Eukaryotic cells produce over 1,000 different lipid species that tune organelle membrane properties, control signalling and store energy1,2. How lipid species are selectively sorted between organelles to maintain specific membrane identities is largely unclear, owing to the difficulty of imaging lipid transport in cells3. Here we measured the retrograde transport and metabolism of individual lipid species in mammalian cells using time-resolved fluorescence imaging of bifunctional lipid probes in combination with ultra-high-resolution mass spectrometry and mathematical modelling. Quantification of lipid flux between organelles revealed that directional, non-vesicular lipid transport is responsible for fast, species-selective lipid sorting, in contrast to the slow, unspecific vesicular membrane trafficking. Using genetic perturbations, we found that coupling between energy-dependent lipid flipping and non-vesicular transport is a mechanism for directional lipid transport. Comparison of metabolic conversion and transport rates showed that non-vesicular transport dominates the organelle distribution of lipids, while species-specific phospholipid metabolism controls neutral lipid accumulation. Our results provide the first quantitative map of retrograde lipid flux in cells4. We anticipate that our pipeline for mapping of lipid flux through physical and chemical space in cells will boost our understanding of lipids in cell biology and disease.

Cluster of Excellence Physics of Life TU Dresden Dresden Germany

École polytechnique fédérale de Lausanne Lausanne Switzerland

Helmholtz Zentrum Dresden Rossendorf Institute of Resource Ecology Dresden Germany

J Heyrovský Institute of Physical Chemistry Academy of Sciences of the Czech Republic v v i Prague Czech Republic

Max Planck Institute of Molecular Cell Biology and Genetics Dresden Germany

Technische Universität Dresden Biotechnologisches Zentrum Center for Molecular and Cellular Bioengineering Dresden Germany

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Harayama, T. & Riezman, H. Understanding the diversity of membrane lipid composition. Nat. Rev. Mol. Cell Biol. 19, 281–296 (2018). PubMed DOI

van Meer, G., Voelker, D. R. & Feigenson, G. W. Membrane lipids: where they are and how they behave. Nat. Rev. Mol. Cell Biol. 9, 112–124 (2008). PubMed DOI PMC

Kim, Y. & Burd, C. G. Lipid Sorting and organelle identity. Cold Spring Harb. Perspect. Biol. 15, a041397 (2023). PubMed DOI

Moon, H., Iglesias-Artola, J. M., Hersemann, L. & Nadler, A. Lipid imaging: quantitative imaging of species-specific lipid transport in mammalian cells. https://doi.org/21.11101/0000-0007-FCE5-B (Max Planck Institute of Molecular Cell Biology and Genetics, 2025).

Klose, C., Surma, M. A. & Simons, K. Organellar lipidomics—background and perspectives. Curr. Opin. Cell Biol. 25, 406–413 (2013). PubMed DOI

Sampaio, J. L. et al. Membrane lipidome of an epithelial cell line. Proc. Natl Acad. Sci. USA 108, 1903–1907 (2011). PubMed DOI PMC

Holthuis, J. C. M. & Menon, A. K. Lipid landscapes and pipelines in membrane homeostasis. Nature 510, 48–57 (2014). PubMed DOI

Reinisch, K. M. & Prinz, W. A. Mechanisms of nonvesicular lipid transport. J. Cell Biol. 220, e202012058 (2021). PubMed DOI PMC

Koivusalo, M., Jansen, M., Somerharju, P. & Ikonen, E. Endocytic trafficking of sphingomyelin depends on its acyl chain length. MBoC 18, 5113–5123 (2007). PubMed DOI PMC

Haberkant, P. & Holthuis, J. C. M. Fat & fabulous: bifunctional lipids in the spotlight. Biochim. Biophys. Acta 1841, 1022–1030 (2014). PubMed DOI

Höglinger, D. et al. Trifunctional lipid probes for comprehensive studies of single lipid species in living cells. Proc. Natl Acad. Sci. USA 114, 1566–1571 (2017). PubMed DOI PMC

Haberkant, P. et al. In vivo profiling and visualization of cellular protein-lipid interactions using bifunctional fatty acids. Angew. Chem. Int. Ed. 52, 4033–4038 (2013). DOI

Höglinger, D. in Intracellular Lipid Transport. Methods in Molecular Biology Vol. 1949 (ed. Drin, G.) 95–103 (Humana Press, 2019); https://doi.org/10.1007/978-1-4939-9136-5_8 .

Altuzar, J. et al. Lysosome-targeted multifunctional lipid probes reveal the sterol transporter NPC1 as a sphingosine interactor. Proc. Natl Acad. Sci. USA 120, e2213886120 (2023). PubMed DOI PMC

Farley, S., Stein, F., Haberkant, P., Tafesse, F. G. & Schultz, C. Trifunctional sphinganine: a new tool to dissect sphingolipid function. ACS Chem. Biol. 19, 336–347 (2024). PubMed DOI PMC

Schuhmacher, M. et al. Live-cell lipid biochemistry reveals a role of diacylglycerol side-chain composition for cellular lipid dynamics and protein affinities. Proc. Natl Acad. Sci. USA 117, 7729–7738 (2020). PubMed DOI PMC

Höglinger, D., Nadler, A. & Schultz, C. Caged lipids as tools for investigating cellular signaling. Biochim. Biophys. Acta 1841, 1085–1096 (2014). PubMed DOI

Jiménez-López, C. & Nadler, A. Caged lipid probes for controlling lipid levels on subcellular scales. Curr. Opin. Chem. Biol. 72, 102234 (2023). PubMed DOI

Frank, J. A. et al. Photoswitchable diacylglycerols enable optical control of protein kinase C. Nat. Chem. Biol. 12, 755 (2016). PubMed DOI PMC

Morstein, J., Impastato, A. C. & Trauner, D. Photoswitchable lipids. ChemBioChem 22, 73–83 (2021). PubMed DOI

Haldar, S. & Chattopadhyay, A. in Fluorescent Methods to Study Biological Membranes (eds Mély, Y. & Duportail, G.) 37–50 (Springer, 2013); https://doi.org/10.1007/4243_2012_43 .

Klymchenko, A. S. & Kreder, R. Fluorescent probes for lipid rafts: from model membranes to living cells. Chem. Biol. 21, 97–113 (2014). PubMed DOI

Triebl, A. & Wenk, M. R. Analytical considerations of stable isotope labelling in lipidomics. Biomolecules 8, 151 (2018). PubMed DOI PMC

Postle, A. D. & Hunt, A. N. Dynamic lipidomics with stable isotope labelling. J. Chromatogr. B 877, 2716–2721 (2009). DOI

Thiele, C. et al. Tracing fatty acid metabolism by click chemistry. ACS Chem. Biol. 7, 2004–2011 (2012). PubMed DOI

Thiele, C., Wunderling, K. & Leyendecker, P. Multiplexed and single cell tracing of lipid metabolism. Nat. Methods 16, 1123–1130 (2019). PubMed DOI

Wunderling, K., Zurkovic, J., Zink, F., Kuerschner, L. & Thiele, C. Triglyceride cycling enables modification of stored fatty acids. Nat. Metab. 5, 699–709 (2023). PubMed DOI PMC

Koukalová, A. et al. Lipid driven nanodomains in giant lipid vesicles are fluid and disordered. Sci. Rep. 7, 5460 (2017). PubMed DOI PMC

Sarmento, M. J. et al. The impact of the glycan headgroup on the nanoscopic segregation of gangliosides. Biophys. J. 120, 5530–5543 (2021). PubMed DOI PMC

Li, G. et al. Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids. Proc. Natl Acad. Sci. USA 113, 14025–14030 (2016). PubMed DOI PMC

Berg, S. et al. ilastik: interactive machine learning for (bio)image analysis. Nat. Methods 16, 1226–1232 (2019). PubMed DOI

Merrill, A. H. Jr Sphingolipid and glycosphingolipid metabolic pathways in the era of sphingolipidomics. Chem. Rev. 111, 6387–6422 (2011). PubMed DOI PMC

Titeca, K. et al. A system-wide analysis of lipid transfer proteins delineates lipid mobility in human cells. Preprint at bioRxiv https://doi.org/10.1101/2023.12.21.572821 (2023).

Chang, C.-L. & Liou, J. Phosphatidylinositol 4,5-bisphosphate homeostasis regulated by Nir2 and Nir3 proteins at endoplasmic reticulum-plasma membrane junctions. J. Biol. Chem. 290, 14289–14301 (2015). PubMed DOI PMC

Lees, J. A. & Reinisch, K. M. Inter-organelle lipid transfer: a channel model for Vps13 and chorein-N motif proteins. Curr. Opin. Cell Biol. 65, 66–71 (2020). PubMed DOI PMC

Hanna, M., Guillén-Samander, A. & Camilli, P. D. RBG motif bridge-like lipid transport proteins: structure, functions, and open questions. Ann. Rev. Cell Dev. Biol. 39, 409–434 (2023). DOI

Guillén-Samander, A. et al. A partnership between the lipid scramblase XK and the lipid transfer protein VPS13A at the plasma membrane. Proc. Natl Acad. Sci. USA 119, e2205425119 (2022). PubMed DOI PMC

Matoba, K. et al. Atg9 is a lipid scramblase that mediates autophagosomal membrane expansion. Nat. Struct. Mol. Biol. 27, 1185–1193 (2020). PubMed DOI

Li, Y. E. et al. TMEM41B and VMP1 are scramblases and regulate the distribution of cholesterol and phosphatidylserine. J. Cell Biol. 220, e202103105 (2021). PubMed DOI PMC

Lorent, J. H. et al. Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape. Nat. Chem. Biol. 16, 644–652 (2020). PubMed DOI PMC

van der Velden, L. M. et al. Heteromeric interactions required for abundance and subcellular localization of human CDC50 proteins and class 1 P4-ATPases*. J. Biol. Chem. 285, 40088–40096 (2010). PubMed DOI PMC

Bryde, S. et al. CDC50 proteins are critical components of the human class-1 P 4-ATPase transport machinery. J. Biol. Chem. 285, 40562–40572 (2010). PubMed DOI PMC

Harayama, T. Metabolic bias: Lipid structures as determinants of their metabolic fates. Biochimie 215, 34–41 (2023). PubMed DOI

Vance, J. E., Aasman, E. J. & Szarka, R. Brefeldin A does not inhibit the movement of phosphatidylethanolamine from its sites for synthesis to the cell surface. J. Biol. Chem. 266, 8241–8247 (1991). PubMed DOI

Kaplan, M. R. & Simoni, R. D. Intracellular transport of phosphatidylcholine to the plasma membrane. J. Cell Biol. 101, 441–445 (1985). PubMed DOI

Wong, L. H., Čopič, A. & Levine, T. P. Advances on the transfer of lipids by lipid transfer proteins. Trends Biochem. Sci 42, 516–530 (2017). PubMed DOI PMC

Farley, S. E. et al. Trifunctional fatty acid derivatives: the impact of diazirine placement. Chem. Commun. 60, 6651–6654 (2024). DOI