Fig 2: Characterization of the domains responsible for the interactions between MICAL3, ELKS and Rab8A. Results of streptavidin pull down assays. In all panels, triangles indicate signals at the expected molecular weight. Dashed lines indicate cropped lanes irrelevant for this study. Full images are presented in Supplementary Figure S3. (A,B) Pull downs are performed by mixing the lysates of cells co-expressing biotin ligase BirA and differently truncated BioGFP-tagged MICAL3, with the lysates of cells expressing mCherry-ELKS-FL. Rabbit-anti-ELKS antibody and mouse-anti-GFP antibody were used for western blotting, depicted in the upper and lower boxes respectively. (C) Illustrated pull down is performed by mixing the lysates of cells co-expressing biotin ligase BirA and differently truncated BioGFP-tagged MICAL3, with the lysates of cells expressing mStrawberry-Rab8A. The blot is incubated with rabbit-anti-Rab8A and mouse-anti-GFP antibodies for the upper and lower boxes. (D) The pull down is performed by mixing the lysates of cells co-expressing biotin ligase BirA and indicated BioGFP-tagged MICAL3 constructs with lysates of cells expressing BFP-Rab8A. Western blot is performed using rabbit-anti-Rab8A and mouse-anti-GFP antibodies for the upper and lower boxes. (E) Pull down is performed by the mixing of lysates of cells co-expressing biotin ligase BirA with indicated BioGFP-tagged proteins with lysates of cells expressing GFP-MKLP1. Mouse-anti-GFP antibody is used in both upper and lower boxes.

Fig 3: Mapping of the MICAL3-interacting site in ELKS. (A) An overview of ELKS truncation and deletions constructs and their interactions with MICAL3. (B,C) Results of streptavidin pull down assays, performed by mixing the lysates of cells co-expressing biotin ligase BirA and differently truncated or deleted BioGFP-tagged ELKS, with the lysates of cells expressing mCherry-MICAL3-WT. Rabbit-anti-MICAL3 and mouse-anti-GFP antibodies were used for the upper and lower boxes. Triangles indicate signals at the expected molecular weight. Dashed lines indicate cropped lanes irrelevant for this study. Full images are presented in Supplementary Figure S4. *Published information from reference20.

Fig 4: Proposed model to explain the role of RAB6 in post-Golgi secretory pathway. All RAB6 effectors associated to post-Golgi secretion, myosin II, KIF5B, and ELKS/RAB6IP2, are involved in the secretion of various cargos. The different events can be envisioned as follows. RAB6 is present on CD59-, TNFa-, and ColX-containing vesicles exiting the Golgi complex. Myosin II is implicated in the fission of these vesicles from Golgi membranes (Miserey-Lenkei et al., 2010). Vesicles are transported to the plasma membrane in a KIF5B-dependent manner (Grigoriev et al., 2007; Miserey-Lenkei et al., 2010), and ELKS ensures their docking to exocytic hotspots close to FAs (Grigoriev et al., 2007, 2011).

Fig 5: 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, TNFa-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.