Figure 4.
Cdc14 activity is required for Gic2-PBD redistribution.(A) Representative time-lapse images showing Cdc14 nucleolar release and Gic2-PBD redistribution in a wild-type cell released from a G1 α factor arrest. Numbers indicate time in minutes, counted from the time of initial Cdc42 polarization. Scale bar, 5 µm. (B) Timing of Cdc14 nucleolar release and Gic2-PBD-RFP redistribution in wild-type cells released from a G1 α factor synchronization (n = 100 cells from three experiments). (C) Representative images showing Gic2-PBD localization in wild-type cells at 37°C. Scale bars, 5 µm. Cells were released from a G1 α factor block and then arrested at metaphase by depletion of Cdc20 using a methionine-repressible MET3 promoter. Cells were incubated in methionine medium for 2 h at 25°C and then released in media lacking methionine at 37°C. Numbers indicate time in minutes, counted from the transfer to methionine-free medium. (D) The timing of progression through anaphase, Gic2-PBD redistribution, and spindle breakdown in wild-type cells released from a metaphase arrest at 37°C. Time was counted from the transfer to methionine-free medium. n = 150 cells from three experiments. (E) Representative images showing Gic2-PBD localization in cdc14-1 mutant cells released from metaphase arrest at 37°C. Cells were treated as in C. Numbers indicate time in minutes, counted from the transfer to methionine-free medium. Scale bars, 5 µm. (F) The timing of progression through anaphase, Gic2-PBD redistribution, and spindle breakdown in cdc14-1 mutant cells released from a metaphase arrest at 37°C. Scale bar, 5 µm. n = 150 cells from three experiments. (G) Representative time-lapse images showing the mitotic spindle and Gic2-PBD in a wild-type cell treated with CMK. Numbers indicate time in minutes from the initial Cdc42 polarization in G1. Scale bar, 5 µm. (H) The percentage of wild-type and cdc5-as1 cells treated with CMK with bud-concentrated Gic2-PBD. For each background, n = 100 cells from three experiments. (I) Representative time-lapse images showing the mitotic spindle and the Gic2-PBD biosensor in a cdc5-as1 cell treated with the CMK inhibitor. Numbers indicate time in minutes from the initial Cdc42 polarization in G1. Scale bar, 5 µm. (J) Gic2-PBD-RFP fluorescence intensity in cdc5-as1 cells treated with CMK for 3 h. Average intensity in each cell compartment was normalized to the total intensity in the cell (n = 50 cells from three experiments; error bars indicate SEM). Asterisks indicate statistically significant differences (****, P < 0.0001, Mann-Whitney t test).

Cdc14 activity is required for Gic2-PBD redistribution. (A) Representative time-lapse images showing Cdc14 nucleolar release and Gic2-PBD redistribution in a wild-type cell released from a G1 α factor arrest. Numbers indicate time in minutes, counted from the time of initial Cdc42 polarization. Scale bar, 5 µm. (B) Timing of Cdc14 nucleolar release and Gic2-PBD-RFP redistribution in wild-type cells released from a G1 α factor synchronization (n = 100 cells from three experiments). (C) Representative images showing Gic2-PBD localization in wild-type cells at 37°C. Scale bars, 5 µm. Cells were released from a G1 α factor block and then arrested at metaphase by depletion of Cdc20 using a methionine-repressible MET3 promoter. Cells were incubated in methionine medium for 2 h at 25°C and then released in media lacking methionine at 37°C. Numbers indicate time in minutes, counted from the transfer to methionine-free medium. (D) The timing of progression through anaphase, Gic2-PBD redistribution, and spindle breakdown in wild-type cells released from a metaphase arrest at 37°C. Time was counted from the transfer to methionine-free medium. n = 150 cells from three experiments. (E) Representative images showing Gic2-PBD localization in cdc14-1 mutant cells released from metaphase arrest at 37°C. Cells were treated as in C. Numbers indicate time in minutes, counted from the transfer to methionine-free medium. Scale bars, 5 µm. (F) The timing of progression through anaphase, Gic2-PBD redistribution, and spindle breakdown in cdc14-1 mutant cells released from a metaphase arrest at 37°C. Scale bar, 5 µm. n = 150 cells from three experiments. (G) Representative time-lapse images showing the mitotic spindle and Gic2-PBD in a wild-type cell treated with CMK. Numbers indicate time in minutes from the initial Cdc42 polarization in G1. Scale bar, 5 µm. (H) The percentage of wild-type and cdc5-as1 cells treated with CMK with bud-concentrated Gic2-PBD. For each background, n = 100 cells from three experiments. (I) Representative time-lapse images showing the mitotic spindle and the Gic2-PBD biosensor in a cdc5-as1 cell treated with the CMK inhibitor. Numbers indicate time in minutes from the initial Cdc42 polarization in G1. Scale bar, 5 µm. (J) Gic2-PBD-RFP fluorescence intensity in cdc5-as1 cells treated with CMK for 3 h. Average intensity in each cell compartment was normalized to the total intensity in the cell (n = 50 cells from three experiments; error bars indicate SEM). Asterisks indicate statistically significant differences (****, P < 0.0001, Mann-Whitney t test).

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