Figure 1.
CD63 compartments fusing with PM represent an early non-degradative stage of late-endosomes. (A) Example of DC-TIRF-M analysis on CD63-(pHuji)fusion event in HeLa cells co-expressing LAMP1-GFP. (B) Fluorescent signal pattern for CD63-pHuji/LAMP1-GFP averaged over >12 events, synchronized using peak-intensity of CD63-pHuji signal at 60 s. Inset is 3.5 µm. (C) Immunofluorescent co-labeling of endogenous CD63 (red) and LAMP1 (green) in HeLa cells. (D) Fluorescent intensity of endogenous CD63 and LAMP1 levels (as in A), plotted for individual endosomes. R-squared shown for linear regression (light blue). (E) Fusion activity of apilimod treated CD63-pHluorin HeLa cells. Analysis >40 cells from n = 3 experiments, using Student’s two-tailed two-sample t test. Red line indicates median. (F) Fluorescent analysis of overlap degradative compartments labeled by MagicRed with late-endo/lysosomal markers LAMP1(-GFP) or CD63(-pHluorin) in HeLa cells untreated or treated with apilimod. (G) Quantification of analysis as in (F). Shown is the ratio of degradative MagicRed compartments that overlap with LAMP1 and CD63, respectively (measured over >12 imaging fields using Student’s two-tailed two-sample t test; red line indicates median). (H) Example of DC-TIRF-M analysis on CD63-(pHluorin)fusion event in HeLa cells incubated with MagicRed. Inset is 3.4 µm. (I) Example of DC-TIRF-M analysis on CD63-(pHuji)fusion event in HeLa cells co-expressing TRPML1-GFP. Inset is 5.6 µm. (J) Example of DC-TIRF-M analysis on CD63-(pHluorin)fusion event in HeLa cells co-expressing LC3B-mRFP. Inset is 3.4 µm. (K) Fluorescent signal patterns for MagicRed, LC3, and TRPML1 averaged over >12 events, synchronized using peak-intensity of CD63 signal at 60 s. Bars: (A, C, F, and H–J) 5 µm. N indicates nucleus.

CD63 compartments fusing with PM represent an early non-degradative stage of late-endosomes. (A) Example of DC-TIRF-M analysis on CD63-(pHuji)fusion event in HeLa cells co-expressing LAMP1-GFP. (B) Fluorescent signal pattern for CD63-pHuji/LAMP1-GFP averaged over >12 events, synchronized using peak-intensity of CD63-pHuji signal at 60 s. Inset is 3.5 µm. (C) Immunofluorescent co-labeling of endogenous CD63 (red) and LAMP1 (green) in HeLa cells. (D) Fluorescent intensity of endogenous CD63 and LAMP1 levels (as in A), plotted for individual endosomes. R-squared shown for linear regression (light blue). (E) Fusion activity of apilimod treated CD63-pHluorin HeLa cells. Analysis >40 cells from n = 3 experiments, using Student’s two-tailed two-sample t test. Red line indicates median. (F) Fluorescent analysis of overlap degradative compartments labeled by MagicRed with late-endo/lysosomal markers LAMP1(-GFP) or CD63(-pHluorin) in HeLa cells untreated or treated with apilimod. (G) Quantification of analysis as in (F). Shown is the ratio of degradative MagicRed compartments that overlap with LAMP1 and CD63, respectively (measured over >12 imaging fields using Student’s two-tailed two-sample t test; red line indicates median). (H) Example of DC-TIRF-M analysis on CD63-(pHluorin)fusion event in HeLa cells incubated with MagicRed. Inset is 3.4 µm. (I) Example of DC-TIRF-M analysis on CD63-(pHuji)fusion event in HeLa cells co-expressing TRPML1-GFP. Inset is 5.6 µm. (J) Example of DC-TIRF-M analysis on CD63-(pHluorin)fusion event in HeLa cells co-expressing LC3B-mRFP. Inset is 3.4 µm. (K) Fluorescent signal patterns for MagicRed, LC3, and TRPML1 averaged over >12 events, synchronized using peak-intensity of CD63 signal at 60 s. Bars: (A, C, F, and H–J) 5 µm. N indicates nucleus.

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