Figure S4.
Relationship between lysosome movement and focal adhesions.(A) Representative results of live cell imaging with Lysotracker recorded using wide-field epifluorescent microscopy. Top: z-projection of 3-min time series (maximum intensity). Images are inverted to get a better contrast. Bottom: Kymographs represent the lysosome traffic along the magenta segmented line indicated on the top panel. The direction of lysosome traffic was color-coded. Lysosomes that underwent anterograde movement were coded in yellow, retrograde movement in cyan, and static lysosomes in magenta. White arrows indicate stable lysosomes near the WT cell surface, which are not found in MYO18B-KO cells. Related to Fig. 6. (B) Left: Representative confocal microscopic images of WT and MYO18B-KO HeLa cells stained with LAMP1 and vinculin. Bars, 10 µm in original images and 3 µm in magnified images. Right: fluorescent intensity of LAMP1 and vinculin along the white line in the magnified images on the left. Related to Fig. 6. (C) Top: Representative CLSM images of WT and MYO18B-KO U2OS cells co-stained with vinculin and LAMP1. Bars, 10 µm. Bottom: Quantitative analysis of lysosome size in WT and MYO18B-KO U2OS cells (mean ± SD; n = 10 images; N = 2 biological replicates; unpaired two-tailed t test; ***P ≤ 0.001). Related to Fig. 6. (D) Top: Representative CLSM images of U2OS cells grown on non-coated or poly-L-lysine coated cover glass stained with vinculin and LAMP1 (x-y view) Bars, 10 µm. Bottom: The x-z view at the dotted line in the x-y view. The white arrows indicate the lysosomes distributed at the top of nuclei in cells grown on poly-L-lysine coated cover glass. A total of 13 z-series were captured at 0.365 intervals and the x-z view was reconstituted using re-slice tool of ImageJ. The spatial ratio of x-z view was kept unchanged during processing. Related to Fig. 6. (E) Top: Representative CLSM images of U2OS cells grown on non-coated or poly-L-lysine coated cover glass stained with LAMP1 under unpermeabilized conditions. Bars, 10 µm. Bottom: Quantitative analysis of cell-surface LAMP1 level in U2OS cells grown on non-coated or poly-L-lysine coated cover glass. The mean ± SD fluorescent intensity of non-coated group was set to unity (n = 20 images; N = 4 biological replicates; unpaired two-tailed t test; ***P ≤ 0.001). Related to Fig. 6. (F) Representative result of plasma membrane repairing assay in HeLa cells under FAK inhibited or deleted condition. Related to Fig. 6.

Relationship between lysosome movement and focal adhesions. (A) Representative results of live cell imaging with Lysotracker recorded using wide-field epifluorescent microscopy. Top: z-projection of 3-min time series (maximum intensity). Images are inverted to get a better contrast. Bottom: Kymographs represent the lysosome traffic along the magenta segmented line indicated on the top panel. The direction of lysosome traffic was color-coded. Lysosomes that underwent anterograde movement were coded in yellow, retrograde movement in cyan, and static lysosomes in magenta. White arrows indicate stable lysosomes near the WT cell surface, which are not found in MYO18B-KO cells. Related to Fig. 6. (B) Left: Representative confocal microscopic images of WT and MYO18B-KO HeLa cells stained with LAMP1 and vinculin. Bars, 10 µm in original images and 3 µm in magnified images. Right: fluorescent intensity of LAMP1 and vinculin along the white line in the magnified images on the left. Related to Fig. 6. (C) Top: Representative CLSM images of WT and MYO18B-KO U2OS cells co-stained with vinculin and LAMP1. Bars, 10 µm. Bottom: Quantitative analysis of lysosome size in WT and MYO18B-KO U2OS cells (mean ± SD; n = 10 images; N = 2 biological replicates; unpaired two-tailed t test; ***P ≤ 0.001). Related to Fig. 6. (D) Top: Representative CLSM images of U2OS cells grown on non-coated or poly-L-lysine coated cover glass stained with vinculin and LAMP1 (x-y view) Bars, 10 µm. Bottom: The x-z view at the dotted line in the x-y view. The white arrows indicate the lysosomes distributed at the top of nuclei in cells grown on poly-L-lysine coated cover glass. A total of 13 z-series were captured at 0.365 intervals and the x-z view was reconstituted using re-slice tool of ImageJ. The spatial ratio of x-z view was kept unchanged during processing. Related to Fig. 6. (E) Top: Representative CLSM images of U2OS cells grown on non-coated or poly-L-lysine coated cover glass stained with LAMP1 under unpermeabilized conditions. Bars, 10 µm. Bottom: Quantitative analysis of cell-surface LAMP1 level in U2OS cells grown on non-coated or poly-L-lysine coated cover glass. The mean ± SD fluorescent intensity of non-coated group was set to unity (n = 20 images; N = 4 biological replicates; unpaired two-tailed t test; ***P ≤ 0.001). Related to Fig. 6. (F) Representative result of plasma membrane repairing assay in HeLa cells under FAK inhibited or deleted condition. Related to Fig. 6.

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