GTP-Cdc42 remains restricted to the bud during S and M phase cell cycle arrest. (A) Representative time-lapse images of a cell arrested in S phase with hydroxyurea. Numbers indicate time in minutes from the initial Cdc42 polarization. Cells were synchronized in G1 with α factor. Scale bar, 5 µm. (B) GTP-Cdc42 concentration in the buds of cells that are not arrested, cells in S phase arrest, and cells in M phase arrest (n = 100 cells from three experiments). (C) Representative time-lapse images of a cell arrested in metaphase by CDC20 depletion. Numbers indicate time in minutes from the initial Cdc42 polarization. Cells were synchronized in G1 with α factor. Scale bar, 5 µm. (D) Graph of relative Gic2-PBD-RFP fluorescence intensity. The average intensity from each cell compartment was normalized to the total intensity in the cell ± SEM. Intensity was measured at 2 h from α factor release (n = 50 cells from three experiments). Asterisks indicate statistically significant differences (****, P < 0.0001, Mann-Whitney t test). (E) Bud/mother volume ratio measurement in arrested cells, calculated at 180 min from α factor release. For cells that were not arrested, the ratio was calculated at cytokinesis (n = 30 from three experiments for each; average ± SEM). Asterisks indicate statistically significant differences (****, P < 0.0001, Mann-Whitney test). (F) Volume of cells arrested in S phase (n = 30 cells from three assays for each time point; average ± SEM). (G) Volume of cells arrested in M phase (n = 30 cells from three assays for every time point; average ± SEM). (H) Growth rate of cells arrested in S and in M phase (n = 30 cells from three experiments; average ± SEM). The growth rate was measured between 30 min and 180 min. Asterisks indicate statistically significant differences (****, P < 0.0001, Mann-Whitney test).