![Figure 7. Ectopic PtdIns(4,5)P2 localization and up-regulation of PIPKIs in PI4KIIIα KO MEFs. (A) Imaging of PtdIns(4,5)P2 distribution in control and PI4KIIIα KO MEFs using RFP-PHPLCδ. (inset) Note the presence of PtdIns(4,5)P2-positive vesicles only in KO MEFs. (B) Quantification of the plasma membrane/cytosol (PM/cyt) GFP-PHPLCδ fluorescence ratio in control and PI4KIIIα KO MEFs based on line scans (control, n = 22; KO, n = 23; *, P < 0.0001). (C) PIPKIγ and PtdIns(4,5)P2 colocalize on intracellular vesicles in cells lacking PI4KIIIα. PI4KIIIα KO MEFs were transfected with RFP-PHPLCδ and GFP-PIPKIγ87 and imaged by confocal microscopy. Insets show the region outlined with dotted lines at higher magnification. (D) Western blot analysis of PIPKIβ and PIPKIγ levels in control MEFs, PI4KIIIα KO MEFs, and PI4KIIIα KO MEFs transfected with GFP-M1-PI4KIIIα (KO + rescue). *, P < 0.0002 (n = 3); **, P < 0.003 (n = 3). rel., relative. (E) Actin comet tails in PI4KIIIα KO MEFs visualized by CHUtrophin-mCherry (a marker of F-actin). (F) Actin comet tail in PI4KIIIα KO MEFs double transfected with CHUtrophin-mCherry and GFP-PHPLCδ. Note the presence of a PtdIns(4,5)P2-positive vesicle at the head of the comet. (G) Acute, 488-nm light-mediated depletion of plasma membrane PtdIns(4,5)P2 from HeLa cells (see Materials and methods for details) results in diffuse intracellular fluorescence of the PtdIns(4,5)P2 probe iRFP-PHPLCδ but no detectable PtdIns(4,5)P2-positive intracellular vesicles. (H) Chronic depletion of plasma membrane PtdIns(4,5)P2 from HeLa cells by transfection with a membrane-anchored inositol 5-phosphatase (mRFP-IPP1-CAAX; see cell on the right) results in diffuse intracellular fluorescence of the PtdIns(4,5)P2 probe iRFP-PHPLCδ, but no PtdIns(4,5)P2-positive intracellular vesicles are apparent 16 h after transfection. Note that the myristoylated/palmitoylated plasma membrane marker L10-GFP is retained at the plasma membrane. Ctrl, control. Error bars indicate standard deviations. Bars: (A [inset], C [inset], E, and F) 5 µm; (H) 10 µm; (A [full size], C [full size], and G) 20 µm.](https://cdn.rupress.org/rup/content_public/journal/jcb/199/6/10.1083_jcb.201206095/5/jcb_201206095_fig7.jpeg?Expires=1771874894&Signature=d4OGyrRhlgYBb4EfLvTfnSNSrohITu-Ll0odU7b1Dmi4TXoJ1v7RzthO9Nzde27doJsljNFD~DRdn03OLfDcsjfM7DsMxBZO591z7U6jyOog-3EeiTIET3He0PK3G7newIxARw80k1EgR2Qgw7-EWCZAfBAU6WbB-RuwUCSXb2VySZxvso8vrzvp5FtQeI41gVh~2-UyhjLzzOC8XiTMbZW62osmWBYYvphYFfdcd4VwU6ZXCgVKtyiSCw0gcODXuNuQ9UOkH7AIRRv83h7su~MJmtFXrZvcwsxTlF81cBVOA1diEoPH1eNGwtn10AGbQgrM9xoh-IpmqiAVDkPmSA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Ectopic PtdIns(4,5)P2 localization and up-regulation of PIPKIs in PI4KIIIα KO MEFs. (A) Imaging of PtdIns(4,5)P2 distribution in control and PI4KIIIα KO MEFs using RFP-PHPLCδ. (inset) Note the presence of PtdIns(4,5)P2-positive vesicles only in KO MEFs. (B) Quantification of the plasma membrane/cytosol (PM/cyt) GFP-PHPLCδ fluorescence ratio in control and PI4KIIIα KO MEFs based on line scans (control, n = 22; KO, n = 23; *, P < 0.0001). (C) PIPKIγ and PtdIns(4,5)P2 colocalize on intracellular vesicles in cells lacking PI4KIIIα. PI4KIIIα KO MEFs were transfected with RFP-PHPLCδ and GFP-PIPKIγ87 and imaged by confocal microscopy. Insets show the region outlined with dotted lines at higher magnification. (D) Western blot analysis of PIPKIβ and PIPKIγ levels in control MEFs, PI4KIIIα KO MEFs, and PI4KIIIα KO MEFs transfected with GFP-M1-PI4KIIIα (KO + rescue). *, P < 0.0002 (n = 3); **, P < 0.003 (n = 3). rel., relative. (E) Actin comet tails in PI4KIIIα KO MEFs visualized by CHUtrophin-mCherry (a marker of F-actin). (F) Actin comet tail in PI4KIIIα KO MEFs double transfected with CHUtrophin-mCherry and GFP-PHPLCδ. Note the presence of a PtdIns(4,5)P2-positive vesicle at the head of the comet. (G) Acute, 488-nm light-mediated depletion of plasma membrane PtdIns(4,5)P2 from HeLa cells (see Materials and methods for details) results in diffuse intracellular fluorescence of the PtdIns(4,5)P2 probe iRFP-PHPLCδ but no detectable PtdIns(4,5)P2-positive intracellular vesicles. (H) Chronic depletion of plasma membrane PtdIns(4,5)P2 from HeLa cells by transfection with a membrane-anchored inositol 5-phosphatase (mRFP-IPP1-CAAX; see cell on the right) results in diffuse intracellular fluorescence of the PtdIns(4,5)P2 probe iRFP-PHPLCδ, but no PtdIns(4,5)P2-positive intracellular vesicles are apparent 16 h after transfection. Note that the myristoylated/palmitoylated plasma membrane marker L10-GFP is retained at the plasma membrane. Ctrl, control. Error bars indicate standard deviations. Bars: (A [inset], C [inset], E, and F) 5 µm; (H) 10 µm; (A [full size], C [full size], and G) 20 µm.
