Localized calcium release from lysosomes is required for cytoskeleton normal dynamics. (A and B) Hemocytes expressing LifeAct-GFP were cultured ex vivo for 1 h in control media or supplemented with chloroquine (CQ; 25 µM), after which media were changed to the same as the initial or containing thapsigargin (Tg; 1 µM), and recording began immediately. (A) Representative changes of cell area over time with respect to average (dashed line) in both control and trpml¹ hemocytes. Full videos are included in Video 7. (B) Actin dynamics quantified as SD of the area over time (n = 3 independent cultures, n ≥ 4 cells/condition, 30-min movies, 1-min intervals). Kruskal-Wallis test with Dunn's multiple comparisons post hoc test; statistically equivalent values are represented with the same letter (P < 0.05). (C) GCaMP6(m) calcium reporter was expressed in hemocytes and registered ex vivo to reveal intracellular calcium release upon the addition of thapsigargin (at minute 2). Values are normalized against the first time point (n = 3 independent cultures, n ≥ 6 cells/condition, 12-min movies, 30-s intervals). A.U., arbitrary units. Full video is included in Video 8.