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Significance Phosphatidic acid (PA) is a bioactive lipid that functions as an intracellular second messenger, enabling cells to integrate and react to diverse extracellular cues. Because of PA’s strong and pleiotropic effects, its generation is tightly controlled spatiotemporally. It remains unknown how cells regulate local PA production at individual organelle membranes to control different downstream signaling events. We describe a chemical, activity-based imaging approach for precisely visualizing, in real time, subcellular localizations of PA signaling initiated by phospholipase D (PLD) enzymes. This toolset revealed distinct locations of PLD signaling elicited by G protein-coupled receptor and receptor tyrosine kinase pathways and holds promise to shed light on physiological and pathological effects of localized PLD signaling and, more generally, intracellular lipid transport pathways.

A real-time, click chemistry imaging approach reveals stimulus-specific subcellular locations of phospholipase D activity