A Membrane Tubule Bilayer Assay for Curvature Sorting of Phosphatidic Acid

Abstract

Phosphatidic acid (PA) is a signaling lipid that is thought to support membrane fusion by stabilizing negative curvature. PA is the product of phospholipase D, an enzyme that is essential for exocytosis. Using supported lipid bilayers with tubules to induce curvature, we identify to what extent PA localizes to curvature. Liposomes containing POPC, pegylated DOPE, and dye-labeled lipids with varying concentrations of NaCl (20 mM to 1 M) were deposited on glass to form bilayers with connected tubules. Tubules were then imaged using TIRF or confocal microscopy. The fluorescence intensity is related to the amount of the lipid present, meaning tubules appear brighter than flat regions. Using nitrobenzoxadiazole(NBD)-labeled lipids and its quencher, dithionite, we then selectively quench the outer leaflet of the bilayer, allowing us to determine which curvature a lipid localizes to; the outer leaflet is positively curved and the inner leaflet is negatively curved. Melittin was then added to form pores in the membrane, allowing dithionite to quench the inner leaflet. Current data suggests that PA localizes to the inner leaflet of tubules (negative curvature) in bilayers and that PA could be a way that the curvature observed during membrane fusion is stabilized.