B-Cyclin/CDKs Regulate Mitotic Spindle Assembly by Phosphorylating Kinesins-5 in Budding Yeast
Figure 1
SIC1 deletion allows SPB separation and DNA replication in cdc4-3(ts) cells at the restrictive temperature.
(A) Spot assay showing that cdc4-3 sic1Δ cells arrest at 37°C. Strains growing in log phase at permissive temperature were diluted to 2×106 cells/ml, and further diluted serially to 2×104 cells/ml. An equal volume of cells from each dilution was spotted on YEPD, and plates were incubated at either ambient temperature (∼22°C) or 37°C. (B) Percentages of budded cells, cells with duplicated and separated SPBs, and cells with duplicated but unseparated SPBs are shown for asynchronous log phase cultures shifted from permissive (24°C) to restrictive (37°C) temperature; each percentage shown is a percentage of that cell type over the total number of cells counted. The experiment was done in triplicate and the mean percentages are plotted; error bars indicate the standard deviation. Gray bars indicate cdc4-3 cells; spotted bars, cdc4-3 sic1Δ cells. Times after the shift to the restrictive temperature are shown. (C) Flow cytometric analysis of cells at the indicated times after the shift to the restrictive temperature; histograms show DNA content on the horizontal axis and the number of counts on the vertical axis. (D) Micrographs of arrested cdc4-3 and cdc4-3 sic1Δ cells showing bud morphology, DAPI-stained DNA, and Spc42-GFP-labeled SPBs. Scale bar: 2 µm.