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. 2007 Sep 24;178(7):1193–1206. doi: 10.1083/jcb.200707080

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Copyright © 2007, The Rockefeller University Press

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Figure 4. — Motility analysis of purified myosins. Actin gliding assays were performed by directly adsorbing purified myosins onto the surface of a glass coverslip in a motility chamber. Fluorescently labeled actin filaments were added and movement was recorded within the first 5 min of adding actin filaments to the motility chambers, except at low motor densities, when filament landing was slower. Motor density was determined by quantitative Western blots of the amount of motor input and the amount that passed through the chamber without binding. Myosins were diluted before addition to the motility chambers. (A) The velocities of Myo2p and Myo4p respond differently to changes in motor density. The velocity of actin filaments decreases as Myo2p density decreases, indicative of a nonprocessive motor. In contrast, the velocity of actin filaments increases as Myo4p density decreases. (B) The decreased filament velocity at higher Myo4p density is not due to She3p. The velocity vs. motor density profiles of Myo4p with She3p (top panel) and without She3p (bottom panel) are similar. (C) The velocity versus motor density profile of Myo2/4p resembles Myo4p, whereas the profile of Myo4/2p is similar to Myo2p. Note that the maximum velocity of Myo4/2p is comparable to Myo4p and the maximum velocity of Myo2/4p is equivalent to Myo2p. Shown are representative curves for each myosin, each point is the average filament velocity (μm/s ± SD) for all filaments analyzed in a chamber.