|Budget Amount *help
¥19,500,000 (Direct Cost: ¥19,500,000)
Fiscal Year 1994: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1993: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 1992: ¥12,500,000 (Direct Cost: ¥12,500,000)
We have refined total internal reflection fluorescence microscopy (TIRFM) to visualize single fluorescent dye molecules in aqueous solution at a full video rate (Funatsu, et al., Nature, 374,555 '95). We extened this method to measurements of individual ATP turnovers. Individual ATP turnover events by single kinesin molecules were detected by directly observing association-(hydrolysis)-dissociation of fluorescent ATP analogue, in which Cy3 was attached to ribose. Elementary mechanical events of single kinesin molecules were measured by optical trapping nanometry. 8-nm steps were clearly observed as reported previously (Svoboda, et al. Nature, 365, 721 '92). For simultaneous measurements of single motor mechanics and the ATP turnover, we combined the optics for single molecule imaging with optical trapping nanometry for single motor mechanics. First, we detected individual ATP turnovers by single kinesin molecules attached to a bead, of which position was controlled by an optical trap. When the kinesin molecule was in solution, apart from a microtubule on a glass surface, the ATP turnover rate was very small, -0.2s^<-1>. While, when it was brought into contact with a microtubule, the rate was greatly enhanced to be -10s^<-1>. Thus, it is now possible to measure the ATP turnover rates of single motors under controlled loads. We have tried to measure 8-nm displacement steps and ATP turnovers by single kinesin molecules simultaneously. ATP turnover events appeared to mostly correlated to 8-nm steps but sometimes do not. Because of low affinity of Cy3 ATP for kinesin, however, 8-nm elementary steps were not sufficiently clear. In order to gain the coupling between elementary mechanical events and ATP turnovers, improvement of the apparatus is necessary. It will not take long time. Since Cy3 ATP functions as normal ATP for actomyosin motors, the present method may be more hopeful for actomyosin motors. This project is also in progress.