Objective: Lipids exert dynamic biological functions which are determined both by their fatty acyl compositions and spatiotemporal distributions inside the cell. However, it remains a daunting task to investigate any of these features for each of the more than 1000 lipid species due to a lack of a universal labeling method for individual lipid moieties in live cells. Here we report a de novo lipid labeling method for individual lipid species with precise acyl compositions in live cells. The method is based on the principle of de novo lipid remodeling of exogenously added lysolipids with fluorescent acyl-CoA, leading to the re-synthesis of fluorescence-labeled lipids which can be imaged by confocal microscopy.
Methods: The cells were incubated with lysolipids and a nitro-benzoxadiazolyl (NBD) labeled acyl-CoA. The newly remodeled NBD-labeled lipids and their subcellular localization were analyzed by confocal imaging in live cells. Thin layer chromatography was carried out to verify the synthesis of NBD-labeled lipids. The mitochondrial trafficking of NBD-labeled lipids was validated in live cells with targeted deletion of phospholipids transporters, including TRIAP1/PRELI protein complex and StarD7.
Results: Incubation cells with lysolipids and NBD-acyl-CoA successfully labeled major lipid species with precise acyl compositions, including phospholipids, cholesterol esters, and neutral lipids, which can be analyzed by confocal imaging in live cells. In contrast to exogenously labeled lipids, the de novo labeled lipids retained full biological properties of their endogenous counterparts, including subcellular localization, trafficking, and recognition by lipid transporters. This method also uncovered some unexpected features of newly remodeled lipids and their transporters.
Conclusions: The de novo lipid labeling method not only provides a powerful tool for functional analysis of individual lipid species and lipid transporters, but also calls for re-evaluation of previously published results using exogenously labeled lipids.
Keywords: Lipid remodeling; Lipid trafficking; NBD; Phospholipid transporters.
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