Fig. 2. CAR formation occurs after entry into anaphase. (A) The timing of cell cycle events in myo2-gc cen1-gfp cdc25-22 cells synchronised by transient temperature shift. The peak of cells containing separated centromeres (gold filled triangles) precedes that of cells also containing Myo2 rings (filled green circles), that is, Myo2 rings are seen only in cells in which centromere separation has occurred. Binucleate cells (filled blue diamonds) and septa (filled black squares) appear subsequently. (B) myo2-gc cen1-GFP cdc25-22 cells from the 40 minute time point in A. Left panel: Myo2-GFP and Cen1 GFP fluorescence; the middle cell contains two Cen1 dots but no Myo2 ring; middle panel: DAPI staining of DNA; right hand panel: DNA (blue) and GFP (green) signals overlayed. (C) myo2-gc cells expressing Mad2 for 16 hours from the plasmid pREP3Xmad2⁺ contain no Myo2 rings. The left panel shows Myo2-GFP signal; middle panel, DAPI staining (arrows point to condensed chromatin); right hand panel shows combined DAPI staining of DNA and phase contrast. Bar, 10 µm.

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