Fluorescent Method Images Cellular Phosphatidic Acid Synthesis

A new study describes how an innovative chemoenzymatic strategy can be used to image cellular synthesis sites of the potent lipid secondary messenger phosphatidic acid (PA).

Developed at Cornell University (Cornell; Ithaca, NY, USA), the new method is based on a two-step process, which involves first attaching an ethanol-like molecule to phospholipase D (PLD), an enzyme that facilitates that process by catalyzing phospholipid head-group exchange with alkynols to generate alkyne-labeled PA analogues within cells. A second azide–alkyne cycloaddition reaction that is catalyzed by copper is then used to attach a fluorescent tag to PA when it is produced.

The fluorophore tagging allowed the researchers to image PA at the moment and location where it is created using fluorescence microscopy and quantification by high performance liquid chromatography (HPLC). The researchers were thus able to reveal the intracellular sites of PLD-mediated PA synthesis. Future applications include dissecting PA-dependent signaling pathways, imaging PLD activity in disease, and remodeling intracellular membranes with new functionality. The study was published on September 16, 2016, in Angewandte Chemie.

“Our imaging approach enables us to directly visualize in cells the locations where the lipid is produced; we believe that where PA is produced has tremendous impact on subsequent downstream biology that occurs,” said senior author Jeremy Baskin, of the department of chemistry and chemical biology at the Cornell Weill Institute for Cell and Molecular Biology. “Also, from an engineering perspective, the click reaction with PLD may allow bioengineers to attach other components onto PA to remodel or re-engineer membranes inside of cells and change their properties and functions.”

Phosphatidic acid is the acid form of phosphatidate, a part of common phospholipids, which are major constituents of cell membranes. PA fulfills three major functions: it is the precursor for the biosynthesis of many other lipids; it’s physical properties influence membrane curvature; and it acts as a signaling lipid, recruiting cytosolic proteins to appropriate membranes.

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