Figure 4.

Plo1 localization to the SPB is not driven by the premature activation of Spg1 that drives inappropriate septation but is affected in cdc7. A20 mutants. (A) Induction of cytokinesis by overexpressing spg1⁺ from an integrated version of the gene that was controlled by the nmt1 promoter (IH1292). Samples were taken for immunofluorescence every hour after gene induction by removal of thiamine from the medium and stained with HN184 antibodies and DAPI. Overproduction of Spg1 led to a reduction in the number of cells with Plo1 on their SPB. (B) cdc16.116 cells were synchronized with respect to cell size by elutrient centrifugation and immediately incubated at 36°C at time 0 on the graph in B. Correlative HN184 immunofluoresence, DAPI, and calcofluor staining showed that the septation started to occur within 1 h of the shift to the restrictive temperature. In contrast, Plo1 association with the SPB correlated with the normal timing of mitosis in such an experiment and did not rise once the second round of septation was initiated (filled squares). (C) cdc7. A20 cells were synchronized with respect to cell size by elutrient centrifugation, and the isolated small G2 cells were immediately cultured at 36°C. Samples were taken for immunofluoresence and stained with HN184 and DAPI. Scoring for one or two dots of intense Plo1 staining showed one dot of Plo1 staining on the nuclei of G2 cells much earlier than in wild-type cells. The appearance of two dots per nucleus coincided with the normal timing of mitosis. n = 200 for each data point on the graphs. Each experiment gave similar results when repeated.