Torsional rigidity of single actin filaments and actin–actin bond breaking force under torsion measured directly by in vitro  micromanipulation

Abstract

Knowledge of the elastic properties of actin filaments is crucial for considering its role in muscle contraction, cellular motile events, and formation of cell shape. The stiffness of actin filaments in the directions of stretching and bending has been determined. In this study, we have directly determined the torsional rigidity and breaking force of single actin filaments by measuring the rotational Brownian motion and tensile strength using optical tweezers and microneedles, respectively. Rotational angular f luctuations of filaments supplied the torsional rigidity as (8.0 ؎ 1.2) ؋ 10 ؊26 Nm 2 . This value is similar to that deduced from the longitudinal rigidity, assuming the actin filament to be a homogeneous rod. The breaking force of the actin-actin bond was measured while twisting a filament through various angles using microneedles. The breaking force decreased greatly under twist, e.g., from 600-320 pN when filaments were turned through 90؇, independent of the rotational direction. Our results indicate that an actin filament exhibits comparable f lexibility in the rotational and longitudinal directions, but breaks more easily under torsional load.Actin is a major protein involved in a variety of cellular motile events and in the maintenance of cell shape and form. Determining its elastic properties in the polymeric state is central to an understanding of its function (1, 2). Recently, actin filaments have been measured to be several-fold more flexible longitudinally in vitro (3) and in muscle (4-6) than many models of muscle contraction have assumed (7). This finding would seem to require reconsideration of aspects of such models (8-10). Because of its helical structure, an actin filament should experience not only longitudinal but also torsional loads during interactions with myosin (11). To explain the mechanism of force generation, it is also important to know the elastic behavior of actin filaments during torsion. The torsional rigidity of actin filament can be estimated based on its bending rigidity, assuming the actin filament to be a homogenous rod (12). However, this assumption is not necessarily correct, because the actin filament is a double-helical polymer of globular actin monomers (13,14). Spectroscopic (12,(15)(16)(17) and electron microscopic studies (18) have suggested that the elastic property of an actin filament is largely anisotropic in the directions of twisting, bending, and stretching, i.e., the torsional rigidity is much smaller than the bending and longitudinal ones, assuming the actin filament to be a homogeneous rod, whereas the normal mode analysis based on the atomic structure of actin has shown much larger torsional rigidity than those suggested by these studies (19).Here, we have directly determined the torsional rigidity of actin filaments and the actin-actin bond breaking force under torsion by manipulating single actin filaments with optical tweezers and microneedles, respectively. Developments in video-assist fluorescence microscopy have enabled direct observat...

Direct Measurement of the Torsional Rigidity of Single Actin Filaments

“…With direct imaging, on the other hand, Suzuki et al (1996) have analyzed the motion of bead aggregates bound to the tail of an actin filament and estimated the torsional rigidity of F-actin to be of a magnitude similar to our values. Tsuda et al (1996) have also suggested a similar value.…”

Mechanical Strength of Sarcomere Structures of Skeletal Myofibrils Studied by Submicromanipulation

“…These would be either ruptures of thin actin filaments from Z-disks and/or ruptures of Z-disk components as comparable ruptures were not observed for calpaintreated myofibrils. Alternatively, they could be due to the splitting of actin filaments, 250-550 pN (Tsuda et al, 1996), and/or intramolecular ruptures of connectin filaments, about 200 pN (Li et al, 2002). Notably, in the AFM experiments, the ruptures that took place in trypsin-treated and calpain-treated myofibrils were significantly smaller in magnitude than those that took place in intact myofibrils in rigor and relaxed states (Fig.…”

“…The actin filaments were stretched and eventually severed into two filaments, in which each end of the filament remained attached to the beads in all the cases examined. This indicates that the binding strength between actin filaments and α-actinin-coated beads is greater than the tensile force to split single actin filaments, 250-550 pN (Tsuda et al, 1996).…”

“…Unfortunately we could not find any such regular rupture patterns in the present experiments. The possible reasons would be that (1) the actin filaments located at the very surface of myofibril preparations could be partly damaged during preparations, and (2) the rupture force of breaking rigor complexes depends on the mutual configurations of the complex formed (Tsuda et al, 1996) as actin filaments are incorporated in thick filaments array in complicated steric configurations. As these points are of interest and importance, further detailed studies are required.…”

Helical Birods: An Elastic Model of Helically Wound Double-Stranded Rods

“…The zeroth order variables for the effective rod frame U (0) ir are obtained by substitution of the U (0) i into (43) - (45). Note that θ has no expansion and hence no labelling, it is the zeroth order form of the angles α i which we do expand.…”