Figure 8. Loss of plasma membrane... | Rockefeller University Press

Figure 8.

$Figure 8. Loss of plasma membrane identity in PI4KIIIα KO MEFs. Proteins typically restricted to the plasma membrane are found on intracellular PtdIns(4,5)P2-positive vesicles, which are negative for a variety of endosomal and Golgi markers. (A) PtdIns(4,5)P2-positive intracellular vesicles, visualized with GFP- or RFP-PHPLCδ, colocalize with endocytic clathrin machinery components (endogenous AP-2, epsin 1, and dynamin 2–GFP) but not with markers of endosomes (endogenous EEA1, GFP-Rab5, and GFP-Rab9), the endosomal phosphoinositide PtdIns3P (GFP-2×FYVEHrs), and Golgi-localized PtdIns4P (GFP-PHOSBP). For clarity, PHPLCδ is false colored green, and the second marker is false colored red in all images. (B) Mislocalization of plasma membrane proteins in cells lacking PI4KIIIα. PI4KIIIα KO MEFs were transfected with M1R-YFP, L10-GFP, or EFR3B-GFP and imaged by confocal microscopy. Note the accumulation of all markers in intracellular vesicles highlighted in the inset images. (C) Measurement of total and plasma membrane fraction of free cholesterol in control and PI4KIIIα KO MEFs. Total free cholesterol levels were measured by HPLC, after lipid extraction, and normalized to total cellular phospholipid levels. *, P < 0.0001 (n = 12 for control and KO). Plasma membrane free cholesterol was measured by treatment of cells with 10 mM MβCD for 1 min followed by quantification of extracted cholesterol using an enzyme-coupled assay and normalization to total protein content. Error bars indicate standard deviations. *, P < 0.02 (n = 6 for control and KO). Bars: (A and B, insets) 5 µm; (B, full size images) 20 µm.$

Loss of plasma membrane identity in PI4KIIIα KO MEFs. Proteins typically restricted to the plasma membrane are found on intracellular PtdIns(4,5)P₂-positive vesicles, which are negative for a variety of endosomal and Golgi markers. (A) PtdIns(4,5)P₂-positive intracellular vesicles, visualized with GFP- or RFP-PH^PLCδ, colocalize with endocytic clathrin machinery components (endogenous AP-2, epsin 1, and dynamin 2–GFP) but not with markers of endosomes (endogenous EEA1, GFP-Rab5, and GFP-Rab9), the endosomal phosphoinositide PtdIns3P (GFP-2×FYVE^Hrs), and Golgi-localized PtdIns4P (GFP-PH^OSBP). For clarity, PH^PLCδ is false colored green, and the second marker is false colored red in all images. (B) Mislocalization of plasma membrane proteins in cells lacking PI4KIIIα. PI4KIIIα KO MEFs were transfected with M1R-YFP, L₁₀-GFP, or EFR3B-GFP and imaged by confocal microscopy. Note the accumulation of all markers in intracellular vesicles highlighted in the inset images. (C) Measurement of total and plasma membrane fraction of free cholesterol in control and PI4KIIIα KO MEFs. Total free cholesterol levels were measured by HPLC, after lipid extraction, and normalized to total cellular phospholipid levels. *, P < 0.0001 (n = 12 for control and KO). Plasma membrane free cholesterol was measured by treatment of cells with 10 mM MβCD for 1 min followed by quantification of extracted cholesterol using an enzyme-coupled assay and normalization to total protein content. Error bars indicate standard deviations. *, P < 0.02 (n = 6 for control and KO). Bars: (A and B, insets) 5 µm; (B, full size images) 20 µm.