Preprint Subcellular imaging of lipids and sugars using genetically encoded proximity sensors, 2024, Moore et al.

[SNT Gatchaman]

Subcellular imaging of lipids and sugars using genetically encoded proximity sensors
William M. Moore; Roberto J. Brea; Caroline Knittel; Ellen Wrightsman; Brandon Hui; Jinchao Loui; Christelle F. Ancajas; Michael D. Best; Neal K. Devaraj; Itay Budin

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.


Link | PDF (Preprint: BioRxiv) [Open Access]

 

[SNT Gatchaman]

We hypothesized that challenges for lipid imaging could be overcome using protein-based lipid sensors, whose localization is precisely controlled through signal sequences or fusion to other protein components.

Here we validate the FACES strategy through imaging of discrete N3-lipid pools in the ER, mitochondria, and trans-Golgi network (TGN).

It has long been recognized that membrane compartmentalization inherently generates directionality between exoplasmic and cytoplasmic leaflets, which is reflected both in membrane protein topology and the transbilayer distribution of lipids. Detailed lipidomic investigation of membrane asymmetry has only been carried out in the PM of erythrocytes, whose lack of intracellular compartments allow for calculation of lipid accessibility to externally added phospholipases. While asymmetry is an emerging concept in membrane biology, the transbilayer distribution of phospholipids in intracellular membranes is poorly understood. There exists few, if any, tools capable of measuring membrane asymmetry in living cells and we asked if the spatial specificity of FACES could provide this capacity.

Our experiments demonstrate that FACES is an especially powerful tool for investigating cellular mechanisms by which lipids are transported between organelles and across individual membranes, an intense area of investigation. A plethora of shuttle and bridge-like LTPs that perform lipid exchange at contact sites and flippases/floppases/scramblases that act on bilayer asymmetry have been identified and biochemically tested.

 

[SNT Gatchaman]

https://en.wikipedia.org/wiki/Flippase
https://en.wikipedia.org/wiki/Phospholipid_scramblase