Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time

Martini J, Schmied K, Palmisano R, Tönsing K, Anselmetti D, Merkle T (2007)
Journal of Structural Biology 158(3): 401-409.

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Abstract
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.
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Martini J, Schmied K, Palmisano R, Tönsing K, Anselmetti D, Merkle T. Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time. Journal of Structural Biology. 2007;158(3):401-409.
Martini, J., Schmied, K., Palmisano, R., Tönsing, K., Anselmetti, D., & Merkle, T. (2007). Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time. Journal of Structural Biology, 158(3), 401-409.
Martini, J., Schmied, K., Palmisano, R., Tönsing, K., Anselmetti, D., and Merkle, T. (2007). Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time. Journal of Structural Biology 158, 401-409.
Martini, J., et al., 2007. Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time. Journal of Structural Biology, 158(3), p 401-409.
J. Martini, et al., “Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time”, Journal of Structural Biology, vol. 158, 2007, pp. 401-409.
Martini, J., Schmied, K., Palmisano, R., Tönsing, K., Anselmetti, D., Merkle, T.: Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time. Journal of Structural Biology. 158, 401-409 (2007).
Martini, Joerg, Schmied, Katja, Palmisano, Ralf, Tönsing, Katja, Anselmetti, Dario, and Merkle, Thomas. “Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time”. Journal of Structural Biology 158.3 (2007): 401-409.
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