Absence of CLASP2 reduces the density of GFP-CLIP-170–decorated MTs at the synaptic membrane of the NMJ in vivo. (a) The localization of MT plus-ends (visualized by GFP labeling) at synaptic AChR clusters is reduced in Clasp2^−/−;GFP-Clip170^ki/ki myotubes. Bar, 5 µm. Graph shows percentage of synapses with GFP-CLIP-170 enriched at the edge of the AChR cluster in wild-type and Clasp2^−/− synapses (means ± SEM, n = 13 wild-type and 18 CLASP2^−/− synapses from 3 muscles each (*, P < 0.05; **, P < 0.01, Mann Whitney U-Test). A synapse was classified “blindly” by visual inspection as enriched in CLIP-170 (arrowheads), when edges of AChR clusters were decorated with GFP puncta along >80% of their lengths. Note that further information on, e.g., number and intensity of CLIP dots cannot be extracted from these confocal images (for details, see Materials and methods). (b) Analysis by structured illumination microscopy (SIMELRYA S.1; Carl Zeiss) of the localization of GFP-CLIP-170–decorated MTs at the synaptic membrane (marked by AChRs) in wild-type and Clasp2^−/−;GFP-Clip-170^ki/ki NMJs. NMJs stained for AChRs (red) and GFP (green). Bars, 1 µm, 3 µm, and 1.5 µm. Stacks of images of NMJs en face were taken through the entire depth of the synapse at 0.125-µm steps and processed. 10–20 µm of contour lines of AChRs (as the one illustrated by the white line in the 3D-reconstructed cluster in top panel) at each of three z-levels for each synapse were selected, and CLIP-170 puncta per length of contour line were counted (arrowheads). Bottom panels show sample AChR contour lines and CLIP-170 immunoreactivity, including enlarged insets. Graph shows combined data from three synapses each in wild-type and Clasp2^−/− muscle (*, P < 0.05; **, P < 0.01, two-sided t test). Note the marked reduction in the density of CLIP-170 puncta on AChR contour lines in the absence of CLASP2.