Figure 4.
Pls3 depletion results in morphological changes and aberrant TrkB localization and phosphorylation. (A) Control and Smn−/−;SMN2 growth cones stained against Pls3 (gray) and F-actin (Phalloidin, yellow). Normalized mean gray values of Pls3 (N = 3, n = 106; U-Mann–Whitney, ***P = 0.001). (B) Relative expression of Pls3 in whole-cell lysates of shLuci and shPls3 motoneurons. Quantification of Pls3 relative to Gapdh (N = 5, n = 8; two-tailed unpaired t test, ****P ≤ 0.0001). Bars represent the mean ± SD. (C and D) Pls3 knockdown results in (C) increased axon length (µm; N = 3; n = 400; shLuci: 443.1 ± 6.7 µm, shPls3: 532.9 ± 9.8 µm; U-Mann–Whitney, ****P ≤ 0.0001) and (D) decreased growth cone size (µm2; N = 8; n = 350; shLuci: 46.48 ± 1.52 µm2, shPls3: 26.63 ± 0.77 µm2; U-Mann–Whitney, ****P ≤ 0.0001). (E–G) shLuci and shPls3 growth cones with SiR-actin were monitored for 20 min. ROI (purple dotted line) of a single filopodia with the corresponding kymograph. Quantification of (E) the moved distance (amplitude in µm) of single filopodia (N = 5; n = 100; U-Mann–Whitney, **P = 0.0016) and (F) the velocity (distance [µm] over time [min]) of single filopodia (N = 5; n = 100; U-Mann–Whitney, ****P ≤ 0.0001). (H) shLuci and shPls3 growth cones stained against TrkB (gray) and Synaptophysin-1 (SYP, magenta). Normalized mean gray values of TrkB (N = 5, n = 115; U-Mann–Whitney, ***P = 0.0002). (I) shLuci and shPls3 growth cones unstimulated and 15 min BDNF stimulated stained against p-TrkB (gray) and F-actin (Phalloidin, yellow). Normalized mean gray values of p-TrkB (N = 3, n = 100; ANOVA Kruskal–Wallis, **P ≤ 0.01; ****P ≤ 0.0001). (J) shLuci and shPls3 growth cones stimulated and stained against surface TrkB (gray) and SYP (magenta). Normalized mean gray values of surface TrkB upon 15 min BDNF stimulation (N = 4, n = 90; ANOVA Kruskal–Wallis, ***P ≤ 0.001; ****P ≤ 0.0001) and 20 min 8-CPT-cAMP stimulation within axon terminals from shLuci and shPls3 motoneurons (N = 4, n = 95; ANOVA Kruskal–Wallis, **P ≤ 0.01; ****P ≤ 0.0001). (K) shLuci and shPls3 growth cones stained against Cav2.2 (gray) and F-actin (Phalloidin, yellow). Normalized mean gray values of Cav2.2 (N = 4, n = 100; U-Mann–Whitney, ****P ≤ 0.0001). (L) Maximum intensity projections of structured illumination microscopy (SIM) images of shLuci and shPls3 growth cones stained against Cav2.2 (cyan) and F-actin (Phalloidin, yellow). Arrows indicate “cluster-like” accumulations. (M) Representative plots of Cal-590 AM fluorescent changes over 1 min indicating spontaneous Ca2+ spikes of shLuci and shPls3 growth cones. Quantification of spontaneous Ca2+ spikes per minute (N = 5; n = 89; shLuci 4.782 ± 0.4410 vs. shPls3: 1.483 ± 0.2137; U-Mann–Whitney, ****P ≤ 0.0001). Bar represent the mean ± SEM. Data are presented as scatter dot plot with bar. Bar represent the mean ± SEM (except for B). Scale bars: 5 µm.

Pls3 depletion results in morphological changes and aberrant TrkB localization and phosphorylation. (A) Control and Smn−/−;SMN2 growth cones stained against Pls3 (gray) and F-actin (Phalloidin, yellow). Normalized mean gray values of Pls3 (N = 3, n = 106; U-Mann–Whitney, ***P = 0.001). (B) Relative expression of Pls3 in whole-cell lysates of shLuci and shPls3 motoneurons. Quantification of Pls3 relative to Gapdh (N = 5, n = 8; two-tailed unpaired t test, ****P ≤ 0.0001). Bars represent the mean ± SD. (C and D) Pls3 knockdown results in (C) increased axon length (µm; N = 3; n = 400; shLuci: 443.1 ± 6.7 µm, shPls3: 532.9 ± 9.8 µm; U-Mann–Whitney, ****P ≤ 0.0001) and (D) decreased growth cone size (µm2; N = 8; n = 350; shLuci: 46.48 ± 1.52 µm2, shPls3: 26.63 ± 0.77 µm2; U-Mann–Whitney, ****P ≤ 0.0001). (E–G) shLuci and shPls3 growth cones with SiR-actin were monitored for 20 min. ROI (purple dotted line) of a single filopodia with the corresponding kymograph. Quantification of (E) the moved distance (amplitude in µm) of single filopodia (N = 5; n = 100; U-Mann–Whitney, **P = 0.0016) and (F) the velocity (distance [µm] over time [min]) of single filopodia (N = 5; n = 100; U-Mann–Whitney, ****P ≤ 0.0001). (H) shLuci and shPls3 growth cones stained against TrkB (gray) and Synaptophysin-1 (SYP, magenta). Normalized mean gray values of TrkB (N = 5, n = 115; U-Mann–Whitney, ***P = 0.0002). (I) shLuci and shPls3 growth cones unstimulated and 15 min BDNF stimulated stained against p-TrkB (gray) and F-actin (Phalloidin, yellow). Normalized mean gray values of p-TrkB (N = 3, n = 100; ANOVA Kruskal–Wallis, **P ≤ 0.01; ****P ≤ 0.0001). (J) shLuci and shPls3 growth cones stimulated and stained against surface TrkB (gray) and SYP (magenta). Normalized mean gray values of surface TrkB upon 15 min BDNF stimulation (N = 4, n = 90; ANOVA Kruskal–Wallis, ***P ≤ 0.001; ****P ≤ 0.0001) and 20 min 8-CPT-cAMP stimulation within axon terminals from shLuci and shPls3 motoneurons (N = 4, n = 95; ANOVA Kruskal–Wallis, **P ≤ 0.01; ****P ≤ 0.0001). (K) shLuci and shPls3 growth cones stained against Cav2.2 (gray) and F-actin (Phalloidin, yellow). Normalized mean gray values of Cav2.2 (N = 4, n = 100; U-Mann–Whitney, ****P ≤ 0.0001). (L) Maximum intensity projections of structured illumination microscopy (SIM) images of shLuci and shPls3 growth cones stained against Cav2.2 (cyan) and F-actin (Phalloidin, yellow). Arrows indicate “cluster-like” accumulations. (M) Representative plots of Cal-590 AM fluorescent changes over 1 min indicating spontaneous Ca2+ spikes of shLuci and shPls3 growth cones. Quantification of spontaneous Ca2+ spikes per minute (N = 5; n = 89; shLuci 4.782 ± 0.4410 vs. shPls3: 1.483 ± 0.2137; U-Mann–Whitney, ****P ≤ 0.0001). Bar represent the mean ± SEM. Data are presented as scatter dot plot with bar. Bar represent the mean ± SEM (except for B). Scale bars: 5 µm.

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