Figure 2.

The SAC prevents multipolar mitoses in S2 cells. (A) Mad2 requirement for centrosome clustering. Centrosome clustering defects were scored in S2 cells upon RNAi of EGFP, Mad2 alone, and EGFP or Mad2 plus 7 h of MG132 treatment. Graph shows the average of three independent experiments; mean ± SD. (*) P < 0.05; (***) P < 0.001, Student’s t-test. (B) Cell cycle delay associated with multipolar mitoses. Comparison of the time (minutes) taken from NEBD to bipolar spindle formation in S2 cells that have two centrosomes (2c) or extra centrosomes (>2c) in mitosis. Dots and bars indicate individual cells and the average, respectively, in minutes. (**) P < 0.002, Student’s t-test. (C) Examples of time-lapse images from mitotic S2 cells with two (top panels) or more than two (bottom panels; Supplemental Movie S1) centrosomes. Centrosomes and MTs are labeled by GFP-SAS-6 (green) and mCherry-α-tubulin (red). (D) Delay induced by extra centrosomes requires Mad2. Time taken from NEBD to anaphase onset was obtained from S2 cells expressing GFP-Cid and mCherry-α-tubulin upon RNAi of EGFP control or Mad2 (average times are shown with red and green bars from cells with two and more than two centrosomes, respectively. (*) P < 0.02; (**) P < 0.002, Student’s t-test. Anaphase delay is not due to cell undergoing mitotic slippage because it is observed in cells with normal number of kinetochores: data were only obtained from S2 cells containing 20–26 Cid dots per cell. (E) Examples of time lapse images (see Supplemental Movies S3, S4). (Bottom panels) Premature anaphase onset occurs before centrosome clustering in Mad2 depleted cells. Arrows indicate centrosomes. Bar, 10 μm.