A Chemoenzymatic Strategy for Imaging Cellular Phosphatidic Acid Synthesis

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 chemoenzy...

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Bibliographic Details
Published in:Angewandte Chemie International Edition 2016-10, Vol.55 (42), p.13155-13158
Main Authors: Bumpus, Timothy W., Baskin, Jeremy M.
Format: Article
Language:English
Subjects:
Alkynes
click chemistry
Cycloaddition
Enzymes
Exchanging
Fluorescence
Fluorescence microscopy
High-performance liquid chromatography
hydrolases
Imaging
imaging agents
Information dissemination
Intracellular
Intracellular signalling
Liquid chromatography
Marking
Membranes
Microscopy
Phosphatidic acid
Phospholipase D
Phospholipids
Signal transduction
Strategy
Visualization
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Summary: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. It takes alkynes: Phosphatidic acid (PA) is a potent lipid secondary messenger that initiates many cellular signaling events. To visualize sites of PA biosynthesis by phospholipase D enzymes, alkynols were used as chemical reporters in a transphosphatidylation reaction, followed by Cu‐catalyzed azide–alkyne cycloaddition to append fluorescent probes for imaging within cells.
ISSN:1433-7851
1521-3773
DOI:10.1002/anie.201607443