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. 2004 Mar 1;18(5):512–527. doi: 10.1101/gad.1177304

Figure 4.

Figure 4.

Phosphorylation of RCC1 is required for spindle assembly and chromosome segregation. (A) Swiss 3T3 cells overexpressing either RCC1-GFP or RCC1S2,11A-GFP were fixed and stained with an anti-α-tubulin antibody (DM1-α) and DAPI. Examples of cells expressing RCC1-GFP with normal spindles and chromosomes or expressing RCC1S2,11A-GFP with defects in chromosome arm congression (white arrowhead), metaphase spindle (blue arrowhead), and chromosome segregation are shown (white arrow pointing to a lagging chromosome). (B) Time-lapse microscopy of Swiss 3T3 cells overexpressing RCC1-GFP or RCC1S2,11A-GFP from prometaphase to anaphase. Chromosomes in RCC1S2,11A-GFP-expressing cells failed to congress properly before segregation. (C) Temperature-shift experiments. tsBN2 cells stably expressing RCC1-GFP or RCC1S2,11A-GFP were arrested with nocodazole at the permissive temperature (37°C). After a further incubation at 39.5°C (to inactivate the endogenous RCC1), nocodazole was washed out and spindle assembly and chromosome segregation were analyzed by immunofluorescence microscopy. Representative metaphase, anaphase, and telophase cells fixed 40min after nocodazole washout are shown (arrow pointing to lagging chromosomes). Bars, 10μm.