S.Ishiwata1,2,3, H.Kato1, T.Nishizaka1, T.Iga1, Y.Kumaki1, H.Tadakuma1 and K.Kinosita,Jr.4
1Department of Physics, School of Science and Engineering, 2Advanced Research Institute for Science and Engineering, and 3Materials Research Laboratory for Bioscience and Photonics, Waseda University; 4Department of Physics, Faculty of Science and Technology, Keio University.
It is now possible to manipulate and characterize single actomyosin motors under dual-view microscope (phase-contrast and fluorescence) equipped with laser optical tweezers. We have made it possible to microscopically image temperature on single actin filaments in an in vitro motility assay system by monitoring the fluorescence intensity of rhodamine phalloidin attached to actin filaments. Temperature pulse could be locally introduced by illuminating metal particles with an infrared laser light. Combining these techniques, reversible increase and decrease of an order of magnitude of sliding velocity and tension generation were observed with repetitive temperature modulation between about 18 and 50 °C. On the other hand, the unbinding force (rupture force) between an actin filament and a single HMM molecule was measured by pulling the filament toward the barbed end or the pointed end, in the absence of ATP at about 28 °C. The histogram of the unbinding force showed two peaks, at approximately 7 and 17 pN, which probably corresponds to a single-head binding and a double-head binding, respectively. The rupture force did not depend on the direction of the imposed loads. The life time of single rigor bonds, approximately 1000s for HMM and 100s for S1, largely decreased with increasing external loads. Mechanics and thermodynamics of single actomyosin bonds will be discussed. Supported by Ministry of Education, Science, Sports and Culture of Japan, and CREST.