High-speed atomic force microscopy shows that annexin V stabilizes membranes on the second timescale.

Annexins are abundant cytoplasmic proteins that can bind to negatively charged phospholipids in a Ca(2+)-dependent manner, and are known to play a role in the storage of Ca(2+) and membrane healing. Little is known, however, about the dynamic processes of protein-Ca(2+)-membrane assembly and disassembly. Here we show that high-speed atomic force microscopy (HS-AFM) can be used to repeatedly induce and disrupt annexin assemblies and study their structure, dynamics and interactions. Our HS-AFM set-up is adapted for such biological applications through the integration of a pumping system for buffer exchange and a pulsed laser system for uncaging caged compounds. We find that biochemically identical annexins (annexin V) display different effective Ca(2+) and membrane affinities depending on the assembly location, providing a wide Ca(2+) buffering regime while maintaining membrane stabilization. We also show that annexin is membrane-recruited and forms stable supramolecular assemblies within ∼5 s in conditions that are comparable to a membrane lesion in a cell. Molecular dynamics simulations provide atomic detail of the role played by Ca(2+) in the reversible binding of annexin to the membrane surface.