Fig. 3. IRSP53 precedes PODXL localization to the apical membrane.

a Schematic representation of IRSp53 and PODXL trafficking during the early phases of lumenogenesis. b MDCK cells expressing GFP-IRSp53 were seeded as single cells on a Matrigel layer and left to grow to form three-dimensional (3D) cysts. The cysts were fixed at the indicated time points, processed for epifluorescence to visualize GFP-IRSp53 (green) and stained with anti-PODXL (red) and DAPI (blue). Arrowheads, IRSp53 preceding PODXL relocalization at the AMIS at early time points; colocalization of IRSp53 and PODXL at the AMIS at later time points; and enrichment at the luminal side in the mature cyst. Arrows, IRSp53 and PODXL colocalization in vesicle-like structures. Scale bar, 18 µm. c, d Still images of time lapse of GFP-IRSp53 during early phases of cystogenesis. MDCK expressing GFP-IRSp53 were seeded as single cells on a Matrigel. 6 h after seeding cells were subjected to time-lapse analysis using Confocal Spinning Disk microscope. Images (Bright Field and GFP channel respectively) were acquired every 5 min for 15 h (see also Supplementary Movies 2 and 3). White arrowheads indicate GFP-IRSp53 vesicular-like structures that emerge from the peripheral plasma membrane and move toward the inner (c) or the forming apical side at cleavage furrow immediately after the first cell division (d). Scale bar, 10 µm.