Figure 2.

Inhibition of MuSK endocytosis attenuates AChR clustering. A, Dynamin K44A attenuated MuSK endocytosis. C2C12 cells were transfected with HA-tagged wild-type or K44A dynamin. MuSK endocytosis was assayed as in Figure 1. B, Dynamin K44A attenuated AChR clustering. C2C12 cells were transfected with HA-tagged wild-type or K44A dynamin along with EGFP expressing construct. Fully differentiated myotubes were treated with agrin (1 nm; 16 h) and then were incubated with Alexa 594-BTX to stain AChR clusters (arrows). Myotube segments (200 μm in length) were viewed at 40× magnification with a Nikon (Tokyo, Japan) Optiphot microscope equipped with phase and epifluorescence optics, and the number of AChR aggregates was counted. AChR clusters with the shortest axis, >4 μm, were counted in 10 fields of each experiment. Scale bar, 20 μm.