Probing nucleocytoplasmic transport by two-photon activation of PA-GFP
✍ Scribed by Yan Chen; Patrick J. Macdonald; Joseph P. Skinner; George H. Patterson; Joachim D. Müller
- Book ID
- 102333133
- Publisher
- John Wiley and Sons
- Year
- 2006
- Tongue
- English
- Weight
- 283 KB
- Volume
- 69
- Category
- Article
- ISSN
- 1059-910X
No coin nor oath required. For personal study only.
✦ Synopsis
Abstract
Two‐photon activation of photoactivatable green fluorescent protein (PA‐GFP) provides a unique tool for probing cellular transport processes, because activation is strictly limited to the subfemtoliter optical volume of the two‐photon spot. We demonstrate two‐photon activation of PA‐GFP immobilized in a gel and freely diffusing within cells and recover a quadratic power dependence. Illumination at 820 nm allows simultaneous activation and fluorescence monitoring by two‐photon excitation. Alternatively, we activate PA‐GFP using two‐photon excitation and monitor the fluorescence of the photoconverted product with one‐photon excitation. We probe nucleocytoplasmic transport through the nuclear pore complex of COS‐1 cells, by observing the time‐dependent fluorescence at various locations within the cell after two‐photon activation of PA‐GFP in the nucleus and in the cytoplasm. Two‐photon activation of a tandem construct of two PA‐GFPs showed a markedly slower rate of crossing through the nuclear pore. Analysis based on a restricted diffusion model yields a nuclear pore radius of 4.5 nm, which is in good agreement with previously reported values. This application demonstrates the attractive features of two‐photon photoactivation over traditional techniques, such as photobleaching, for studying transport processes in cells. Microsc. Res. Tech. 69:220–226, 2006. © 2006 Wiley‐Liss, Inc.