Skip to main content
. 2022 Apr 29;8(17):eabj5716. doi: 10.1126/sciadv.abj5716

Fig. 1. TRPML1-mediated Ca2+ efflux selectively modulates retrograde transport of LEs/amphisomes.

Fig. 1.

(A to C) Time-lapse imaging in Ctrl axons ± ML-SA1 or Ned-19 treatment and PSEN1 KO axons expressing LAMP1-YFP and colabeled with LT-Red. Scale bars, 5 μm. (A) Representative still images (left side; proximal to soma). Closed arrowheads, retrograde; open arrowheads, nonmotile. (B) Corresponding kymographs (time = 135 s). Arrowheads, trajectories of marked vesicles in (A). (C) Quantitative analyses. Overall motility: % retrograde (retro), nonmotile (<0.1 μm/s), and anterograde (antero) vesicles in LAMP1 vesicle subpopulations. Bars = means + SEM (n = 29, 21, 20, and 29 neurons). Percent time in pause of LAMP1+ LT+ vesicles is shown by Tukey’s box-and-whisker plot (n = 462, 326, 293, and 380 vesicles). Pause frequency of LAMP1+ LT+ vesicles with at least one motile segment is shown by Tukey’s box-and-whisker plot (n = 328, 152, 195, and 184 vesicles). *P < 0.05 and **P < 0.01 against Ctrl, Kruskal-Wallis test with Dunn’s multiple comparisons. (D) Morphometric analysis of neurites in untreated (NoTx) or ML-SA1–treated Ctrl neurons and PSEN1 KO neurons by electron microscopy. Representative images of normal and dystrophic neurites. Graph shows number of dystrophic swellings per 1 mm of neurites analyzed. Bars = means + SEM (n = 57 to 59 images per group; Ctrl and PSEN1 KO data also shown in Fig. 7C). *P < 0.05 and **P < 0.01, Kruskal-Wallis test with Dunn’s multiple comparisons. (E and F) Time-lapse imaging in LAMP1-mCherry–expressing Ctrl and PSEN1 KO axons loaded with dextran–Alexa Fluor 647 (AF647) and dextran–Oregon Green 488 (OG488). (E) Representative kymographs. Closed arrowheads, retrograde; open arrowheads, nonmotile. (F) Quantitative analyses of the most acidic 10% of vesicles (LAMP1+ AF647+ OG488+/−) per group. Tukey’s box-and-whisker plots show their acidification (OG488:AF647 ratio) and average velocity (n = 37 vesicles per group; individual data points, see fig. S4B). *P < 0.05 and ***P < 0.001 against Ctrl, Mann-Whitney U test.