Agrin induces alpha-actinin, filamin, and vinculin to co-localize with AChR clusters on cultured chick myotubes.

Agrin induces discrete high-density patches of acetylcholine receptors (AChRs) and other synaptic components on cultured myotubes in a manner that resembles synaptic differentiation. Furthermore, agrin-like molecules are present at developing neuromuscular junctions in vivo. This provides us with a unique opportunity to manipulate AChR patching in order to examine the role of cytoskeletal components. Cultured chick myotubes were fixed and labeled to visualize the distributions of actin, alpha-actinin, filamin, tropomyosin, and vinculin. Overnight exposure to agrin caused a small amount of alpha-actinin, filamin, and vinculin to reorganize into discrete clusters. Double-labeling studies revealed that 78% of the AChR clusters were associated with detectable concentrations of filamin, 70% with alpha-actinin, and 58% with vinculin. Filamin even showed congruence to AChRs within clustered regions. By contrast, actin (visualized with fluorescein-phalloidin) and tropomyosin did not show specific associations with agrin-induced AChR clusters. The accumulation of cytoskeletal components at AChRs clusters raised the possibility that cytoskeletal rearrangements direct AChR clustering. However, a time course of agrin-induced clustering that focused on filamin revealed that most of the early AChR clusters (3-6 h) were not associated with detectable amounts of cytoskeletal material. The accumulation of cytoskeletal material at later times (12-18 h) may imply a role in maintenance and stabilization, but it appears unlikely that these cytoskeletal elements initiate AChR clustering on myotubes.

Agrin-induced reorganization of extracellular matrix components on cultured myotubes: relationship to AChR aggregation.

Agrin, an extracellular matrix-associated protein extracted from synapse-rich tissues, induces the accumulation of acetylcholine receptors (AChRs) and other synaptic components into discrete patches on cultured myotubes. The appearance of agrin-like molecules at neuromuscular junctions suggests that it may direct synaptic organization in vivo. In the present study we examined the role of extracellular matrix components in agrin-induced differentiation. We used immunohistochemical techniques to visualize the spatial and temporal distribution of laminin, a heparan sulfate proteoglycan (HSPG), fibronectin, and type IV collagen on cultured chick myotubes during agrin-induced aggregation of AChRs. Myotubes displayed significant amounts of laminin and HSPG, lesser amounts of type IV collagen, and little, if any, fibronectin. Agrin treatment caused cell surface laminin and HSPG to patch, while collagen and fibronectin distributions were generally unaffected. Many of the agrin-induced laminin and HSPG patches colocalized with AChR patches, raising the possibility of a causal relationship between matrix patching and AChR accumulations. However, patching of AChRs (complete within a few hours) preceded that of laminin or HSPG (not complete until 15-20 h), making it unlikely that matrix accumulations initiate AChR patching at agrin-induced sites. Conversely, when AChR patching was blocked by treatment with anti-AChR antibody mAb 35, agrin was still able to effect patching of laminin and HSPG. Taken together, these findings suggest that agrin-induced accumulations of AChR and laminin/HSPG are not mechanistically linked.

Identification of agrin, a synaptic organizing protein from Torpedo electric organ.

Extracts of the electric organ of Torpedo californica contain a proteinaceous factor that causes the formation of patches on cultured myotubes at which acetylcholine receptors (AChR), acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE) are concentrated. Results of previous experiments indicate that this factor is similar to the molecules in the synaptic basal lamina that direct the aggregation of AChR and AChE at regenerating neuromuscular junctions in vivo. We have purified the active components in the extracts 9,000-fold. mAbs against four different epitopes on the AChR/AChE/BuChE-aggregating molecules each immunoprecipitated four polypeptides from electric organ extracts, with molecular masses of 150, 135, 95, and 70 kD. Gel filtration chromatography of electric organ extracts revealed two peaks of AChR/AChE/BuChE-aggregation activity; one comigrated with the 150-kD polypeptide, the other with the 95-kD polypeptide. The 135- and 70-kD polypeptides did not cause AChR/AChE/BuChE aggregation. Based on these molecular characteristics and on the pattern of staining seen in sections of muscle labeled with the mAbs, we conclude that the electric organ-aggregating factor is distinct from previously identified molecules, and we have named it "agrin."

Acetylcholine receptor-aggregating factor is similar to molecules concentrated at neuromuscular junctions.

The basal lamina in the synaptic cleft of the vertebrate skeletal neuromuscular junction contains molecules that direct the formation of synaptic specializations in regenerating axons and muscle fibres. We have undertaken a series of experiments aimed at identifying and characterizing the molecules responsible for the formation of one of these specializations, the aggregates of acetylcholine receptors (AChRs) in the muscle fibre plasma membrane. We began by preparing an insoluble, basal lamina-containing fraction from Torpedo californica electric organ, a tissue which has a far higher concentration of cholinergic synapses than muscle, and showing that this fraction caused AChRs on cultured chick myotubes to aggregate. A critical step is learning whether or not the electric organ factor is similar to the receptor-aggregating molecule in the basal lamina at the neuromuscular junction. The importance of this problem is emphasized by reports that clearly non-physiological agents, such as positively charged latex beads, can cause AChR aggregation on cultured muscle cells. We have already shown that Torpedo muscle contains an AChR-aggregating factor similar to that of electric organ, although in much lower amounts. Here we demonstrate, using monoclonal antibodies, that the AChR-aggregating factor in our extracts of electric organ is, in fact, antigenically related to molecules concentrated in the synaptic cleft at the neuromuscular junction.

Aggregates of acetylcholinesterase induced by acetylcholine receptor-aggregating factor.

Basal lamina-rich extracts of Torpedo californica electric organ contain a factor that causes acetylcholine receptors (AChRs) on cultured myotubes to aggregate into patches. Our previous studies have indicated that the active component of these extracts is similar to the molecules in the basal lamina which direct the aggregation of AChRs in the muscle fibre plasma membrane at regenerating neuromuscular junctions in vivo. Because it can be obtained in large amounts and assayed in controlled conditions in cell culture, the AChR-aggregating factor from electric organ may be especially useful for examining in detail how the postsynaptic apparatus of regenerating muscle is assembled. Here we demonstrate that the electric organ factor causes not only the formation of AChR aggregates on cultured myotubes, but also the formation of patches of acetylcholinesterase (AChE). This finding, together with the observation that basal lamina directs the formation of both AChR and AChE aggregates at regenerating neuromuscular junctions in vivo, leads us to hypothesize that a single component of the synaptic basal lamina causes the formation of both these synaptic specializations on regenerating myofibres.

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.

Molecular components of the synaptic basal lamina that direct differentiation of regenerating neuromuscular junctions.

Results of experiments outlined here provide evidence that components of the myofiber basal lamina sheath direct the formation of active zones in regenerating motor nerve terminals and the development of infoldings and the aggregation of AChRs in the plasma membrane of regenerating myofibers. As a step toward identifying the basal lamina molecules that aggregate AChRs, we are now studying an ECM fraction from the Torpedo electric organ that causes AChRs to aggregate on cultured myotubes. We have solubilized and purified the electric organ AChR-aggregating molecules over 1000-fold. Only nanogram amounts of the most purified extracts are required to cause detectable AChR aggregation. We have also shown that similar activity can be extracted in relatively small amounts from muscle. Antiserum raised against the partially purified electric organ material completely blocked and immunoprecipitated the AChR-aggregating activity in extracts of the electric organ and muscle and bound to components of the basal lamina of frog muscle fibers. Although several polypeptides are present in our most purified extracts, an antiserum against polypeptides in the range of 80 kD completely blocked AChR aggregation by soluble extracts of the electric organ. These findings demonstrate the feasibility of isolating molecules from the synapse-rich electric organ that cause AChR aggregation and comparing them by immunological techniques with those in basal lamina at the neuromuscular junction.

Internalization of alpha-bungarotoxin on neurons induced by a neurotoxin that blocks neuronal acetylcholine sensitivity.

A protein neurotoxin (Bgt 3.1) present as a minor component in the venom of Bungarus multicinctus has been shown previously to block acetylcholine (ACh) sensitivity on chick ciliary ganglion (CG) neurons in cell culture. Alpha-bungarotoxin (Bgt. 2.2) binds to the neurons but does not block ACh sensitivity; the function of the Bgt. 2.2 binding site is unknown. The present studies demonstrate that Bgt 3.1 can induce the rapid internalization of Bgt 2.2 bound on the surface of CG and sympathetic neurons. The rapid internalization of bound Bgt 2.2 caused by Bgt. 3.1 can be seen with fluoresence microscopy using rhodamine-labeled Bgt 2.2 as the probe and by immunological techniques using anti-Bgt 2.2 antiserum to locate the bound 125I-Bgt 2.2. The rapid internalization is blocked by low temperature or by high concentrations of Bgt 2.2 and is not induced by Bgt 2.2 itself or by small cholinergic ligands. Bound 125I-Bgt 2.2 is released into the medium as degraded material after internalization is induced. Bgt 3.1 does not induce internalization of Bgt 2.2 bound to skeletal myotubes in culture nor does it induce the internalizaton of rhodamine-labeled nerve growth factor bound to sympathetic neurons, suggesting that its effect on neuronally bound Bgt 2.2 might be a specific one. Competition binding studies suggest that Bgt 3.1 may trigger the internalization of bound Bgt 2.2 by direct interaction with a Bgt 2.2 binding site. The effect of Bgt 3.1 on neuronal ACh sensitivity, however, does not depend on internalization of Bgt 2.2 binding sites since full inhibition of ACh sensitivity is still achieved by Bgt 3.1 under conditions where internalization is blocked. Neurons may have more than one class of Bgt 2.2 on the neurons. The internalization of Bgt 2.2 binding sites induced by Bgt 3.1 provides an unusual opportunity to study cellular mechanisms by which neurons can regulate the number and distribution of their surface components.