Mechanical unfolding of single filamin A (ABP‐280) molecules detected by atomic force microscopy

Abstract

Filamin A (ABP-280), which is an actin-binding protein of 560 kDa as a dimer, can, together with actin filaments, produce an isotropic cross-linked three-dimensional network (actin/filamin A gel) that plays an important role in mechanical responses of cells in processes such as maintenance of membrane stability and translational locomotion. In this study, we investigated the mechanical properties of single filamin A molecules using atomic force microscopy. In force^extension curves, we observed sawtooth patterns corresponding to the unfolding of individual immunoglobulin (Ig)-fold domains of filamin A. At a pulling speed of 0.37 W Wm/s, the unfolding interval was sharply distributed around 30 nm, while the unfolding force ranged from 50 to 220 pN. This wide distribution of the unfolding force can be explained by variation in values of activation energy and the width of activation barrier of 24 Ig-fold domains of the filamin A at the unfolding transition. This unfolding can endow filamin A with great extensibility. The refolding of the unfolded chain of filamin A occurred when the force applied to the protein was reduced to near zero, indicating that its unfolding is reversible. Based on these results, we discuss here the physiological implications of the mechanical properties of single filamin A molecules. ß 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Stress-Dependent Elasticity of Composite Actin Networks as a Model for Cell Behavior

“…The mechanical response of the composite FLNa-F-actin networks eludes either the current theoretical models for entangled F-actin solutions or those for rigidly cross-linked F-actin networks. Instead, we speculate that the FLNa cross-links mediate the dynamic nonlinear mechanical response of these networks through an interplay between the nonlinear divergence of the stiffness of individual semiflexible FLNa cross-links with a force-and time-dependent unfolding of sheets within the FLNa molecule [15] as well as binding kinetics of the FLNa molecule with F-actin. Alternatively, current models of prestressed structures suggest that their mechanical response depends very weakly on the elasticity of the individual elements [23].…”

“…It is a homodimer consisting of an actin-binding domain, 24 -sheet repeats, and two unstructured sequences of 32 amino acids [14]. Atomic force measurements show that, at small forces, FLNa can be modeled as a wormlike chain with a persistence length of 20 nm [15]. At higher forces of 50 -100 pN, the -sheet repeat sequences reversibly unfold, doubling the contour length [15].…”

“…Atomic force measurements show that, at small forces, FLNa can be modeled as a wormlike chain with a persistence length of 20 nm [15]. At higher forces of 50 -100 pN, the -sheet repeat sequences reversibly unfold, doubling the contour length [15]. We use recombinant human FLNa purified from Sf9 cell lysates [16] and globular actin purified from rabbit skeletal muscle.…”

Nonlinear Elasticity of Composite Networks of Stiff Biopolymers with Flexible Linkers

“…The compliance of this network is dominated by the flexible cross-linkers, while the much stiffer filaments act mainly as a scaffold for the cross-linkers, ensuring rigidity of the network as a whole. Recent experiments have demonstrated that flexible biological cross-linkers such as filamin can be described as a semiflexible polymer using the wormlike chain (WLC) model [17,18]. The cross-linkers are characterized by their contour length ℓ 0 and persistence length ℓ p [15].…”

Filamin-regulated F-actin Assembly Is Essential for Morphogenesis and Controls Phototaxis in Dictyostelium

“…Using atomic force microscopy 50 -220 pN have been reported; this unfolding is reversible, and the unfolded chains fold back when the external force is removed (31)(32)(33). The unfolding of the ddFLN rod was particularly interesting as one of the repeats, repeat 4, unfolded in two steps and refolded along the same pathway (33,34).…”