Figure 2.
Fusion between LAMP1-positive compartments and the basal membrane mediates the extracellular release of LAMP1-positive structures. (A) CLEM data of TSPAN4-mCherry stably expressing MEF treated with 2 mM LLOMe for 22 h then stained by Cy5. The marked region in the fluorescent graph was enlarged and demonstrated on the right. Scale bars denoted 5 μm for the large-view fluorescent graphs, 1 μm for both the small-view fluorescent graph and large-view electron microscope graph, and 500 nm for other electron microscope graphs (1–3). (B) TSPAN4-BFP MEF stable cell line was treated with or without LLOMe for 22 h and then stained by Cy5 and observed by confocal microscopy. The marked regions in the fluorescent graphs were enlarged and demonstrated on the right. Scale bars denoted 20 μm for the large-view graphs, 5 μm for the small-view graphs. (C) Time-lapse imaging of LAMP1-mCherry stably expressing MEF cells treated with 2 mM LLOMe and stained with Cy5. Cells had been treated about 7 h before the first frame was captured. The last frame was captured almost 12 h later. The marked regions were enlarged and demonstrated on the right. Scale bars denoted 20 μm for the large-view graphs, 2 μm for the small-view graphs. The complete process could be observed in Video 2. (D) Time-lapse imaging of MEF cells treated with 2 mM LLOMe, stained with Cy5 and observed by TIRF microscope. The first frame was captured about 7 h after the addition of LLOMe. The last frame was captured nearly 11 h later. The marked regions were enlarged and demonstrated on the right. Scale bars denoted 20 μm for the large-view graphs, 2 μm for the small-view graphs. The complete process can be observed in Video 3. (E) The sketch diagram of the structure of pHluorin-LAMP1-mCherry protein. The protein was started with a signal peptide sequence of bovine preprolactin at N-terminal, followed by pH-sensitive GFP protein pHluorin. Thus pHluorin should be located at the lysosomal lumen side. The transmembrane fragment sequence of mouse LAMP1 was inserted to ensure the lysosomal membrane localization of the protein. MCherry was at the C-terminal, meaning the protein would be located at the cytosolic side of the lysosomal membrane. (F) Time-lapse imaging of pHluorin-LAMP1 TM-mCherry stably expressing MEF cells treated without or with 2 mM LLOMe, and both stained with Cy5. The first frame was captured about 13 h after addition of LLOMe. Time interval was 12 min. The large-view image was captured at the last frame. The marked regions were enlarged and demonstrated on the right. Scale bars denoted 20 μm for the large-view graphs, 2 μm for the small-view graphs. The complete processes could be observed in Video 4 (Control) and Video 5 (LLOMe). (G) A cartoon diagram showing the pHluorin-LAMP1 TM-mCherry fusion assay, illustrating the phenomenon observed in F. The intact intracellular autolysosome containing damaged lysosomes would show the color of mCherry in the cytosol. Then the autolysosome would turn yellow, the merged color of both mCherry and pHluorin proteins due to the fusion process. The damaged lysosomes coming from the autolysosomal lumen would turn bright blue because these lysosomal structures could be specifically marked with Cy5 in the extracellular environment.

Fusion between LAMP1-positive compartments and the basal membrane mediates the extracellular release of LAMP1-positive structures. (A) CLEM data of TSPAN4-mCherry stably expressing MEF treated with 2 mM LLOMe for 22 h then stained by Cy5. The marked region in the fluorescent graph was enlarged and demonstrated on the right. Scale bars denoted 5 μm for the large-view fluorescent graphs, 1 μm for both the small-view fluorescent graph and large-view electron microscope graph, and 500 nm for other electron microscope graphs (1–3). (B) TSPAN4-BFP MEF stable cell line was treated with or without LLOMe for 22 h and then stained by Cy5 and observed by confocal microscopy. The marked regions in the fluorescent graphs were enlarged and demonstrated on the right. Scale bars denoted 20 μm for the large-view graphs, 5 μm for the small-view graphs. (C) Time-lapse imaging of LAMP1-mCherry stably expressing MEF cells treated with 2 mM LLOMe and stained with Cy5. Cells had been treated about 7 h before the first frame was captured. The last frame was captured almost 12 h later. The marked regions were enlarged and demonstrated on the right. Scale bars denoted 20 μm for the large-view graphs, 2 μm for the small-view graphs. The complete process could be observed in Video 2. (D) Time-lapse imaging of MEF cells treated with 2 mM LLOMe, stained with Cy5 and observed by TIRF microscope. The first frame was captured about 7 h after the addition of LLOMe. The last frame was captured nearly 11 h later. The marked regions were enlarged and demonstrated on the right. Scale bars denoted 20 μm for the large-view graphs, 2 μm for the small-view graphs. The complete process can be observed in Video 3. (E) The sketch diagram of the structure of pHluorin-LAMP1-mCherry protein. The protein was started with a signal peptide sequence of bovine preprolactin at N-terminal, followed by pH-sensitive GFP protein pHluorin. Thus pHluorin should be located at the lysosomal lumen side. The transmembrane fragment sequence of mouse LAMP1 was inserted to ensure the lysosomal membrane localization of the protein. MCherry was at the C-terminal, meaning the protein would be located at the cytosolic side of the lysosomal membrane. (F) Time-lapse imaging of pHluorin-LAMP1 TM-mCherry stably expressing MEF cells treated without or with 2 mM LLOMe, and both stained with Cy5. The first frame was captured about 13 h after addition of LLOMe. Time interval was 12 min. The large-view image was captured at the last frame. The marked regions were enlarged and demonstrated on the right. Scale bars denoted 20 μm for the large-view graphs, 2 μm for the small-view graphs. The complete processes could be observed in Video 4 (Control) and Video 5 (LLOMe). (G) A cartoon diagram showing the pHluorin-LAMP1 TM-mCherry fusion assay, illustrating the phenomenon observed in F. The intact intracellular autolysosome containing damaged lysosomes would show the color of mCherry in the cytosol. Then the autolysosome would turn yellow, the merged color of both mCherry and pHluorin proteins due to the fusion process. The damaged lysosomes coming from the autolysosomal lumen would turn bright blue because these lysosomal structures could be specifically marked with Cy5 in the extracellular environment.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal