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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.