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Fig. 8. Suppression of cdc28-4 clb5 spindle polarity defects by spc110¹³. (A) Western blot analysis of paired extracts from independent isolates of asynchronous cdc28-4 clb5 cells expressing HA₃-tagged Spc110¹³p or Spc110^13-36A91Ap at permissive temperature. Phosphorylation of this construct (arrowheads) was still dependent on the two CDK consensus sites indicating that other B-type cyclins might still direct phosphorylation of this truncation. Phosphorylation of Spc110¹³p dependent upon S36 and S91 within CDK sites in otherwise wild-type cells is shown in supplementary material Fig. S4. (B,C) Selected frames from time-lapse sequences for Dyn1p-GFP accumulation at the SPBs and astral MT behaviour during spindle assembly in cdc28-4 clb5 DYN1:GFP cells expressing Spc110¹³p or Spc110^13-36A91Ap. (B) In a cell expressing Spc110¹³p, Dyn1p-GFP initially marked the old SPB and associated astral MTs (that interacted with the bud cortex). After SPBs separated, the label began to accumulate at the new SPB (0 minutes, arrowhead). Consistent with this intrinsic SPB asymmetry, astral MTs emerging from the new SPB correctly interacted with the mother cortex away from the bud neck. The lag in Dyn1p-GFP acquisition was observed in 60% of cells recorded, n=15 cells. (C) In a cell expressing Spc110^13-36A91Ap, both SPBs were marked by the Dyn1p-GFP fusion during separation (0 minutes, arrowheads). In agreement with this lack of intrinsic asymmetry, both SPBs established dynamic astral MT interactions with the bud cortex. A lag in Dyn1p-GFP acquisition was observed in 5% of cells recorded, n=19 cells. Numbers indicate time elapsed in minutes relative to the first frame in which both SPBs were visible with this label. Bars, 2 µm.

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