| Budget Amount *help |
¥15,100,000 (Direct Cost: ¥15,100,000)
Fiscal Year 2005: ¥6,100,000 (Direct Cost: ¥6,100,000)
Fiscal Year 2004: ¥9,000,000 (Direct Cost: ¥9,000,000)
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| Research Abstract |
Structural differences between dynein and kinesin predict (suggest) a unique molecular mechanism of dynein motility. Measuring the mechanical properties of single molecule of dynein is crucial to reveal the mechanisms underlying its movement. We measured the step size and force produced by single molecules of active cytoplasmic dynein using an optical trap and fluorescence imaging with a high temporal resolution. The velocity of dynein movement, 800 nm/s, is consistent with that reported in cells. The maximum force of 7-8 pN was independent of the ATP concentration and similar to that of kinesin. Dynein exhibited forward and occasional backwards steps of 〜8 nm, independent of load. Visualization of dynein by negative stain electron microscopy shows that the ring domains partially overlap. This indicates that the large dynein heads take 16-nm steps using an overlapping hand-over-hand mechanism.
Myosin V is a double-headed processive molecular motor that moves along an actin filament by taking 36-nm steps. Using optical trapping nanometry with high spatiotemporal resolution, we discovered that there are two possible pathways for the 36-nm steps, one with 12- and 24-nm substeps, in this order, and the other without substeps. Based on the analyses of effects of ATP, ADP and 2,3-butanedione 2-monoxime (a reagent shown here to slow ADP release from actomyosin V) on the dwell time and the occurrence frequency of the main and the intermediate states, we propose that the 12-nm substep occurs after ATP binding to the bound trailing head and the 24-nm substep results from a mechanical step following the isomerization of an actornyosin-ADP state on the bound leading head. When the isomerization precedes the 12-nm substep, the 36-nm step occurs without substeps.
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