title: Heterogeneity of phosphatidic acid levels and distribution at the plasma membrane in living cells as visualized by a Föster resonance energy transfer (FRET) biosensor.
abstract: Phosphatidic acid (PA) is one of the major phospholipids in the plasma membrane. Although it has been reported that PA plays key roles in cell survival and morphology, it remains unknown when and where PA is produced in the living cell. Based on the principle of Förster resonance energy transfer (FRET), we generated PA biosensor, and named Pii (phosphatidic acid indicator). In these biosensors, the lipid-binding domain of DOCK2 is sandwiched with the cyan fluorescent protein and yellow fluorescent protein and is tagged with the plasma membrane-targeting sequence of K-Ras. The addition of synthetic PA, or the activation of phospholipase D or diacylglycerol kinase at the plasma membrane, changed the level of FRET in Pii-expressing cells, demonstrating the response of Pii to PA. The biosensor also detected divergent PA content among various cell lines as well as within one cell line. Interestingly, the growth factor-induced increment in PA content correlated negatively with the basal PA content before stimulation, suggesting the presence of an upper threshold in the PA concentration at the plasma membrane. The biosensor also revealed uneven PA distribution within the cell, i.e. the basal level and growth factor-induced accumulation of PA was higher at the cell-free edges than at the cell-cell contact region. An insufficient increase in PA may account for ineffective Ras activation at areas of cell-cell contact. In conclusion, the PA biosensor Pii is a versatile tool for examining heterogeneity in the content and distribution of PA in single cells as well as among different cells.
authors.affiliation: Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Teruko
authors.initials: T
authors.lastname: Nishioka
authors.forename: Michael A
authors.initials: MA
authors.lastname: Frohman
authors.forename: Michiyuki
authors.initials: M
authors.lastname: Matsuda
authors.forename: Etsuko
authors.initials: E
authors.lastname: Kiyokawa
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015374:Biosensing Techniques / Q000379:methods*; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D002522:Chlorocebus aethiops; D019852:Diacylglycerol Kinase / Q000235:genetics / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007365:Intercellular Junctions / Q000737:chemistry / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D041681:NIH 3T3 Cells; D010712:Phosphatidic Acids / Q000032:analysis* / Q000378:metabolism; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: C402110:DOCK2 protein, human; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D008164:Luminescent Proteins; D010712:Phosphatidic Acids; D004815:Epidermal Growth Factor; D019852:Diacylglycerol Kinase; D010739:Phospholipase D
references.citation: Prog Lipid Res. 2006 May;45(3):250-78
references.pmid: 16574237
references.citation: Science. 2009 Apr 17;324(5925):384-7
references.pmid: 19325080
references.citation: Proc Natl Acad Sci U S A. 2006 Jul 11;103(28):10817-22
references.pmid: 16818883
references.citation: Nature. 2001 Jun 28;411(6841):1065-8
references.pmid: 11429608
references.citation: Biochim Biophys Acta. 2009 Sep;1791(9):876-80
references.pmid: 19376267
references.citation: Biochim Biophys Acta. 2009 Sep;1791(9):862-8
references.pmid: 19410013
references.citation: J Lipid Res. 2006 Jul;47(7):1588-96
references.pmid: 16632799
references.citation: Nat Cell Biol. 2003 Nov;5(11):1016-22
references.pmid: 14528311
references.citation: Biochim Biophys Acta. 2006 Aug;1761(8):913-26
references.pmid: 16624617
references.citation: Cell Mol Life Sci. 2005 Oct;62(19-20):2305-16
references.pmid: 16143829
references.citation: Clin Cancer Res. 2008 Jul 1;14(13):4267-74
references.pmid: 18594009
references.citation: J Biol Chem. 1999 Jan 1;274(1):494-502
references.pmid: 9867870
references.citation: Mol Cell Biol. 1991 Oct;11(10):4903-8
references.pmid: 1656217
references.citation: Proc Natl Acad Sci U S A. 2009 Nov 17;106(46):19352-7
references.pmid: 19858485
references.citation: J Biol Chem. 2000 May 5;275(18):13621-7
references.pmid: 10788479
references.citation: Mol Biol Cell. 2008 Jul;19(7):3111-23
references.pmid: 18480413
references.citation: Mol Biol Cell. 2005 May;16(5):2207-17
references.pmid: 15728722
references.citation: Nat Methods. 2006 Oct;3(10):797-9
references.pmid: 16990811
references.citation: J Biol Chem. 2000 Aug 4;275(31):23911-8
references.pmid: 10801816
references.citation: Trends Biochem Sci. 2007 Sep;32(9):407-14
references.pmid: 17764955
references.citation: Sci Signal. 2009 Sep 08;2(87):ra52
references.pmid: 19738202
references.citation: J Biol Chem. 2006 Oct 20;281(42):31627-37
references.pmid: 16905533
references.citation: Mol Biol Cell. 2004 Mar;15(3):1024-30
references.pmid: 14718562
references.citation: Mol Biol Cell. 2004 Apr;15(4):1802-15
references.pmid: 14742704
references.citation: J Biol Chem. 2006 Jun 9;281(23):15862-8
references.pmid: 16595654
references.citation: Mol Biol Cell. 2007 Jan;18(1):119-28
references.pmid: 17079732
references.citation: Mol Cell Biol. 2003 Jan;23(2):645-54
references.pmid: 12509462
references.citation: Biochim Biophys Acta. 2009 Sep;1791(9):850-5
references.pmid: 19540930
references.citation: Mol Biol Cell. 2008 Oct;19(10):4213-23
references.pmid: 18685081
references.citation: Biochim Biophys Acta. 2009 Sep;1791(9):942-8
references.pmid: 19264149
references.citation: J Cell Biol. 2007 Jun 4;177(5):817-27
references.pmid: 17535963
references.citation: J Cell Biol. 2003 Jul 21;162(2):223-32
references.pmid: 12860967
references.citation: J Biol Chem. 2007 Jul 27;282(30):21746-57
references.pmid: 17540765
references.citation: J Biol Chem. 2001 Aug 17;276(33):31305-10
references.pmid: 11406630
references.citation: J Cell Biol. 2006 Nov 6;175(3):377-82
references.pmid: 17088424
references.citation: J Cell Biol. 2005 Sep 26;170(7):1147-58
references.pmid: 16186260
references.citation: Biochem Biophys Res Commun. 1999 Feb 16;255(2):502-7
references.pmid: 10049738
references.citation: Traffic. 2006 Mar;7(3):365-77
references.pmid: 16497229
references.citation: Trends Neurosci. 2009 Jan;32(1):19-29
references.pmid: 19008000
references.citation: Nat Cell Biol. 2007 Jun;9(6):706-12
references.pmid: 17486115
references.citation: Curr Biol. 2005 Oct 11;15(19):1749-54
references.pmid: 16213822
references.citation: Trends Cell Biol. 2003 Oct;13(10):540-6
references.pmid: 14507482
references.citation: Mol Biol Cell. 2009 Jan;20(1):200-8
references.pmid: 18946083
references.citation: Mol Pharmacol. 2009 Mar;75(3):437-46
references.pmid: 19064628
references.citation: J Biol Chem. 1998 Dec 11;273(50):33722-7
references.pmid: 9837959
references.citation: Nat Cell Biol. 2006 Nov;8(11):1255-62
references.pmid: 17028579
references.citation: J Biol Chem. 2002 Feb 22;277(8):5823-31
references.pmid: 11744730