We used a novel mammalian coculture system to study ACh receptor (AChR) redistribution and synaptic structure at nerve-muscle contacts. Ventral spinal cord (VSC) neurons were plated on cultures containing extensive myotubes but few fibroblasts. Neurite-induced redistribution of AChRs occurred within 6 hr after plating neurons and was maximal between 36-48 hr. This AChR redistribution appeared in two patterns: (1) AChR density at sites directly apposed to the neurite where neurites crossed preexisting AChR patches was sharply reduced, (2) Newly aggregated AChRs formed swaths lateral to the neurite path. VSC neurons induced more AChR aggregation than hippocampal, superior cervical ganglion and dorsal root ganglion neurons. The 43 and 58 kDa postsynaptic proteins were colocalized with AChR-enriched domains in all VSC neurite-induced aggregates whereas the colocalization of laminin was variable. Electron microscopy of regions with neurite-induced AChR aggregation showed postsynaptic membrane specializations characteristic of developing synapses and, in older cultures, features of more mature synaptic structure. Thus, the coculture system is useful for studying early stages of neuromuscular junction (NMJ) formation. Neurites in these cocultures were identified as axons or dendrites by morphological criteria and by their immunoreactivity for synaptophysin and phosphorylated heavy neurofilament subunits or for microtubule associated protein 2 (MAP2), respectively. Axons showed a 10-fold higher induction of AChR aggregation than did dendrites. Thus, at least one essential signaling molecule necessary for the induction of AChR aggregation at sites of interaction with muscle appears to be expressed in a polarized fashion in developing VSC neurons.

Abstract. We used quick-freeze, deep-etch, rotary replication and immunogold cytochemistry to identify a new structure at focal contacts. In Xenopus fibroblasts, elongated aggregates of particles project from the membrane to contact bundles of actin microfilaments. Before terminating, a single bundle of microfilaments interacts with several aggregates that appear intermittently over a distance of several microns. Aggregates are enriched in proteins believed to mediate actin-membrane interactions at focal contacts, including B~-integrin, vinculin, and talin, but they appear to contain less t~-actinin and filamin. We also identified a second, smaller class of aggregates of membrane particles that contained/~-integrin but not vinculin or talin and that were not associated with actin microfilaments. Our results indicate that vinculin, talin, and ~-integrin are assembled into distinctive structures that mediate multiple lateral interactions between microfilaments and the membrane at focal contacts.F OCAL contacts are specialized structures where actin filaments converge and terminate at the plasma membrane (16,32,44,63,65). Interactions between the membrane and microfilaments involve several proteins that are concentrated at focal contacts, including integrin (e.g., references 17, 21, 22; reviewed in 1, 61) and cytoskeletal proteins such as vinculin (26), talin (13), fimbrin (9), tensin (71), paxillin (68), zyxin (5), and ot-actinin (40). Biochemical studies have suggested several ways in which these proteins could anchor microfilaments to the cytoplasmic surface of the plasma membrane at focal contacts. Early studies suggested that particular isoforms of integrin ("CSAT'; 47) bound talin, and that this complex then bound vinculin (35; reviewed in 15). Later studies showed that integrins could also bind tx-actinin (53). Thus, integrin could anchor actin filaments indirectly through the actin-binding protein, tx-actinin, as well as through talin and vinculin. If both of these mechanisms are important for actin-membrane interactions, one would expect all these proteins to be concentrated at focal contacts.Structural studies also suggest two models for microfilament-membrane interactions at focal contacts. Focal contacts are several microns long, and individual actin filaments course over this entire distance before terminating (8). These filaments could bind to the membrane laterally as they approach their termini. In this case, vinculin, talin, integrin, and other proteins of focal contacts would be expected to concentrate at many points along the filament length. Alter- natively, filaments could attach only at their termini. In this case, the proteins of focal contacts would be expected to occur only at the ends of microfilaments.Predictions of the relationship between actin microfilaments and the membrane, and of the distribution of proteins at focal contacts, can be tested by ultrastructural methods coupled with immunocytochemistry. We exposed the cytoplasmic surfaces of ventral membrane by mechanical shearing, the...