Choline phospholipids: Now you see them

Lipidomics Gateway (23 September 2009) [doi:10.1038/lipidmaps.2009.25]

A new metabolic labeling technique allows in vivo imaging of choline phospholipids.

Cryostat section of small intestine from a propargyl-Cho-injected mouse stained in parallel with TMR-azide in red, overlayed with DNA (blue) and DIC micrographs. For full figure and legend see Jao C. Y. et al. PNAS 2009, 106, 15332-15337 10.1073/pnas.0907864106

Choline (Cho) is the most common phospholipid head group in eukaryotic cell membranes and is found in both structural and signaling lipids. Although the biochemistry of Cho phospholipid metabolism is well understood, much remains unknown about their intracellular behavior, including how their synthesis is regulated, and how they move within and between membranes. Adrian Salic and colleagues, reporting in Proceedings of the National Academy of Sciences of the USA, have developed a new metabolic labeling technique that allows direct in vivo imaging of Cho phospholipids, and will facilitate study of their cell biology.

Cho analogs with a short alkyl chain in place of a methyl group incorporate efficiently into phospholipids. Using this knowledge, Salic and colleagues realized that Cho modified with a three-carbon propargyl group should be equally well incorporated. Importantly, the propargyl group terminates with an alkyne moiety, which allows covalent attachment of a labeled azide using copper-catalyzed 'click' chemistry. The labeled azide can be imaged microscopically, and this technique has been used to visualize proteins, DNA, and other macromolecules.

The authors synthesized propargyl-Cho and incubated it with cultured cells. Fixed and stained with fluorescent azide, the cells produced an intense signal that was abolished by incubation with phospholipase C, a Cho hydrolyzing enzyme.

To measure the incorporation of propargyl-Cho into cellular phospholipids, total cell lipid extracts were analyzed with electrospray ionization-tandem mass spectrometry (ESI-MS/MS). All Cho phospholipid classes showed efficient incorporation of propargyl-Cho, which did not perturb the levels of Cho or non-Cho phospholipids, and the labeled molecules were stable. Fluorescence microscopy showed co-localization of the labeled phospholipids with markers for each of the expected membrane compartments. Furthermore, immunogold labeling and electron microscopy allowed direct visualization of the Cho phospholipids, which again were detected in the expected locations.

Having established that proparagyl-Cho is a good Cho analog, and that the modified phospholipids behave as normal within the cell, the authors tested its use in animals. Organs harvested from mice injected with propargyl-Cho, sectioned and stained, were all strongly labeled with fluorescent azide. Thus, the synthesis of Cho phospholipids in organs and tissues can be visually assayed using this method.

Fluorescence and electron microscopy have proven to be invaluable tools for the study of protein trafficking and function in cells. This study brings Cho phospholipids into the reach of these techniques, and promises great insights into their detailed cell biology.

Emma Leah