Subcellular imaging of lipids and sugars using genetically encoded proximity sensors

Abstract

Live cell imaging of lipids and other metabolites is a long-standing challenge in cell biology. Bioorthogonal labeling tools allow for the conjugation of fluorophores to several phospholipid classes, but cannot discern their trafficking between adjacent organelles or asymmetry across individual membrane leaflets. 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 validate this approach for quantifying discrete phosphatidylcholine pools in the ER and mitochondria that are trafficked by lipid transfer proteins. We then show that transmembrane domain-containing FAPs can be used to reveal the membrane asymmetry of multiple lipid classes that are generated in the trans-Golgi network. Lastly, we show that FACES is a generalizable tool for subcellular bioorthogonal imaging by measuring changes in mitochondrial N-acetylhexosamine levels. These results demonstrate the use of fluorogenic tags for spatially-defined molecular imaging.

Imaging Interorganelle Phospholipid Transport by Extended Synaptotagmins Using Bioorthogonally Tagged Lipids

“…In sum, these data support that E-Syts can transport unnatural fluorescent lipids that are rapidly generated on the cytosolic leaflet of the PM, using PLD transphosphatidylation and bioorthogonal tagging, to the ER. As such, IMPACT constitutes a tool for directly monitoring PM-to-ER interorganelle lipid transport by E-Syts and other LTPs, complementing other emerging toolsets such as METALIC, time-resolved imaging of bifunctional lipid probes, and visualization of metabolically labeled lipids using bioorthogonal chemistry and fluorogen-activated proteins. ,, The broad substrate specificity of E-Syts and likely other LTPs toward unnatural lipids with bulky head groups is intriguing and suggests that these LTPs may be capable of transporting a wide array of glycerolipids in cells depending on experimental perturbations, to restore lipid homeostasis. Beyond E-Syts and ER-PM contact sites, we envision that our approach may be generalizable to LTPs with broad substrate scope  i.e., those that are headgroup-agnostic  acting at other membrane contact sites.…”