Program in Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada.
SickKids IBD Centre, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada.
J Cell Sci. 2021 Apr 1;134(7). doi: 10.1242/jcs.252411. Epub 2021 Apr 15.
Rab5 is required for macropinosome formation, but its site and mode of action remain unknown. We report that Rab5 acts at the plasma membrane, downstream of ruffling, to promote macropinosome sealing and scission. Dominant-negative Rab5, which obliterates macropinocytosis, had no effect on the development of membrane ruffles. However, Rab5-containing vesicles were recruited to circular membrane ruffles, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent endomembrane fusion was necessary for the completion of macropinocytosis. This fusion event coincided with the disappearance of PtdIns(4,5)P2 that accompanies macropinosome closure. Counteracting the depletion of PtdIns(4,5)P2 by expression of phosphatidylinositol-4-phosphate 5-kinase impaired macropinosome formation. Importantly, we found that the removal of PtdIns(4,5)P2 is dependent on Rab5, through the Rab5-mediated recruitment of the inositol 5-phosphatases OCRL and Inpp5b, via APPL1. Knockdown of OCRL and Inpp5b, or APPL1, prevented macropinosome closure without affecting ruffling. We therefore propose that Rab5 is essential for the clearance of PtdIns(4,5)P2 needed to complete the scission of macropinosomes or to prevent their back-fusion with the plasmalemma.
Rab5 是形成大胞饮体所必需的，但它的作用位点和作用方式仍不清楚。我们报告称，Rab5 在质膜上发挥作用，位于皱襞之后，促进大胞饮体的封闭和分裂。消除大胞饮作用的显性失活 Rab5 对膜皱襞的发育没有影响。然而，Rab5 包含的囊泡被募集到环状膜皱襞，可溶性 N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）依赖性内膜融合对于完成大胞饮作用是必需的。这一融合事件与伴随大胞饮体闭合的 PtdIns(4,5)P2 的消失同时发生。通过表达磷酸肌醇-4-磷酸 5-激酶来抵消 PtdIns(4,5)P2 的耗竭会损害大胞饮体的形成。重要的是，我们发现 PtdIns(4,5)P2 的去除依赖于 Rab5，通过 Rab5 介导的招募肌醇 5-磷酸酶 OCRL 和 Inpp5b，通过 APPL1。敲低 OCRL 和 Inpp5b 或 APPL1 可防止大胞饮体闭合，而不影响皱襞。因此，我们提出 Rab5 对于清除完成大胞饮体分裂或防止其与质膜重新融合所需的 PtdIns(4,5)P2 是必需的。
