Research Article: Estimation of actomyosin active force maintained by tropomyosin and troponin complex under vertical forces in the in vitro motility assay system

Date Published: February 8, 2018

Publisher: Public Library of Science

Author(s): Shuya Ishii, Masataka Kawai, Shin’ichi Ishiwata, Madoka Suzuki, Young-Hwa Song.


The interaction between actin filaments and myosin molecular motors is a power source of a variety of cellular functions including cell division, cell motility, and muscular contraction. In vitro motility assay examines actin filaments interacting with myosin molecules that are adhered to a substrate (e.g., glass surface). This assay has been the standard method of studying the molecular mechanisms of contraction under an optical microscope. While the force generation has been measured through an optically trapped bead to which an actin filament is attached, a force vector vertical to the glass surface has been largely ignored with the in vitro motility assay. The vertical vector is created by the gap (distance) between the trapped bead and the glass surface. In this report, we propose a method to estimate the angle between the actin filament and the glass surface by optically determining the gap size. This determination requires a motorized stage in a standard epi-fluorescence microscope equipped with optical tweezers. This facile method is applied to force measurements using both pure actin filaments, and thin filaments reconstituted from actin, tropomyosin and troponin. We find that the angle-corrected force per unit filament length in the active condition (pCa = 5.0) decreases as the angle between the filament and the glass surface increases; i.e. as the force in the vertical direction increases. At the same time, we demonstrate that the force on reconstituted thin filaments is approximately 1.5 times larger than that on pure actin filaments. The range of angles we tested was between 11° and 36° with the estimated measurement error less than 6°. These results suggest the ability of cytoplasmic tropomyosin isoforms maintaining actomyosin active force to stabilize cytoskeletal architecture.

Partial Text

Force produced by actomyosin interaction is essential in a wide variety of cellular functions [1,2], hence the organization of actin filament and myosin is diverse. While the contractile system is stable and regularly aligned in muscle cells, continuous modulation in structures and functions is essential in cell migration, cell division and tissue morphogenesis in non-muscle cells. In vitro motility assay has been a powerful experimental system to study the actomyosin interaction. This assay reconstitutes the actin and myosin interaction on a substrate (typically a glass surface) under an optical microscope by using purified contractile proteins [3–5]. It is often combined with additional techniques to observe interactions at the single molecule level. Optical tweezers are a prominent example being used among these techniques. With optical tweezers, microscopic particles such as polystyrene beads and bacteria (size can range from 20 nm to tens of μm) can be handled in a non-invasive manner and the developed force can be quantified up to tens of pN [6–9]. Optical tweezers have been successfully used to characterize molecular motors [10–17]. In particular, the in vitro motility assay utilizing optical tweezers has been successful in revealing the properties of actomyosin interaction: force generation and consequent motile mechanisms [10,13,14,18,19], and intra- and inter-molecular cooperativity [17,20,21]. However, in the past the force measurement was carried out only in a two-dimensional plane. Recently, Pollari and Milstein reported a method to measure vertical force by using optical tweezers, and to correct the vertical component of the trap stiffness that is affected by aberrations and interferences of laser light [22,23]. In fact, measuring the force of actomyosin interaction in three-dimensional space is essential to characterize under the various cellular conditions in which the contractile system is placed.




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