Figure 2. Nigericin-induced enlarged compartments originate at the Golgi and contain trans-Golgi marker GalT, later found in ILVs, with most enlarged compartments resolved by 48 hr.

Nigericin was added to HeLa cells for 20 min and washed away, and cells were processed for electron microscopy (A–C), imaged by time-lapse microscopy (D) or harvested for counting (E) at specified times after the wash. (A) Cells stained with osmium tetroxide and potassium hexacyanoferrate reveal large spherical compartments (cyan arrows) originating at the trans-face of the Golgi (magenta arrows) in nigericin-treated cells and, occasionally, in untreated cells. (B,C) Cells stably expressing GalT-GFP were stained with anti-GFP and 12 nM Gold-conjugated secondary antibody to reveal GalT-GFP at the Golgi (magenta arrows), the limiting membrane of the enlarged compartments (cyan arrows) as well as in ILVs of the enlarged MVBs at later time points (yellow arrows).

Figure 2—figure supplement 1. Cell growth is unaffected by nigericin treatment, which induces reversible Golgi vesiculation HeLa cells stably expressing mApple-Rab5 (A,B) or GalT-GFP (C–E) were treated for 20 min with 10 μM nigericin or 5 μM monensin, or left untreated.

(A) Images of cells stained with LC3b antibody to show absence of autophagy induction or presence of LC3B at the enlarged endosomes of nigericin-treated cells. Bafilomycin-treated cells are shown here as positive control. (B) Images to show cells dividing shortly after nigericin treatment, with recovery times specified. Scale bar = 10 μm. (C) Cells were plated in 12-well plates at 10⁴ cells per well 24 hr prior to nigericin treatment, trypsinised and counted at specified recovery times. Triplicate wells were counted per time point per condition. Actual cells numbers and doubling times are presented. Representative graph of three independent experiments. Source data is available in Figure 2—figure supplement 1—source data 1. (D) Images to reveal extensive formation of GalT-positive enlarged compartments upon either treatment following 2 hr recovery. (E) Images to show Golgi vesiculation and return to ribbon morphology within 48 hr of recovery from nigericin treatment.