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Structural and Mechanical Heterogeneity of the Erythrocyte Membrane Reveals Hallmarks of Membrane Stability

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U1006 INSERM, Aix-Marseille Université, Parc Scientifique de Luminy, Marseille, F-13009 France
PhysicalChemistry Curie—Institut Curie/CNRS UMR 168/Paris, France
*Address correspondence to [email protected]
Cite this: ACS Nano 2013, 7, 2, 1054–1063
Publication Date (Web):January 24, 2013
https://doi.org/10.1021/nn303824j
Copyright © 2013 American Chemical Society
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Abstract

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The erythrocyte membrane, a metabolically regulated active structure that comprises lipid molecules, junctional complexes, and the spectrin network, enables the cell to undergo large passive deformations when passing through the microvascular system. Here we use atomic force microscopy (AFM) imaging and quantitative mechanical mapping at nanometer resolution to correlate structure and mechanics of key components of the erythrocyte membrane, crucial for cell integrity and function. Our data reveal structural and mechanical heterogeneity modulated by the metabolic state at unprecedented nanometer resolution. ATP-depletion, reducing skeletal junction phosphorylation in RBC cells, leads to membrane stiffening. Analysis of ghosts and shear-force opened erythrocytes show that, in the absence of cytosolic kinases, spectrin phosporylation results in membrane stiffening at the extracellular face and a reduced junction remodeling in response to loading forces. Topography and mechanical mapping of single components at the cytoplasmic face reveal that, surprisingly, spectrin phosphorylation by ATP softens individual filaments. Our findings suggest that, besides the mechanical signature of each component, the RBC membrane mechanics is regulated by the metabolic state and the assembly of its structural elements.

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Materials and methods of samples shown in Supporting Figure S1 and details on the Peak Force Quantitative Nanomechanics Imaging and calculations, the characterization of the different samples prepared (Supporting Figure S1), the effect of ATP on the topographic and mechanical remodeling of intact RBCs at different loading forces (300, 400, 500, and retrieved 300 pN) (Supporting Figure S2), profile analysis of images shown in Supporting Figure S2 (Supporting Figure S3), structural insight of the RBC membrane on intact erythrocytes (Supporting Figure S4), the effect of MgATP on the topography and mechanics of the extracellular and cytoplasmic face of RBC membrane at different loading forces (300, 400, 500, and retrieved 300 pN) (Supporting Figures S5 and S7, respectively), an extended version of raw images obtained in these series is displayed in Figure 4 (Supporting Figure S6), a large scan size AFM image acquired on the cytoplasmic face of RBC membrane (Supporting Figure S8), and a series of images showing the control conditions of the different samples presented (Supporting Figure S9). This material is available free of charge via the Internet at http://pubs.acs.org.

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