Annexin-A5 Stabilizes Membrane Defects via Modulating Lipid Order

Abstract

Annexins are abundant cytoplasmic proteins that bind in a Ca2+-dependent manner to the inner leaflet of the plasma membranes (or bio-mimetic membranes) that expose negatively charged phospholipids. Annexins have been implicated in several cell biological processes including membrane stabilization and repair, and endocytosis and exocytosis. However, due to the lack of structure-function correlation at molecular-level, the roles of the annexin-membrane interactions involved in these processes are still not clear. Here, we report the direct monitoring of annexin-V (A5) interactions with membranes in real time using high-speed atomic force microscopy (HS-AFM) and confocal laser scanning microscopy (CLSM). We show that PS-rich membranes can be stabilized by the formation of A5 two-dimensional (2D)-lattices, and ultimately transformed into ordered structures. We also demonstrate that the A5 2D-lattice can slow down lipid diffusivity and increase lipid order using fluorescence recovery after photobleaching (FRAP) and environmental-sensitive, fluorescent probes, respectively. Molecular dynamic simulation further provides atomistic details on how the order of the A5 2D-lattice translates into the underlying lipids through a layer of Ca2+-ions. Finally, we provide evidence that the A5 2D-lattice promotes membrane repair via the patch resealing mechanism. Overall, our results suggest that the A5 2D-lattice can stabilize membrane and prevent wound expansion in the seconds time scale, which is an early key step in cell membrane repair.