Abstract
During neuromuscular junction formation, agrin secreted from motor neurons causes muscle cell surface acetylcholine receptors (AChRs) to cluster at synaptic sites by mechanisms that are insufficiently understood. The Rho family of small guanosine triphosphatases (GTPases), including Rac and Cdc42, can mediate focal reorganization of the cell periphery in response to extracellular signals. Here, we investigated the role of Rac and Cdc42 in coupling agrin signaling to AChR clustering. We found that agrin causes marked muscle-specific activation of Rac and Cdc42 in differentiated myotubes, as detected by biochemical measurements. Moreover, this activation is crucial for AChR clustering, since the expression of dominant interfering mutants of either Rac or Cdc42 in myotubes blocks agrin-induced AChR clustering. In contrast, constitutively active Rac and Cdc42 mutants cause AChR to aggregate in the absence of agrin. By indicating that agrin-dependent activation of Rac and Cdc42 constitutes a critical step in the signaling pathway leading to AChR clustering, these findings suggest a novel role for these Rho-GTPases: the coupling of neuronal signaling to a key step in neuromuscular synaptogenesis.
Highlights
Acetylcholine receptors (AChRs)1 that are diffusely distributed on the surface of cultured C2C12 muscle cells become aggregated into clusters upon treatment with neural agrin (Bowen et al, 1996; Ferns et al, 1996)
We show that agrin triggers the activation of Rac and Cdc42, and present evidence that this activation is crucial for agrininduced AChR clustering
Tants of Rac and Cdc42, and muscle differentiation was subsequently induced by switching the cultures from growth medium to differentiation medium, as described in Materials and Methods. 3 d after transfection, the effects of these mutants on AChR surface distribution in agrintreated and -untreated myotubes were examined by fluorescence microscopy of TMR-Bgt–labeled cells
Summary
Acetylcholine receptors (AChRs) that are diffusely distributed on the surface of cultured C2C12 muscle cells become aggregated into clusters upon treatment with neural agrin (Bowen et al, 1996; Ferns et al, 1996). This mimics the action of agrin secreted by motor neurons during the formation of neuromuscular junctions (Bowe and Fallon, 1995; Burden, 1998; Sanes and Lichtman, 1999). ␣-actinin (Shadiack and Nitkin, 1991; Apel et al, 1997) These findings support the possibility that the clustering of AChR and other cell surface components can be mediated through agrin-induced cytoskeletal reorganization (Hoch et al, 1994)
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