Bifunctional Probes Reveal the Rules of Intracellular Ether Lipid Transport

Ether glycerophospholipids bear a long chain alcohol attached via an alkyl or vinyl ether bond at the sn1 position of the glycerol backbone. Ether lipids play a significant role in physiology and human health. However, their cellular functions remain largely unknown due to a lack of tools for identifying their subcellular localization and interacting proteins. Here, we address this methodological gap by synthesizing minimally modified bifunctional ether lipid probes by introducing diazirine and alkyne groups. To interrogate the subcellular kinetics of intracellular ether lipid transport in mammalian cells, we used a combination of fluorescence imaging, machine learning-assisted image analysis, and mathematical modelling. We find that alkyl-linked ether lipids are transported up to twofold faster than vinyl-linked species (plasmalogens), pointing to yet undiscovered cellular lipid transport machinery able to distinguish between linkage types differing by as little as two hydrogen atoms. We find that ether lipid transport predominantly occurs via non-vesicular pathways, with varying contributions from vesicular mechanisms between cell types. Altogether, our results suggest that differential recognition of alkyl- and vinyl ether lipids by lipid transfer proteins contributes to their distinct biological functions. In the future, the probes reported here will enable studying ether lipid biology in much greater detail through identification of interacting proteins and in-depth characterization of intracellular ether lipid dynamics.