| 研究課題/領域番号 |
19K23737
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| 研究機関 | 大学共同利用機関法人自然科学研究機構(新分野創成センター、アストロバイオロジーセンター、生命創成探究 |
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研究代表者 |
Ganser Christian 大学共同利用機関法人自然科学研究機構(新分野創成センター、アストロバイオロジーセンター、生命創成探究, 生命創成探究センター, 特任助教 (50846095)
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| 研究期間 (年度) |
2019-08-30 – 2021-03-31
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| キーワード | HS-AFM / TIRFM / AFM force curves / Kinesin / Motor proteins / Microtubules |
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| 研究実績の概要 |
A procedure to stably observe the motor protein kinesin moving along microtubules was established with high-speed atomic force microscopy (HS-AFM) and total internal reflectance fluorescence microscopy (TIRFM). While HS-AFM observation had been previously achieved, the procedure was optimized in terms of buffer conditions and concentrations. The motility of kinesin in dependence of adenosine triphosphate (ATP), the "fuel" of motor protein movement, was observed with HS-AFM and TIRFM and was found to be in an expected range. Force curve measurements have been implemented to the combined HS-AFM/TIRFM machine and are ready to use. The performance of the system (in terms of observation range and invasiveness) has been improved in preparation for observing kinesin motility and defect creation.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
1: 当初の計画以上に進展している
理由
Currently, the combined HS-AFM/TIRFM system is being improved further to allow a higher degree of control during force measurements. This will be necessary to create defects as precise and localized as possible. Furthermore, the invasiveness of the system is being reduced further. Such improvements are achieved by overhauling the system from top to bottom. Already previous difficulties to scan areas larger than 500 nm × 500 nm were overcome by fine-tuning: stable observation of 10,000 nm × 10,000 nm areas were achieved on a calibration grating. Such a feature was not believed possible previously. Measurement of such large areas will be necessary to properly correlate TIRFM images with HS-AFM images by visualizing microtubule networks.
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| 今後の研究の推進方策 |
The next steps will start with imaging microtubule networks and making defects in microtubules, followed by observation of kinesin moving on microtubules without and with defects. First, separate observation of HS-AFM and TIRFM will be conducted with the goal to create defects using the HS-AFM functionality while observing kinesin motility using the TIRFM. After these observations were successful, details of kinesin movement around defects will be studied by the HS-AFM/TIRFM combination. In these experiments, also the effect of HS-AFM scanning on kinesin motility will be investigated by comparing TIRFM observation with and without HS-AFM scanning. The next part will be to conduct such experiments using dynein, which is believed to show behavior different from kinesin around obstacles.
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| 次年度使用額が生じた理由 |
The remaining 533,610 JPY will be used to purchase a digital oscilloscope that has been borrowed from another machine, so far. After all the testing is complete it will be purchased according to the original plan. Furthermore, cantilevers for HS-AFM use will be purchased to replace used ones. Originally, it was planned to attend a conference, though it is currently not clear if it will be possible to attend said conference due to the COVID-19 outbreak and accompanied restrictions. Alternatively, the remainder will be used to purchase cantilevers, which are necessary for continued research.
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