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J Struct Biol. 2007 Jun;158(3):401-9. Epub 2007 Jan 17.

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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Department of Experimental Biophysics and Applied Nanoscience, Faculty of Physics, University of Bielefeld, 33615 Bielefeld, Germany.


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 after its photo-activation in the nucleus. In contrast, an export-negative mutant of LCL1 remained trapped inside the nucleus. We determined average time constants of 51 s and 125 s 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.

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