| 研究課題/領域番号 |
14F04910
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| 研究機関 | 大阪大学 |
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研究代表者 |
永井 健治 大阪大学, 産業科学研究所, 教授 (20311350)
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| 研究分担者 |
TIWARI Dhermendra 大阪大学, 産業科学研究所, 外国人特別研究員
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| 研究期間 (年度) |
2014-04-25 – 2016-03-31
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| キーワード | Nanoscopy / Photoswitching / Functional imaging / Calcium / Fluorescent protein / Super resolution |
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| 研究実績の概要 |
Functional imaging in a living system at nanoscopic level is difficult due to unavailability of suitable fluorescent protein (FP)-based probe. Reversibly photoswitchable fluorescent proteins (RSFPs)-based probe is the best option. However due to the poor performance of conventional RSFPs, we tried to develop an RSFP that has fast photoswitching, high photostability and superior brightness. After site-directed mutagenesis and several random mutagenesis cycles, finally, we developed an RSFP, Kohinoor that contains seven mutations in its parental RSFP Padron. Kohinoor has the fastest switching speed and the highest photostability, which produce ~25 times more switching on-off cycles than its parental Padron RSFP. The quantum yield of Kohinoor was 0.71, which is highest among all RSFPs. Using kohinooor, we successfully observed nanoscale paxillin dynamics in focal adhesion by PALM method. In reversible saturable optical fluorescence transition nanoscopy, Kohinoor could obtain super-resolved images of vimentin using a simpler optical setup and much lower laser power than has previously been possible. We have now started developing RSFP based Ca+2 indicator using Kohinoor for functional Ca+2 imaging.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
The project was divided into two categories: 1, development of fast RSFPs and 2, utilization of those RSFPs for fast reversibly photoswitching Ca+2 indicator. We have already achieved the first goal, i.e. we succeeded development of very fast switching, highly photostable and bright RSFP, Kohinoor, and submitted a paper (Tiwari D.K. et al. Nat. Methods, in revision). Therefore, we already achieved more than half of our task. Now, we are developing a RSFP-based Ca+2 indicator, which could be implemented for functional imaging.
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| 今後の研究の推進方策 |
We will use our new RSFP Kohinoor for construction of reversibly switchable FRET based Ca+2 indicator for superresolution Ca2+ imaging. For this purpose, Kohinoor and Venus, a yellow fluorescent protein, will be used for FRET donor and acceptor, respectively. Photoswitching on/off will be controlled by 488/405 nm light. Ca+2 binding domain will be inserted using RE sites between donor and acceptor with insertion of desired length of linker. We will search the best indicator by screening colonies for both high photoswitching and high Ca2+ response using our laboratory build screening system. To further ensure that the selected variants will have satisfactory Ca2+ response, they will be again analyzed in spectrofluorometer for Ca2+ response. This RSFP based indicator will selectively control the switching on and off at single cell level. After successful development of RSFP indicator, we will perform superresolution imaging of Ca+2 dynamics in diffraction unlimited sub-cellular compartments such as postsynapse.
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