title: Metabolic labeling and direct imaging of choline phospholipids in vivo.
abstract: Choline (Cho)-containing phospholipids are the most abundant phospholipids in cellular membranes and play fundamental structural as well as regulatory roles in cell metabolism and signaling. Although much is known about the biochemistry and metabolism of Cho phospholipids, their cell biology has remained obscure, due to the lack of methods for their direct microscopic visualization in cells. We developed a simple and robust method to label Cho phospholipids in vivo, based on the metabolic incorporation of the Cho analog propargylcholine (propargyl-Cho) into phospholipids. The resulting propargyl-labeled phospholipid molecules can be visualized with high sensitivity and spatial resolution in cells via a Cu(I)-catalyzed cycloaddition reaction between the terminal alkyne group of propargyl-Cho and a labeled azide. Total lipid analysis of labeled cells shows strong incorporation of propargyl-Cho into all classes of Cho phospholipids; furthermore, the fatty acid composition of propargyl-Cho-labeled phospholipids is very similar to that of normal Cho phospholipids. We demonstrate the use of propargyl-Cho in cultured cells, by imaging phospholipid synthesis, turnover, and subcellular localization by both fluorescence and electron microscopy. Finally, we use propargyl-Cho to assay microscopically phospholipid synthesis in vivo in mouse tissues.
authors.affiliation: Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
authors.forename: Cindy Y
authors.initials: CY
authors.lastname: Jao
authors.forename: Mary
authors.initials: M
authors.lastname: Roth
authors.forename: Ruth
authors.initials: R
authors.lastname: Welti
authors.forename: Adrian
authors.initials: A
authors.lastname: Salic
mesh_terms: D000480:Alkynes / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002794:Choline / Q000031:analogs & derivatives* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D010743:Phospholipids / Q000096:biosynthesis / Q000378:metabolism*; D013194:Staining and Labeling
chemical_list: D000480:Alkynes; D010743:Phospholipids; C000603143:propargylcholine; D002794:Choline
references.citation: J Org Chem. 2002 May 3;67(9):3057-64
references.pmid: 11975567
references.citation: Proc Natl Acad Sci U S A. 2008 Oct 14;105(41):15779-84
references.pmid: 18840688
references.citation: Biochem Cell Biol. 2004 Feb;82(1):113-28
references.pmid: 15052332
references.citation: J Lipid Res. 2007 Apr;48(4):837-47
references.pmid: 17210984
references.citation: Angew Chem Int Ed Engl. 2006 Nov 13;45(44):7364-7
references.pmid: 17036290
references.citation: J Lipid Res. 2000 Sep;41(9):1448-54
references.pmid: 10974052
references.citation: Phytochemistry. 2006 Sep;67(17):1907-24
references.pmid: 16843506
references.citation: Angew Chem Int Ed Engl. 2002 Jul 15;41(14):2596-9
references.pmid: 12203546
references.citation: J Biol Chem. 2002 Aug 30;277(35):31994-2002
references.pmid: 12077151
references.citation: Mol Cell Biol. 1989 Jan;9(1):325-8
references.pmid: 2538723
references.citation: Prog Lipid Res. 2001 May;40(3):199-229
references.pmid: 11275267
references.citation: J Biol Chem. 1958 Mar;231(1):53-64
references.pmid: 13538947
references.citation: Proc Natl Acad Sci U S A. 2008 Feb 19;105(7):2415-20
references.pmid: 18272492
references.citation: Science. 2000 Mar 17;287(5460):2007-10
references.pmid: 10720325
references.citation: Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9116-21
references.pmid: 12874386
references.citation: J Insect Physiol. 1970 Apr;16(4):579-93
references.pmid: 5443995
references.citation: Biochem J. 1970 Sep;119(3):481-8
references.pmid: 5500307
references.citation: Proc Natl Acad Sci U S A. 2002 Jan 8;99(1):19-24
references.pmid: 11752401
references.citation: J Lipid Res. 1963 Oct;4:397-401
references.pmid: 14168181