Practical Labeling Methodology for Choline‐Derived Lipids and Applications in Live Cell Fluorescence Imaging

Abstract

Lipids of the plasma membrane participate in a variety of biological processes, and methods to probe their function and cellular location are essential to understanding biochemical mechanisms. Previous reports have established that phosphocholine-containing lipids can be labeled by alkyne groups through metabolic incorporation. Herein, we have tested alkyne, azide and ketone-containing derivatives of choline as metabolic labels of choline-containing lipids in cells. We also show that 17-octadecynoic acid can be used as a complementary metabolic label for lipid acyl chains. We provide methods for the synthesis of cyanine-based dyes that are reactive with alkyne, azide and ketone metabolic labels. Using an improved method for fluorophore conjugation to azide or alkyne-modified lipids by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), we apply this methodology in cells. Lipid-labeled cell membranes were then interrogated using flow cytometry and fluorescence microscopy. Furthermore, we explored the utility of this labeling strategy for use in live cell experiments. We demonstrate measurements of lipid dynamics (lateral mobility) by fluorescence photobleaching recovery (FPR). In addition, we show that adhesion of cells to specific surfaces can be accomplished by chemically linking membrane lipids to a functionalized surface. The strategies described provide robust methods for introducing bioorthogonal labels into native lipids.

Click-EM for imaging metabolically tagged nonprotein biomolecules

“…Cho-phospholipids can be tracked via fluorescence microscopy using choline analogs such as propargyl-choline [ 11 ] and azido-choline ( AzCho, Fig. 4a ) [ 31 ]. Azide-functionalized analogs such as AzCho are especially versatile metabolic labeling substrates, since in addition to modification by CuAAC, they are also susceptible to strain-promoted ligation with cyclooctynes—a reaction referred to as “copper free click chemistry” that is compatible with live cells [ 16 – 17 ].…”

Metabolic labeling of glycerophospholipids via clickable analogs derivatized at the lipid headgroup

“…Multiple reports have showcased the efficacy of labeling strategies for elucidating these complex processes. Cairo and co-workers followed up on lipid metabolic labeling using PCho, AECho and a ketone-containing choline (Li et al, 2014). In doing so, they explored various live cell imaging protocols and showed that lipid dynamics could be studied via fluorescence recovery by photobleaching, and that cells could be attached onto functionalized surfaces by click chemistry.…”

Metabolic phospholipid labeling of intact bacteria enables a fluorescence assay that detects compromised outer membranes

“…Two analogs of Cho, PCho and 1-azidoethyl-choline (AECho) ( Fig. 1B), have been used to metabolically label PC in mammalian cells (27,39,40). Both analogs react with an azide or alkyne after incorporation into PC to allow further detection.…”

“…M9 minimal medium with maltose (M9+malt, per liter containing 0.3% KH2PO4, 0.6% Na2HPO4, 0.5% NaCl, 0.1% NH4Cl, 2 mM MgSO4, 0.1 mM CaCl2, 0.2% maltose, sterile filtered) was used for washing or flow cytometry medium. Choline chloride (Cho) was purchased from Sigma Aldrich and propargylcholine bromide (PCho) from Aobious; 1-azidoethyl-choline iodide (AECho) was synthesized as described (40). AlexaFluor488 DIBO alkyne (Alexa488-DIBO) and AlexaFluor488 sDIBO alkyne (Alexa488-sDIBO) were obtained from Thermo Fisher Scientific.…”