Fig. 8. Full Plo1 SPB association is microtubule and Mad2 dependent. plo1-gfp cdc25-22 mad2⁺ (A-C) or plo1-gfp cdc25-22 mad2 (D-F) cells were synchronised by transient temperature shift and released in the presence or absence of 100 µg/ml TBZ. (A,D) Time course of appearance of cells with Plo1 on the SPB (filled green diamonds, DMSO control; open green diamonds, TBZ) and septa (filled black squares, DMSO control; open black squares, TBZ). Initial recruitment of Plo1 to the SPB is microtubule independent. Disassociation of Plo1 from the SPB is, however, microtubule dependent, and this relationship is Mad2 dependent. (B,E) Fluorescence intensity of Plo1-GFP at the SPBs. Fluorescence was measured in 100 cells at each time point in the absence (green bars) or presence (open bars) of TBZ. Plo1 is only partially loaded onto the SPBs in TBZ, and this inhibition is Mad2 dependent. (C,F) Micrographs of cells from the 40 minute time points in A and D, respectively, showing the marked difference in Plo1 ntensity at the SPBs. (Upper panels, in presence of DMSO; lower panels, in presence of TBZ). (G) 10% of normal SBP associated Plo1 fluorescence was observed when plo1-gfp cells bearing the pREP3Xmad2⁺ plasmid overexpressed the Mad2 protein for 17 hours. Arrows point to the faint Plo1 SPB fluorescence. Bars, 10 µm.