Class III Phosphoinositide 3‐Kinase/VPS34 and Dynamin are Critical for Apical Endocytic Recycling

Synopsis Recycling is a limiting step for receptor‐mediated endocytosis. We here show that class III PI3K is the key isoform involved, based on differential pharmacological inhibition (substractive approach) and PI‐3P sequestration by overexpressed GFP‐(FYVE)x2 in cultured kidney epithelial cells, as well as conditional PI3K‐III/Vps34 KO in developing mice kidneys. Acute reversal of PI3K inhibition induced a spectacular burst of recycling tubules that recruited dynamin‐GFP and depended on its GTPase activity. This simple procedure may help further dissect recycling machineries. Recycling is a limiting step for receptor‐mediated endocytosis. We first report three in vitro or in vivo evidences that class III PI3K/VPS34 is the key PI3K isoform regulating apical recycling. A substractive approach, comparing in Opossum Kidney (OK) cells a pan‐class I/II/III PI3K inhibitor (LY294002) with a class I/II PI3K inhibitor (ZSTK474), suggested that class III PI3K/VPS34 inhibition induced selective apical endosome swelling and sequestration of the endocytic receptor, megalin/LRP‐2, causing surface down‐regulation. GFP‐(FYVE)x2 overexpression to sequester PI(3)P caused undistinguishable apical endosome swelling. In mouse kidney proximal tubular cells, conditional Vps34 inactivation also led to vacuolation and intracellular megalin redistribution. We next report that removal of LY294002 from LY294002‐treated OK cells induced a spectacular burst of recycling tubules and restoration of megalin surface pool. Acute triggering of recycling tubules revealed recruitment of dynamin‐GFP and dependence of dynamin‐GTPase, guidance directionality by microtubules, and suggested that a microfilamentous net constrained endosomal swelling. We conclude that (i) besides its role in endosome fusion, PI3K‐III is essential for endosome fission/recycling; and (ii) besides its role in endocytic entry, dynamin also supports tubulation of recycling endosomes. The unleashing of recycling upon acute reversal of PI3K inhibition may help study its dynamics and associated machineries.