMA increased the rate of degradation and decreased the rate of synthesis of AChR. The consequent reduction in steady state receptor levels suggests that oncogenic transformation and tumor promoter significantly alter the metabolism of cell surface membranes. We also observed that parameters of AChR metabolism in control cultures changed systematically in a pattern which depended upon the age of the culture as well as the use of embryo extract or fetal bovine serum as medium supplements. The muscle cell system allows quantitative measurement of an integral membrane protein and its metabolism, and may serve as a more general model for alterations in membrane and surface receptor metabolism associated with the transformed state.     

Chronic stimulation of the spinal cord in developing chick embryo causes the differentiation of multiple clusters of acetylcholine receptor in the posterior latissimus dorsi muscle

Electrodes were implanted around the spinal cord of 7-day-old chick embryos and electric pulses delivered at 0.5-Hz frequency from the 10th to 15th day of incubation. At Day 15, the posterior latissimus dorsi (PLD) muscle, which, in control animals, is focally innervated, was dissected. The number and distribution of AChR clusters revealed by autoradiography after labeling with ¹²⁵I-α-bungarotoxin was quantitatively studied on isolated muscle fiber fragments and on serial sections of the whole muscle. After chronic stimulation, muscle fibers with multiple AChR clusters were observed. The distribution of the clusters appeared less regular than in the anterior latissimus dorsi muscle which, in control embryos, receives a multiple innervation. The total number of AChR clusters per PLD muscle increased about 1.8 times as a consequence of the stimulation without significant change of the total number of muscle fibers.     

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.     

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.     

Detection of the nicotinic acetylcholine receptor alpha-subunit mRNA by in situ hybridization at neuromuscular junctions of 15-day-old chick striated muscles.

In adult vertebrate striated muscle, the nicotinic acetylcholine receptor (AChR) is almost exclusively localized in the postsynaptic membrane of the neuromuscular junction. Using in situ hybridization, we show that, in two different chicken muscles [the slow multi-innervated anterior latissimus dorsi (ALD) and the fast singly innervated posterior latissimus dorsi (PLD)], the AChR alpha-subunit mRNA is detected at discrete regions on myofibres and that these regions co-localize (80% correspondence) with neuromuscular junctions identified by histochemical staining for acetylcholinesterase. Moreover, autoradiographic grains densely accumulate on and around subsynaptic nuclei. In contrast, hybridization with an actin probe results in a strong signal distributed over the entire length of the myofibres. Denervation increases the level of AChR alpha-subunit mRNA both in the PLD and to a lesser extent in the ALD. By in situ hybridization we observe that, although a perinuclear pattern is maintained, the labelled nuclei appear randomly distributed among approximately 10% of the nuclei. These results are discussed in a model of AChR gene expression in vertebrate striated muscle fibres.     

Hemodynamic effects of acetylcholine in the chick embryo and differences from those in the rat embryo

It has been reported that acetylcholine induces cardiac anomalies in the chick embryo. Thus, we studied hemodynamic effects of this drug in the chick embryo and also compared them with those in the rat embryo since we found that the effect of caffeine was different between the chick and rat embryos. Acetylcholine was given at doses of 5, 0.5, and 0.05 micrograms into the vitelline vein in chick embryos at Hamburger-Hamilton stage 21 and at a dose of 0.5 micrograms into the placenta in rat embryos at gestational day 12. In the chick embryo, heart rate was reduced to 91, 88, and 87% of control at the end of injection of 0.05, 0.5, and 5 micrograms, respectively, then returned to the baseline level. Vitelline arterial blood pressure was 110% of control with 0.05 micrograms, 134% with 0.5 micrograms, and 142% with 5 micrograms at 1 min after injection. The dorsal aortic blood flow decreased with time after injection, but it was increased only by a 5 micrograms dose at the end of injection. The vascular resistance increased in a dose-dependent manner. In the rat embryo, the change of heart rate was qualitatively similar to that of the chick embryo. The blood pressure did not change significantly. The blood flow velocity at the outflow tract decreased at the end of injection, which indicated the decrease in cardiac output, along with slowing of heart rate, then returned to the control level thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microfilaments and actin-associated proteins at sites of membrane-substrate attachment within acetylcholine receptor clusters

Rat myotubes in tissue culture form broad areas of close contact with the substrate. These areas often display two distinct, interdigitating sets of membrane domains. One, the "contact domain", is close to the substrate; the other, termed the "AChR domain", is further from the substrate and is rich in acetylcholine receptors (AChR). We have used fluorescence techniques to study the organization of the cytoskeleton in these areas. Substrate-apposed membrane of the myotubes was exposed either by shearing or by permeabilizing the cells with a neutral detergent. Phalloidin derivatives and affinity-purified polyclonal or monoclonal antibodies specific for cytoskeletal proteins were then applied to the samples. Sheared samples were observed by epifluorescence microscopy; detergent-permeabilized samples were observed by total internal reflection fluorescence microscopy. We found that, like antivinculin, fluorescent phalloidin derivatives and antibodies to alpha-actinin, filamin, and talin preferentially labeled the contact domains. This suggests that bundles of microfilaments associate with the membrane at sites of myotube-substrate attachment. In contrast, a 43K protein, closely associated with AChR, was present only at AChR domains. A monoclonal antibody to actin labeled both AChR and contact domains, suggesting that actin is enriched over both regions. Our results suggest that, like the plasma membrane of AChR clusters, the underlying membrane skeleton is organized into at least two distinct domains.     

The distribution of acetylcholine receptor clusters and sites of transmitter release along chick ciliary ganglion neurite-myotube contacts in culture

Acetylcholine receptors accumulate along the length of cholinergic neuron-skeletal muscle contacts in vitro. The main purpose of this study was to describe, in a quantitative way, the distribution of acetylcholine receptor clusters induced by ciliary ganglion neurons over a period of time extending from hours to weeks after contacts are established. Neurites were filled with Lucifer Yellow and receptor clusters were identified with rhodamine-bungarotoxin. A cluster located within 5 micron of a nerve process or 10 micron of the base of a growth cone was considered to be a neurite-associated receptor patch (NARP). The first synaptic potentials were evoked 20 min after growth cone-myotube contact, and, after 24 h of co-culture, greater than 60% of the nerve-muscle pairs tested were functionally connected. NARPs appear rapidly; the first clusters were detected approximately 6 h after the neurons were plated. They were composed of several small subclusters or speckles of rhodamine-bungarotoxin fluorescence. The initial accumulation of receptors may occur at the advancing tips of nerve processes because NARPs were found at greater than 80% of the growth cone-muscle contacts examined between 12 and 24 h of co-culture. Over the 3-wk period examined, the mean incidence of NARPs ranged between 1.0 and 2.6 per 100 micron of neurite-myotube contact, with the peak observed on the second day of co-culture. During the first 3 d in culture, when the neurons were multipolar, nearly all of the primary processes induced one or more clusters. With time, as the neurons become unipolar (Role and Fischbach, 1987) NARPs persisted along the remaining dominant process. Measurements made during the third day of co-culture suggest that NARPs disappear along shorter neurites before they retract. Synaptic currents were detected by focal extracellular recording at 55% of the NARPs. The fact that spontaneous or evoked responses were not recorded at 45% suggests that contacts with clusters exhibit two functional states. Two types of presynaptic specialization at identified NARPs observed by scanning electron microscopy appear to be correlated with the functional state.     

Sphingolipids are necessary for nicotinic acetylcholine receptor export in the early secretory pathway

The nicotinic acetylcholine receptor (AChR) is the prototype ligand-gated ion channel, and its function is dependent on its lipid environment. In order to study the involvement of sphingolipids (SL) in AChR trafficking, we used pharmacological approaches to dissect the SL biosynthetic pathway in CHO-K1/A5 cells heterologously expressing the muscle-type AChR. When SL biosynthesis was impaired, the cell surface targeting of AChR diminished with a concomitant increase in the intracellular receptor pool. The SL-inhibiting drugs increased unassembled AChR forms, which were retained at the endoplasmic reticulum (ER). These effects on AChR biogenesis and trafficking could be reversed by the addition of exogenous SL, such as sphingomyelin. On the basis of these effects we propose a 'chaperone-like' SL intervention at early stages of the AChR biosynthetic pathway, affecting both the efficiency of the assembly process and subsequent receptor trafficking to the cell surface.     

A novel AP180-related protein in vesicles that concentrate at acetylcholine receptor clusters

Monoclonal antibodies were generated to vesicular membranes of clathrin coated vesicles enriched for acetylcholinesterase (AChE). One of these, C172, recognizes vesicles which accumulate in muscle cells around nuclei associated with acetylcholine receptor AChR clusters. Immunoblots of muscle extracts and brain purified clathrin coated vesicles show that C172 recognizes a 100 kd band in muscle, but a 180 kd band in brain. Western blots of purified AP180 protein stained with the two antibodies AP180.1 and C172 displayed the same staining pattern. Tryptic digests probed with peptide antibodies (PS26 and PS27) generated to known sequences of AP180 were used to map the epitope for C172 within the brain AP180 sequence. On immunoblots of digested AP180, all AP180 antibodies and C172 recognized a 100 kd tryptic fragment, however only C172 recognized a smaller 60 kd. Our results suggest that the C172 epitope is located within amino acids 305–598 of the AP180 sequence. Confocal fluorescence microscopy of myoblasts and myotubes stained with the C172 antibody gives a punctate immunofluorescence pattern. Myoblasts stained with C172 revealed a polarized distribution of vesicles distinct from that observed when cells are stained with γ adaptin antibody which is known to localize to trans Golgi network. Myotubes stained with C172 antibody reveal a linear array of vesicular staining. Quantitative analysis of C172 reactive vesicles revealed a significant increase in number of vesicles present around the nuclei associated with the acetylcholine receptor clusters. These vesicles did not colocalize with the Golgi cisternae. These results indicate that a protein with homology to the neuron-specific coated vesicle protein AP180, is present in muscle cells associated with vesicles showing significant concentration around postsynaptic nuclei present in close proximity to AChR clusters. J. Cell. Biochem. 68:457–471, 1998. © 1998 Wiley-Liss, Inc.