MyTH4 (1) and FERM domain deletion mutants and Myo15 localization to presynaptic MTs. (A) Quantification of Myo15C2 protein level in deletion mutants: mean pixel intensity wild type 100.0 ± 2.57%, n = 8; Myo15ΔFERM 105.6 ± 8.06%, n = 12, Myo15ΔMyTH4 (1) 96.78 ± 4.55%, n = 12, Myo15ΔMyTH4 (1+2) 91.63 ± 5.58%, n = 13). (B) Confocal images of Myo15ΔFERM deletion mutant and wild-type NMJs stained for BRP (green) and HRP (magenta), scale bar overview 5 µm, zoom 2 µm. (C–I) Quantification of B. (C) AZ area (control 100.0 ± 7.48% n = 10; Myo15ΔFERM 87.43 ± 5.61%, n = 11). (D) Relative frequency histogram of AZ areas (total number of AZs: control 2675 and Myo15ΔFERM 3296), bin width 0.02. (E) Density of AZs, as number of AZ per NMJ area (control 100.0 ± 2.37% n = 10; Myo15ΔFERM 101.1 ± 5.35%, n = 11). (F) BRP mean pixel intensity of single AZs (control 100.0 ± 4.83% n = 10; Myo15ΔFERM 103.1 ± 6.15%, n = 11). (G) NMJ area (control 100.0 ± 2.84% n = 10; Myo15ΔFERM 112.8 ± 5.31%, n = 11). (H) Total number of all AZs per NMJ (control 100.0 ± 3.86% n = 10; Myo15ΔFERM 112.0 ± 4.90%, n = 11). (I) Total BRP intensity as integral of the BRP mean pixel intensity over the entire NMJ (control 100.0 ± 5.56% n = 10; Myo15ΔFERM 98.93 ± 4.94%, n = 11). (J) Confocal images of Myo15ΔMyTH4 (1) deletion mutant and wild-type NMJs stained for BRP (green) and HRP (magenta), scale bar overview 5 µm, zoom 2 µm. (K–Q) Quantification of J. (K) AZ area (control 100.0 ± 1.86% n = 12; Myo15ΔMyTH4 (1) 90.21 ± 2.86%, n = 11). (L) Relative frequency histogram of AZ areas (total number of AZs control 4038 and Myo15DMyTH4 (1) 3928), bin width 0.02. (M) Density of AZs, as number of AZ per NMJ area (control 100.0 ± 2.55% n = 12; Myo15ΔMyTH4 (1) 108.5 ± 5.02%, n = 11). (N) BRP mean pixel intensity of single AZs (control 100.0 ± 6.49% n = 12; Myo15ΔMyTH4 (1) 102.9 ± 7.54%, n = 11). (O) NMJ area (control 100.0 ± 5.95% n = 12; Myo15ΔMyTH4 (1) 100.2 ± 7.90%, n = 11). (P) Total number of all AZs per NMJ (control 100.0 ± 5.79% n = 12; Myo15ΔMyTH4 (1) 106.1 ± 5.70%, n = 11). (Q) Total BRP intensity as integral of the BRP mean pixel intensity over the entire NMJ (control 100.0 ± 8.20% n = 12; Myo15ΔMyTH4 (1) 100.8 ± 9.17%, n = 11). (R) Confocal images of Myo15ΔMyTH4 (1+2)/Δmyo15 trans-heterozygous mutant and wild-type NMJs stained for BRP (green) and HRP (magenta), scale bar overview 5 µm, zoom 2 µm. (S–V) Quantification of R. (S) AZ area (control 100.0 ± 3.90% n = 11; Myo15ΔMyTH4 (1+2)/Δmyo15 89.71 ± 2.17%, n = 12). (T) Density of AZs, as number of AZ per NMJ area (control 100.0 ± 5.45% n = 11; Myo15ΔMyTH4 (1+2)/Δmyo15 120.4 ± 2.82%, n = 12). (U) BRP mean pixel intensity of single AZs (control 100.0 ± 15.98% n = 11; Myo15ΔMyTH4 (1+2)/Δmyo15 95.75 ± 3.85%, n = 11). (V) NMJ area (control 100.0 ± 6.61% n = 11; Myo15ΔMyTH4 (1+2)/Δmyo15 99.32 ± 4.6%, n = 11). (W and X) Confocal images of wild-type muscles stained for (W) Myo15C1 (green) or (X) Myo15C2 (green) and acetylated tubulin (magenta), white arrowheads mark Myo15 spots localizing to MTs, scale bar 2 µm. (Y) STED images of wild-type and Myo15ΔMyTH4 (1+2) deletion mutant stained for Myo15C2 (green), MTs (acetylated tubulin, magenta), scale bar overview 0.5 µm, zoom 0.2 µm. White square mark zoom area. (Z) Quantification of Y. 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 25/28 (wild type/mutant) images are shown. (a) STED imaging of wild-type NMJs stained for Myo15C2 (green), MTs (futsch, blue), BRP (magenta), bouton scale bar 1 µm, zoom 0.1 µm. White square indicates zoom region, white dashed line in zoom marks the plane of line profile. Lower panel shows line profile, green Myo15C2, blue futsch, magenta BRP. All data are provided as mean ± SEM. (A, C–I, K–Q, and S–V)n = NMJs with 1–2 NMJs/animal, four to six animals, Z: 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 S4.

MyTH4 (1) and FERM domain deletion mutants and Myo15 localization to presynaptic MTs. (A) Quantification of Myo15C2 protein level in deletion mutants: mean pixel intensity wild type 100.0 ± 2.57%, n = 8; Myo15ΔFERM 105.6 ± 8.06%, n = 12, Myo15ΔMyTH4 (1) 96.78 ± 4.55%, n = 12, Myo15ΔMyTH4 (1+2) 91.63 ± 5.58%, n = 13). (B) Confocal images of Myo15ΔFERM deletion mutant and wild-type NMJs stained for BRP (green) and HRP (magenta), scale bar overview 5 µm, zoom 2 µm. (C–I) Quantification of B. (C) AZ area (control 100.0 ± 7.48% n = 10; Myo15ΔFERM 87.43 ± 5.61%, n = 11). (D) Relative frequency histogram of AZ areas (total number of AZs: control 2675 and Myo15ΔFERM 3296), bin width 0.02. (E) Density of AZs, as number of AZ per NMJ area (control 100.0 ± 2.37% n = 10; Myo15ΔFERM 101.1 ± 5.35%, n = 11). (F) BRP mean pixel intensity of single AZs (control 100.0 ± 4.83% n = 10; Myo15ΔFERM 103.1 ± 6.15%, n = 11). (G) NMJ area (control 100.0 ± 2.84% n = 10; Myo15ΔFERM 112.8 ± 5.31%, n = 11). (H) Total number of all AZs per NMJ (control 100.0 ± 3.86% n = 10; Myo15ΔFERM 112.0 ± 4.90%, n = 11). (I) Total BRP intensity as integral of the BRP mean pixel intensity over the entire NMJ (control 100.0 ± 5.56% n = 10; Myo15ΔFERM 98.93 ± 4.94%, n = 11). (J) Confocal images of Myo15ΔMyTH4 (1) deletion mutant and wild-type NMJs stained for BRP (green) and HRP (magenta), scale bar overview 5 µm, zoom 2 µm. (K–Q) Quantification of J. (K) AZ area (control 100.0 ± 1.86% n = 12; Myo15ΔMyTH4 (1) 90.21 ± 2.86%, n = 11). (L) Relative frequency histogram of AZ areas (total number of AZs control 4038 and Myo15DMyTH4 (1) 3928), bin width 0.02. (M) Density of AZs, as number of AZ per NMJ area (control 100.0 ± 2.55% n = 12; Myo15ΔMyTH4 (1) 108.5 ± 5.02%, n = 11). (N) BRP mean pixel intensity of single AZs (control 100.0 ± 6.49% n = 12; Myo15ΔMyTH4 (1) 102.9 ± 7.54%, n = 11). (O) NMJ area (control 100.0 ± 5.95% n = 12; Myo15ΔMyTH4 (1) 100.2 ± 7.90%, n = 11). (P) Total number of all AZs per NMJ (control 100.0 ± 5.79% n = 12; Myo15ΔMyTH4 (1) 106.1 ± 5.70%, n = 11). (Q) Total BRP intensity as integral of the BRP mean pixel intensity over the entire NMJ (control 100.0 ± 8.20% n = 12; Myo15ΔMyTH4 (1) 100.8 ± 9.17%, n = 11). (R) Confocal images of Myo15ΔMyTH4 (1+2)/Δmyo15 trans-heterozygous mutant and wild-type NMJs stained for BRP (green) and HRP (magenta), scale bar overview 5 µm, zoom 2 µm. (S–V) Quantification of R. (S) AZ area (control 100.0 ± 3.90% n = 11; Myo15ΔMyTH4 (1+2)/Δmyo15 89.71 ± 2.17%, n = 12). (T) Density of AZs, as number of AZ per NMJ area (control 100.0 ± 5.45% n = 11; Myo15ΔMyTH4 (1+2)/Δmyo15 120.4 ± 2.82%, n = 12). (U) BRP mean pixel intensity of single AZs (control 100.0 ± 15.98% n = 11; Myo15ΔMyTH4 (1+2)/Δmyo15 95.75 ± 3.85%, n = 11). (V) NMJ area (control 100.0 ± 6.61% n = 11; Myo15ΔMyTH4 (1+2)/Δmyo15 99.32 ± 4.6%, n = 11). (W and X) Confocal images of wild-type muscles stained for (W) Myo15C1 (green) or (X) Myo15C2 (green) and acetylated tubulin (magenta), white arrowheads mark Myo15 spots localizing to MTs, scale bar 2 µm. (Y) STED images of wild-type and Myo15ΔMyTH4 (1+2) deletion mutant stained for Myo15C2 (green), MTs (acetylated tubulin, magenta), scale bar overview 0.5 µm, zoom 0.2 µm. White square mark zoom area. (Z) Quantification of Y. 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 25/28 (wild type/mutant) images are shown. (a) STED imaging of wild-type NMJs stained for Myo15C2 (green), MTs (futsch, blue), BRP (magenta), bouton scale bar 1 µm, zoom 0.1 µm. White square indicates zoom region, white dashed line in zoom marks the plane of line profile. Lower panel shows line profile, green Myo15C2, blue futsch, magenta BRP. All data are provided as mean ± SEM. (A, C–I, K–Q, and S–V)n = NMJs with 1–2 NMJs/animal, four to six animals, Z: 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.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal