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Equilibrium physics breakdown reveals the active nature of red blood cell flickering

Abstract

Red blood cells, or erythrocytes, are seen to flicker under optical microscopy, a phenomenon initially described as thermal fluctuations of the cell membrane. But recent studies have suggested the involvement of non-equilibrium processes, without definitively ruling out equilibrium interpretations. Using active and passive microrheology to directly compare the membrane response and fluctuations on single erythrocytes, we report here a violation of the fluctuation–dissipation relation, which is a direct demonstration of the non-equilibrium nature of flickering. With an analytical model of the composite erythrocyte membrane and realistic stochastic simulations, we show that several molecular mechanisms may explain the active fluctuations, and we predict their kinetics. We demonstrate that tangential metabolic activity in the network formed by spectrin, a cytoskeletal protein, can generate curvature-mediated active membrane motions. We also show that other active membrane processes represented by direct normal force dipoles may explain the observed membrane activity. Our findings provide solid experimental and theoretical frameworks for future investigations of the origin and function of active motion in cells.

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Figure 1: Experimental set-up.
Figure 2: Membrane fluctuations and apparent response.
Figure 3: Analytic composite model of the red blood cell membrane.
Figure 4: Simulations mimicking the experimental conditions.

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Acknowledgements

We thank V. L. Lorman and J. Prost for careful reading of the analytical model. T.B. was founded by ANR-11-JSV5-0002 and by the Deutsche Forschungsgemeinschaft (DFG), Cells-in-Motion Cluster of Excellence (EXC 1003—CiM), University of Münster, Germany. H.T. acknowledges funding from the EMBL Interdisciplinary Postdoc program under Marie Curie Actions FP7-PEOPLE-2011-COFUND and generous support from the Bettencourt Schueller Foundation. D.A.F. acknowledges funding by the Alexander von Humboldt Foundation. D.A.F., T.A. and G.G. gratefully acknowledge a CPU time grant by the Jülich Supercomputing Center. N.S.G. acknowledges the Institut Curie’s Mayent-Rothschild Visiting Professor fund and Labex CelTisPhyBio for their support during the stay at the Institut Curie. N.S.G. is the incumbent of the Lee and William Abramowitz Professorial Chair of Biophysics, and thanks ISF Grant 580/12 for support.

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T.B. and C.S. designed experiments; T.B. performed experiments; H.T., B.A. and J.-F.J. derived the analytical model; D.A.F., T.A. and G.G. designed the simulation set-up; D.A.F. performed simulations; T.B., H.T. and D.A.F. analysed the data; all authors discussed and interpreted results; all authors wrote the manuscript.

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Correspondence to T. Betz.

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Turlier, H., Fedosov, D., Audoly, B. et al. Equilibrium physics breakdown reveals the active nature of red blood cell flickering. Nature Phys 12, 513–519 (2016). https://doi.org/10.1038/nphys3621

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