Fig. 3.

PI(3,4)P₂ is an apical- and recycling endosome-enriched lipid. a, b PI(3,4)P₂ localization during different stages of lumen formation (12–48 h) in MDCK cells stably co-expressing EGFP-2xPH-TAPP1 [PI(3,4)P₂ sensor, green] and either a TagRFPT-Rab11a WT (red) and stained for F-actin (blue), or b Podxl (red) and stained for β-catenin (blue). White arrows, PI(3,4)P₂ co-localization with Rab11a vesicles at all stages; white arrowheads, cortical PI(3,4)P₂ localization; red arrowheads, PI(3,4)P₂-positive, Podxl-negative vesicles below the cell–cell contact at the AMIS stage; yellow arrowheads, PI(3,4)P₂ present at low levels at the basolateral PM at the AMIS stage. In all instances, bottom panels are higher magnifications of split color images from the boxed regions. c Localization of Rab11a (TagRFPT-Rab11a, left; endogenous antibody staining, right; both red) in either 24 or 48 h cysts in relation to either PI(3)P [EGFP-2xFYVE, top] or PI(4)P [EGFP-P4M-SidM, bottom] (both green). In blue, Podxl (left panel) or nuclear staining (right panel). Yellow arrowheads, Rab11a-negative PIP reporter vesicles. White arrowheads (red in inverted color panels), Rab11a endosomes. White arrows (red in inverted color panels), triple Rab11a/Podxl/PIP reporter-positive vesicles. Yellow arrows (magenta in inverted color panels), Rab11a-negative, Podxl/PIP reporter-positive vesicles. Pink arrows, Rab11a/PI(4)P overlapping endosomes. d PI(3,4)P₂ and caveolin-1 localization during lumen formation (12–48 h) in EGFP-2xPH-TAPP1 (green) MDCK cells stained with Caveolin-1 (red). Note the cortical co-localization of Caveolin-1 and PI(3,4)P₂ at all stages (white arrowheads) and the absence of intracellular co-localization (yellow arrowheads). All scale bars, 10 µm