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PIP 5 KI b Selectively Modulates Apical Endocytosis in Polarized Renal Epithelial Cells

Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at clathrin coated pits (CCPs) is crucial for the recruitment of adaptors and other components of the internalization machinery, as well as for regulating actin dynamics during endocytosis. PtdIns(4,5)P2 is synthesized from phosphatidylinositol 4-phosphate by any of three phosphatidylinositol 5-kinase type I (PIP5KI) isoforms (a, b or c). PIP5KIb localizes almost exclusively to the apical surface in polarized mouse cortical collecting duct cells, whereas the other isoforms have a less polarized membrane distribution. We therefore investigated the role of PIP5KI isoforms in endocytosis at the apical and basolateral domains. Endocytosis at the apical surface is known to occur more slowly than at the basolateral surface. Apical endocytosis was selectively stimulated by overexpression of PIP5KIb whereas the other isoforms had no effect on either apical or basolateral internalization. We found no difference in the affinity for PtdIns(4,5)P2-containing liposomes of the PtdIns(4,5)P2 binding domains of epsin and Dab2, consistent with a generic effect of elevated PtdIns(4,5)P2 on apical endocytosis. Additionally, using apical total internal reflection fluorescence imaging and electron microscopy we found that cells overexpressing PIP5KIb have fewer apical CCPs but more internalized coated structures than control cells, consistent with enhanced maturation of apical CCPs. Together, our results suggest that synthesis of PtdIns(4,5)P2 mediated by PIP5KIb is rate limiting for apical but not basolateral endocytosis in polarized kidney cells. PtdIns(4,5)P2 may be required to overcome specific structural constraints that limit the efficiency of apical endocytosis. Citation: Szalinski CM, Guerriero CJ, Ruiz WG, Docter BE, Rbaibi Y, et al. (2013) PIP5KIb Selectively Modulates Apical Endocytosis in Polarized Renal Epithelial Cells. PLoS ONE 8(1): e53790. doi:10.1371/journal.pone.0053790 Editor: Joshua Z. Rappoport, University of Birmingham, United Kingdom Received November 5, 2012; Accepted December 5, 2012; Published January 16, 2013 Copyright: 2013 Szalinski et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health (NIH) grants R01 DK064613 (to OAW), DA024698 (to MAP), R37DK54425 (to GA), and by Cores A and C of the Pittsburgh Center for Kidney Research (P30 DK079307). CMS was supported in part by NIH T32 DK061296. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: weisz@pitt.edu