Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time.
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.
Martini
Joerg
J
Department of Experimental Biophysics and Applied Nanoscience, Faculty of Physics, University of Bielefeld, 33615 Bielefeld, Germany.
Schmied
Katja
K
Palmisano
Ralf
R
Toensing
Katja
K
Anselmetti
Dario
D
Merkle
Thomas
T
D021581:Active Transport, Cell Nucleus; D002467:Cell Nucleus; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000528:radiation effects*; D008856:Microscopy, Fluorescence / Q000379:methods*; D009154:Mutation; D049790:Nuclear Export Signals / Q000235:genetics; D019913:Nuclear Localization Signals / Q000032:analysis / Q000378:metabolism; D017785:Photons; D010957:Plasmids / Q000235:genetics; D011506:Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D011523:Protoplasts / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014026:Nicotiana / Q000166:cytology; D014157:Transcription Factors / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014466:Ultraviolet Rays
D004268:DNA-Binding Proteins; C113192:LHY protein, Arabidopsis; D049790:Nuclear Export Signals; D019913:Nuclear Localization Signals; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins