title: A Chemoenzymatic Strategy for Imaging Cellular Phosphatidic Acid Synthesis.
abstract: Phosphatidic acid (PA) is a potent lipid secondary messenger, the synthesis of which is tightly regulated in both space and time. Established tools for detecting PA involve ex vivo analysis and do not provide information on the subcellular locations where this lipid is synthesized. Here, a chemoenzymatic strategy for imaging sites of cellular PA synthesis by phospholipase D (PLD) enzymes is reported. PLDs were found to be able to catalyze phospholipid head-group exchange with alkynols to generate alkyne-labeled PA analogues within cells. Subsequent fluorophore tagging through Cu-catalyzed azide-alkyne cycloaddition enabled both visualization by fluorescence microscopy and quantification by HPLC. Our studies revealed several intracellular sites of PLD-mediated PA synthesis. We envision applications of this approach to dissect PA-dependent signaling pathways, image PLD activity in disease, and remodel intracellular membranes with new functionality.
authors.affiliation: Department of Chemistry and Chemical Biology and Weill Institute for Cell and Molecular Biology, Cornell University, 464 Weill Hall, Ithaca, NY, 14853, USA.
authors.forename: Timothy W
authors.identifier: 0000-0001-5866-5361
authors.initials: TW
authors.lastname: Bumpus
authors.affiliation: Department of Chemistry and Chemical Biology and Weill Institute for Cell and Molecular Biology, Cornell University, 464 Weill Hall, Ithaca, NY, 14853, USA. jeremy.baskin@cornell.edu.
authors.forename: Jeremy M
authors.identifier: 0000-0003-2939-3138
authors.initials: JM
authors.lastname: Baskin
mesh_terms: D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D015394:Molecular Structure; D061848:Optical Imaging; D010712:Phosphatidic Acids / Q000096:biosynthesis* / Q000737:chemistry; D010739:Phospholipase D / Q000737:chemistry / Q000378:metabolism*
chemical_list: D010712:Phosphatidic Acids; D010739:Phospholipase D
keywords: click chemistry; hydrolases; imaging agents; phosphatidic acid; phospholipids