Synaptic localization of CLASP2, Tyr-tubulin, and Glu-tubulin in wt and Clasp2^−/− NMJs. (a) Staining of CLIP-170 and CLASP2 at NMJ of wild-type muscle. Like CLIP-170 (left, green), CLASP2-decorated MT plus-ends (right, green) are enriched at synaptic AChR clusters primarily at their edges and at the crests of synaptic folds (arrowheads; troughs of folds marked by the AChRs). Bar, 5 µm. Note that images in panel a are from the level of the synaptic folds, i.e., deeper in the synaptic gutter; in contrast, maximum intensity projections from optical slices taken at the level of the edge of the synaptic AChR cluster and thus of the synaptic gutter (such as in Fig. 1 d, top images) reveal pronounced CLIP-170 enrichment compared with adjacent perisynaptic membrane. This explains the apparent difference in CLIP-170 intensity along the edge of the clusters in the two images (Fig. 2 a vs. 1 d). (b) Double staining of CLIP-170 and CLASP2 at NMJ of wild-type muscle. Accumulation of both +TIPs at edge of synaptic AChR cluster (blue) and overlap of the two +TIPs (marked by asterisks) is consistent with cooperation between CLIP-170 and CLASP2 at MT plus-ends. Note that some of the CLIP-170 and CLASP2 staining does not overlap, which may reflect differences in antibody accessibility and staining, or, alternatively, independent functions of these proteins in the NMJ. For localization of CLIP-170 in Clasp2^−/− NMJ, see Fig. 4. Bar, 2.5 µm. (c) Top: CLASP2 is enriched at edge of synaptic AChR cluster at wild-type NMJ. Bottom: absence of CLASP2 staining of NMJ from Clasp2^−/− muscle shows specificity of antibody used. Bars, 5 µm. (d) Both Tyr-tubulin (top) and Glu-tubulin (bottom) are enriched at NMJs of Clasp2^−/− muscle. Bars, 10 µm.