Auto-luminescent genetically-encoded ratiometric indicator for real-time Ca2+ imaging at the single cell level.
Auto-luminescent genetically-encoded ratiometric indicator for real-time Ca2+ imaging at the single cell level.
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DOI：
10.1371/journal.pone.0009935
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发表时间：
2010-04-01
期刊：
影响因子：
3.7
通讯作者：
Nagai T
中科院分区：
文献类型：
--
作者：
Saito K;Hatsugai N;Horikawa K;Kobayashi K;Matsu-Ura T;Mikoshiba K;Nagai T
从生物发光蛋白质到荧光蛋白质的高效生物发光共振能量转移(BRET)被用来提高发光强度，使单细胞成像能够在没有外部光照的情况下接近实时。我们应用Bret开发了一种自发光钙指示剂BRAC，它由钙离子结合蛋白钙调蛋白及其靶肽M13组成，夹在黄色荧光蛋白变体Venus和增强型Renilla荧光素酶RLuc8之间。调节发光蛋白质发色团的相对偶极取向，BRAC中Bret信号变化的动态范围最高可达60%，这是迄今报道的基于Bret的指示物中最大的动态范围。利用BRAC，我们成功地展示了单细胞水平的钙动态可视化，时间分辨率为1赫兹。此外，BRAC信号是通过比率成像获得的，能够抵消由于细胞形状变化、焦点移动等引起的钙非依赖性信号漂移。BRAC的亮度和大的动态范围不仅有利于在单个活细胞中进行高灵敏度的钙成像，而且在小的活体受试者中也可以进行高灵敏度的钙成像。
Efficient bioluminescence resonance energy transfer (BRET) from a bioluminescent protein to a fluorescent protein with high fluorescent quantum yield has been utilized to enhance luminescence intensity, allowing single-cell imaging in near real time without external light illumination. We applied BRET to develop an autoluminescent Ca2+ indicator, BRAC, which is composed of Ca2+-binding protein, calmodulin, and its target peptide, M13, sandwiched between a yellow fluorescent protein variant, Venus, and an enhanced Renilla luciferase, RLuc8. Adjusting the relative dipole orientation of the luminescent protein's chromophores improved the dynamic range of BRET signal change in BRAC up to 60%, which is the largest dynamic range among BRET-based indicators reported so far. Using BRAC, we demonstrated successful visualization of Ca2+ dynamics at the single-cell level with temporal resolution at 1 Hz. Moreover, BRAC signals were acquired by ratiometric imaging capable of canceling out Ca2+-independent signal drifts due to change in cell shape, focus shift, etc. The brightness and large dynamic range of BRAC should facilitate high-sensitive Ca2+ imaging not only in single live cells but also in small living subjects.
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影响因子：
2.9
作者：
Dacres, Helen;Dumancic, Mira M.;Trowell, Stephen C.
通讯作者：
Trowell, Stephen C.
影响因子：
46.9
作者：
Nagai, T;Ibata, K;Miyawaki, A
通讯作者：
Miyawaki, A
DOI：
10.1073/pnas.97.13.7260
发表时间：
2000-06-20
影响因子：
11.1
作者：
Baubet, V;Le Mouellic, H;Brûlet, P
通讯作者：
Brûlet, P
影响因子：
46.2
作者：
Frommer WB;Davidson MW;Campbell RE
通讯作者：
Campbell RE
影响因子：
4.8
作者：
De, Abhijit;Ray, Pritha;Gambhir, Sanjiv Sam
通讯作者：
Gambhir, Sanjiv Sam