Distribution of ot-Dystroglycan during Embryonic Nerve-Muscle Synaptogenesis

The distribution of ot-dystroglycan (aDG) relative to acetylcholine receptors (ACHEs) and neural agrin was examined by immunofluorescent staining with mAb IIH6 in cultures of nerve and muscle cells derived from Xenopus embryos. In Western blots • probed with mAb IIH6, o~DG was evident in membrane extracts of Xenopus muscle but not brain, c~DG immunofluorescence was present at virtually all synaptic clusters of ACHEs and neural agrin. Even microclusters of ACHEs and agrin at synapses no older than 1-2 h (the earliest examined) had ctDG associated with them. c~DG was also colocalized at the submicrometer level with ACHES at nonsynaptic clusters that have little or no agrin. The number of large (>4 gin) nonsynaptic clusters of oLDG, like the number of large nonsynaptic clusters of ACHES, was much lower on innervated than on noninnervated cells. When mAb IIH6 was included in the culture medium, the large nonsynaptic clusters appeared fragmented and less compact, but the accumulation of agrin and ACHES along nerve-muscle contacts was not prevented. It is concluded that during nerve-muscle synaptogenesis, txDG undergoes the same nerve-induced changes in distribution as ACHES. We propose a diffusion trap model in which the otDG-transmembrane complex participates in the anchoring and recruitment of AChEs and txDG during the formation of synaptic as well as nonsynaptic AChR clusters. ~ -DYSTROGLYCAN (otDG), ~ an extracellular peripheral membrane protein, is part of the dystrophin receptor complex, which comprises at least six proteins that are tightly associated with dystrophin or dystrophin-related protein (DRP; also called utrophin) at the cell surface in muscle, nerve, and a variety of other tissues (Ervasti and Campbell, 1991). c~DG binds to the extracellular matrix proteins laminin and merosin (Douville et al., 1988; IbraghimovBeskrovnaya et al., 1992; Ervasti and Campbell, 1993a; Gee et al., 1993) and is thought to forge a structurally important link from the basal lamina surrounding skeletal muscle cells to their submembranous cytoskeleton (Ervasti and Campbell, 1993b; Lindenbaum and Carbonetto, 1993). Mutations in dystrophin, adhalin (a transmembrane member of the complex), or merosin can lead to muscular dystrophy (Anderson and Kunkel, 1992; Roberds et al., 1994; Sunada et al., 1994; Tom6 et al., 1994; Xu et al., 1994). Address all correspondence to M. W. Cohen, Dept. of Physiology, McGill University, 3655 Drummond St., Montreal, Quebec H3G IY6, Canada. Tel.: (514) 398-4342. Fax: (514) 398-7452. 1. Abbreviations used in this paper: AChR, acetylcholine receptor; aDG, a,-dystroglycan; DRP, dystrophin-related protein; SC, spinal cord. Immunocytochemical studies have indicated that the otDG complex and dystrophin are present over the entire surface of mature skeletal muscle cells (Ervasti and Campbell, 1991; Matsumura et al., 1992). At the neuromuscular junction, c~DG is concentrated with other members of the complex, but dystrophin is replaced by DRP at the tops of the junctional folds where acetylcholine receptors (AChRs) are clustered (Ohlendieck et al., 1991a; Bewick et al., 1992; Bowe et al., 1994). o~DG, other members of its complex, and DRP are also concentrated at agrin-induced AChR clusters on cultured muscle cells (Campanelli et al., 1994; Gee et al., 1994; Sugiyama et al., 1994). Agrin, in addition to causing AChRs to cluster (Godfrey et al., 1984; Nitldn et al., 1987; Campanelli et al., 1991; Ferns et al., 1992; Tsim et al., 1992), is deposited by neurites at newly forming nerve-muscle synapses (Cohen and Godfrey, 1992), and some anti-agrin antibodies inhibit nerveinduced AChR clustering (Reist et al., 1992). This evidence suggests that agrin is the primary neural agent that triggers clustering of AChP, s during embryonic nerve-muscle synaptogenesis. That the c~DG complex is concentrated at the mature neuromuscular junction and at agrin-induced AChR clusters implies that it too plays a role in nerve-muscle synaptogenesis. A direct role for ctDG itself, as a receptor for © The Rockefeller University Press, 0021-9525/95/05/1093/9 $2.00 The Journal of Cell Biology, Volume 129, Number 4, May 1995 1093-1101 1093 on A ril 7, 2017 D ow nladed fom Published May 15, 1995