Myo15 partially co-localizes with and regulates actin at the synaptic terminal. (A) Single optical slide of confocal images of NMJs expressing actin5CGFP using the UAS–Gal4 expression system, stained for Myo15C1 (green) and GFP (actin, magenta), scale bar overview 2 µm, zoom 1 µm, white arrowheads mark overlapping Myo15 and actin signals. White square marks the zoom area, white line in zoom marks the plane of line profile. Right panel shows line profile, green Myo15C1, magenta actin5CGFP. (B) Quantification of A. Pearson’s R value of Myo15C1 and actin5CGFP (Pearson’s R above threshold 0.1159 ± 0.0211; inverted images −0.0124 ± 0.0236, n = 10). (C) Confocal images of NMJs expressing actin5CGFP, Myo15 (OE), and control stained for GFP (actin, green) and HRP (magenta), scale bar 5 µm. (D) Quantification of (C), actin mean pixel intensity at the NMJ (control 100.0 ± 9.06%, n = 10; Myo15 OE 136.9 ± 13.24%, n = 12). (E) Confocal images of NMJs expressing actin5CGFP, Myo15-RNAi, and control stained for GFP (actin, green) and HRP (magenta), white arrowheads mark actin accumulations in ectopic satellite boutons, scale bar 5 µm. (F) Quantification of E. actin mean pixel intensity at the NMJ (control 100.0 ± 7.88%, n = 10; Myo15-RNAi 105.5 ± 8.38%, n = 11). (G) STED imaging of NMJs expressing actin5CGFP (control) and Myo15-RNAi or Myo15 (OE) labeled for actin (green), white arrowheads mark actin accumulations in ectopic satellite boutons, scale bar 1 µm. (H–J) Quantification of G. (H) Single actin cluster area (control 100.0 ± 2.21%, n = 41; Myo15-RNAi 106.4 ± 2.51%, n = 35; Myo15 OE 136.9 ± 3.69%, n = 45), (I) actin cluster density as the number of actin clusters per bouton area (control 100.0 ± 6.81%, n = 37; Myo15-RNAi 124.5 ± 9.39%, n = 35; Myo15 OE 134.0 ± 8.63%, n = 47), (J) single actin cluster mean pixel intensity (control 100.0 ± 2.8%, n = 41; Myo15-RNAi 99.07 ± 3.41%, n = 37; Myo15 OE 103.1 ± 2.60%, n = 47). All data are provided as mean ± SEM. (D and F)n represents NMJs with 1–2 NMJs/animal from five to six animals, G: 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.
Figure 5.

Myo15 partially co-localizes with and regulates actin at the synaptic terminal. (A) Single optical slide of confocal images of NMJs expressing actin5CGFP using the UAS–Gal4 expression system, stained for Myo15C1 (green) and GFP (actin, magenta), scale bar overview 2 µm, zoom 1 µm, white arrowheads mark overlapping Myo15 and actin signals. White square marks the zoom area, white line in zoom marks the plane of line profile. Right panel shows line profile, green Myo15C1, magenta actin5CGFP. (B) Quantification of A. Pearson’s R value of Myo15C1 and actin5CGFP (Pearson’s R above threshold 0.1159 ± 0.0211; inverted images −0.0124 ± 0.0236, n = 10). (C) Confocal images of NMJs expressing actin5CGFP, Myo15 (OE), and control stained for GFP (actin, green) and HRP (magenta), scale bar 5 µm. (D) Quantification of (C), actin mean pixel intensity at the NMJ (control 100.0 ± 9.06%, n = 10; Myo15 OE 136.9 ± 13.24%, n = 12). (E) Confocal images of NMJs expressing actin5CGFP, Myo15-RNAi, and control stained for GFP (actin, green) and HRP (magenta), white arrowheads mark actin accumulations in ectopic satellite boutons, scale bar 5 µm. (F) Quantification of E. actin mean pixel intensity at the NMJ (control 100.0 ± 7.88%, n = 10; Myo15-RNAi 105.5 ± 8.38%, n = 11). (G) STED imaging of NMJs expressing actin5CGFP (control) and Myo15-RNAi or Myo15 (OE) labeled for actin (green), white arrowheads mark actin accumulations in ectopic satellite boutons, scale bar 1 µm. (H–J) Quantification of G. (H) Single actin cluster area (control 100.0 ± 2.21%, n = 41; Myo15-RNAi 106.4 ± 2.51%, n = 35; Myo15 OE 136.9 ± 3.69%, n = 45), (I) actin cluster density as the number of actin clusters per bouton area (control 100.0 ± 6.81%, n = 37; Myo15-RNAi 124.5 ± 9.39%, n = 35; Myo15 OE 134.0 ± 8.63%, n = 47), (J) single actin cluster mean pixel intensity (control 100.0 ± 2.8%, n = 41; Myo15-RNAi 99.07 ± 3.41%, n = 37; Myo15 OE 103.1 ± 2.60%, n = 47). All data are provided as mean ± SEM. (D and F)n represents NMJs with 1–2 NMJs/animal from five to six animals, G: 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.

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