Cargo binding makes a wild-type single-headed myosin-VI move processively

¹ Graduate School of Frontier Biosciences, Osaka University
² Japan Science and Technology Agency
³ Institute of Medical Science, The University of Tokyo
⁴ University of Massachusetts Medical School

^* To whom correspondence should be addressed. E-mail: yanagida{at}phys1.med.osaka-u.ac.jp.

Submitted on October 20, 2005
Revised on November 28, 2005
Accepted on 18 January 2006

	Abstract

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 GFP-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 (40nm) steps. The characteristics of this monomer driven movement were different to that of artificial dimer diriven movement: Compared to artificial dimer, monomer-bead complex had a reduced stall force (1 pN compared to 2 pN), a shorter average run length 2.5-fold less (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 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 it rebinding following detachment and supporting processive movement of the bead-monomer complexes. While 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.

Key Words: biased Brownian motion, endocytosis, single molecule, unconventional myosin, vesicle transport

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