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This imaging assay shows the differences in nucleo-cytoplasmic trafficking rates of two mutant variants of the small regulatory protein Rev of HIV-1 in living cells. Translocation of the tagged proteins was induced by PEG-mediated polykaryon formation. While the GFP tagged mutant shows rapid translocation into the nuclei, the DsRed coupled mutant shows a strongly reduced nuclear import functionality allowing conclusions about the necessary import signals.
Image kindly provided by Dr. Horst Wolff, GSF-Institute for Molecular Virology, Retroviral Regulation group (Dr. R. Brack), Neuherberg, Germany
The movies are provided as Video for Windows (avi) or QuickTime format (mov):
HeLa-Fusion.mov (2.1 MB)
HeLa-Fusion.avi (2.3 MB)
Description: HeLa cells stably transfected with the green variant Rev protein were mixed with a population of stably transfected HeLa cells expressing the red variant Rev protein. Cells were washed and polykaryon formation was induced by treatment with PEG (Polyethylene glycol). After several washes the cells were refed with medium and transferred to the microscope stage and multichannel time lapse imaging with both fluorescent and a Nomarski (DIC) channel started.
In the movie one can clearly observe the import of the green labelled mutant into the nuclei of the red acceptor cells within 10 minutes as indicated by the appearance of a yellow colour in the merge image. The red mutant readily traffic's into the cytoplasm of the green acceptor cells but fails to enter the green nuclei within the time frame observed. This allows the conclusion, that the mutations in the red labelled Rev variant do influence the import capabilities of the shuttling protein Rev.
Technical details
HeLa cells were grown on glass bottom dishes and transfected with the appropriate expression plasmids. Image acquisition was started 24 hours post transfection in a fully automated Cell Observer System with a Plan Neofluar 40x/1.30 Oil Ph3 objective, incubator XL, heating insert P and CO2 cover HP for best pH and humidity control. The following filtersets were used: GFP with filterset #44; DsRed with filter set #26).
Imaging was performed using an AxioCam HRm under control by AxioVision using the modules Multichannel, Time-lapse and Autofocus. A three channel experiment including a DIC contrast channel was set up including an autofocus run for every time-point in the GFP channel to guarantee the capture of focused images even during polykaryon formation. Images were acquired every 2 minutes for a total time of 100 minutes. The resulting time lapse image was contrast adjusted, annotated with scale bar and relative time tag and exported to movie formats using AxioVision’s powerful export functionality. | |
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