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Fig. 2. Nuclear cycle arrest is insufficient to allow completion of cytokinesis upon perturbation of the cell division machinery. (A) cdc25-22 and clp1
cdc25-22 cells were grown to early log phase at 24°C and then shifted to 36°C for three hours to arrest cells at the G2/M transition. Cells were subsequently shifted down to 24°C for 30 minutes, treated with a low dose of Lat A (0.2 µM) (t=0) to perturb the actomyosin ring and then shifted back to 36°C at t=30 minutes in the continuing presence of the drug. (B) Quantitative data for cdc25-22 and clp1cdc25-22 cells treated as in Fig. 2A and then fixed with methanol, washed twice with PBS, and stained with DAPI (nuclei) and aniline blue (septa) at the indicated time points. Septa were classified into three groups: normal (similar to septa formed in wild type cells during logarithmic growth); imperfect and complete (functional septa that appeared thicker and more disorganized but bisected the cell); and spotty and incomplete (non-functional deposits of septal material that failed to form a linear structure across the width of the cell). (C) cdc25-22 and clp1cdc25-22 cells treated as in Fig. 2A (at t=4 hours) and stained with both DAPI (nuclei) and aniline blue (cell wall/septa). Imperfect but functional, septa are indicated with arrowheads. (D) cdc25-22 and clp1cdc25-22 cells treated as in Fig. 2A (at t=4 hours) and stained with aniline blue (cell wall/septa). Z-series were obtained and deconvolved as described in Materials and Methods. Max projections of the entire cell are shown to the left of each panel, whereas three alternate views of 3D reconstructions of septa are shown to the right.
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