A Fluorescence-based Assay for Measuring Phospholipid Scramblase Activity in Giant Unilamellar Vesicles

A Fluorescence-based Assay for Measuring Phospholipid Scramblase Activity in Giant Unilamellar Vesicles

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Transbilayer movement of phospholipids in biological membranes is mediated by a diverse set of lipid arcade smokey the bear belt transporters.Among them are scramblases that facilitate rapid bi-directional movement of lipids without metabolic energy input.In this protocol, we describe the incorporation of phospholipid scramblases into giant unilamellar vesicles (GUVs) formed from scramblase-containing large unilamellar vesicles by electroformation.

We also describe how to analyze their activity using membrane-impermeant sodium dithionite, to bleach symmetrically incorporated fluorescent ATTO488-conjugated phospholipids.The fluorescence-based readout allows single vesicle tracking for a large number of settled/immobilized GUVs, and provides a well-defined experimental setup to directly characterize these lipid transporters at the molecular level.Graphic abstract: Giant unilamellar vesicles (GUVs) are formed by electroformation from large unilamellar vesicles (LUVs) containing phospholipid scramblases (purple) and trace amounts of a fluorescent lipid reporter (green).

The scramblase activity is analyzed by a fluorescence-based assay of single GUVs, using the membrane-impermeant quencher dithionite.Sizes not to scale.Modified moondrop quarks from Mathiassen et al.

(2021).