Figure 4.
Figure 4. Spastin and IST1 regulate lysosomal enzyme traffic and lysosome morphology. (A) HeLa cells were subjected to spastin or IST1 KD, then protein precipitated from collected culture medium was immunoblotted for pro–cathepsin D (Pro-CatD). Coomassie staining validates equal loading. Blots were quantified, and the mean ± SEM fold-change in Pro-CatD secretion was plotted (n = 5). (B) HeLa cells were subjected to spastin KD, fixed, and labeled for LAMP1, then the diameter of the largest lysosome was measured in 100 cells/condition. The mean ± SEM percentage of cells with largest lysosome >1.8 µm and mean diameter of the largest lysosome are shown (n = 4). (C) HeLa cells were subjected to spastin KD and incubated in culture medium with dextran-Oregon Green (DexOG; fluorescence is acid quenched) or dextran-tetramethylrhodamine (DexRhod; fluorescence is pH insensitive) for a 4-h pulse, which was chased into the terminal degradative compartment for 20 h with dextran-free medium. Living cells were then imaged, and the ratio of red to green pixels in 300 labeled puncta was measured and used to calculate pH, as described in Materials and methods. In the image, the differential interference contrast channel is shown to allow definition of the cell boundaries. The pH distribution of puncta from a single experiment is shown in the corresponding histograms. (D) Top, EM of HeLa cells subjected to spastin or IST1 KD, showing abnormal endolysosomal structures with abnormal dense membrane (arrowheads). Bottom, EM of MEFs from spastinN384K mice with genotypes indicated. Abnormal lysosomes indicated by arrowheads. (E) EM after BSA-gold uptake in mock-transfected or spastin-depleted HeLa cells. Right panels show the boxed areas indicated in the left panels; gold indicated by arrowheads. (F and G) Wild-type HeLa cells or HeLa cells stably expressing the siRNA-resistant spastin proteins indicated were subjected to endogenous spastin KD, fixed, and labeled for LAMP1. Mean ± SEM percentage of cells with largest lysosome >1.8 µm is plotted (n = 6, 100 cells/condition in each). All p-values generated by two-tailed Student’s t test, except C, in which differences in pH were analyzed using a two-tailed Mann–Whitney U test. Bars in light micrographs: (main image) 10 µm; (magnified insets) 5 µm. Bars in EM: 500 nm; (magnified images in E) 250 nm.

Spastin and IST1 regulate lysosomal enzyme traffic and lysosome morphology. (A) HeLa cells were subjected to spastin or IST1 KD, then protein precipitated from collected culture medium was immunoblotted for pro–cathepsin D (Pro-CatD). Coomassie staining validates equal loading. Blots were quantified, and the mean ± SEM fold-change in Pro-CatD secretion was plotted (n = 5). (B) HeLa cells were subjected to spastin KD, fixed, and labeled for LAMP1, then the diameter of the largest lysosome was measured in 100 cells/condition. The mean ± SEM percentage of cells with largest lysosome >1.8 µm and mean diameter of the largest lysosome are shown (n = 4). (C) HeLa cells were subjected to spastin KD and incubated in culture medium with dextran-Oregon Green (DexOG; fluorescence is acid quenched) or dextran-tetramethylrhodamine (DexRhod; fluorescence is pH insensitive) for a 4-h pulse, which was chased into the terminal degradative compartment for 20 h with dextran-free medium. Living cells were then imaged, and the ratio of red to green pixels in 300 labeled puncta was measured and used to calculate pH, as described in Materials and methods. In the image, the differential interference contrast channel is shown to allow definition of the cell boundaries. The pH distribution of puncta from a single experiment is shown in the corresponding histograms. (D) Top, EM of HeLa cells subjected to spastin or IST1 KD, showing abnormal endolysosomal structures with abnormal dense membrane (arrowheads). Bottom, EM of MEFs from spastinN384K mice with genotypes indicated. Abnormal lysosomes indicated by arrowheads. (E) EM after BSA-gold uptake in mock-transfected or spastin-depleted HeLa cells. Right panels show the boxed areas indicated in the left panels; gold indicated by arrowheads. (F and G) Wild-type HeLa cells or HeLa cells stably expressing the siRNA-resistant spastin proteins indicated were subjected to endogenous spastin KD, fixed, and labeled for LAMP1. Mean ± SEM percentage of cells with largest lysosome >1.8 µm is plotted (n = 6, 100 cells/condition in each). All p-values generated by two-tailed Student’s t test, except C, in which differences in pH were analyzed using a two-tailed Mann–Whitney U test. Bars in light micrographs: (main image) 10 µm; (magnified insets) 5 µm. Bars in EM: 500 nm; (magnified images in E) 250 nm.

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