Takayuki Nishizaka

Characterization of single actomyosin rigor bonds: load dependence of lifetime and mechanical properties.

Load dependence of the lifetime of the rigor bonds formed between a single myosin molecule (either heavy meromyosin, HMM, or myosin subfragment-1, S1) and actin filament was examined in the absence of nucleotide by pulling the barbed end of the actin filament with optical tweezers. For S1, the relationship between the lifetime (tau) and the externally imposed load (F) at absolute temperature T could be expressed as tau(F) = tau(0).exp(-F.d/k(B)T) with tau(0) of 67 s and an apparent interaction distance d of 2.4 nm (k(B) is the Boltzmann constant). The relationship for HMM was expressed by the sum of two exponentials, with two sets of tau(0) and d being, respectively, 62 s and 2.7 nm, and 950 s and 1.4 nm. The fast component of HMM coincides with tau(F) for S1, suggesting that the fast component corresponds to single-headed binding and the slow component to double-headed binding. These large interaction distances, which may be a common characteristic of motor proteins, are attributed to the geometry for applying an external load. The pulling experiment has also allowed direct estimation of the number of myosin molecules interacting with an actin filament. Actin filaments tethered to a single HMM molecule underwent extensive rotational Brownian motion, indicating a low torsional stiffness for HMM. From these results, we discuss the characteristics of interaction between actin and myosin, with the focus on the manner of binding of myosin.

Orientation of actin monomers in moving actin filaments

We have visualized, under an optical microscope, the orientations of actin monomers in individual actin filaments undergoing Brownian motion in solution, actively sliding past myosin molecules, or immobile on a surface. For the visualization, two strategies have been adopted. One is to exploit the fluorescence polarization of a fluorescent probe firmly attached to actin. Using the probe phalloidin-tetramethylrhodamine, the fluorescence was clearly polarized along the filament axis, showing alignment of the probe molecules along the filament axis. Within our temporal resolution of 33 ms and spatial resolution of better than 1 micron (average over approximately 10(2) actin monomers), the orientation of the probe (hence of actin monomers) did not change upon interaction of the filament with heavy meromyosin; myosin-induced reorientation was estimated to be a few degrees at most. This first method, while highly sensitive to small reorientations of monomers off or toward the filament axis, does not report on reorientations around the axis. To detect rotation around the filament axis, we adopted the second strategy in which we attached small plastic beads to the actin filaments. Axial turns would be immediately apparent from the movement of the beads. Preliminary observations indicate that actin filaments can slide over a heavy meromyosin-coated surface without axial rotations. Since rotations have been implicated in different experiments, we are currently investigating the source of the apparent discrepancy. The attached bead also serves as a handle through which we can apply force, via optical tweezers, on the filament. By letting the sliding actin filament pull the bead against the optical force, we were able to estimate the sliding force and its fluctuation.

3-D Single Particle Tracking Using Dual Images Divided by Prism: Method and Application to Optical Trapping

We here describe the three-dimensional optical tracking method, which is realized with a simple optical component, a quadrangular wedge prism. Additional two lenses located between a conventional optical microscope and a camera enable to track single particles in 3-D. Because of the simplicity of its rationale and construction, any laboratory equipped with 2-D tracking method, under either fluorescence, phase-contrast, bright-field or dark-field illumination, can adopt our method with the same analysis procedure and thus the same precision. Applications to a molecular motor, kinesin-microtubule system, and optical trapping, are also demonstrated, verifying the advantage of our approach to assess the movement of tiny objects, with the size ranging from ten nanometers to a few microns, in an aqueous solution. T.A. Katoh • S. Fujimura • T. Nishizaka (*) Department of Physics, Gakushuin University, Tokyo, Japan e-mail: 14141007@gakushuin.ac.jp; shokotan0421@gmail.com; takayuki.nishizaka@gakushuin. ac.jp # Springer Science+Business Media Dordrecht 2015 A. H.-P. Ho et al. (eds.), Handbook of Photonics for Biomedical Engineering, DOI 10.1007/978-94-007-6174-2_2-1 1