Chemical imaging techniques have played instrumental roles in dissecting the spatiotemporal regulation of signal transduction pathways. Phospholipase D (PLD) enzymes affect cell signaling by producing the pleiotropic lipid second messenger phosphatidic acid via hydrolysis of phosphatidylcholine. It remains a mystery how this one lipid signal can cause such diverse physiological and pathological signaling outcomes, due in large part to a lack of suitable tools for visualizing the spatial and temporal dynamics of its production within cells. Here, we report a chemical method for imaging phosphatidic acid synthesis by PLD enzymes in live cells. Our approach capitalizes upon the enzymatic promiscuity of PLDs, which we show can accept azidoalcohols as reporters in a transphosphatidylation reaction. The resultant azidolipids are then fluorescently tagged using the strain-promoted azide–alkyne cycloaddition, enabling visualization of cellular membranes bearing active PLD enzymes. Our method, termed IMPACT (Imaging Phospholipase D Activity with Clickable Alcohols via Transphosphatidylation), reveals pools of basal and stimulated PLD activities in expected and unexpected locations. As well, we reveal a striking heterogeneity in PLD activities at both the cellular and subcellular levels. Collectively, our studies highlight the importance of using chemical tools to directly visualize, with high spatial and temporal resolution, the subset of signaling enzymes that are active.

The fidelity of signal transduction requires spatiotemporal control of the production of signaling agents. Phosphatidic acid (PA) is a pleiotropic lipid second messenger whose modes of action differ based on upstream stimulus, biosynthetic source, and site of production. How cells regulate the local production of PA to effect diverse signaling outcomes remains elusive. Unlike other second messengers, sites of PA biosynthesis cannot be accurately visualized with subcellular precision. Here, we describe a rapid, chemoenzymatic approach for imaging physiological PA production by phospholipase D (PLD) enzymes. Our method capitalizes on the remarkable discovery that bulky, hydrophilic trans-cyclooctene–containing primary alcohols can supplant water as the nucleophile in the PLD active site in a transphosphatidylation reaction of PLD’s lipid substrate, phosphatidylcholine. The resultant trans-cyclooctene–containing lipids are tagged with a fluorogenic tetrazine reagent via a no-rinse, inverse electron-demand Diels–Alder (IEDDA) reaction, enabling their immediate visualization by confocal microscopy in real time. Strikingly, the fluorescent reporter lipids initially produced at the plasma membrane (PM) induced by phorbol ester stimulation of PLD were rapidly internalized via apparent nonvesicular pathways rather than endocytosis, suggesting applications of this activity-based imaging toolset for probing mechanisms of intracellular phospholipid transport. By instead focusing on the initial 10 s of the IEDDA reaction, we precisely pinpointed the subcellular locations of endogenous PLD activity as elicited by physiological agonists of G protein-coupled receptor and receptor tyrosine kinase signaling. These tools hold promise to shed light on both lipid trafficking pathways and physiological and pathological effects of localized PLD signaling.

Biological lipids are a structurally diverse and historically vexing group of hydrophobic metabolites. Here, we review recent advances in chemical imaging techniques that reveal changes in lipid biosynthesis, metabolism, dynamics, and interactions. We highlight tools for tagging many lipid classes via metabolic incorporation of bioorthogonally functionalized precursors, detectable via click chemistry, and photocaged, photoswitchable, and photocrosslinkable variants of different lipids. Certain lipid probes can supplant traditional protein-based markers of organelle membranes in super-resolution microscopy, and emerging vibrational imaging methods, such as stimulated Raman spectroscopy (SRS), enable simultaneous imaging of more than a dozen different types of target molecule, including lipids. Collectively, these chemical imaging techniques will illuminate, in living color, previously hidden aspects of lipid biology.

SUMMARY Insulin stimulates the conversion of phosphatidylino-sitol-4,5-bisphosphate (PI(4,5)P 2 ) to phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P 3 ), which mediates downstream cellular responses. PI(4,5)P 2 is produced by phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) and by phosphatidylinositol-5-phos-phate 4-kinases (PIP4Ks). Here, we show that the loss of PIP4Ks ( PIP4K2A, PIP4K2B , and PIP4K2C) in vitro results in a paradoxical increase in PI(4,5)P 2 and a concomitant increase in insulin-stimulated production of PI(3,4,5)P 3 . The reintroduction of either wild-type or kinase-dead mutants of the PIP4Ks restored cellular PI(4,5)P 2 levels and insulin stimulation of the PI3K pathway, suggesting a catalytic-independent role of PIP4Ks in regulating PI(4,5)P 2 levels. These effects are explained by an increase in PIP5K activity upon the deletion of PIP4Ks, which normally suppresses PIP5K activity through a direct binding interaction mediated by the N-terminal motif VMLϕFPDD of PIP4K. Our work uncovers an allosteric function of PIP4Ks in suppressing PIP5K-mediated PI(4,5)P 2 synthesis and insulin-dependent conversion to PI(3,4,5)P 3 and suggests that the pharmacological depletion of PIP4K enzymes could represent a strategy for enhancing insulin signaling.

Phosphatidic acid (PA) is a potent lipid secondary messenger whose synthesis 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 we report a chemoenzymatic strategy for imaging sites of cellular PA synthesis by phospholipase D (PLD) enzymes. We find that PLDs can catalyze phospholipid head group exchange with alkynols to generate alkyne-labeled PA analogs within cells. Subsequent fluorophore tagging using the 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.