Figure 4. Induction of AChR Clustering by Tid1 in C2C12 Myotubes.

(A) Schematic diagram of full-length and four truncated fragments of Tid1S. (B) The N-terminal half of Tid1 induces AChR clustering. C2C12 myoblasts were transfected with the control plasmid pCS2+MT, which expresses 6 myc tags (Vector), or plasmid cDNAs encoding the various myc-tagged (fused in-frame to the N-terminus) Tid1S constructs shown in (A). At ~72 h after transfection and fusion, myotubes were incubated with or without neural agrin for 4 h, fixed, permeabilized, and double-stained with Rhodamine-conjugated αBTX and the anti-myc antibody mAb 9E10 followed by FITC-conjugated goat anti-mouse IgG. Arrows highlight transfected cells. Scale bar = 100 μm. (C) The effect of full-length and truncated fragments of Tid1S on the number of spontaneous and agrin-induced AChR clusters. n = 60 myotubes for each experimental group. *, 0.01 < p < 0.05; **, p < 0.01. (D) Overexpression of the N-terminal half of Tid1 does not induce tyrosine phosphorylation of MuSK. C2C12 myoblasts were transfected and fused as in (B), then incubated without (lanes 1 – 6), or with, neural agrin (lane 7) for 20 min, lysed, immunoprecipitated with anti-MuSK, and immunoblotted with the phosphotyrosine-specific antibodies mAb 4G10 and PY20. The blot was stripped and re-probed for MuSK. Crude lysates were immunoblotted with mAb 9E10. (E) MuSK is dispensable for the AChR clustering induced by overexpression of Tid1(1–222). Myoblasts from wildtype or MuSK^−/−mice were transfected with plasmid cDNAs encoding DsRed alone (control) or DsRed-tagged Tid1(1–222), respectively, and incubated in fusion medium for 48–72 h. Myotubes were fixed and stained with Alexa Fluor 488-conjugated αBTX. The results represent mean ± SEM for the number of AChR clusters per DsRed-positive myotube from four independent experiments. **, p < 0.01. n = 60 myotubes for each of the experimental groups.