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.

由于缺乏合适的基于荧光蛋白（FP）的探针，在纳米级的生命系统中进行功能成像很困难。基于可逆光开关荧光蛋白 (RSFP) 的探针是最佳选择。然而，由于传统RSFP的性能较差，我们尝试开发一种具有快速光切换、高光稳定性和优异亮度的RSFP。经过定点诱变和几次随机诱变循环后，最终，我们开发了 RSFP Kohinoor，其亲本 RSFP Padron 包含七个突变。 Kohinoor 具有最快的开关速度和最高的光稳定性，其开关开关周期比其父代 Padron RSFP 多约 25 倍。 Kohinoor的量子产率为0.71，是所有RSFP中最高的。使用 kohinooor，我们通过 PALM 方法成功观察了粘着斑中的纳米级桩蛋白动力学。在可逆可饱和光学荧光跃迁纳米显微镜中，Kohinoor 可以使用比以前更简单的光学装置和低得多的激光功率来获得波形蛋白的超分辨率图像。我们现在已经开始使用 Kohinoor 开发基于 RSFP 的 Ca+2 指示剂，用于功能性 Ca+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