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. 2021 Sep 9;9:729444. doi: 10.3389/fcell.2021.729444

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Copyright © 2021 Sluysmans, Méan, Jond and Citi.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Phospholipid binding and cellular localizations of the PH domains of WW-PLEKHAs. (A–C) Scheme of the structural organization of PLEKHA5 (A), PLEKHA6 (B), and PLEKHA7 (C) showing the domains: WW (Trp-Trp) (W) in green, PH (pleckstrin homology) in red, Proline-rich (Pro-rich or Pr) in blue, coiled-coil (C) in pink. Red box indicates the structural domain analyzed. (D–F) In vitro interaction of PH domains of WW-PLEKHAs with phospholipids. Coomassie staining (D) of purified GST fusions of PH domains of PLEKHA5 (P5), PLEKHA6 (P6) and PLEKHA7 (P7) (framed bands), and (E,F) IB analysis of lipid-protein overlay assay (GST-PH domain of phospholipase C-∂1 (PLC∂) as positive control, GST alone as negative control). Green frames highlight binding, red frames indicate low/undetectable interaction. LPA, lysophosphatidic acid; LPC, lysophosphocholine; PtdIns, phosphatidylinositol; P, phosphate; P2, biphosphate; P3, triphosphate; PE, phosphatidylethanolamine; PC, phosphatidylcholine. (G–I) IF analysis of the localization of exogenous GFP-tagged constructs of PLEKHA5 (G), PLEKHA6 (H), and PLEKHA7 (I) in WT mCCD cells. Either full-length (FL), or FL with a deletion of the PH domain (ΔPH), or PH domain alone (PH) were used. ZO-1 and PLEKHA7 are used as junctional markers. Junctional (junct.) and fibrillar microtubules-like [(MT)] localizations are indicated. Arrows show labeling, arrowheads indicate low/undetectable labeling. (J,K) IF analysis of the localization of either GFP-tagged PLEKHA7 (P7) (J) or PLEKHA5 (P5) (K) constructs in WT mCCD cells. Either full-length (FL) proteins, or chimeras where the PH domain was replaced [either from P5 or P6 in PLEKHA7 (J), or from P6 or P7 for PLEKHA5 (K)] are shown. mCCD cells were polarized on transwells and XZ section were taken at the horizontal middle of the XY plane (square panels). Cytoplasmic sub-apical (cyt. apic.), junctional (junct.), and lateral (lat.) localizations are indicated. E-cadherin and ZO-1 are used as lateral and junctional markers, respectively. Scale bar = 20 μm.