Figure 3.

Figure 3.

ER-Golgi trafficking of pro-α1(I) is unperturbed in giantin KO RPE1 cells. (A) Schematic of the pro-SBP-GFP-COL1A1 construct used in this study. SS, signal peptide. (B and C) Single-plane confocal stills taken from live imaging movies of a WT (B) and giantin KO (C) cell stably expressing GFP-COL1A1 (green) and transiently transfected with mCh-ST (magenta) and an ER RUSH hook. Frame times in the top left corner indicate the time (minutes) after biotin addition, which releases GFP-COL1A1 from the ER hook. Scale bars = 10 µm on overview and 1 µm on zoom. White arrows indicate the appearance of peripheral GFP-COL1A1 punctate. Orange arrows indicate emerging post-Golgi carriers. See also videos. (D) Quantification of the time taken for GFP-COL1A1 to first appear at the Golgi in videos represented in B and C. Each dot represents one cell (16 WT cells and 13 giantin KO cells), and videos were collected over seven independent experiments. Bars represent median and interquartile range. Results are not significant using a Mann-Whitney U test. (E) Maximum projections of confocal z-stacks of WT and giantin KO cells imaged live as in B and C and then fixed with PFA as soon as the GFP-COL1A1 appeared around the Golgi. Cells were then immunolabeled for the Golgi marker GM130 (blue). Scale bars = 10 µm.