Figure 3. Ceapins retain tagged-ATF6α as foci in the ER and prevent trafficking of tagged ATF6α to the Golgi apparatus.
(A-–D) U2-OS cells stably expressing GFP-ATF6α were treated either with vehicle (A, DMSO), ER stress (B, 100 nM Tg), ER stress and site-1-protease inhibitor (C, 1 μM Pf-429242) or ER stress and Ceapin-A1 (D, 10 μM Ceapin-A1) for thirty minutes prior to fixation and fluorescent imaging of GFP-ATF6α (green), anti-Giantin to mark the Golgi apparatus (red in RGB, purple in GM insets) and DNA (blue). (A) Unstressed cells have minimal co-localization of GFP-ATF6α and Giantin. (B) ER stress induces trafficking of GFP-ATF6α to the Golgi apparatus where it colocalizes with Giantin. (C) ER stress combined with the site-1 protease inhibitor inhibits cleavage of GFP-ATF6α in the Golgi apparatus causing GFP-ATF6α to accumulate there. (D) ER stress combined with Ceapin-A1 shows minimal colocalization of GFP-ATF6α with Giantin, indicating GFP-ATF6α has not trafficked to the Golgi apparatus in the presence of the Ceapin-A1. (E–L) 293 T-REx cells stably expressing doxycycline inducible 3xFLAG-ATF6α were treated either with vehicle (E,I, DMSO), ER stress (F,J, 100 nM Tg), ER stress and Ceapin-A1 (G,K, 5 μM Ceapin-A1) or Ceapin-A1 alone (H,L, 5 μM Ceapin-A1) for thirty minutes prior to fixation and fluorescent imaging of 3xFLAG-ATF6α (green) and DNA (blue) and either an ER marker Calnexin (E–H, red in RGB, purple in GM inset) or a Golgi apparatus marker Giantin (I–L, red in RGB, purple in GM inset). ER stress induced Golgi trafficking of 3xFLAG-ATF6α (J, arrowheads) is prevented by the addition of the Ceapin-A1 (K). Ceapin-A1 either in combination with ER stress (G) or alone (H) induces formation of 3xFLAG-ATF6α foci that co-localize with ER tubules (arrowheads). (M–P) U2-OS cells stably expressing GFP-ATF6α were treated either with vehicle (M, DMSO), ER stress (N, 100 nM Tg), ER stress and active Ceapin analogs (O, 5 μM Ceapin-A1), (P, 5 μM Ceapin-A7). After time-lapse imaging for 2.4 hr, cells were fixed and stained for GFP-ATF6α (green), anti-GRP94 to mark the ER (red) and anti-Giantin to mark the Golgi apparatus (blue). ER stress induced trafficking to the Golgi apparatus (N) is blocked by the Ceapin analogs, and the induced GFP-ATF6α foci remain co-localized with ER tubules even after almost 2.5 hr of ER stress (O,P). Note that fixation conditions to visualize the ER and Golgi apparatus are not suitable for imaging the nuclear translocated fraction of GFP-ATF6α (see Materials and methods). Higher magnification panels underneath each image show each channel singly in greyscale (middle row), pairwise merges bottom row) of GFP-ATF6α (green) with either ER (magenta, bottom left) or Golgi markers in (magenta, and triple merge (bottom row, right) of GFP-ATF6α (green), ER (red) and Golgi (blue). In each panel, scale bars are 10 μm and boxed inserts are 7 x 7 μm (A–L) or 11.8 x 11.8 μm (M–P).