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Leaflet-specific phospholipid imaging using genetically encoded proximity sensors

Nature Chemical Biology volume 22, pages 128–139 (2026) Cite this article

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Abstract

The lipid composition of cells varies widely across organelles and between individual membrane leaflets. Transport proteins are thought to generate this heterogeneity, but measuring their functions in vivo has been hampered by limited tools for imaging lipids at relevant spatial resolutions. Here we present fluorogen-activating coincidence encounter sensing (FACES), a chemogenetic tool capable of quantitatively imaging subcellular lipid pools and reporting their transbilayer orientation in living cells. FACES combines bioorthogonal chemistry with genetically encoded fluorogen-activating proteins (FAPs) for reversible proximity sensing of conjugated molecules. We first apply this approach to identify roles for lipid transfer proteins that traffic phosphatidylcholine pools between the ER and mitochondria. We then show that transmembrane domain-containing FAPs can reveal the membrane asymmetry of multiple lipid classes in the trans-Golgi network and be used to investigate the mechanisms that generate it. Finally, we present that FACES can be applied to measure glycans and other molecule classes.

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Fig. 1: Conceptual development of FACES.
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Fig. 2: Using FACES to analyze PC trafficking between the mitochondrion and the ER.
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Fig. 3: Imaging lipid asymmetry generated at the TGN with FACES.
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Fig. 4: Mechanisms contributing to PS asymmetry at the TGN.
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Fig. 5: Intracellular imaging of N-acetylhexosamine sugars with FACES.
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Data availability

All data are available in the article, Supplementary Information and Source data files. Plasmids generated in this study have been deposited in Addgene (242990, 242991, 242992, 242993, 242994). Source data are provided with this paper.

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Acknowledgements

N.-F. Lipp provided discussions and comments. G. Perkunnaz (UCSD, Dept. of Neurobiology) provided discussions and reagents. J. Santini and the UCSD Microscopy Core (NS047101 and OD030505) provided assistance with microscopy. C. Burd (Yale University, School of Medicine) and J. Lippincott-Schwartz (Janelia Research Campus) provided plasmids. The work was supported by the National Institutes of Health (NIH; grants R35-GM142960 to I.B. and R35-GM141939 to N.K.D.), the Allen Family Philanthropies and Frontiers Group, and the National Science Foundation (grant CHE-2310263 to M.D.B.). R.J.B. acknowledges Agencia Estatal de Investigación and the Ministerio de Ciencia e Innovación (RYC2020-030065-I). The global polar phospholipid analyses described in this work were performed at the Kansas Lipidomics Research Center Analytical Laboratory. Instrument acquisition and lipidomics method development were supported by the National Science Foundation (including support from the Major Research Instrumentation program; most recent award DBI-1726527), K-IDeA Networks of Biomedical Research Excellence of the NIH (P20GM103418), USDA National Institute of Food and Agriculture (Hatch/Multi-State project 1013013) and Kansas State University.

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Author notes

  1. Roberto J. Brea

    Present address: Centro Interdisciplinar de Química e Bioloxía (CICA), Universidade da Coruña, A Coruña, Spain

  2. These authors contributed equally: Roberto J. Brea, Caroline H. Knittel.

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA

    William M. Moore, Roberto J. Brea, Caroline H. Knittel, Ellen Wrightsman, Brandon Hui, Christopher J. Obara, Neal K. Devaraj & Itay Budin

  2. Department of Chemistry, University of Tennessee, Knoxville, TN, USA

    Jinchao Lou, Christelle F. Ancajas & Michael D. Best

  3. Department of Pharmacology, University of California, San Diego, La Jolla, CA, USA

    Christopher J. Obara

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Contributions

W.M.M. and I.B. conceived the project and designed all experiments. All experiments were conducted by W.M.M. unless otherwise noted. R.J.B. synthesized the MG probes. C.H.K. conducted LC–MS/MS experiments. E.W. and B.H. assisted W.M.M. in cloning constructs. J.L., C.F.A. and M.D.B. synthesized and provided the inositol and serine probes. C.J.O. assisted in microscopy. N.K.D. provided expertise in bioorthogonal chemistry and support to R.J.B. and C.H.K.

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Correspondence to Itay Budin.

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Supplementary Figs. 1–11 and Notes 1 and 2.

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Supplementary Video 1 (download AVI )

Mobility of TGN vesicles containing the soluble secretory cargo marker SP-mCherry.

Supplementary Video 2 (download AVI )

Mobility of TGN vesicles containing cytosolic PC and exoplasmic SM.

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Mobility of TGN vesicles containing cytosolic PC and protein cargoes.

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Moore, W.M., Brea, R.J., Knittel, C.H. et al. Leaflet-specific phospholipid imaging using genetically encoded proximity sensors. Nat Chem Biol 22, 128–139 (2026). https://doi.org/10.1038/s41589-025-02021-z

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