Annexin V Drives Stabilization of Damaged Asymmetric Phospholipid Bilayers

Annexins are soluble membrane-binding proteins that associate in a calcium dependent manner with anionic phospholipids. They play roles in membrane organization, signaling and vesicle transport and in several disease states including thrombosis and inflammation. Annexin V is believed to be involved in membrane repair. Mediated through binding to phosphatidylserine exposed at damaged plasma membrane, the protein forms crystalline networks that seal or stabilize small membrane tears. Herein, we model this biochemical mechanism to simulate membrane healing at microcavity array supported, transversally asymmetric, lipid bilayers (MSLBs) comprising 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS). Varying annexin V concentration, lipid composition, and DOPS presence at each leaflet, fluorescence imaging and correlation spectroscopy confirmed that when DOPS was present at the external, annexin V, contacting leaflet, the protein assembled rapidly at the membrane interface to form a layer. From electrochemical impedance studies, the annexin layer decreased membrane capacitance while reducing resistance. With DOPS incorporated only at the lower (proximal) leaflet, no appreciable annexin assembly was observed over the first 21 h. This suggests that membrane asymmetry is preserved over this window and transversal diffusion of DOPS is slow. Intense laser light applied to the membrane, in which DOPS is initially isolated at the lower leaflet, was found to simulate membrane damage, stimulating the rapid assembly of annexin V at the membrane interface confirmed by fluorescence imaging, correlation spectroscopy, and electrochemical impedance measurements. The damage induced by light increased impedance and decreased membrane resistance. The resulting bilayer annexin V patched bilayer showed better temporal stability toward impedance changes when compared with that of the parent membrane. In summary, this simple model of annexin V assembly in a fluidic lipid membrane provides new insights into the assembly of annexins as well as an empirical basis for building patch-repair mechanisms into interfacial bilayer membrane assemblies.