Date Published: July 24, 2017
Publisher: Public Library of Science
Author(s): Kohei Iwase, Masateru Tanaka, Keiko Hirose, Taro Q. P. Uyeda, Hajime Honda, Miklos S. Kellermayer.
We examined the movement of an actin filament sliding on a mixture of normal and genetically modified myosin molecules that were attached to a glass surface. For this purpose, we used a Dictyostelium G680V mutant myosin II whose release rates of Pi and ADP were highly suppressed relative to normal myosin, leading to a significantly extended life-time of the strongly bound state with actin and virtually no motility. When the mixing ratio of G680V mutant myosin II to skeletal muscle HMM (heavy myosin) was 0.01%, the actin filaments moved intermittently. When they moved, their sliding velocities were about two-fold faster than the velocity of skeletal HMM alone. Furthermore, sliding movements were also faster when the actin filaments were allowed to slide on skeletal muscle HMM-coated glass surfaces in the motility buffer solution containing G680V HMM. In this case no intermittent movement was observed. When the actin filaments used were copolymerized with a fusion protein consisting of Dictyostelium actin and Dictyostelium G680V myosin II motor domain, similar faster sliding movements were observed on skeletal muscle HMM-coated surfaces. The filament sliding velocities were about two-fold greater than the velocities of normal actin filaments. We found that the velocity of actin filaments sliding on skeletal muscle myosin molecules increased in the presence of a non-motile G680V mutant myosin motor.
Actin filaments are one of the major and ubiquitous cytoskeletal filaments that play multiple important functions in various types of cells, including the contraction of muscles driven by the sliding movement of actin filaments along myosin molecules within a series of sarcomeres. This type of motion can be reconstituted in vitro by allowing actin filaments to move over surfaces coated with myosin molecules, then visualized and quantified under a fluorescent microscope [1, 2]. The movements are unidirectional and the speeds remain largely invariable under constant conditions. Moreover, the same skeletal muscle actin filaments move at different velocities on surfaces coated with different types of myosin, leading to the notion that the velocity of an actin filament is primarily determined by the property of driving myosin molecules . Indeed, in the prevailing swinging lever arm theory, the sliding velocity is primarily determined by the step size divided by the duration of the strongly bound state .