The MyTH4 domain is required for Myo15 function, and Myo15 partially localizes to presynaptic MTs. (A) Confocal images of Myo15ΔMyTH4 (1+2) deletion mutant and wild-type NMJs stained for BRP (green) and HRP (magenta), scale bar overview 5 µm, zoom 2 µm, white arrowheads mark satellite boutons, Is and Ib NMJs indicated. (B–H) Quantification of A. (B) AZ area (control 100.0 ± 3.32% n = 12; Myo15ΔMyTH4 (1+2) 86.71 ± 2.47%, n = 12). (C) Relative frequency histogram of AZ areas (total number of AZs: control 3864 and Myo15ΔMyTH4 (1+2) 4220), bin width 0.02. (D) Density of AZs, as number of AZ per NMJ area (control 100.0 ± 3.83%, n = 12; Myo15ΔMyTH4 (1+2) 118.3 ± 4.52%, n = 12). (E) NMJ area (control 100.0 ± 5.92%, n = 12; Myo15ΔMyTH4 (1+2) 91.46 ± 4.07%, n = 12). (F) Total number of all AZs per NMJ (control 100.0 ± 4.05%, n = 12; Myo15ΔMyTH4 (1+2) 112.2 ± 3.76%, n = 11). (G) BRP mean pixel intensity of single AZs (control 100.0 ± 2.84%, n = 12; Myo15ΔMyTH4 (1+2) 86.09 ± 5.80%, n = 12). (H) Total BRP intensity as integral of the BRP mean pixel intensity over the entire NMJ (control 100.0 ± 5.61% n = 12; Myo15ΔMyTH4 (1+2) 78.61 ± 6.19%, n = 12). (I) Confocal images of wild-type NMJs stained for MTs (acetylated tubulin, magenta) and Myo15C1 (green), scale bar overview 2 µm, white arrowheads mark overlapping Myo15 and MT signal. (J) STED images of wild-type and Myo15ΔMyTH4 (1+2) deletion mutant stained for Myo15C1 (green), MTs (acetylated tubulin, magenta), scale bar overview 0.5 µm, zoom 0.2 µm. White square mark zoom area. (K) Quantification of J. Normalized radial intensity distribution of MT and myosin signal around the centers of myosin clusters for Myo15ΔMyTH4 (1+2) deletion mutant and wild type. Green as control compares Myo15 to Myo15 distances in both phenotypes, magenta compares Myo15 to MT distance. Means ± 95% confidence intervals (dashed lines) from 23/18 (wild type/mutant) images are shown. (L) STED imaging of wild-type NMJs stained for the MAP1 homolog futsch, localizing to MTs (green) and BRP (magenta), scale bar overview 2 µm, zoom 0.5 and 0.1 µm. White square mark zoom area, white dashed line in zoom marks the plane of line profile. Lower panel shows line profile, green futsch, magenta BRP. All data are provided as mean ± SEM. (B–H): n represents NMJs with 1–2 NMJs/animal from five to six animals, K: n = image, with 1–2 boutons/image and 2–3 images/NMJ, 2 NMJs/animal, three to four animals in total. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, n.s. not significant.
Figure 6.

The MyTH4 domain is required for Myo15 function, and Myo15 partially localizes to presynaptic MTs. (A) Confocal images of Myo15ΔMyTH4 (1+2) deletion mutant and wild-type NMJs stained for BRP (green) and HRP (magenta), scale bar overview 5 µm, zoom 2 µm, white arrowheads mark satellite boutons, Is and Ib NMJs indicated. (B–H) Quantification of A. (B) AZ area (control 100.0 ± 3.32% n = 12; Myo15ΔMyTH4 (1+2) 86.71 ± 2.47%, n = 12). (C) Relative frequency histogram of AZ areas (total number of AZs: control 3864 and Myo15ΔMyTH4 (1+2) 4220), bin width 0.02. (D) Density of AZs, as number of AZ per NMJ area (control 100.0 ± 3.83%, n = 12; Myo15ΔMyTH4 (1+2) 118.3 ± 4.52%, n = 12). (E) NMJ area (control 100.0 ± 5.92%, n = 12; Myo15ΔMyTH4 (1+2) 91.46 ± 4.07%, n = 12). (F) Total number of all AZs per NMJ (control 100.0 ± 4.05%, n = 12; Myo15ΔMyTH4 (1+2) 112.2 ± 3.76%, n = 11). (G) BRP mean pixel intensity of single AZs (control 100.0 ± 2.84%, n = 12; Myo15ΔMyTH4 (1+2) 86.09 ± 5.80%, n = 12). (H) Total BRP intensity as integral of the BRP mean pixel intensity over the entire NMJ (control 100.0 ± 5.61% n = 12; Myo15ΔMyTH4 (1+2) 78.61 ± 6.19%, n = 12). (I) Confocal images of wild-type NMJs stained for MTs (acetylated tubulin, magenta) and Myo15C1 (green), scale bar overview 2 µm, white arrowheads mark overlapping Myo15 and MT signal. (J) STED images of wild-type and Myo15ΔMyTH4 (1+2) deletion mutant stained for Myo15C1 (green), MTs (acetylated tubulin, magenta), scale bar overview 0.5 µm, zoom 0.2 µm. White square mark zoom area. (K) Quantification of J. Normalized radial intensity distribution of MT and myosin signal around the centers of myosin clusters for Myo15ΔMyTH4 (1+2) deletion mutant and wild type. Green as control compares Myo15 to Myo15 distances in both phenotypes, magenta compares Myo15 to MT distance. Means ± 95% confidence intervals (dashed lines) from 23/18 (wild type/mutant) images are shown. (L) STED imaging of wild-type NMJs stained for the MAP1 homolog futsch, localizing to MTs (green) and BRP (magenta), scale bar overview 2 µm, zoom 0.5 and 0.1 µm. White square mark zoom area, white dashed line in zoom marks the plane of line profile. Lower panel shows line profile, green futsch, magenta BRP. All data are provided as mean ± SEM. (B–H): n represents NMJs with 1–2 NMJs/animal from five to six animals, K: n = image, with 1–2 boutons/image and 2–3 images/NMJ, 2 NMJs/animal, three to four animals in total. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, n.s. not significant.

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