Class III Phosphoinositide 3-Kinase/VPS34 and Dynamin are Critical for Apical Endocytic Recycling
Class III Phosphoinositide 3-Kinase/VPS34 and Dynamin are Critical for Apical Endocytic Recycling
复制标题
DOI：
10.1111/tra.12079
复制
发表时间：
2013-08-01
期刊：
影响因子：
4.5
通讯作者：
Courtoy, Pierre J.
中科院分区：
文献类型：
--
作者：
Carpentier, Sarah;N'Kuli, Francisca;Courtoy, Pierre J.
循环是受体介导的内吞作用的限制步骤。我们首先报道了三个体外或体内证据，即III类PI3K/Vps34是调节心尖循环的关键PI3K亚型。在负鼠肾(Opossum Kidney，OK)细胞中，比较I/II/III类PI3K抑制剂(LY294002)和I/II类PI3K抑制剂(ZSTK474)的差减方法表明，III类PI3K/Vps34抑制诱导选择性顶端内吞肿胀和内吞受体megalin/LRP-2的隔离，导致表面下调。GFP-(FYVE)x2过表达以隔离PI(3)P导致无法区分的顶端内涵体肿胀。在小鼠肾小管上皮细胞中，Vps34的条件性失活也导致空泡化和细胞内巨球蛋白的重新分布。我们接下来报道，从LY294002处理的OK细胞中移除LY294002导致了壮观的回收小管破裂和巨蛋白表面池的恢复。循环小管的急性触发揭示了Dynamin-GFP的募集和Dynamin-GTP酶的依赖，以及微管的导向方向性，并提示微丝网抑制了内小体的肿胀。我们的结论是：(I)PI3K-III除了在内小体融合中发挥作用外，对内小体的分裂/再循环也是必不可少的；(Ii)除了在内吞进入中发挥作用外，Dynamin还支持再循环内小体的管状化。在急性逆转PI3K抑制后释放再循环，可能有助于研究其动力学和相关机制。
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.