Class XV Myo15 regulates NMJ morphology, presynaptic AZ scaffold size, and number. (A) Schematic representation of class XV Myo15 in D. melanogaster and human domain structure. (B) Confocal images of RNAi-mediated Myo15-knock-down (UAS–Gal4 system using ok6-Gal4 driver) and control NMJs (driver control) stained for BRP (green) and HRP (magenta), scale bar overview 5 µm, zoom 2 µm, white arrowheads mark ectopic satellite boutons. (C–I) Quantifications of B. (C) AZ area (control 100.0 ± 3.63% n = 14; Myo15-RNAi 77.31 ± 2.06%, n = 16). (D) Relative frequency histogram of AZ areas (total number of AZs control 3034 and Myo15-RNAi 4738), bin width 0.02. (E) Density of AZs, as number of AZ per NMJ area (control 100.0 ± 4.77% n = 14; Myo15-RNAi 125.1 ± 4.38%, n = 16). (F) BRP mean pixel intensity of single AZs (control 100.0 ± 4.64% n = 14; Myo15-RNAi 104.8 ± 3.04%, n = 16). (G) NMJ length (control 100.0 ± 7.37% n = 15; Myo15-RNAi 101.6 ± 6.40%, n = 15). (H) Total number of all AZs per NMJ (control 100.0 ± 3.92% n = 14; Myo15-RNAi 132.9 ± 5.77%, n = 16). (I) Total BRP intensity as integral of the BRP mean pixel intensity over the entire NMJ (control 100.0 ± 6.68% n = 14; Myo15-RNAi 123.6 ± 7.27%, n = 16). (J) Confocal images of gRNA (CRISPR/Cas9)-mediated knock-down of Myo15 and control NMJs stained for BRP as AZ marker (green) and HRP outlining the synaptic terminal (magenta), scale bar overview 5 µm, zoom 2 µm, white arrowheads mark satellite boutons. (K–Q) Quantification of J. (K) AZ area (control 100.0 ± 3.75% n = 12; Myo15gRNA 71.09 ± 1.94%, n = 12). (L) Relative frequency histogram of AZ areas (total number of AZs: control 3026 and Myo15gRNA 5265), bin width 0.02. (M) Density of AZs, as number of AZ per NMJ area (control 100.0 ± 2.57%, n = 12; Myo15gRNA 121.1 ± 3.27%, n = 12). (N) BRP mean pixel intensity of single AZs (control 100.0 ± 4.59%, n = 12; Myo15gRNA 92.90 ± 3.52%, n = 12). (O) NMJ length (control 100.0 ± 11.95%, n = 12; Myo15gRNA 101.1 ± 12.25%, n = 12). (P) Total number of all AZs per NMJ (control 100.0 ± 4.81%, n = 12; Myo15gRNA 171.0 ± 5.11%, n = 12). (Q) Total BRP intensity as integral of the BRP mean pixel intensity over the entire NMJ (control 100.0 ± 7.02%, n = 12; Myo15gRNA 123.9 ± 5.11%, n = 12). (R) STED imaging of AZs labeled with BRP (green) upon RNAi-mediated Myo15-knock-down (scale bar 0.5 µm). (S–U) Quantification of R. (S) AZ area (control 100.0 ± 2.9%, n = 22; Myo15-RNAi 81.01 ± 2.04%, n = 24), (T) relative frequency histogram of AZ areas (total number of AZs: control 520 and Myo15-RNAi 810), bin width 0.007, (U) density of AZs, as number of AZ per NMJ area (control 100.0 ± 4.79%, n = 22; Myo15-RNAi 151.7 ± 7.57%, n = 24). All data are provided as mean ± SEM. (C–I and K–Q)n represents NMJs with 1–2 NMJs/animal from five to six animals, R: n = image, with 1–2 boutons/image and two to three images/NMJ, 2 NMJs/animal, three to four animals in total. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. SH3, Src homology 3.
Figure 1.

Class XV Myo15 regulates NMJ morphology, presynaptic AZ scaffold size, and number. (A) Schematic representation of class XV Myo15 in D. melanogaster and human domain structure. (B) Confocal images of RNAi-mediated Myo15-knock-down (UAS–Gal4 system using ok6-Gal4 driver) and control NMJs (driver control) stained for BRP (green) and HRP (magenta), scale bar overview 5 µm, zoom 2 µm, white arrowheads mark ectopic satellite boutons. (C–I) Quantifications of B. (C) AZ area (control 100.0 ± 3.63% n = 14; Myo15-RNAi 77.31 ± 2.06%, n = 16). (D) Relative frequency histogram of AZ areas (total number of AZs control 3034 and Myo15-RNAi 4738), bin width 0.02. (E) Density of AZs, as number of AZ per NMJ area (control 100.0 ± 4.77% n = 14; Myo15-RNAi 125.1 ± 4.38%, n = 16). (F) BRP mean pixel intensity of single AZs (control 100.0 ± 4.64% n = 14; Myo15-RNAi 104.8 ± 3.04%, n = 16). (G) NMJ length (control 100.0 ± 7.37% n = 15; Myo15-RNAi 101.6 ± 6.40%, n = 15). (H) Total number of all AZs per NMJ (control 100.0 ± 3.92% n = 14; Myo15-RNAi 132.9 ± 5.77%, n = 16). (I) Total BRP intensity as integral of the BRP mean pixel intensity over the entire NMJ (control 100.0 ± 6.68% n = 14; Myo15-RNAi 123.6 ± 7.27%, n = 16). (J) Confocal images of gRNA (CRISPR/Cas9)-mediated knock-down of Myo15 and control NMJs stained for BRP as AZ marker (green) and HRP outlining the synaptic terminal (magenta), scale bar overview 5 µm, zoom 2 µm, white arrowheads mark satellite boutons. (K–Q) Quantification of J. (K) AZ area (control 100.0 ± 3.75% n = 12; Myo15gRNA 71.09 ± 1.94%, n = 12). (L) Relative frequency histogram of AZ areas (total number of AZs: control 3026 and Myo15gRNA 5265), bin width 0.02. (M) Density of AZs, as number of AZ per NMJ area (control 100.0 ± 2.57%, n = 12; Myo15gRNA 121.1 ± 3.27%, n = 12). (N) BRP mean pixel intensity of single AZs (control 100.0 ± 4.59%, n = 12; Myo15gRNA 92.90 ± 3.52%, n = 12). (O) NMJ length (control 100.0 ± 11.95%, n = 12; Myo15gRNA 101.1 ± 12.25%, n = 12). (P) Total number of all AZs per NMJ (control 100.0 ± 4.81%, n = 12; Myo15gRNA 171.0 ± 5.11%, n = 12). (Q) Total BRP intensity as integral of the BRP mean pixel intensity over the entire NMJ (control 100.0 ± 7.02%, n = 12; Myo15gRNA 123.9 ± 5.11%, n = 12). (R) STED imaging of AZs labeled with BRP (green) upon RNAi-mediated Myo15-knock-down (scale bar 0.5 µm). (S–U) Quantification of R. (S) AZ area (control 100.0 ± 2.9%, n = 22; Myo15-RNAi 81.01 ± 2.04%, n = 24), (T) relative frequency histogram of AZ areas (total number of AZs: control 520 and Myo15-RNAi 810), bin width 0.007, (U) density of AZs, as number of AZ per NMJ area (control 100.0 ± 4.79%, n = 22; Myo15-RNAi 151.7 ± 7.57%, n = 24). All data are provided as mean ± SEM. (C–I and K–Q)n represents NMJs with 1–2 NMJs/animal from five to six animals, R: n = image, with 1–2 boutons/image and two to three images/NMJ, 2 NMJs/animal, three to four animals in total. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. SH3, Src homology 3.

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