Direct measurement of stiffness of single actin filaments with and without tropomyosin by in vitro nanomanipulation.

Abstract

In order to explain the molecular mechanism of muscle contraction, it is crucial to know the distribution of the sarcomere compliance of active muscle. Here, we directly measure the stiffness of single actin filaments with and without tropomyosin, using a recently developed technique for nanomanipulation of single actin filaments with microneedles.The results show that the stiffness for 1-,Im-long actin filaments with and without tropomyosin is 65.3 ± 6.3 and 43.7 ± 4.6 pN/nm, respectively. When the distribution of crossbridge forces along the actin ifiament is taken into account, the elongation of a l-,um-long thin rilament during development of isometric contraction is calculated to be -0.23%. The time constant of force in response to a sudden length change is <0.2 ms, indicating that the viscoelasticity is negligible in the millisecond time range. These results suggest that '50% of the sarcomere compliance of active muscle is due to extensibility of the thin riaments.In striated muscles, contraction is due to cyclic interactions of myosin crossbridges with thin filaments in the sarcomere (1, 2). The mechanical properties ofthe interaction have often been studied by analyzing tension transients of contracting muscle fibers. In 1971, Huxley and Simmons (3) deduced an ingenious mechanical model to fit the kinetics of these transients. In this model, the force produced by the crossbridges is transmitted to thick and thin filaments by a series elastic element. The model assumed for simplification that the mechanical compliance (reciprocal of the stiffness) in the sarcomere is mostly localized in the crossbridges and that the thin and thick filaments are rigid to transmit the force produced by the crossbridges to the next sarcomere along the muscle fiber axis. This assumption has allowed us to lump the mechanical behavior of the crossbridges distributed along the thin filaments. Indeed, many subsequent experiments have been interpreted on the basis of this simplifying assumption.The assumption that compliance resides primarily within the crossbridges has been tested by many mechanical and structural studies. Ford et al. (4) measured the instantaneous stiffness of isometrically contracting muscle fibers at various sarcomere lengths and concluded that .80% of the sarcomere compliance is due to the crossbridges. A similar result was obtained from the dependence of the rigor stiffness on the sarcomere length (6). Huxley and coworkers (7,8) showed that the strong 5.9-nm x-ray diffraction peak from actin filaments did not change spacing significantly during contraction. These results suggested that the thin filaments are rigid, but the experiments are not conclusive (see Discussion). On the other hand, measurements of the elastic modulus for bending of actin filaments have suggested that the actin filament is quite flexible and, hence, might contribute significantly to muscle compliance (see ref. 9 for review).Recently, we have developed a nanomanipulation technique for single actin filaments that involves attach...

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