Modulation of Annexin-Induced\nMembrane Curvature by\nCholesterol and the Anionic Lipid Headgroup during Plasma Membrane\nRepair

Annexins (ANXAs), calcium-sensitive phospholipid-binding\nproteins,\nare pivotal for cellular membrane repair, which is crucial for eukaryotic\ncell survival under membrane stress. With their unique trimeric arrangements\nand crystalline arrays on the membrane surface, ANXA4 and ANXA5 induce\nmembrane curvature and rapidly orchestrate plasma membrane resealing.\nHowever, the influence of cholesterol and anionic lipid headgroups\non annexin-induced membrane curvature remains poorly understood at\nthe molecular level. Using all-atom molecular dynamics simulations,\nwe measured the local curvature-induced underneath ANXA4 and ANXA5\nmonomers and trimers when they bind to lipid bilayers of distinct\nlipid compositions: PSPC (20% POPS, 80% POPC), PAPC (20% POPA, 80%\nPOPC), and PSPCCHL (14% POPS, 56% POPC, 30% cholesterol). Laser injury\nexperiments were conducted on MCF7 cells transfected to transiently\nexpress fluorescently labeled ANXA4 or ANXA5 to facilitate the examination\nof protein and lipid accumulation at the damage site. Annexin trimers\ninduce higher curvature than monomers, particularly with cholesterol\npresent. Annexin trimers induce similar curvatures on both PAPC and\nPSPC membranes. Notably, among monomers, ANXA5 induces the highest\ncurvature on PAPC, suggesting more efficient recruitment of ANXA5\ncompared with ANXA4 in the early stages of membrane repair near a\nlesion. Laser injury experiments confirm rapid coaccumulation of phosphatidic\nacid lipids with ANXA4 and ANXA5 at repair sites, potentially enhancing\nthe accumulation of annexins in the early stages of membrane repair.