Abstract
Polarity is a central feature of eukaryotic cells and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) has a central role in the polarization of neurons and chemotaxing cells. In polarized epithelial cells, PtdIns(3,4,5)P3 is stably localized at the basolateral plasma membrane, but excluded from the apical plasma membrane, as shown by localization of GFP fused to the PtdIns(3,4,5)P3-binding pleckstrin-homology domain of Akt (GFP-PH–Akt), a fusion protein that indicates the location of PtdIns(3,4,5)P3. Here, we ectopically inserted exogenous PtdIns(3,4,5)P3 into the apical plasma membrane of polarized Madin-Darby canine kidney (MDCK) cells. Within 5 min many cells formed protrusions that extended above the apical surface. These protrusions contained basolateral plasma membrane proteins and excluded apical proteins, indicating that their plasma membrane was transformed from apical to basolateral. Addition of PtdIns(3,4,5)P3 to the basolateral surface of MDCK cells grown as cysts caused basolateral protrusions. MDCK cells grown in the presence of a phosphatidylinositol 3-kinase inhibitor had abnormally short lateral surfaces, indicating that PtdIns(3,4,5)P3 regulates the formation of the basolateral surface.
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Acknowledgements
We thank K. Matlin, G. Ojakian and B. Stevenson for reagents. We thank H. Bourne, O. Weiner, M. Zegers, P. Brakeman and members of our laboratory for advice and comments on the paper. and D. Mills for help with the manuscript. This work was supported by National Institutes of Health (NIH) grants to K.M. and J.E. W.Y. is supported by a fellowship from the National Kidney Foundation. F.M.-B. is supported by the Human Frontiers Science Program (HFSP).
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A.G. designed, performed and interpreted all of the experiments. W.Y. helped with the design of some experiments. M.t.B. and F.M.-B. made invaluable reagents. A.K. and J.E. helped with the interpretation of data and writing of the paper. K.M. conceived the experiments, helped with interpretation of data and wrote the paper.
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Gassama-Diagne, A., Yu, W., ter Beest, M. et al. Phosphatidylinositol-3,4,5-trisphosphate regulates the formation of the basolateral plasma membrane in epithelial cells. Nat Cell Biol 8, 963–970 (2006). https://doi.org/10.1038/ncb1461
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DOI: https://doi.org/10.1038/ncb1461
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