Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time
Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time
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DOI：
10.1016/j.jsb.2006.12.012
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发表时间：
2007-06-01
影响因子：
3
通讯作者：
Merkle, Thomas
中科院分区：
文献类型：
--
作者：
Martini, Joerg;Schmied, Katja;Merkle, Thomas
我们使用多焦点双光子激光扫描显微镜进行局部和选择性蛋白质激活和细胞内蛋白质动力学的定量研究。利用光激活绿色荧光蛋白（pa-GFP）和光双光子激发实现局部激活，以研究细胞内实时动态。这些过程对于深入理解和模拟活细胞中的调节和代谢过程至关重要。例如，我们定量研究了含有核输入和核输出信号的拟南芥MYB转录因子LHY/CCA1-like 1 （LCL1）的细胞内动力学。利用烟草BY-2原生质体共转染编码光激活绿色荧光蛋白（pa-GFP）融合蛋白的质粒和红色荧光转染标记物，测定了pa-GFP- lcl1 I在细胞核内光激活后的快速核输出。相比之下，LCL1的输出阴性突变体仍然被困在细胞核内。我们测定了pa-GFP- lcl1活跃的双向核转运和pa-GFP扩散导致细胞核内荧光下降的平均时间常数分别为51s和125s。(c) 2007爱思唯尔公司版权所有。
We used multifocal two-photon laser scanning microscopy for local and selective protein activation and quantitative investigation of intracellular protein dynamics. The localized activation was realized with photo-activatable green-fluorescent-proteins (pa-GFP) and optical two-photon excitation in order to investigate the real-time intracellular dynamics in vivo. Such processes are of crucial importance for a deep understanding and modelling of regulatory and metabolic processes in living cells. Exemplarily, the intracellular dynamics of the Arabidopsis MYB transcription factor LHY/CCA1-like 1 (LCL1) that contains both a nuclear import and a nuclear export signal was quantitatively investigated. We used tobacco BY-2 protoplasts co-transfected with plasmids encoding photo-activatable green fluorescent protein (pa-GFP) fusion proteins and a red fluorescing transfection marker and measured the rapid nuclear export of pa-GFP-LCL1 I after its photo-activation in the nucleus. In contrast, an export-negative mutant of LCL1 remained trapped inside the nucleus. We detemined average time constants of 51s and 125s for the decrease of fluorescence in the nucleus due to active bi-directional nuclear transport of pa-GFP-LCL1 and diffusion of pa-GFP, respectively. (c) 2007 Elsevier Inc. All rights reserved.