Figure 4.

Cdk1 Activity Is Required for Mad2 Recruitment and Retention at Kinetochores upon Microtubule Depolymerization

(A) IF analysis of Mad2-EGFP kinetochore localization. HeLa cells were arrested in metaphase by addition of MG132 for 2 hr and subsequently released (DMSO) or kept in presence of MG132 during the experiment. DMSO or flavopiridol (15 μM) was added 2 min prior to addition of nocodazole (1 μg/ml) or at the time of nocodazole addition. Kinetochore localization of Mad2-EGFP was analyzed in cells with disrupted chromosome alignment 10 min after treatment with nocodazole (n > 300 cells from three independent experiments).

(B) Mad2-EGFP kinetochore localization in live cells. HeLa cells expressing H2B-mCherry and Mad2-EGFP were arrested in metaphase with MG132 (30–90 min) and imaged by three-dimensional confocal live-cell microscopy. Nocodazole (1 μg/ml) and flavopiridol (20 μM) were acutely added during imaging as indicated by the arrowheads and vertical lines in black and blue, respectively. Images show single confocal z sections of representative cells. Graphs show quantified kinetics of Mad2 intensity on chromatin regions over cytoplasm in individual cells (n > 15 cells from three independent experiments). Time = 0:00 min:s at the first drug addition.

Scale bars represent 10 μm. See also Figure S4 for microtubule depolymerization kinetics.