Myo15−/−mutants show defects in NMJ morphology and AZ scaffold assembly, which are rescued by presynaptic restoration of Myo15 level. (A) Confocal images of overexpressed Myo15RFP and simultaneous RNAi-mediated Myo15-knock-down show RNAi efficacy and specificity, stained for RFP with aDsRed (green) and HRP (magenta), scale bar overview 5 µm. (B) Quantifications of RFP mean pixel intensity of A (control 0.0 ± 2,26%, n = 11; Myo15RFP (OE) 100 ± 4,98%, n = 12; Myo15RFP (OE) and Myo15-RNAi 35,61 ± 3,21, n = 13). (C) Locus of Myo15 deletion (Df(1)ΔMyo15), red squares mark P-Elements used for flip-out (see Materials and methods for details, image adapted from https://flybase.org). (D) Confocal images of myo15−/− mutant escapers and control NMJs stained for BRP (green) and HRP (magenta), scale bar overview 5 µm, zoom 2 µm, white arrowheads mark satellite boutons. (E–G) Quantifications of D. (E) AZ area (wild type 100.0 ± 3.20%, n = 12; myo15−/− mutant 88.29 ± 3.61%, n = 12). (F) NMJ length (wild type 100.0 ± 9.31%, n = 12; myo15−/− mutant 78.04 ± 6.04%, n = 11). (G) Total number of all AZs per NMJ (wild type 100.0 ± 4.45%, n = 12; myo15−/− mutant 118.7 ± 6.34%, n = 12). (H) Confocal images of control, myo15−/− mutant, and presynaptic rescue NMJs stained for BRP (green) and HRP (magenta), scale bar overview 5 µm, zoom 2 µm, white arrowheads mark satellite boutons. (I–M) Quantifications of H. (I) AZ area (wild type 100.0 ± 2.19%, n = 17; myo15−/− mutant 89.70 ± 1.88%, n = 19; rescue 100.10 ± 2.26, n = 19). (J) Relative frequency histogram of AZ areas (total number of AZs control 1279, myo15−/− mutant 1110, rescue 1237), bin width 0.02. (K) Density of AZs, as number of AZ per NMJ area (control 100.0 ± 2.25%, n = 17; myo15−/− mutant 110.9 ± 2.08%, n = 19, rescue 104.40 ± 2.15, n = 20). (L) BRP mean pixel intensity of NMJ (control 100.0 ± 3.67%, n = 16; myo15−/− mutant 108.80 ± 7.82%, n = 18, rescue 90.96 ± 6.29, n = 19). (M) NMJ length (control 100.0 ± 3.79%, n = 17; myo15−/− mutant 92.76 ± 4.49%, n = 19, rescue 91.16 ± 6.34, n = 20). All data are provided as mean ± SEM. For all quantifications, n represents NMJs with 1–3 NMJs/animal from five to seven animals. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. OE, overexpression.
Figure S1.

Myo15 −/− mutants show defects in NMJ morphology and AZ scaffold assembly, which are rescued by presynaptic restoration of Myo15 level. (A) Confocal images of overexpressed Myo15RFP and simultaneous RNAi-mediated Myo15-knock-down show RNAi efficacy and specificity, stained for RFP with aDsRed (green) and HRP (magenta), scale bar overview 5 µm. (B) Quantifications of RFP mean pixel intensity of A (control 0.0 ± 2,26%, n = 11; Myo15RFP (OE) 100 ± 4,98%, n = 12; Myo15RFP (OE) and Myo15-RNAi 35,61 ± 3,21, n = 13). (C) Locus of Myo15 deletion (Df(1)ΔMyo15), red squares mark P-Elements used for flip-out (see Materials and methods for details, image adapted from https://flybase.org). (D) Confocal images of myo15−/− mutant escapers and control NMJs stained for BRP (green) and HRP (magenta), scale bar overview 5 µm, zoom 2 µm, white arrowheads mark satellite boutons. (E–G) Quantifications of D. (E) AZ area (wild type 100.0 ± 3.20%, n = 12; myo15−/− mutant 88.29 ± 3.61%, n = 12). (F) NMJ length (wild type 100.0 ± 9.31%, n = 12; myo15−/− mutant 78.04 ± 6.04%, n = 11). (G) Total number of all AZs per NMJ (wild type 100.0 ± 4.45%, n = 12; myo15−/− mutant 118.7 ± 6.34%, n = 12). (H) Confocal images of control, myo15−/− mutant, and presynaptic rescue NMJs stained for BRP (green) and HRP (magenta), scale bar overview 5 µm, zoom 2 µm, white arrowheads mark satellite boutons. (I–M) Quantifications of H. (I) AZ area (wild type 100.0 ± 2.19%, n = 17; myo15−/− mutant 89.70 ± 1.88%, n = 19; rescue 100.10 ± 2.26, n = 19). (J) Relative frequency histogram of AZ areas (total number of AZs control 1279, myo15−/− mutant 1110, rescue 1237), bin width 0.02. (K) Density of AZs, as number of AZ per NMJ area (control 100.0 ± 2.25%, n = 17; myo15−/− mutant 110.9 ± 2.08%, n = 19, rescue 104.40 ± 2.15, n = 20). (L) BRP mean pixel intensity of NMJ (control 100.0 ± 3.67%, n = 16; myo15−/− mutant 108.80 ± 7.82%, n = 18, rescue 90.96 ± 6.29, n = 19). (M) NMJ length (control 100.0 ± 3.79%, n = 17; myo15−/− mutant 92.76 ± 4.49%, n = 19, rescue 91.16 ± 6.34, n = 20). All data are provided as mean ± SEM. For all quantifications, n represents NMJs with 1–3 NMJs/animal from five to seven animals. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. OE, overexpression.

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