Capturing of dynamic MTs at agrin-induced AChR clusters in cultured myotubes is abolished by pharmacological inhibition of PI3-K and by genetic elimination of Clasp2. (a) Scheme illustrating focal impregnation of culture substrate with neural agrin. Transfected COS1 cells secrete and locally deposit neural agrin on a laminin substrate, and the cells are then lysed. Stable AChR clusters form where GFP-Clip-170^ki/ki myotubes contact agrin deposits. (b) MT plus-ends decorated with GFP-CLIP-170 as observed by TIRF microscopy at 1-s intervals for 160 s. Shown are AChR cluster, surface reflective interference contrast (SRIC) image of same myotube, first frame and maximum intensity projection of all 160 images of the stack. Note higher density and brighter GFP signal at AChR cluster (marked in red). Bars, 10 µm. (c) Representative examples of kymograms of type I and type II comet behavior. See text for discussion of comet behavior and criteria for comet classification. (d) Same as in b, except that myotubes had been incubated for 90 min in ZSTK474 (5 µM) before GFP analysis. Note that “cluster”-specific comet behavior is abolished. Bars, 10 µm. (e) Same as in b, except that myotubes derived from Clasp2^−/−;GFP-Clip-170^ki/ki mice were used. Note that “cluster”-specific comet behavior is abolished. Bars, 10 µm. (f) Quantification of comet behavior inside and outside agrin-induced AChR clusters. Comet immobilization by neural agrin is abolished by inhibition of PI3-K or the absence of CLASP2. Comets are grouped from kymograms into type I (mobile) and type II (stable) comets as described in the text, and the percentages of stable comets, both inside and outside agrin-induced AChR clusters, is given (means ± SEM, n = 4 myotubes for each condition; number of comets analyzed: wild type: 374; wild type, ZSTK474-treated: 342; Clasp2^−/−: 323; *, P < 0.05; **, P < 0.01, two-sided t test).