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.
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