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.
U1006 INSERM, Université Aix-Marseille, Parc Scientifique et Technologique de Luminy, 163 avenue de Luminy, 13009 Marseille, France.
Atsushi
A
Miyagi
Laboratoire International Associé Centre National de la Recherche Scientifique et University of Illinois at Urbana-Champaign, UMR 7565, Université de Lorraine, BP 70239, 54506 Vandœuvre-lès-Nancy cedex, France.|Department of Physics, University of Illinois at Urbana-Champaign, 1110 West Green Street, Urbana, Illinois 61801, USA.
Christophe
C
Chipot
U1006 INSERM, Université Aix-Marseille, Parc Scientifique et Technologique de Luminy, 163 avenue de Luminy, 13009 Marseille, France.
Martina
M
Rangl
U1006 INSERM, Université Aix-Marseille, Parc Scientifique et Technologique de Luminy, 163 avenue de Luminy, 13009 Marseille, France.
Simon
S
Scheuring
D017304:Annexin A5 / Q000737:chemistry* / Q000378:metabolism*; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism*; D018625:Microscopy, Atomic Force / Q000379:methods*; D056004:Molecular Dynamics Simulation
D017304:Annexin A5; D008051:Lipid Bilayers; D002118:Calcium