Probing nucleocytoplasmic transport by two-photon activation of PA-GFP.
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 sub-femtoliter 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.
School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota 55455, USA. Chen@physics.umn.edu
Yan
Y
Chen
Patrick J
PJ
MacDonald
Joseph P
JP
Skinner
George H
GH
Patterson
Joachim D
JD
Müller
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D049452:Green Fluorescent Proteins / Q000378:metabolism / Q000528:radiation effects*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods; D017785:Photons
D049452:Green Fluorescent Proteins