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

Nature Nanotechnology volume 11, pages 783–790 (2016)Cite this article

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Abstract

Annexins are abundant cytoplasmic proteins that can bind to negatively charged phospholipids in a Ca2+-dependent manner, and are known to play a role in the storage of Ca2+ and membrane healing. Little is known, however, about the dynamic processes of protein–Ca2+–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 Ca2+ and membrane affinities depending on the assembly location, providing a wide Ca2+ 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 Ca2+ in the reversible binding of annexin to the membrane surface.

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Figure 1: The structure of 2D A5 arrays grown in the presence of 2 mM Ca2+ on a lipid bilayer containing 20% negatively charged PS.
Figure 2: Rotational freedom of the non-p6 A5 trimers.
Figure 3: Bio-enhanced HS-AFM featuring a fluid exchange pumping system and an optical pathway for pulsed UV laser uncaging of caged compounds.
Figure 4: Controlled buffer modifications allow detailed analysis of the A5–Ca2+–membrane interaction.
Figure 5: A5 Ca2+-mediated buffering and apparent assembly-related Ca2+ affinity.
Figure 6: Molecular dynamics simulations of A5 binding to the surface of a DOPC:DOPS bilayer.

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Acknowledgements

The authors thank H. Haigler for sharing insights in annexin biophysics and structural biology and for valuable comments on the manuscript, and A. Karner for assistance with the setting up of the fluid exchange system. This work was funded by the ANR grants ANR-Nano (ANR-12-BS10-009-01) and ANR-BBMS (ANR-12-BSV8-0006-01) and a European Research Council (ERC) Grant (No. 310080). The GENCI and CINES, Montpellier, France, are acknowledged for the provision of computer time.

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Authors and Affiliations

  1. U1006 INSERM, Université Aix-Marseille, Parc Scientifique et Technologique de Luminy, 163 avenue de Luminy, Marseille, 13009, France

    Atsushi Miyagi, Martina Rangl & Simon Scheuring

  2. Laboratoire International Associé Centre National de la Recherche Scientifique et University of Illinois at Urbana-Champaign, UMR 7565, Université de Lorraine, BP 70239, Vandœuvre-lès-Nancy cedex, 54506, France

    Christophe Chipot

  3. Department of Physics, University of Illinois at Urbana−Champaign, 1110 West Green Street, Urbana, 61801, Illinois, USA

    Christophe Chipot

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  1. Atsushi Miyagi
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Contributions

S.S., A.M and C.C conceived and designed the experiments. A.M., C.C. and M.R. performed the experiments. S.S., C.C and A.M analysed the data. S.S., C.C and A.M wrote the paper.

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Correspondence to Simon Scheuring.

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The authors declare no competing financial interests.

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Miyagi, A., Chipot, C., Rangl, M. et al. High-speed atomic force microscopy shows that annexin V stabilizes membranes on the second timescale. Nature Nanotech 11, 783–790 (2016). https://doi.org/10.1038/nnano.2016.89

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