Estimation of the number of V-ATPases per lysosome. (A) Photobleaching of monodisperse SidK-AL568. Representative intensity-time traces for a single SidK-AL568 labeled with one (top) or two (bottom) Alexa Fluor 568 moieties. Loss of fluorescence after complete photobleaching shown by the dotted red lines. (B) Histogram of the measured number of dye molecules per SidK-AL568 monomer determined analyzing >5,000 single-molecule photobleaching traces. Number of SidK-AL568 molecules in each category is indicated above the bar. (C) HeLa cells stained with SidK-AL568 at varying concentrations. Lysosomal fluorescence was plotted to determine the concentration of SidK-AL568 required to reach maximal lysosome fluorescence, indicative of saturation of binding sites. (D) HeLa cells stained with SidK-AL568 using the near-saturation conditions determined in C (1:50). Total fluorescence per lysosome is compared with the average fluorescence of a single molecule of SidK-AL568; the average number of SidK-AL568 molecules bound per lysosome is indicated by the horizontal line, as is the SE (whiskers). A total of 5,715 lysosomes from two independent experiments were quantified. (E) HeLa cells stained with SidK-AL568 (magenta) using the near-saturating conditions determined in C (1:50), and costained for LAMP1 (green). Side panels show individual LAMP1 (top) and SidK-AL568:LAMP1 colocalization channels (bottom) for juxtanuclear (left) or peripheral (right) lysosomes in the areas denoted by dotted squares. Cell and lysosome outlines indicated by dotted and solid lines, respectively. (F) SidK-AL568:LAMP1 fluorescence ratio (RatioSidK-AL568:LAMP1) corresponding to image in E. The fluorescence ratio was pseudocolored, representing ratio values from 0 to 2. (G) Shell analysis of SidK distribution as a function of distance from the cell outer edge. HeLa cells were stained using SidK-AL568 and costained for LAMP1. Cell outlines were drawn and degraded iteratively inward by 4 µm to create concentric shells (colored differentially) within each cell that were used to subgroup LAMP1+ lysosomes for subsequent analysis. Images in E-G are compressions of confocal stacks representative of ≥10 fields from two or more experiments of each type. (H) For each shell, total fluorescence per lysosome was compared with the average fluorescence of a single molecule of SidK-AL568, and the average number of SidK-AL568 molecules bound per lysosome calculated as a function of distance from the cell outer edge. Data are mean ± SEM of two independent experiments with five or more cells per replicate. n = 791, 1,112, 1,203, 1,275, and 1,334 lysosomes analyzed for shells 0–4, 4–8, 8–12, 12–16, and >16 µm from the cell edge, respectively. (I) For the data in H, the number of SidK-AL568 molecules bound per lysosome was divided by the lysosome surface area (μm2), and the ratio graphed as a function of distance from the cell outer edge. Data are mean ± SEM of two experiments with five or more cells per replicate. Scale bars: 5 µm.
Figure 8.

Estimation of the number of V-ATPases per lysosome. (A) Photobleaching of monodisperse SidK-AL568. Representative intensity-time traces for a single SidK-AL568 labeled with one (top) or two (bottom) Alexa Fluor 568 moieties. Loss of fluorescence after complete photobleaching shown by the dotted red lines. (B) Histogram of the measured number of dye molecules per SidK-AL568 monomer determined analyzing >5,000 single-molecule photobleaching traces. Number of SidK-AL568 molecules in each category is indicated above the bar. (C) HeLa cells stained with SidK-AL568 at varying concentrations. Lysosomal fluorescence was plotted to determine the concentration of SidK-AL568 required to reach maximal lysosome fluorescence, indicative of saturation of binding sites. (D) HeLa cells stained with SidK-AL568 using the near-saturation conditions determined in C (1:50). Total fluorescence per lysosome is compared with the average fluorescence of a single molecule of SidK-AL568; the average number of SidK-AL568 molecules bound per lysosome is indicated by the horizontal line, as is the SE (whiskers). A total of 5,715 lysosomes from two independent experiments were quantified. (E) HeLa cells stained with SidK-AL568 (magenta) using the near-saturating conditions determined in C (1:50), and costained for LAMP1 (green). Side panels show individual LAMP1 (top) and SidK-AL568:LAMP1 colocalization channels (bottom) for juxtanuclear (left) or peripheral (right) lysosomes in the areas denoted by dotted squares. Cell and lysosome outlines indicated by dotted and solid lines, respectively. (F) SidK-AL568:LAMP1 fluorescence ratio (RatioSidK-AL568:LAMP1) corresponding to image in E. The fluorescence ratio was pseudocolored, representing ratio values from 0 to 2. (G) Shell analysis of SidK distribution as a function of distance from the cell outer edge. HeLa cells were stained using SidK-AL568 and costained for LAMP1. Cell outlines were drawn and degraded iteratively inward by 4 µm to create concentric shells (colored differentially) within each cell that were used to subgroup LAMP1+ lysosomes for subsequent analysis. Images in E-G are compressions of confocal stacks representative of ≥10 fields from two or more experiments of each type. (H) For each shell, total fluorescence per lysosome was compared with the average fluorescence of a single molecule of SidK-AL568, and the average number of SidK-AL568 molecules bound per lysosome calculated as a function of distance from the cell outer edge. Data are mean ± SEM of two independent experiments with five or more cells per replicate. n = 791, 1,112, 1,203, 1,275, and 1,334 lysosomes analyzed for shells 0–4, 4–8, 8–12, 12–16, and >16 µm from the cell edge, respectively. (I) For the data in H, the number of SidK-AL568 molecules bound per lysosome was divided by the lysosome surface area (μm2), and the ratio graphed as a function of distance from the cell outer edge. Data are mean ± SEM of two experiments with five or more cells per replicate. Scale bars: 5 µm.

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