Nanoscale analysis reveals agonist-sensitive and heterogeneous pools of phosphatidylinositol 4-phosphate in the plasma membrane

• Published in: Biochimica et biophysica acta Vol. 1858; no. 6; pp. 1298 - 1305
• Main Authors: Yoshida, Akane, Shigekuni, Mikiko, Tanabe, Kenji, Fujita, Akikazu
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-06-2016
• Subjects: Angiotensin II - pharmacology; Caveolae; Cell membrane; Cell Membrane - drug effects; Cell Membrane - metabolism; Electron microscopy; Freeze-fracture; G protein-coupled receptor; HeLa Cells; Humans; Microscopy, Fluorescence; Nanotechnology; Phosphatidylinositol Phosphates - metabolism; GFP; G protein-coupled receptor; Cell membrane; Caveolae; OSH; OSBP; P4M; PtdIns(4,5)P2; PtdInsP; Electron microscopy; Freeze-fracture; FAPP1
• ISSN: 0005-2736; 0006-3002; 1879-2642
• DOI: 10.1016/j.bbamem.2016.03.011

Phosphatidylinositol 4-phosphate [PtdIns(4)P] is the immediate precursor of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], which is localized to the cytoplasmic leaflet of the plasma membrane and has been reported to possess multiple cell biological functions. Direct evidence showing the distribution of PtdIns(4)P pools at a nanoscale when the plasma membrane PtdIns(4,5)P2 is hydrolyzed by agonist stimulation is lacking. To analyze the distribution of PtdIns(4)P at a nanoscale, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the plasma membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilized in situ. Using this technique, we observed no PtdIns(4)P in the caveolae of normal cultured human fibroblasts, although PtdIns(4,5)P2 has been shown to be highly concentrated in them in our previous report. When cells were stimulated with angiotensin II, the level of PtdIns(4)P in the undifferentiated membrane transiently decreased to 64.3% at 10s, began to increase at 30s and largely increased to 341.9% at 40s, and then returned to the initial level at 130s after the stimulation. Interestingly, PtdIns(4)P localized at the caveolae at 70 and 130s after the stimulation. These results suggest that the level of the PtdIns(4)P pool in the plasma membrane is sensitive and the distribution of PtdIns(4)P dramatically changes by agonist stimulation, and there are active sites of production or replenishment of PtdIns(4)P at undifferentiated membrane and caveolar areas.
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•PtdIns(4)P distribution is shown at a nanoscale in the plasma membrane.•PtdIns(4)P was not detected in caveolae in unstimulated cells.•Angiotensin II induced a transient PtdIns(4)P increase in undifferentiated flat plasma membrane.•PtdIns(4)P localized at caveolae after angiotensin II stimulation.