Impairment of MT function by nocodazole, by inhibition of PI3-K and AKT, and by genetic deletion of CLASP2 impairs AChR insertion into agrin-induced AChR clusters in cultured myotubes. (a) Areas of agrin-induced AChR clusters in primary myotubes appear smaller when AChRs are stained after 3 h of treatment with the following drugs: nocodazole (10 µM); inhibitors of AKT (A6730, 0.5 µM); PI3-K (ZSTK474, 5 µM; only the effect of ZSTK474 is illustrated). However, the density of AChRs in clusters remains unchanged. Conversely, inhibiting GSK3β activity (CHIR99021, 0.1 µM) increases AChR cluster size. Bar, 30 µm. Means ± SEM. Total numbers of AChR clusters analyzed in two independent experiments: Control (wild type): 78; nocodazole: 67; A6730: 68; ZSTK474: 38; CHIR99021: 54 (*, P < 0.05; **, P < 0.01, two-sided t test). (b) In contrast, AChR clusters appear unchanged after 3 h of drug treatment when AChRs are stained at the time of drug addition; again, AChR densities in clusters remain unchanged. Bar, 60 µm. Means ± SEM. Total numbers of AChR clusters analyzed in two independent experiments: Control (wild type): 30; nocodazole: 46; ZSTK474: 27; CHIR99021: 21 (*, P < 0.05; **, P < 0.01, two-sided t test). (c) AChR clusters are smaller in Clasp2^−/− than in wild-type muscle, and unlike in wild-type muscle (see panel a), cluster size is not affected by ZSTK474 (5 µM) or CHIR99021 (0.1 µM). AChRs were stained after drug treatment. Bar: 30 µm. Graphs give means ± SEM, 20–78 clusters analyzed from 2 cultures for each condition (*, P < 0.05; **, P < 0.01, two-sided t test). AChR clusters marked by arrowheads.