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. 2021 Jan 22;10:e63645. doi: 10.7554/eLife.63645

Figure 6. Mitotic exit network (MEN) and Cdc5 promote release of Cdc14 from the nucleolus by phosphorylating Cfi1/Net1.

(A) Cdc14 nucleolar release kinetics in wild-type (A41387, n = 134, 123, and 96 cells for each condition), slk19Δ (A41410, n = 86 cells), cdc15-as1 (A41408, n = 38 cells), or cdc5-as1 mutant (A41409, n = 61 cells). Cells were grown at 25°C in SC medium + 2% glucose with corresponding inhibitors and imaged every 5 min for 5 hr. Release of Cdc14 from the nucleolus was quantified as the ratio of fluorescence intensity of Cdc14-eGFP to Cfi1/Net1-mScarlet-I in the nucleolus (ICdc14/ICfi1). Relative degree of Cdc14 release from the nucleolus was calculated with the normalized minimal Cdc14 level in the nucleolus as 1 - (ICdc14(tmin)/ICfi1(tmin))/ (ICdc14(t-20)/ICfi1(t-20)), where tmin represents the frame with minimal Cdc14 level in the nucleolus and t-20 represents 20 min before movement of the spindle pole body (SPB) into bud. (B) Cdc14 nucleolar release kinetics in cells harboring wild-type CFI1/NET1 (A41387, n = 102 and 114 cells) or CFI1/NET1 phospho-mutants for CDK sites (A41420, n = 95 cells), Cdc15 sites (A41587, n = 104 cells), Cdc5 sites (A41588, n = 86 cells), and Cdc15&Cdc5 sites (A41589, n = 131 cells). Cells were grown at 25°C in SC medium + 2% glucose and imaged every 5 min for 5 hr. (C) Distribution of anaphase duration and relative delay of nucleolar segregation for different CFI1/NET1 phospho-mutants (A41436, A41590, A41591, and A41592; n = 76, 85, 99, and 92 cells respectively) measured using the SPB marker Spc42-eGFP and the nucleolar marker Cfi1/Net1-mScarlet-I (see Figure 6—figure supplement 3 for details). Cells were grown at 25°C in SC medium + 2% glucose and imaged every 3 min for 4 hr. (D) Intensities of Cdc14-eGFP at dSPBs in different CFI1/NET1 phospho-mutant cells (A41387, A41587, A41588, and A41589; n = 80, 82, 77, and 89 cells respectively). Cells were grown and imaged as in (B). (E) Genetic interactions between different CFI1/NET1 phospho-mutants and slk19Δ (A41645, A41646, A41647, A41648, A41649) or spo12Δ (A41650, A41651, A41652, A41653, A41654) analyzed by plasmid shuffling (see Materials and methods for details). Fivefold serial dilutions were spotted onto plates with or without 5’-fluoroorotic acid (5-FOA) and incubated at 25°C for 2–3 days. The presence of 5-FOA selects cells that are viable after losing the CFI1(URA3/CEN) plasmid. For all graphs, single cell traces were aligned to the frame where the dSPB entered the bud and averaged. Solid lines represent the average. Shaded areas represent 95% confidence intervals. For distributions, each dot represents a single cell. Solid lines represent the median for (A and B) and the mean for (C). ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05 by two-sided Wilcoxon rank sum test.

Figure 6.

Figure 6—figure supplement 1. Phosphorylation in zone 2 of Cfi1/Net1 regulates Cdc14 release from the nucleolus.

Figure 6—figure supplement 1.

(A and B) Kinetics of Cdc14 release from the nucleolus in cells harboring wild-type CFI1/NET1 (A41387, n = 103 cells for A, n = 130 cells for B) or different CFI1/NET1 phospho-null mutants (A41398, A41399, A41400, A41401, A41404, A41405, A41397, A41402 and A41403; n = 95, 103, 146, 59, 102, 113, 128, 114, and 102 cells respectively). Cells were grown at 25°C in SC medium + 2% glucose and imaged every 5 min for 5 hr. Release of Cdc14 from the nucleolus was quantified as in Figure 6A. Each dot represents a single cell and the solid lines represent the median. ****p<0.0001; **p<0.01; *p<0.05 by two-sided Wilcoxon rank sum test. (C) Representative images showing Cdc14 nucleolar release kinetics for cells harboring wild-type CFI1/NET1 (A41387) or CFI1/NET1 phospho-mutants with all 91 sites (A41404) or only sites in zone 2 (A41399) mutated to alanine. Yellow arrows highlight localization of Cdc14 at dSPB prior to anaphase.
Figure 6—figure supplement 2. The mitotic exit network (MEN) and CDC5 promote release of Cdc14 from the nucleolus by phosphorylating Cfi1/Net1.

Figure 6—figure supplement 2.

Representative images showing Cdc14 nucleolar release kinetics for cells harboring wild-type CFI1/NET1 (A41387) or CFI1/NET1 phospho-mutants for Cdc15 sites (A41587), Cdc5 sites (A441588), and Cdc15 and Cdc5 sites (A41589). Red box highlights anaphase when Cdc14 is fully released in WT cells. Yellow arrows highlight localization of Cdc14 at daughter spindle pole body (dSPB) prior to anaphase.
Figure 6—figure supplement 3. Cfi1/Net1 phospho-mutants delay mitotic exit and nucleolar segregation.

Figure 6—figure supplement 3.

(A) Tracking anaphase progression and nucleolar segregation using Spc42-eGFP and Cfi1/Net1-mScarlet-I as markers, respectively. A41436 cells were grown at 25°C in SC medium + 2% glucose and imaged every 3 min for 4 hr. (B) Quantification of spindle elongation (spindle length was estimated by measuring the distance between the two spindle pole bodies [SPBs]) and nucleolar segregation (nucleoli length was estimated by measuring the length of the major axis of the nucleolar mass) for CFI1/NET1 mutants using the SPB marker Spc42-eGFP and the nucleolar marker Cfi1/Net1-mScarlet-I (A41436, A41590, A41591, and A41592; same dataset as in Figure 6C). Single cell traces were aligned to anaphase onset (spindle length >3 μm) and averaged. Solid lines represent the average. Shaded areas represent 95% confidence intervals. (C) Cumulative density of anaphase duration (left) and delay of nucleolar segregation (right) for cells in (B). Anaphase duration was defined as the time from anaphase onset (spindle length >3 μm) to mitotic exit (spindle breakdown, determined as relaxation of the distance between SPBs). Delay of nucleolar segregation was defined as the time of nucleolar segregation (clear separation of two nucleolar masses) relative to anaphase onset.
Figure 6—figure supplement 4. Phosphorylation of the Cdc14 nuclear localization signal (NLS) does not play a major role in promoting mitotic exit.

Figure 6—figure supplement 4.

(A) Profiles (left) and relative degrees (right) of nuclear release of wild-type (A41584, n = 69 cells) and mutant NLSCdc14 reporters where all three potential Dbf2-Mob1 target sites (A41585, n = 59 cells) or only two out of three sites (A41586 and A41623, n = 77 and 55 cells) were mutated to alanine. Cells were grown at 25°C in SC medium + 2% glucose and imaged every 5 min for 4 hr. Solid lines (right) represent the median. (B) Distribution of anaphase duration for CDC14 phospho-mutants in combination with CFI1 or cfi1-Cdc15&Cdc5(z2) measured using the spindle pole body (SPB) marker Spc42-eGFP (A41436, A41707, A41592, and A41708; n = 64, 65, 61, and 58 cells, respectively). Cells were grown at 25°C in SC medium + 2% glucose and imaged every 3 min for 4 hr. Anaphase duration was defined as the time from anaphase onset (spindle length >3 μm) to mitotic exit (spindle breakdown). Solid lines represent the mean. ****p<0.0001; *p<0.05; ns, not significant (p>0.05) by two-sided Wilcoxon rank sum test.
Figure 6—figure supplement 5. Mutating both CDK and Cdc5 sites in Cfi1/Net1 results in severe delays in mitotic exit and nucleolar segregation.

Figure 6—figure supplement 5.

(A) Cdc14 nucleolar release kinetics in cells harboring wild-type CFI1/NET1 (A41387, n = 39 cells) or CFI1/NET1 phospho-mutants for CDK sites combined with Cdc5 sites in zone 2 (A41691, n = 79 cells). Cells were grown at 25°C in SC medium + 2% glucose and imaged every 5 min for 5 hr. Single cell traces were aligned to the frame where the daughter spindle pole body (dSPB) entered the bud and averaged. Solid lines represent the average. Shaded areas represent 95% confidence intervals. Each dot represents a single cell. Solid lines represent the median. ****p<0.0001 by two-sided Wilcoxon rank sum test. (B) Distribution of anaphase duration and relative delay of nucleolar segregation for different CFI1/NET1 phospho-mutants (A41436, A41692, and A41694; n = 58, 55, and 44 cells respectively) measured using the SPB marker Spc42-eGFP and the nucleolar marker Cfi1/Net1-mScarlet-I. Cells were grown at 25°C in SC medium + 2% glucose and imaged every 3 min for 4 hr. Each dot represents a single cell. Solid lines represent the median. ****p<0.0001 by two-sided Wilcoxon rank sum test.