title: Nanoscale analysis reveals agonist-sensitive and heterogeneous pools of phosphatidylinositol 4-phosphate in the plasma membrane.
abstract: 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 10 s, began to increase at 30 s and largely increased to 341.9% at 40 s, and then returned to the initial level at 130 s after the stimulation. Interestingly, PtdIns(4)P localized at the caveolae at 70 and 130 s 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.
authors.affiliation: Field of Veterinary Pathobiology, Basic Veterinary Science, Department of Veterinary Medicine, Joint Faculty of Veterinary Medicine, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan.
authors.forename: Akane
authors.initials: A
authors.lastname: Yoshida
authors.affiliation: Field of Veterinary Pathobiology, Basic Veterinary Science, Department of Veterinary Medicine, Joint Faculty of Veterinary Medicine, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan.
authors.forename: Mikiko
authors.initials: M
authors.lastname: Shigekuni
authors.affiliation: Medical Research Institute, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Tanabe
authors.affiliation: Field of Veterinary Pathobiology, Basic Veterinary Science, Department of Veterinary Medicine, Joint Faculty of Veterinary Medicine, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan. Electronic address: afujita2854@gmail.com.
authors.forename: Akikazu
authors.initials: A
authors.lastname: Fujita
mesh_terms: D000804:Angiotensin II / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D036103:Nanotechnology; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*
chemical_list: D018129:Phosphatidylinositol Phosphates; C037178:phosphatidylinositol 4-phosphate; D000804:Angiotensin II
keywords: Caveolae; Cell membrane; Electron microscopy; Freeze-fracture; G protein-coupled receptor