Figure 4.
Migration-dependent autolysosome exocytosis could release sEVs and maintain lysosomal homeostasis in response to lysosomal damage. (A) TSPAN4-BFP stably expressing MEF cell line was treated with 3 mM LLOMe for 22 h and then stained with ALIX. The marked region was enlarged and demonstrated on the right. Scale bars denoted 20 μm for the large-view graph, 5 μm for the small-view graphs. (B) TSPAN4-BFP stably expressing MEF cell line was treated with 3 mM LLOMe for 22 h then stained with Syntenin1. The marked region was enlarged and demonstrated on the right. Scale bars denoted 20 μm for the large-view graph, 5 μm for the small-view graphs. (C) CLEM data of TSPAN4-BFP stably expressing MEF cell line treated with LLOMe for 22 h and stained with Syntenin1. The marked region in the fluorescent graph was enlarged and demonstrated on the right, stacked with the electron microscope graph. Scale bar was 10 μm for the large-view fluorescent graph, 5 μm for the small-view fluorescent graph. About electron microscope graphs, scale bars denoted 1 μm for graph 1, 500 nm for graph 2, and 200 nm for graph 3. (D) Control, Sec22b−/−, Syntaxin4−/−, and SNAP23−/− MEF cells stably expressing GFP-Galectin3 were seeded in fibronectin-coated confocal dishes. Then cells were treated with 2.5 mM LLOMe for 22 h and fixed. The marked regions in the large-view graphs were enlarged and demonstrated on the right. Scale bars denoted 20 μm for the large-view graphs, and 5 μm for the small-view graphs. (E) Statistic result of the normalized ratio of intracellular galectin3 puncta area in D. The measured puncta area was normalized to cell area then standardized to the control group (NC). Error bars denoted ± SEM from three independent fixed-cell imaging experiments, >50 cells were analyzed per experiment and >190 cells were analyzed in total for each group. T test, ****P < 0.0001. (F) TSPAN4-BFP stably expressing MEF cell line was seeded in fibronectin-coated, and none-coated confocal dishes. Then cells were treated with 2.5 mM LLOMe along with or without 2.5 μM GLPG0187 for 22 h and then stained with Cy5 and captured with live-cell mode. The marked regions in the large graphs were enlarged and demonstrated beneath. Scale bars denoted 20 μm for the large-view graphs, 5 μm for the small-view graphs. (G) Statistic result of the area of extracellular lysosomal structure indicated by Cy5 in F. Error bars denoted ± SEM from three independent live-cell imaging experiments, >30 cells were analyzed per experiment and >105 cells were analyzed in total. T test, ****P < 0.0001. (H) GFP-Galectin3 stably expressed MEF cell line was seeded in fibronectin-coated and none-coated confocal dishes. Then cells were treated with 2.5 mM LLOMe along with or without 2.5 μM GLPG0187 for 22 h and fixed. The marked regions in the large graphs were enlarged and demonstrated beneath. Scale bars denoted 20 μm for the large-view graphs, and 5 μm for the small-view graphs. (I) Statistic result of the normalized ratio of intracellular galectin3 puncta area in H. The counted puncta numbers were normalized to cell area then standardized to the control group (+FN). Error bars denoted ± SEM from three independent fixed-cell imaging experiments, >40 cells were analyzed per experiment and >130 cells were analyzed in total for each group. T test, **P < 0.01, ****P < 0.0001. (J) The schematic diagram of the process of MAD pathway. (i) Formation of autophagosomes after LLOMe treatment. (ii) Autolysosomes fused with the basal plasma membranes. Cargoes including damaged lysosomes and exosomes were released to the extracellular environment. (iii) Cells migrated far away and MAD cargoes released in process ii were left behind.

Migration-dependent autolysosome exocytosis could release sEVs and maintain lysosomal homeostasis in response to lysosomal damage. (A) TSPAN4-BFP stably expressing MEF cell line was treated with 3 mM LLOMe for 22 h and then stained with ALIX. The marked region was enlarged and demonstrated on the right. Scale bars denoted 20 μm for the large-view graph, 5 μm for the small-view graphs. (B) TSPAN4-BFP stably expressing MEF cell line was treated with 3 mM LLOMe for 22 h then stained with Syntenin1. The marked region was enlarged and demonstrated on the right. Scale bars denoted 20 μm for the large-view graph, 5 μm for the small-view graphs. (C) CLEM data of TSPAN4-BFP stably expressing MEF cell line treated with LLOMe for 22 h and stained with Syntenin1. The marked region in the fluorescent graph was enlarged and demonstrated on the right, stacked with the electron microscope graph. Scale bar was 10 μm for the large-view fluorescent graph, 5 μm for the small-view fluorescent graph. About electron microscope graphs, scale bars denoted 1 μm for graph 1, 500 nm for graph 2, and 200 nm for graph 3. (D) Control, Sec22b−/−, Syntaxin4−/−, and SNAP23−/− MEF cells stably expressing GFP-Galectin3 were seeded in fibronectin-coated confocal dishes. Then cells were treated with 2.5 mM LLOMe for 22 h and fixed. The marked regions in the large-view graphs were enlarged and demonstrated on the right. Scale bars denoted 20 μm for the large-view graphs, and 5 μm for the small-view graphs. (E) Statistic result of the normalized ratio of intracellular galectin3 puncta area in D. The measured puncta area was normalized to cell area then standardized to the control group (NC). Error bars denoted ± SEM from three independent fixed-cell imaging experiments, >50 cells were analyzed per experiment and >190 cells were analyzed in total for each group. T test, ****P < 0.0001. (F) TSPAN4-BFP stably expressing MEF cell line was seeded in fibronectin-coated, and none-coated confocal dishes. Then cells were treated with 2.5 mM LLOMe along with or without 2.5 μM GLPG0187 for 22 h and then stained with Cy5 and captured with live-cell mode. The marked regions in the large graphs were enlarged and demonstrated beneath. Scale bars denoted 20 μm for the large-view graphs, 5 μm for the small-view graphs. (G) Statistic result of the area of extracellular lysosomal structure indicated by Cy5 in F. Error bars denoted ± SEM from three independent live-cell imaging experiments, >30 cells were analyzed per experiment and >105 cells were analyzed in total. T test, ****P < 0.0001. (H) GFP-Galectin3 stably expressed MEF cell line was seeded in fibronectin-coated and none-coated confocal dishes. Then cells were treated with 2.5 mM LLOMe along with or without 2.5 μM GLPG0187 for 22 h and fixed. The marked regions in the large graphs were enlarged and demonstrated beneath. Scale bars denoted 20 μm for the large-view graphs, and 5 μm for the small-view graphs. (I) Statistic result of the normalized ratio of intracellular galectin3 puncta area in H. The counted puncta numbers were normalized to cell area then standardized to the control group (+FN). Error bars denoted ± SEM from three independent fixed-cell imaging experiments, >40 cells were analyzed per experiment and >130 cells were analyzed in total for each group. T test, **P < 0.01, ****P < 0.0001. (J) The schematic diagram of the process of MAD pathway. (i) Formation of autophagosomes after LLOMe treatment. (ii) Autolysosomes fused with the basal plasma membranes. Cargoes including damaged lysosomes and exosomes were released to the extracellular environment. (iii) Cells migrated far away and MAD cargoes released in process ii were left behind.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal