Leaflet specific phospholipid imaging using genetically encoded proximity sensors.

The lipid composition of cells varies widely across organelles and between individual membrane leaflets. Transport proteins acting within and between membranes are thought to generate this heterogeneity, but measuring their functions has been hampered by limited tools for imaging lipid composition at relevant spatial resolutions. Here we present fluorogen-activating coincidence 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 (LTP) that traffic phosphatidylcholine pools between the ER and mitochondria. We then show that transmembrane domain-containing FAPs can be used to reveal the membrane asymmetry of multiple lipid classes that is generated at the trans-Golgi network (TGN). Using FACES, we show how the asymmetry of a phosphatidylserine analogue at the TGN is supported by the activity of both cytosolic LTPs and transmembrane flippases. Lastly, we demonstrate that FACES is a generalizable tool for subcellular detection of other molecule classes by measuring changes in mitochondrial -acetylhexosamine levels. These results introduce the application of fluorogenic tags for spatially-defined molecular imaging.