Figure 3.
ELKS- and RAB6-dependent arrival of secreted cargos at exocytosis hotspots. (A) HeLa cells were transfected with SBP-mCherry-ColX and GFP-ELKS. After 30- or 45-min incubation with biotin, cells were processed for immunofluorescence and stained with an anti-paxillin antibody. Coverslips were coated with an anti-GFP antibody (SPI assay). Higher magnifications of the images are shown on the right. (B) HeLa cells expressing SBP-EGFP-CD59, TNFα-SBP-EGFP, and SBP-EGFP-ColX were treated for 3 d with control or ELKS siRNAs. Cells were incubated for 60–90 min with biotin to allow cargo release from the ER and its arrival to the plasma membrane. Representative images taken from videos. (C) The number of vesicles per cell were quantified using ImageJ (mean ± SEM, n = 23–39 cells). *, P < 0.05; ***, P < 10−4 (Student’s t test). Cells were treated as indicated in B. Scale bars, 10 µm. (D) HeLa cells were treated with control or ELKS siRNA and processed for Western blotting. ELKS signal was revealed using an anti-ELKS antibody. Actin signal was used as a loading control. (E) HeLa cells were treated with control or ELKS siRNA and then cotransfected with SBP-EGFP-gp135 and paxillin-mCherry. Cells were seeded on anti-GFP–coated coverslips (SPI), and trafficking of SBP-EGFP-gp135 was monitored by spinning disk microscope upon addition of biotin. (F) HeLa cells were treated with control or ELKS siRNA and then transfected with SBP-EGFP-gp135. For rescue experiments, cells were cotransfected with BFP-ELKS. Cells were seeded on anti-GFP–coated coverslips (SPI). After a 50-min treatment with biotin, cells were fixed. Representative images are displayed. (G) The number of secreted spots as well as their intensity was quantified using ImageJ or Fiji software (mean ± SEM, n = 39–60 cells). Cells were treated as indicated in F. **, P < 10−2; ***, P < 10−4 (Student’s t test). Scale bars, 10 µm.