Figure 2.
Arl8b localizes to the newly synthesized post-endocytic RUSH-LAMP1 vesicles prior to fusion with active lysosomes. (A) Representative confocal images of Arl8bEN-mStayGold KI HeLa cells expressing SBP-mCherry-LAMP1. Live-cell imaging was performed after biotin addition, and representative images for indicated time points are shown. Images are maximum-intensity projections of z-stack micrographs. The line profiles indicate fluorescence intensity along the white lines for both channels: SBP-mCherry-LAMP1 (red) and Arl8bEN-mStayGold (green). Scale bars: 10 μm (main); 2 μm (inset). (B) Quantification of Manders’ colocalization coefficient of SBP-mCherry-LAMP1 with Arl8bEN-mStayGold at the indicated time points is shown from three independent experiments (n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated by one-way ANOVA with Dunnett’s multiple comparisons test (***P = 0.0006; ****P < 0.0001). The values are represented as the mean ± SEM. (C) Representative confocal micrographs of HeLa cells treated with control or Vps33a siRNA, followed by co-expression of SBP-GFP-LAMP1 (green) and Arl8b-Halo (red). Live-cell imaging was performed after cells were incubated with LTR (blue) dye to label acidic compartments, followed by biotin addition. Blue arrowheads indicate SBP-GFP-LAMP1– and Arl8b-Halo–positive vesicles. The line profiles indicate fluorescence intensity along the blue lines for all channels: SBP-GFP-LAMP1 (green), Arl8b-Halo (red), and LTR (blue). Scale bars: 10 μm (main); 2 μm (inset). (D) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with LTR in HeLa cells treated with the indicated siRNAs is shown from three independent experiments (n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated using unpaired Student’s t test (***P = 0.0003). The values are represented as the mean ± SEM. (E) Quantification of Manders’ colocalization coefficient for SBP-GFP-LAMP1 with Arl8b-Halo in HeLa cells treated with the control or Vps33a siRNA is shown from three independent experiments (n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated using unpaired Student’s t test (n.s., non-significant). The values are represented as the mean ± SEM. Refer to the image caption for details. Panel A shows live-cell confocal microscopy images and fluorescence intensity line profiles of Arl8bEN-mStayGold knock-in HeLa cells expressing SBP-mCherry-LAMP1 at different post-biotin incubation time points. Panel B shows scatter bar plots quantifying Manders colocalization coefficient between SBP-mCherry-LAMP1 and Arl8bEN-mStayGold across multiple post-biotin incubation time points. Panel C shows confocal microscopy images and fluorescence intensity line profiles of control or Vps33a siRNA-treated HeLa cells co-expressing SBP-GFP-LAMP1, Arl8b-Halo, and LysoTracker following biotin addition. Panel D shows scatter bar plots quantifying Manders colocalization coefficient between SBP-GFP-LAMP1 and LysoTracker in control or Vps33a siRNA-treated HeLa cells. Panel E shows scatter bar plots quantifying Manders colocalization coefficient between SBP-GFP-LAMP1 and Arl8b-Halo in control or Vps33a siRNA-treated HeLa cells.

Arl8b localizes to the newly synthesized post-endocytic RUSH-LAMP1 vesicles prior to fusion with active lysosomes. (A) Representative confocal images of Arl8bEN-mStayGold KI HeLa cells expressing SBP-mCherry-LAMP1. Live-cell imaging was performed after biotin addition, and representative images for indicated time points are shown. Images are maximum-intensity projections of z-stack micrographs. The line profiles indicate fluorescence intensity along the white lines for both channels: SBP-mCherry-LAMP1 (red) and Arl8bEN-mStayGold (green). Scale bars: 10 μm (main); 2 μm (inset). (B) Quantification of Manders’ colocalization coefficient of SBP-mCherry-LAMP1 with Arl8bEN-mStayGold at the indicated time points is shown from three independent experiments (n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated by one-way ANOVA with Dunnett’s multiple comparisons test (***P = 0.0006; ****P < 0.0001). The values are represented as the mean ± SEM. (C) Representative confocal micrographs of HeLa cells treated with control or Vps33a siRNA, followed by co-expression of SBP-GFP-LAMP1 (green) and Arl8b-Halo (red). Live-cell imaging was performed after cells were incubated with LTR (blue) dye to label acidic compartments, followed by biotin addition. Blue arrowheads indicate SBP-GFP-LAMP1– and Arl8b-Halo–positive vesicles. The line profiles indicate fluorescence intensity along the blue lines for all channels: SBP-GFP-LAMP1 (green), Arl8b-Halo (red), and LTR (blue). Scale bars: 10 μm (main); 2 μm (inset). (D) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with LTR in HeLa cells treated with the indicated siRNAs is shown from three independent experiments (n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated using unpaired Student’s t test (***P = 0.0003). The values are represented as the mean ± SEM. (E) Quantification of Manders’ colocalization coefficient for SBP-GFP-LAMP1 with Arl8b-Halo in HeLa cells treated with the control or Vps33a siRNA is shown from three independent experiments (n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated using unpaired Student’s t test (n.s., non-significant). The values are represented as the mean ± SEM.

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