超解像機能イメージングを可能にする新規蛍光タンパク質プローブの開発

开发新型荧光蛋白探针，实现超分辨率功能成像

• 批准号: 14F04910
• 负责人: 永井 健治
• 金额: $ 1.47万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for JSPS Fellows
• 财政年份: 2014
• 资助国家: 日本
• 起止时间: 2014-04-25 至 2016-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-14F04910/
• 关键词: Nanoscopy; Photoswitching; Functional imaging; Calcium; Fluorescent protein; Super resolution

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.

由于没有合适的荧光蛋白探针，在纳米水平上对生物系统进行功能成像是很困难的。基于可逆光开关荧光蛋白（rsfp）的探针是最好的选择。然而，由于传统的rrsfp性能不佳，我们试图开发一种具有快速光电开关，高光稳定性和优越亮度的rrsfp。经过定点诱变和几个随机诱变周期后，我们最终培育出亲本RSFP Padron中含有7个突变的RSFP， Kohinoor。Kohinoor具有最快的开关速度和光稳定性，其开关周期是其亲代Padron RSFP的25倍。Kohinoor的量子产率为0.71，在所有rsfp中最高。利用kohinooor，我们成功地用PALM方法观察了纳米尺度paxillin在病灶黏附中的动力学。在可逆饱和光学荧光跃变纳米显微镜中，Kohinoor可以使用比以前更简单的光学装置和更低的激光功率获得超分辨率的波形蛋白图像。我们现在已经开始使用Kohinoor开发基于RSFP的钙+2指示器，用于功能性钙+2成像。

项目成果

FAST PHOTOSWITCHING FLUORESCENT PROTEIN FOR LIVE CELL SUPERRESOLUTION IMAGING.

用于活细胞超分辨率成像的快速光切换荧光蛋白。

• 发表时间: 2014
• 作者: Dhermendra K TIWARI;Yoshiyuki ARAI;Yamanaka MASAHITO;Katsumasa FUJITA;Takeharu NAGAI
• 通讯作者: Takeharu NAGAI

A fast positive photoswitching fluorescent protein for quantitative PALM and other live cell superresolution microscopy imaging.

一种快速正光开关荧光蛋白，用于定量 PALM 和其他活细胞超分辨率显微镜成像。

• 发表时间: 2014
• 作者: D. K. Tiwari;Y. Arai;M. Yamanaka;T. Dertinger;K. Fujita;T. Nagai
• 通讯作者: T. Nagai