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

Abstract

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.

Functional significance of HCM mutants of tropomyosin, V95A and D175N, studied with <i>in vitro</i> motility assays

“…The optical setup used in this study was same as the one described previously [ 43 ]. Briefly, the setup was built around an inverted microscope (IX 71, Olympus, Tokyo, Japan).…”

“…The protocol to prepare the in vitro motility assay was based on our previous report [ 43 ] with modifications ( Fig. 2 ).…”

Bio-Molecular Applications of Recent Developments in Optical Tweezers

“…One field where combined OT and fluorescence techniques really shine is that of molecular motors. Ishii et al [91] used a combination of optical trapping, TIRF, and epifluorescence to enrich a standard assay of motility. In Figure 5, a fluorescently labeled thin filament reconstituted from actin, tropomyosin, and troponin is tethered to a fluorescent polystyrene bead and interacts with heavy meromyosin (HMM) molecules attached to the coverslip.…”

“…Experimental setup used by Ishii et al [91]. The filament made of actin, tropomyosin, and troponin is attached to the coverslip for a length a up to the point P by its interaction with heavy meromyosin molecules (HMM).…”

“…Optical tweezers measure the intensity of the force F /cos θ , while fluorescence allows to estimate both the lengths h and b ; in this way, the z -component of the force can be reconstructed. Reprinted with permission from [91].…”