Phosphoinositides serve crucial roles in cell physiology, ranging from cell signalling to membrane traffic1,2. Among the seven eukaryotic phosphoinositides the best studied species is phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), which is concentrated at the plasma membrane where, among other functions, it is required for the nucleation of endocytic clathrin-coated pits3,4,5,6. No phosphatidylinositol other than PI(4,5)P2 has been implicated in clathrin-mediated endocytosis, whereas the subsequent endosomal stages of the endocytic pathway are dominated by phosphatidylinositol-3-phosphates(PI(3)P)7. How phosphatidylinositol conversion from PI(4,5)P2-positive endocytic intermediates to PI(3)P-containing endosomes is achieved is unclear. Here we show that formation of phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2) by class II phosphatidylinositol-3-kinase C2α (PI(3)K C2α) spatiotemporally controls clathrin-mediated endocytosis. Depletion of PI(3,4)P2 or PI(3)K C2α impairs the maturation of late-stage clathrin-coated pits before fission. Timed formation of PI(3,4)P2 by PI(3)K C2α is required for selective enrichment of the BAR domain protein SNX9 at late-stage endocytic intermediates. These findings provide a mechanistic framework for the role of PI(3,4)P2 in endocytosis and unravel a novel discrete function of PI(3,4)P2 in a central cell physiological process.
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We thank E. Ungewickell, P. Di Fiore, P. De Camilli, H. McMahon, E. Wancker, T. Südhof and S. Carlsson for antibodies, L. Cantley, T. Takenawa, M. Wymann, T. Ross, O. Daumke and W. Yang for plasmids, and O. Daumke, B. Eickolt and F. Wieland for critical comments. Supported by grants from the Deutsche Forschungsgemeinschaft (SFB 740/C8; SFB 740/D7; SFB 958/A04; SFB 958/A07; SFB 958/Z02).
The authors declare no competing financial interests.
This file contains Supplementary Figures 1-8 and Supplementary Table 1. (PDF 10942 kb)
The peripheral regions of two neighbouring eGFP-clathrin light chain expressing cells imaged by TIRF microscopy shown for 3 min. The cell on the right suffers from PI(3,4)P2 depletion due to co-expression of mCherry-INPP4B-CAAX . For clarity, a dotted line has been drawn along the border between the two cells. Note the strikingly attenuated CCPs dynamics in the PI(3,4)P2-depleted cell. (MOV 7431 kb)
Videos 2 and 3 show representative areas from eGFP-clathrin light chain expressing cells treated with scrambled or PI3K C2α siRNAs, respectively, imaged by TIRF microscopy for 3 min. CCPs in control cells (video 2; scrambled siRNA) display a dynamic succession of appearance, growth, and disappearance (internalization). By contrast, CCPs in PI3K C2α-depleted cells (video 3; PI3K C2α-siRNA) are long-lived and stable over time, indicative of defective CCP maturation. (MOV 4251 kb)
Videos 2 and 3 show representative areas from eGFP-clathrin light chain expressing cells treated with scrambled or PI3K C2α siRNAs, respectively, imaged by TIRF microscopy for 3 min. CCPs in control cells (video 2; scrambled siRNA) display a dynamic succession of appearance, growth, and disappearance (internalization). By contrast, CCPs in PI3K C2α-depleted cells (video 3; PI3K C2α-siRNA) are long-lived and stable over time, indicative of defective CCP maturation. (MOV 3910 kb)
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Posor, Y., Eichhorn-Gruenig, M., Puchkov, D. et al. Spatiotemporal control of endocytosis by phosphatidylinositol-3,4-bisphosphate. Nature 499, 233–237 (2013). https://doi.org/10.1038/nature12360
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