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Abstract: PO0619

Membrane Phosphoinositides and Renal Epithelial Cell Polarity Determination in the Xenopus Pronephros In Vivo

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 500 Development, Stem Cells, and Regenerative Medicine

Authors

  • Schena, Giorgia, Yale University School of Medicine, New Haven, Connecticut, United States
  • Rajendran, Vanathy, Yale University School of Medicine, New Haven, Connecticut, United States
  • Khokha, Mustafa, Yale University School of Medicine, New Haven, Connecticut, United States
  • Caplan, Michael J., Yale University School of Medicine, New Haven, Connecticut, United States
Background

Though only minor components of cell membranes, phosphoinositide lipids (PIs) participate in numerous signaling processes and in membrane identity determination. Many studies of cultured MDCK cells found that PIs are distributed with polarity among plasma membrane (PM) domains, and that their polarized distributions are required for the delivery of distinct populations of apical and basolateral membrane proteins. The extent to which PIs drive these processes in actual renal epithelial cells in vivo has never been examined. We investigate the distribution of PIs in vivo in the pronephros of Xenopus Tropicalis tadpoles using MCherry-tagged Pleckstrin Homology (PH) domains that selectively bind different PIs (PH-AKT, PH-PLCD1, which bind to (PI(3,4,5,)P3 and PI(4,5)P2 respectively) with the goal of assessing whether and how PI localizations affect cell polarity determination and the trafficking of proteins to their sites of ultimate functional residence in renal epithelial cells in situ.

Methods

mRNA encoding MCherry-PH-AKT or MCherry-PH-PLCD1 was injected into fertilized oocytes and their distributions were assessed in the developed pronephros at stage NF45 via fluorescence microscopy. Knockdown (KD) of PTEN, a lipid 3’ phosphatase that regulates membrane PI composition, was achieved via injection of targeted morpholinos and confirmed by western blotting. The effects of PTEN KD on MCherry-PH-AKT distribution were assessed by fluorescence microscopy.

Results

In MDCK cells PH-AKT and PH-PLCD1 localize to the basolateral and apical PMs, respectively. Their distributions are quite different in the pronephros, with both sensors showing a markedly apical signal in the proximal portion of the tubule and diffuse cytosolic staining in the distal part. PH-AKT staining dramatically re-distributes to the lateral domain of renal cells upon PTEN KD but this treatment does not alter the localization of protein markers of epithelial PM polarity.

Conclusion

These studies constitute the first effort to assess the role of PIs in establishing PM polarity in renal epithelial cells in vivo. Our data reveal discrepancies with previous reports from in vitro systems. These findings highlight the need to explore the processes that produce renal epithelial cell polarity in vivo and in the context of intact renal tubules.

Funding

  • NIDDK Support