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Updated: Jun 26, 2025

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes
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Leaflet specific phospholipid imaging using genetically encoded proximity sensors.

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    Researchers developed a new tool, fluorogen-activating coincidence sensing (FACES), to image lipid composition and orientation within living cells. This breakthrough enables quantitative analysis of lipid transport and membrane asymmetry, advancing cell biology research.

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    Area of Science:

    • Cell Biology
    • Biochemistry
    • Molecular Imaging

    Background:

    • Cellular lipid composition is heterogeneous across organelles and membrane leaflets.
    • Measuring lipid composition and transbilayer orientation at high resolution is challenging.
    • Existing tools lack the specificity and resolution for quantitative subcellular lipid analysis.

    Purpose of the Study:

    • To introduce fluorogen-activating coincidence sensing (FACES), a novel chemogenetic tool for quantitative lipid imaging in living cells.
    • To investigate the roles of lipid transfer proteins (LTPs) in trafficking phosphatidylcholine between the ER and mitochondria.
    • To reveal membrane asymmetry at the trans-Golgi network (TGN) and identify contributing proteins.

    Main Methods:

    • Development of FACES, combining bioorthogonal chemistry with genetically encoded fluorogen-activating proteins (FAPs).
    • Application of FACES for quantitative imaging of subcellular lipid pools and their transbilayer orientation.
    • Utilizing transmembrane domain-containing FAPs to assess membrane asymmetry of various lipid classes.

    Main Results:

    • FACES successfully imaged subcellular lipid pools and their orientation in living cells.
    • Identified roles for specific LTPs in phosphatidylcholine transport between the ER and mitochondria.
    • Revealed membrane asymmetry at the TGN for multiple lipid classes, driven by cytosolic LTPs and transmembrane flippases.
    • Demonstrated FACES' generalizability for detecting other molecule classes, such as mitochondrial N-acetylhexosamine.

    Conclusions:

    • FACES provides a powerful, generalizable platform for quantitative, spatially-defined molecular imaging in living cells.
    • The tool elucidates the mechanisms generating and maintaining lipid asymmetry at organelle membranes.
    • This work advances our understanding of lipid dynamics and transport in cellular membranes.