Figure 3.

Reaccumulation of Securin and MCC after Sister Chromatid Disjunction in the Absence of Cyclin B1 Proteolysis

(A) Frames of confocal live-cell imaging of H2B-iRFP HeLa cells expressing securin-EGFP and WT or ND cyclin B1-mCherry are shown (left). The integrated intensities of securin-EGFP and cyclin B1-mCherry were measured, background corrected, and normalized to the maximum-intensity value obtained per cell. Measurements in different cells were aligned to t = 0 min as the first frame after anaphase onset. The graph displays the mean intensity of securin and cyclin B1 (right). Error bars represent the SD of the analysis of nine cells per condition (from three experiments). The scale bar represents 10μm.

(B) Coimmunoprecipitation analysis of mitotic checkpoint complex (MCC) formation in cells expressing WT or ND cyclin B1. Cdc20 was immunoprecipitated from extracts prepared from nocodazole-arrested (Noc) and MG132-arrested cells at t = 0 min as well as from cells released from metaphase for the indicated times. Mad2 and Cdc20 intensities were analyzed in whole-cell extracts (WCE) and the precipitated fraction (IP) by fluorescent immunoblotting. The Mad2/Cdc20 ratio in the IP fraction was determined, normalized to the ratio at 0 min, and plotted (right). Due to the degradation of Cdc20 in WT cyclin B1-expressing cells, the amount of IP fraction loaded was adjusted for comparable amounts of Cdc20. The percentage of metaphase cells was determined using time-lapse analysis (n > 102 cells).

(C) Analysis of MCC formation as in (B). Cells expressing ND cyclin B1 were treated with DMSO or 0.5 μM reversine 40 min after MG132 release.

(D) Securin-EGFP accumulation in pseudoanaphase cells depends on Mps1 kinase. One hour after release from nocodazole (30 ng/ml), cells expressing ND cyclin B1 were treated with DMSO or reversine (0.5 μM). The graph is as in (A) (n = 9 cells per condition from three independent experiments).

See also Figure S3.