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* Department of Biophysical Engineering, Osaka University, Suita, Osaka, Japan; Formation of Soft Nanomachines, Core Research for Evolution Science and Technology, Japan Science and Technology Agency, Suita, Osaka, Japan; Soft Biosystem Group, Laboratories for Nanobiology, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan; Division of Biomolecular Imaging, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan; and ¶ University of Massachusetts Medical School, Worcester, Massachusetts
Correspondence: Address reprint requests to Toshio Yanagida, Soft Biosystem Group, Laboratories for Nanobiology, Graduate School of Frontier Biosciences, Osaka University, 1-3,Yamadaoka, Suita, Osaka 565-871, Japan. Tel.: 81-6-6879-4632; Fax: 81-6-6879-4634; E-mail: yanagida{at}phys1.med.osaka-u.ac.jp.
Class VI myosin is an intracellular vesicle and organelle transporter that moves along actin filaments in a direction opposite to most other known myosin classes. The myosin-VI was expected to form a dimer to move processively along actin filaments with a hand-over-hand mechanism like other myosin organelle transporters. Recently, however, wild-type myosin-VI was demonstrated to be monomer and single-headed, casting a doubt on its processivity. By using single molecule techniques, we show that green-fluorescent-protein-tagged single-headed, wild-type myosin-VI does not move processively. However, when coupled to 200-nm polystyrene beads (comparable to intracellular vesicles in size) at a ratio of one head per bead, single-headed myosin-VI moves processively with large (40-nm) steps. The characteristics of this monomer-driven movement were different to that of artificial dimer-driven movement: Compared to the artificial dimer, the monomer-bead complex had a reduced stall force (1 pN compared to 2 pN), an average run length 2.5-fold shorter (91 nm compared to 220 nm) and load-dependent step size. Furthermore, we found that a monomer-bead complex moved more processively in a high viscous solution (40-fold higher than water) similar to cellular environment. Because the diffusion constant of the bead is 60-fold lower than myosin-VI heads alone in water, we propose a model in which the bead acts as a diffusional anchor for the myosin-VI, enhancing its rebinding following detachment and supporting processive movement of the bead-monomer complexes. Although a single-headed myosin-VI was able to move processively with a large cargo, the travel distance was rather short. Multiple molecules may be involved in the cargo transport for a long travel distance in cells.
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