Myo15 localization at the NMJ and salivary gland and antibody verification. (A) Schematic representation of Drosophila class XV Myo15 indicating antibody position (Myo15C1 and Myo15C2). (B) Confocal images of control and Myo15-RNAi knock-down NMJs stained for Myo15 (anti-Myo15C2, green) and HRP (magenta), scale bar overview 5 µm. (C) Quantification of B: Myo15C2 mean pixel intensity (control 100.0 ± 7.82%, n = 9; Myo15-RNAi 67.4 ± 3.24%, n = 11). (D) Western blot of wild-type and myo15−/− mutant larvae for Myo15C2. (E) Confocal images of wild-type and myo15−/− mutant NMJs stained for Myo15 (anti-Myo15C1, green) and HRP (magenta), scale bar overview 5 µm, zoom 2 µm. (F) Quantification of E. Myo15C1 mean pixel intensity (wild type 100.0 ± 11.52%, n = 9; myo15−/− mutant 67.38 ± 3.79%, n = 13). (G) Confocal images of wild-type and myo15−/− mutant NMJs stained for Myo15 (anti-Myo15C2, green) and HRP (magenta), scale bar overview 5 µm, zoom 2 µm. (H) Quantification of G. Myo15C2 mean pixel intensity (wild type 100.0 ± 5.71%, n = 12; myo15−/− mutant 58.05 ± 8.54%, n = 9). (I) Confocal images of NMJs upon motoneuronal Myo15 overexpression and control, stained for Myo15C2 (green) and HRP (magenta), scale bar overview 5 µm. (J) Quantification of I: Myo15C2 mean pixel intensity (control 100.0 ± 4.89%, n = 12; Myo15 OE 282.4 ± 23.21%, n = 12). (K) Western blot of adult brains neuronally overexpressing Myo15 and control brains for Myo15C2. (L) Confocal images of control and Myo15GFP-OE NMJs co-labeled Myo15 (anti-Myo15C1, green) and GFP-tagged Myo15 (magenta), scale bar 2 µm. White line marks the plane of line profile. Lower panel shows the line profile. (M) Confocal images of salivary glands overexpressing Myo15 and control, stained for Myo15C1 (green), acetylated tubulin (blue); RFP (no antibody staining, magenta), scale bar overview 20 µm, zoom 5 µm. (N) Quantification of M. Myo15C1 mean pixel intensity (control 27.59 ± 2.36, n = 11; Myo15 OE 95.61 ± 10.44, n = 10). (O) Confocal images of salivary glands overexpressing Myo15 and control, stained for Myo15C2 (green), acetylated tubulin (blue); RFP (no antibody staining, magenta), scale bar overview 20 µm, zoom 5 µm. (P) Quantification of O: Myo15C2 mean pixel intensity (control 5.49 ± 0.43%, n = 9; Myo15 OE 52.07 ± 6.36%, n = 9). (Q) Confocal images (single optical slides) of wild-type NMJs sequentially stained for Myo15C1 with Fab1 goat–anti-rabbit Al488 (green) and Myo15C2 with Fab1 goat–anti-rabbit Al647 (magenta), for control (right panel) no primary Myo15C2 antibody was applied, scale bar 5 µm, white arrowheads mark overlapping signal of both antibodies. (R) Quantification of Q: Myo15C1/Myo15C2 mean pixel intensity (Myo15C1/Fab1Al488 100 ± 11.3%, n = 10; control 129 ± 13.82%, n = 9; Myo15C2/Fab1Al647 100 ± 9.47%, n = 10; control 35.63 ± 4.05%, n = 9). (S) STED imaging of wild-type NMJs stained for Myo15 (anti-Myo15C2, green) BRP (magenta), scale bar 1 µm, zoom 0.1 µm. White square marks zoom area, white dashed line in zoom marks the plane of line profile. Lower panel shows line profile, green anti-Myo15C2, magenta BRP. (T) Quantification of S. Number of Myo15 spots within 400 nm of AZ center, binned for small (area = 1–159 pixel2, total 227 AZs) with 7.58 ± 0.27 Myo15 dots, middle (160–215 pixel2, total 301 AZs) with 7.29 ± 0.29 Myo15 dots, big (<215 pixel2, total 305 AZs) with 7.32 ± 0.28 Myo15 dots (1 pixel = 18,9 nm2). (U) Quantification of S. K-neighbor analysis (k 1–5) determining Myo15 (aMyo15C2) dot distance from the AZ center (BRP ring center) comparing small (area = 1–159 pixel2, total 227 AZs), middle (160–215 pixel2, total 301 AZs), and big (<215 pixel2, total 305 AZs) AZs (1 pixel = 18,9 nm2). All data are provided as mean ± SEM. (C, F, H, J, and R)n represents NMJs with 1–2 NMJs/animal from five to six animals; N, P: n represents one salivary gland with 1/2 salivary glands per animal from five to six animals, T: n = image, with 1–2 boutons/image and 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. OE, overexpression. Source data are available for this figure: SourceData FS3.
Figure S3.

Myo15 localization at the NMJ and salivary gland and antibody verification. (A) Schematic representation of Drosophila class XV Myo15 indicating antibody position (Myo15C1 and Myo15C2). (B) Confocal images of control and Myo15-RNAi knock-down NMJs stained for Myo15 (anti-Myo15C2, green) and HRP (magenta), scale bar overview 5 µm. (C) Quantification of B: Myo15C2 mean pixel intensity (control 100.0 ± 7.82%, n = 9; Myo15-RNAi 67.4 ± 3.24%, n = 11). (D) Western blot of wild-type and myo15−/− mutant larvae for Myo15C2. (E) Confocal images of wild-type and myo15−/− mutant NMJs stained for Myo15 (anti-Myo15C1, green) and HRP (magenta), scale bar overview 5 µm, zoom 2 µm. (F) Quantification of E. Myo15C1 mean pixel intensity (wild type 100.0 ± 11.52%, n = 9; myo15−/− mutant 67.38 ± 3.79%, n = 13). (G) Confocal images of wild-type and myo15−/− mutant NMJs stained for Myo15 (anti-Myo15C2, green) and HRP (magenta), scale bar overview 5 µm, zoom 2 µm. (H) Quantification of G. Myo15C2 mean pixel intensity (wild type 100.0 ± 5.71%, n = 12; myo15−/− mutant 58.05 ± 8.54%, n = 9). (I) Confocal images of NMJs upon motoneuronal Myo15 overexpression and control, stained for Myo15C2 (green) and HRP (magenta), scale bar overview 5 µm. (J) Quantification of I: Myo15C2 mean pixel intensity (control 100.0 ± 4.89%, n = 12; Myo15 OE 282.4 ± 23.21%, n = 12). (K) Western blot of adult brains neuronally overexpressing Myo15 and control brains for Myo15C2. (L) Confocal images of control and Myo15GFP-OE NMJs co-labeled Myo15 (anti-Myo15C1, green) and GFP-tagged Myo15 (magenta), scale bar 2 µm. White line marks the plane of line profile. Lower panel shows the line profile. (M) Confocal images of salivary glands overexpressing Myo15 and control, stained for Myo15C1 (green), acetylated tubulin (blue); RFP (no antibody staining, magenta), scale bar overview 20 µm, zoom 5 µm. (N) Quantification of M. Myo15C1 mean pixel intensity (control 27.59 ± 2.36, n = 11; Myo15 OE 95.61 ± 10.44, n = 10). (O) Confocal images of salivary glands overexpressing Myo15 and control, stained for Myo15C2 (green), acetylated tubulin (blue); RFP (no antibody staining, magenta), scale bar overview 20 µm, zoom 5 µm. (P) Quantification of O: Myo15C2 mean pixel intensity (control 5.49 ± 0.43%, n = 9; Myo15 OE 52.07 ± 6.36%, n = 9). (Q) Confocal images (single optical slides) of wild-type NMJs sequentially stained for Myo15C1 with Fab1 goat–anti-rabbit Al488 (green) and Myo15C2 with Fab1 goat–anti-rabbit Al647 (magenta), for control (right panel) no primary Myo15C2 antibody was applied, scale bar 5 µm, white arrowheads mark overlapping signal of both antibodies. (R) Quantification of Q: Myo15C1/Myo15C2 mean pixel intensity (Myo15C1/Fab1Al488 100 ± 11.3%, n = 10; control 129 ± 13.82%, n = 9; Myo15C2/Fab1Al647 100 ± 9.47%, n = 10; control 35.63 ± 4.05%, n = 9). (S) STED imaging of wild-type NMJs stained for Myo15 (anti-Myo15C2, green) BRP (magenta), scale bar 1 µm, zoom 0.1 µm. White square marks zoom area, white dashed line in zoom marks the plane of line profile. Lower panel shows line profile, green anti-Myo15C2, magenta BRP. (T) Quantification of S. Number of Myo15 spots within 400 nm of AZ center, binned for small (area = 1–159 pixel2, total 227 AZs) with 7.58 ± 0.27 Myo15 dots, middle (160–215 pixel2, total 301 AZs) with 7.29 ± 0.29 Myo15 dots, big (<215 pixel2, total 305 AZs) with 7.32 ± 0.28 Myo15 dots (1 pixel = 18,9 nm2). (U) Quantification of S. K-neighbor analysis (k 1–5) determining Myo15 (aMyo15C2) dot distance from the AZ center (BRP ring center) comparing small (area = 1–159 pixel2, total 227 AZs), middle (160–215 pixel2, total 301 AZs), and big (<215 pixel2, total 305 AZs) AZs (1 pixel = 18,9 nm2). All data are provided as mean ± SEM. (C, F, H, J, and R)n represents NMJs with 1–2 NMJs/animal from five to six animals; N, P: n represents one salivary gland with 1/2 salivary glands per animal from five to six animals, T: n = image, with 1–2 boutons/image and 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. OE, overexpression. Source data are available for this figure: SourceData FS3.

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