Probing nucleocytoplasmic transport by two-photon activation of PA-GFP

Yan Chen, Patrick J. Macdonald, Joseph P. Skinner, George H. Patterson, Joachim Mueller

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)220-226
Number of pages7
JournalMicroscopy Research and Technique
Volume69
Issue number3
DOIs
StatePublished - Mar 2006

Keywords

  • Green fluorescent protein
  • Nuclear pore complex
  • Photoactivation
  • Restricted diffusion
  • Two-photon microscopy

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