Formation of the apicosome in singly isolated cells. (A–C) Live imaging of H9 cells expressing EZRIN–GFP (A) or PODXL–GFP (B and C) during apicosome formation (see the detailed legend of Video 4 for C). (D) Quantitation of the total volume of the central perinuclear complex in C during imaging, revealing increase in volume over time (a representative of six independent live-imaged samples). Values were obtained from the high-threshold VR image (C, bottom left) acquired every 15 s. (E) H9 cells dissociated from monolayers incubated for 30 min with mCLING–ATTO647N to label apical membrane. Singly plated cells were examined at several time points (30 min to 20 h after plating), after immunostaining for PODXL and p-ERM. (F) Schematic of an isolated hPSC undergoing apicosome formation. Blue indicates DNA staining (HOECHST). Bars: (A and B) 25 µm; (C and E) 10 µm; (E-i) 3 µm. Time stamp: (A and B) h:min; (C) min:s.