Figure 2.
Figure 2. Exocytosis is directed between FAs. (A) HeLa cells were transfected with SBP-EGFP-ColX and paxillin-mCherry. Real-time pictures were acquired using a TIRF microscope at the indicated times. The TIRF angle was chosen based on the paxillin-mCherry signal. Biotin was added at time 0. (B) Fixed images of areas 1 and 2 (see A) taken every 40 s between 20 min 40 s and 47 min 20 s of ColX trafficking. Area 1 is a control condition without ColX signal at the plasma membrane. Area 2 shows secretion in close proximity to FAs. (C) Coverslips were coated with an anti-GFP antibody (SPI assay). Real-time images were acquired using a TIRF microscope at the indicated times. The TIRF angle was determined based on the paxillin-mCherry signal. Biotin was added at time 0. (D) Distance of secreted SBP-EGFP-ColX (green) or myristoylated and palmitoylated (MyrPalm)-EGFP (gray) signal from the closest FAs was measured. The distance of each pixel from the whole cell area from the closest FA was also measured. Ratio of enrichment compared with the distance of pixels of the region of interest to the closest FAs was calculated (n = 585 FAs, 10 cells for secreted SBP-EGFP-ColX and n = 319 FAs, 11 cells for Myr-Palm-EGFP). (E–H) HeLa cells were transfected with SBP-EGFP-gp135 (F and G) or SBP-EGFP-gp135 and paxillin-mCherry (E and H). Coverslips were coated with an anti-GFP antibody (SPI assay; H). Microtubules were stained with SiR-tubulin (G). Cells were observed by time-lapse imaging using a spinning disk microscope and images were acquired at the indicated times. Biotin was added at time 0. After 20 min of incubation with biotin, SBP-EGFP-gp135 localizes in the Golgi apparatus, and fast acquisition imaging was performed. Between 26 and 32 min (E) or 21 and 27 min (F), a temporal projection of EGFP-gp135 signal was performed using Fiji software and is represented with a temporal-color code (left). In F–H, kymographs (time space plots) were performed on the indicated yellow lines using Fiji software. In H, intensity profiles of SBP-EGFP-gp135 (in green) and paxillin-mCherry (in red) were performed using Fiji software at the indicated blue line. To help the interpretation of the kymographs, the passage of distinct post-Golgi carriers has been colorized. px, pixels. Scale bars, 10 µm.

Exocytosis is directed between FAs. (A) HeLa cells were transfected with SBP-EGFP-ColX and paxillin-mCherry. Real-time pictures were acquired using a TIRF microscope at the indicated times. The TIRF angle was chosen based on the paxillin-mCherry signal. Biotin was added at time 0. (B) Fixed images of areas 1 and 2 (see A) taken every 40 s between 20 min 40 s and 47 min 20 s of ColX trafficking. Area 1 is a control condition without ColX signal at the plasma membrane. Area 2 shows secretion in close proximity to FAs. (C) Coverslips were coated with an anti-GFP antibody (SPI assay). Real-time images were acquired using a TIRF microscope at the indicated times. The TIRF angle was determined based on the paxillin-mCherry signal. Biotin was added at time 0. (D) Distance of secreted SBP-EGFP-ColX (green) or myristoylated and palmitoylated (MyrPalm)-EGFP (gray) signal from the closest FAs was measured. The distance of each pixel from the whole cell area from the closest FA was also measured. Ratio of enrichment compared with the distance of pixels of the region of interest to the closest FAs was calculated (n = 585 FAs, 10 cells for secreted SBP-EGFP-ColX and n = 319 FAs, 11 cells for Myr-Palm-EGFP). (E–H) HeLa cells were transfected with SBP-EGFP-gp135 (F and G) or SBP-EGFP-gp135 and paxillin-mCherry (E and H). Coverslips were coated with an anti-GFP antibody (SPI assay; H). Microtubules were stained with SiR-tubulin (G). Cells were observed by time-lapse imaging using a spinning disk microscope and images were acquired at the indicated times. Biotin was added at time 0. After 20 min of incubation with biotin, SBP-EGFP-gp135 localizes in the Golgi apparatus, and fast acquisition imaging was performed. Between 26 and 32 min (E) or 21 and 27 min (F), a temporal projection of EGFP-gp135 signal was performed using Fiji software and is represented with a temporal-color code (left). In F–H, kymographs (time space plots) were performed on the indicated yellow lines using Fiji software. In H, intensity profiles of SBP-EGFP-gp135 (in green) and paxillin-mCherry (in red) were performed using Fiji software at the indicated blue line. To help the interpretation of the kymographs, the passage of distinct post-Golgi carriers has been colorized. px, pixels. Scale bars, 10 µm.

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