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Nucleocytoplasmic transport in human astrocytes: decreased nuclear uptake of the HIV Rev shuttle protein

Markus Neumann^1,2, Elena Afonina^2, Francesca Ceccherini-Silberstein¹, Stefan Schlicht¹, Volker Erfle¹, George N. Pavlakis² and Ruth Brack-Werner¹

¹Institute of Molecular Virology, GSF National Research Center for Environment and Health, D-85764 Neuherberg, Germany
²National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702-1201, USA

Rapid nuclear accumulation of Rev in acceptor nuclei of Hela homo-polykaryons. Hela cells growing at subconfluency were transfected with Rev-GFP expressing plasmid pCsRevsg143. Nontransfected HeLa cells were added to achieve confluency and, 24 hours later, PEG-mediated cell fusions were performed. Imaging started approximately 10 minutes after initiation of fusion. GFP images (shown in black and white on the left) and phase-contrast (shown together with green channel overlay on the right) were taken in 2-minute intervals and combined to form QuickTime movies.

Very slow nuclear accumulation of Rev in acceptor nuclei of astrocyte homo-polykaryons. Astrocytic cell line 85HG66 growing at subconfluency was transfected with Rev-GFP expressing plasmid pCsRevsg143. Nontransfected 85HG66 cells were added to achieve confluency. 24 hours later, cells were treated with Hoechst 33342 nuclear dye and PEG-mediated cell fusions performed. Imaging started approximately 10 minutes after initiation of fusion. GFP images (shown in black and white on the left) and phase-contrast (shown together with green channel and blue channel overlay on the right) were taken in 2-minute intervals and combined to form QuickTime movies.

Slow nuclear accumulation of Rev in astrocyte acceptor nuclei of Hela/astrocyte hetero-polykaryons. Hela cells growing at subconfluency were transfected with Rev-GFP expressing plasmid pCsRevsg143. Nontransfected 85HG66 cells were added to achieve confluency. 24 hours later, cells were treated with Hoechst 33342 nuclear dye and PEG-mediated cell fusions performed. Imaging started approximately 10 minutes after initiation of fusion. GFP images (shown in black and white on the left) and phase-contrast (shown together with green channel and blue channel overlay on the right) were taken in 2-minute intervals and combined to form QuickTime movies.

Reduced nuclear accumulation of Rev in Hela acceptor nuclei of astrocyte/Hela hetero-polykaryons. Astrocytic cell line 85HG66 growing at subconfluency was transfected with Rev-GFP expressing plasmid pCsRevsg143. Nontransfected Hela cells were added to achieve confluency. 24 hours later cells were treated with Hoechst 33342 nuclear dye and PEG-mediated cell fusions performed. Imaging started approximately 10 minutes after initiation of fusion. GFP images (shown in black and white on the left) and phase-contrast (shown together with green channel and blue channel overlay on the right) were taken in 2-minute intervals and combined to form QuickTime movies.

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