Abstract
Myosin movement in vivo takes place on a wide variety of F-actin structures, including single filaments and 2D/3D bundled networks. Using in vitro single molecule motility techniqes, we have investigated the processivity and stepping characteristics of myosin V HMM and myosin X HMM with a leucine zipper on single actin filaments and 2D actin bundles. To answer how myosin V and myosin X step on actin bundles, we observed single molecule motility of fluorescently labeled myosin V and X using total internal reflection fluorescent microscopy, and analyzed the step-size, run length, speed, and direction of the movements on actin-bundles. Actin was polymerized and cross-linked on a charged lipid monolayer in Teflon wells to create regular 2D actin arrays. Two cross-linking proteins were used: alpha-actinin, which produces non-polarized bundles with 40 nm filament spacing, and fascin which produces polarized actin bundles with 13 nm filament spacing. We applied a modified particle tracking program, which allowed us to analyze thousands of simultaneous myosin tracks and determine the run lengths and velocities typical of processive movement on the bundled networks. Myosin V moved processively on all types of in vitro actin structures. Myosin X moved well on polarized fascin cross-linked bundles, but movement was impaired or nonexistent on non-polarized alpha-actinin bundles. We hypothesize that forward runs of myosin X on alpha-actinin cross-linked bundles are inhibited because myosin X might makes “sidesteps” to a neighboring filament, which stalls the run. The presence of an SAH domain in the lever arm of myosin X could increase the working stroke or flexibility of the lever arm allowing it to more easily sidestep across the larger alpha-actinin filament spacing.
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