Figure 4.
Exocytosis of ATG9A-positive vesicles is polarized toward the cell front and induced by chemotactic stimulation. (A) U87 MG cells expressing ATG9A-pHluorin were recorded (one frame every 390 ms) using TIRF microscopy. Upper left: Time-lapse sequence of a representative spreading event. Yellow circle indicates the rapid appearance of the pHluorin signal, likely due to de-acidification upon fusion pore opening. Arrows indicate diffusion of the fluorescence signal in areas of the plasma membrane surrounding the insertion sites. Scale bar, 1 µm. Bottom left: 3D fusion profiles of the indicated images (framed in yellow) from the time-lapse sequence. Middle: Normalized fluorescence intensities, from the analysis of 68 spreading events. For each event, measurements were performed in parallel into a central ROI (5 × 5 pixels; 0.64 × 0.64 µm) and a distant ROI (delimited by 13 × 13–pixel and 17 × 17–pixel squares). Data represent means and SEM. Right: Nonlinear regression analysis (single exponential decay) from the profile obtained in the central ROI, for the determination of spreading event half-life (2.8 s). (B) U87 MG cells expressing ATG9A-pHluorin were recorded as in A. Upper left: Time-lapse sequence of a representative nonspreading event. Scale bar, 1 µm. Bottom left: 3D fusion profiles of the indicated images (framed in yellow) from the time-lapse sequence. Middle: Normalized fluorescence intensities, from the analysis of 13 nonspreading events. For each event, measurements were performed in a central ROI and a distant ROI, as in A. Data represent means and SEM. Right: Nonlinear regression analysis (plateau followed by single exponential decay) from the profile obtained in the central ROI, for the determination of nonspreading event half-life (4 s). (C) Left: Map of all observed (n = 248) ATG9A-pHluorin fusion events from a representative, polarized U87 MG cell, during a 10-min time-lapse sequence. Scale bar, 20 µm. Right: Rose plot from the cell shown on the left, depicting the angular distribution of the fusion events relative to the cell centroid. (D) Quantification, from U87 MG cells displaying clear polarization with unique and large lamellipodia (n = 11 cells), of the number of fusion events occurring at a distance inferior (R1 region) or superior (R2 region) to 10 µm from the leading edge. For each cell, data were normalized to the areas of the respective regions. Data represent means and SEM. (E) Left: Map of all observed (n = 40) ATG9A-pHluorin fusion events from a representative, nonpolarized U87 MG cell, during a 10-min time-lapse sequence. Scale bar, 20 µm. Right: Rose plot from the cell shown on the left, depicting the angular distribution of the fusion events relative to the cell centroid. (F) Left: Representative U87 MG cell with projections of all observed ATG9A-pHluorin fusion events, during 1-min time-lapse sequences, before (−EGF) and 2-min after (+EGF) treatment with EGF (50 ng/ml). Scale bar, 20 µm. Right: Quantification of the effect of EGF, as depicted on the left, on the number ATG9A-pHluorin fusion events (n = 23 cells from three independent experiments; cells from independent experiments were color-coded). Statistical significance was evaluated using Mann–Whitney U test (D), paired t test (F), and Rayleigh test for fusion events distribution (C and E). ***, P < 0.001.

Exocytosis of ATG9A-positive vesicles is polarized toward the cell front and induced by chemotactic stimulation. (A) U87 MG cells expressing ATG9A-pHluorin were recorded (one frame every 390 ms) using TIRF microscopy. Upper left: Time-lapse sequence of a representative spreading event. Yellow circle indicates the rapid appearance of the pHluorin signal, likely due to de-acidification upon fusion pore opening. Arrows indicate diffusion of the fluorescence signal in areas of the plasma membrane surrounding the insertion sites. Scale bar, 1 µm. Bottom left: 3D fusion profiles of the indicated images (framed in yellow) from the time-lapse sequence. Middle: Normalized fluorescence intensities, from the analysis of 68 spreading events. For each event, measurements were performed in parallel into a central ROI (5 × 5 pixels; 0.64 × 0.64 µm) and a distant ROI (delimited by 13 × 13–pixel and 17 × 17–pixel squares). Data represent means and SEM. Right: Nonlinear regression analysis (single exponential decay) from the profile obtained in the central ROI, for the determination of spreading event half-life (2.8 s). (B) U87 MG cells expressing ATG9A-pHluorin were recorded as in A. Upper left: Time-lapse sequence of a representative nonspreading event. Scale bar, 1 µm. Bottom left: 3D fusion profiles of the indicated images (framed in yellow) from the time-lapse sequence. Middle: Normalized fluorescence intensities, from the analysis of 13 nonspreading events. For each event, measurements were performed in a central ROI and a distant ROI, as in A. Data represent means and SEM. Right: Nonlinear regression analysis (plateau followed by single exponential decay) from the profile obtained in the central ROI, for the determination of nonspreading event half-life (4 s). (C) Left: Map of all observed (n = 248) ATG9A-pHluorin fusion events from a representative, polarized U87 MG cell, during a 10-min time-lapse sequence. Scale bar, 20 µm. Right: Rose plot from the cell shown on the left, depicting the angular distribution of the fusion events relative to the cell centroid. (D) Quantification, from U87 MG cells displaying clear polarization with unique and large lamellipodia (n = 11 cells), of the number of fusion events occurring at a distance inferior (R1 region) or superior (R2 region) to 10 µm from the leading edge. For each cell, data were normalized to the areas of the respective regions. Data represent means and SEM. (E) Left: Map of all observed (n = 40) ATG9A-pHluorin fusion events from a representative, nonpolarized U87 MG cell, during a 10-min time-lapse sequence. Scale bar, 20 µm. Right: Rose plot from the cell shown on the left, depicting the angular distribution of the fusion events relative to the cell centroid. (F) Left: Representative U87 MG cell with projections of all observed ATG9A-pHluorin fusion events, during 1-min time-lapse sequences, before (−EGF) and 2-min after (+EGF) treatment with EGF (50 ng/ml). Scale bar, 20 µm. Right: Quantification of the effect of EGF, as depicted on the left, on the number ATG9A-pHluorin fusion events (n = 23 cells from three independent experiments; cells from independent experiments were color-coded). Statistical significance was evaluated using Mann–Whitney U test (D), paired t test (F), and Rayleigh test for fusion events distribution (C and E). ***, P < 0.001.

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