Biophysical Society Thematic Meeting - October 13-15, 2015

Biophysics of Proteins at Surfaces: Assembly, Activation, Signaling

Wednesday Speaker Abstracts

Structural and Nanomechanical Features of Reconstructed Spectrin - Actin Membrane Cytoskeletons for Artificial Cell Realizations: an Atomic Force Microscopy Characterization Ivan Lopez-Montero 1,2 , Mario Encinar 3 , Santiago Casado 4 , Alicia Calzado-Martín 3 , Álvaro San Paulo 3 , Monserrat Calleja 3 , Marisela Velez 4,5 , Francisco Monroy 1,2 . 1 Complutense University, Madrid, Spain, 2 Instituto de Investigación Hospital Doce de Octubre (i+12)., Madrid, Spain, 3 Instituto de Microelectrónica de Madrid, CSIC, Tres Cantos, Spain, 4 IMDEA Nanociencia, Madrid, Spain, 5 Instituto de Catálisis y Petroleoquímica, CSIC, Madrid, Spain. The possibility to fabricate giant unilamellar vesicles (GUVs) composed of native membranes opens exciting opportunities for artificial cell synthesis. In particular, GUVs can be artificially prepared by electroswelling from native erythroid membranes (erythroGUVs). Erythroid membranes are naturally furnished with a spectrin cytoskeleton that supports their mechanical resilience upon blood stream. Previously, we have shown a method to reconstruct spectrin skeletons onto erythroGUVs when incubated with ATP. Here, we present a detailed nano- structural study of artificial erythroGUV skeletons adsorbed onto glass cover slides performed with a combination of Atomic Force Microscopy (AFM) and fluorescence optical microscopy imaging. Three different kinds of filaments have been identified depending on the ATP concentration. At low ATP (  M), separate actin- and spectrin-enriched filaments can be observed. At high ATP concentrations (mM), a highly-connected network is formed, the links between the nodes being complex filaments composed by actin and spectrin. From nano- mechanical AFM measurements, a value of the Young modulus Esp-act = 0.4 MPa is found for the complex filaments, whereas single spectrin- and actin-enriched fibers are found stiffer (Esp = 5 and Eact = 15 MPa, respectively). Upon further ATP, a reconstructed network emerges as a protein cytoskeleton that supports increased membrane rigidity in erythroGUVs.

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