Elimination of preexistent acetylcholine receptor clusters induced by the formation of new clusters in the absence of nerve

Abstract

Acetylcholine receptors (AChRs) form clusters spontaneously in cultured Xenopus myotomal muscle cells in the absence of innervation. Innervation causes a dispersal of these clusters, as well as the formation of new clusters along the neuromuscular contact. To examine whether this dispersal process is dependent upon the presence of the nerve or can be effected by the formation of new clusters alone, we examined the stability of the preexistent clusters during the formation of new AChR clusters induced by basic polypeptide-coated latex beads. These beads cause a rapid development of clusters in cultured Xenopus muscle cells, and the size and the number of clusters can be experimentally manipulated by varying the size and number of beads applied to the culture. Accompanying the bead-induced formation of new clusters was an observed suppression of preexistent clusters, as shown by fluorescence microscopy after staining with rhodamine-conjugated α-bungarotoxin. This process of extrabead cluster removal can be speeded up by treating the cultures with more beads or using beads of larger diameter. Furthermore, if the beads are applied before the spontaneous AChR clustering, no extra-bead clusters are formed. Thus, the latex beads can mimic the nerve in both causing the formation of new clusters and the dispersal of preexistent clusters. Although the receptor clusters are dispersed by the beads, the density of diffuse receptors, as shown by autoradiography with 125I-conjugated α-bungarotoxin does not decrease in the short-term cultures under study. This indicates that the dispersal of preexistent clusters is not due to a depletion of the surface pool of AChRs. We suggest that a change in the cytoskeleton associated with preexistent clusters may be involved in their destabilization.