Kyoto University achieves success in labeling fatty acid-containing lipids using intracellular organelles

Fatty acids taken up into cells are converted into various lipids such as phospholipids and neutral lipids, becoming components of biological membranes and sources of energy. Various types of fatty acids exist, differing in chain length and the number and position of double bonds, which creates lipid diversity while simultaneously making lipid analysis complex and difficult. A research group led by Professor Itaru Hamachi, Associate Professor Tomonori Tamura, and Graduate Students Takami Kimura (at the time of the research), and Seita Kawamoto from the Graduate School of Engineering at Kyoto University has developed a new method that enables labeling of fatty acid-containing lipids, which are components of biological membranes, using intracellular organelles, making it possible to analyze their composition, molecular structure, and dynamics. This is expected to lead to a major breakthrough toward clarifying intracellular lipid metabolic mechanisms, many aspects of which remain unexplored. Their research was published in the Journal of the American Chemical Society.

The research group developed a new fatty acid metabolite labeling method using azido fatty acids (AFA) and organelle-localizing click reagents (OCD) that the research group had developed. In this method, after introducing AFA into cells, OCD containing strained alkynes is added to the cells, and copper-free click reactions (SPAAC) are performed specifically for target organelles, thereby selectively fluorescently labeling fatty acid metabolites within the organelles. The fluorescently labeled fatty acid metabolites can be analyzed qualitatively and quantitatively with TLC and mass spectrometry. Their organelle localization information can be identified by the wavelength of the labeled fluorescent dye.

AzPal (Azido-palmitic acid) and AzOle (Azido-oleic acid) were adopted as azido analogs of fatty acids most abundantly present in mammalian cells, and these were introduced into human K562 cells for 15 minutes. Mass spectrometry results confirmed that these were efficiently incorporated into phosphatidylcholine (PC), phosphatidylethanolamine (PE), triacylglycerol (TAG), diacylglycerol (DAG), and other lipids, similar to natural fatty acids. Subsequently, azidized lipids were selectively fluorescently labeled with OCD within different organelle membranes such as intracellular endoplasmic reticulum/Golgi apparatus, mitochondria, lysosomes, and plasma membrane, after which lipids were extracted from cells and TLC analysis was performed.

The results showed that each lipid species that incorporated AFA was labeled by OCD. Furthermore, it was revealed that the ratios between labeled lipid species differed among organelles. Moreover, structural analysis of fluorescently labeled lipids with mass spectrometry revealed that lipid molecules with various carbon chain lengths and degrees of unsaturation were labeled for each lipid species. Quantitative analysis of these showed that mitochondria contained more PE with polyunsaturated fatty acids compared with the endoplasmic reticulum/Golgi apparatus. These results are in good agreement with previous findings.

Furthermore, pulse-chase analysis was conducted to examine the dynamics of newly biosynthesized fatty acid-containing lipids in more detail. The analysis revealed that the quantitative changes in PE and PC differed between the endoplasmic reticulum/Golgi apparatus and mitochondria.

These results demonstrated that this method is useful for qualitatively and quantitatively analyzing the intracellular dynamics of fatty acid metabolites at the organelle level.

Tamura commented: "This research diverged significantly from our previous fluorescence imaging research on lipids to take on the challenge of structural and quantitative analysis of organelle lipids using mass spectrometry. Comprehensive mass spectrometry of lipids was a completely unknown challenge for our group, but Kimura established the foundation of this method. Kawamoto, who took over from him, persistently refined the data using the most advanced mass spectrometry equipment, leading to excellent results. In addition, for publication, lab graduate Dr. Fujisawa's treasured molecules and Dr. Tsuchiya's advice were key to acceptance. The method developed in this study is expected to serve as a useful research tool for analyzing fatty acid-containing lipids with high spatial resolution, and to greatly accelerate lipid research, which is still poorly understood, from an unprecedented perspective."

Journal Information
Publication: Journal of the American Chemical Society
Title: Subcellular Analysis of Fatty Acid Metabolism Using Organelle-Selective Click Chemistry
DOI: 10.1021/jacs.5c02871