Abstract
A key aspect of neuromuscular synapse formation is the clustering of muscle acetylcholine receptors (AChR) at synaptic sites in response to neurally secreted agrin. Agrin-induced AChR clustering in cultured myotubes proceeds via the initial formation of small microclusters, which then aggregate to form AChR clusters. Here we show that the coupling of agrin signaling to AChR clustering is dependent on the coordinated activities of Rac and Rho GTPases. The addition of agrin induces the sequential activation of Rac and Rho in C2 muscle cells. The activation of Rac is rapid and transient and constitutes a prerequisite for the subsequent activation of Rho. This temporal pattern of agrin-induced Rac and Rho activation reflects their respective roles in AChR cluster formation. Whereas agrin-induced activation of Rac is necessary for the initial phase of AChR cluster formation, which involves the aggregation of diffuse AChR into microclusters, Rho activation is crucial for the subsequent condensation of these microclusters into full-size AChR clusters. Co-expression of constitutively active forms of Rac and Rho is sufficient to induce the formation of mature AChR clusters in the absence of agrin. These results establish that Rac and Rho play distinct but complementary roles in the mechanism of agrin-induced AChR clustering.
Highlights
During embryonic development, innervation induces the anatomical and biochemical specialization of a defined region of the muscle cell membrane immediately under the motor nerve ending
Inhibitors of the Rho Pathway Impair Agrin-induced acetylcholine receptors (AChR) Clustering—We have recently found that constitutively active Rac and Cdc42 mutants cause the formation of AChR microclusters (2–5 m in diameter) but are insufficient to induce full- size AChR clusters (15–20 m in diameter) (14), suggesting that other regulatory components might be required
In view of these findings and the observations implicating the actin cytoskeleton in AChR clustering (3), we have investigated the potential contribution of Rho
Summary
Reagents—Expression plasmids encoding T7 epitope-tagged constitutively active Rac (RacV12), dominant negative Rac (RacN17), wild type Rho (RhoWT), constitutively active Rho (RhoV14), and dominant negative Rho (RhoN19), as well as RacV12, RhoV14, and C3 transferase proteins were generously provided by D. Microscopy—Cells plated on glass coverslips and subsequently either transfected or microinjected were labeled for 1 h with 10 nM tetramethylrhodamine-conjugated ␣-bungarotoxin in Dulbecco’s modified Eagle’s medium with 1 mg/ml bovine serum albumin for 1 h at 37 °C, rinsed with PBS, and fixed in 3.7% formaldehyde with PBS for 30 min to visualize the surface distribution of AChR. To measure Rho activation, an affinity precipitation method was used (36, 37) in which cell lysates prepared with lysis buffer A were incubated with GST fused to the Rho-binding domain from the effector protein Rhotekin (GST-TRBD) bound to glutathione-coupled Sepharose beads for 45 min at 4 °C. In a similar manner Rac activation was measured by affinity precipitation of cellular GTP-bound forms of Rac (39) In this case cell lysates were prepared with lysis buffer B and incubated with GST fused to the Cdc42/Rac (p21)-binding domain of PAK (GST-PBD) bound to glutathione-coupled Sepharose beads for 30 min at 4 °C. Rho activation was assessed using the TRBD pull-down assay described above, using antibody to the T7 epitope to select for transfected cells
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