Figure 1.

Association of Ran with the centrosome. (A) Immunolocalization of Ran on HeLa cells by using the monoclonal Ran antibody after different fixation protocols. Note that after a PAF fixation, a bright fluorescent staining was observed both in the nucleus and in the cytoplasm. The γ-tubulin centrosome staining was poorly visible in these conditions. After PAF fixation followed by a postfixation with methanol at –20°C, the centrosome was detected with Ran antibody. The clearest centrosome staining was obtained using a direct methanol fixation (B) Decoration of nucleus–centrosome complexes from human lymphoblasts KE37 cells with the polyclonal AR12 antibody directed against RanGTP and a monoclonal anti-γ-tubulin antibody. Note that RanGTP was enriched at centrosomes while a faint staining around the nuclear remnant was also observed. (C and D) Ran was localized at the centrosome throughout the cell cycle as observed in centrin 1-GFP–expressing HeLa cells. Ran staining was larger than centrin staining, suggesting its presence in the pericentriolar matrix. In G2-M cells, Ran decorated both centrosomes and the link between them, whereas γ-tubulin was restricted to the centrosomes. (E) In G2-M cells, Ran colocalized perfectly with centrosomal AKAP450 as observed with serum 0013. Bars, 10 μm.