Measuring molecular rupture forces between single actin filaments and actin-binding proteins.
Actin-binding proteins (ABPs) regulate the assembly of actin filaments (F-actin) into networks and bundles that provide the structural integrity of the cell. Two of these ABPs, filamin and alpha-actinin, have been extensively used to model the mechanical properties of actin networks grown in vitro; however, there is a lack in the understanding of how the molecular interactions between ABPs and F-actin regulate the dynamic properties of the cytoskeleton. Here, we present a native-like assay geometry to test the rupture force of a complex formed by an ABP linking two quasiparallel actin filaments. We readily demonstrate the adaptability of this assay by testing it with two different ABPs: filamin and alpha-actinin. For filamin/actin and alpha-actinin/actin, we measured similar rupture forces of 40-80 pN for loading rates between 4 and 50 pN/s. Both ABP unfolding and conformational transition events were observed, demonstrating that both are important and may be a significant mechanism for the temporal regulation of the mechanical properties of the actin cytoskeleton. With this modular, single-molecule assay, a wide range of ABP/actin interactions can be studied to better understand cytoskeletal and cell dynamics.
Ferrer
Jorge M
JM
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Lee
Hyungsuk
H
Chen
Jiong
J
Pelz
Benjamin
B
Nakamura
Fumihiko
F
Kamm
Roger D
RD
Lang
Matthew J
MJ
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000185:Actinin / Q000378:metabolism; D000818:Animals; D001696:Biomechanical Phenomena; D003285:Contractile Proteins / Q000378:metabolism; D064448:Filamins; D018260:Gelsolin / Q000378:metabolism; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism*; D008958:Models, Molecular; D011817:Rabbits
D003285:Contractile Proteins; D064448:Filamins; D018260:Gelsolin; D008840:Microfilament Proteins; D000185:Actinin