Fig 1.
Nuclear-limited Cdc14 release during mitosis.
Wild-type cells in log-phase mitosis were stained with DAPI to reveal DNA (in blue) and by indirect immunofluorescence to localize α-tubulin (green) and Cdc14-7Myc (red). Cell cycle stages were identified as described in Materials and Methods. Cell outlines are shown in solid white, and dashed white lines outline the region of strong DAPI-staining (i.e., the nucleus minus the nucleolus, see Materials and Methods). The white bar represents 5 μm. (A) A metaphase cell with Cdc14 sequestered in the nucleolus. (B) An early anaphase cell with Cdc14 released to the nucleus. (C) A late anaphase cell with Cdc14 exported to the cytoplasm.
Fig 2.
Cdc14-7Myc localization during meiosis I and II.
Cdc14-7Myc cells undergoing meiotic nuclear divisions. Images show the most representative Cdc14 localization at each stage of meiosis I and meiosis II. Fractions represent the number of cells with that localization divided by the total number of cells imaged at that stage. Images show Cdc14-7Myc (red), tubulin (green) and DNA (blue). The white bar represents 5 μm.
Fig 3.
Mitotic arrest reveals nuclear-limited Cdc14 release by the FEAR pathway.
See Materials and Methods for the mitotic growth, synchronization and nocodazole-induced arrest procedure. Images of arrested cells were collected 3.5 hours after release from G1 arrest into nocodazole. Nuclear-localized GFP is shown in green and Cdc14-7Myc protein in red; the white bar represents 5 μm. Cell outlines are shown in solid white, and nuclei in dashed white lines. Cdc14 was scored as nucleolar (filled circles), nuclear (open circles) or cytoplasmic (triangles). (A) Wild-type cells with Cdc14 sequestered in the nucleolus. (B) mad2Δ cells with Cdc14 in the nucleolus. (C) cdc55Δ cells with Cdc14 released into the nucleus only. (D) bub2Δ cells with Cdc14 exported to the cytoplasm. (E) net1-6cdk cells with Cdc14 in the nucleolus. (F) cdc55Δ net1-6cdk cells with Cdc14 in the nucleolus.
Fig 4.
Substrates of APCCdh1 are stable during FEAR.
Cells were synchronized and arrested in mitosis as described for Fig 3 (see S2 Fig for cell synchrony data). (A) Cdc20-13Myc protein stability in wild-type, mad2Δ, cdc55Δ and bub2Δ cells. β-tubulin loading controls are indicated with arrows. (B) Stability of Clb2-9Myc, Cdc5-9Myc and Ipl1-13Myc proteins in wild-type and cdc55Δ cells. (C) β-actin loading controls for Fig 3B.
Fig 5.
Deletion of Cdc55 bypasses FEAR loss-of-function mutants.
Cells were synchronized and arrested in mitosis as described for Figs 3 and 4 (see S2 Fig for synchrony data). Cdc14 protein was scored as nucleolar (black circles), nuclear (gray circles) or cytoplasmic (triangles).
Fig 6.
Genetic interactions between FEAR and MEN mutations.
Interactions of FEAR mutations with lte1Δ were determined by the viability of double-mutant haploid spores germinated at 30°C. To assay interactions with high-temperature-sensitive MEN mutations, cells with the indicated genotypes were grown to saturation in YPD liquid medium at 23°C and a 10-fold dilution series was plated on YPD agar. The plates were incubated at the indicated temperatures for 24–48 before being photographed. (A) Interactions of slk19Δ and spo12Δ with MEN mutations. (B) Interactions of net1-6cdk and cdc55Δ with MEN mutations. The W303 strain background carries a dominant suppressor of mob1-77 temperature-sensitivity at an unknown locus, which we refer to as DSM. We backcrossed the recessive (non-suppressing) allele dsm along with mob1-77 into our W303 strains a minimum of five times to eliminate the suppressor.
Fig 7.
Summary of interactions between FEAR and MEN mutations.
MEN proteins in red had mutations that interacted negatively with the indicated FEAR mutation, enhancing temperature-sensitivity. MEN proteins in green had mutations that interacted positively with the FEAR mutation, suppressing temperature-sensitivity. MEN proteins in blue had mutations that did not significantly interact with the indicated FEAR mutation. The interactions of proteins in black were not determined in this study.
Table 1.
Mitotic doubling times.
Fig 8.
net1-6cdk cells progress efficiently through anaphase and mitotic exit.
Synchronized cells growing at 25°C. (A) Quantitative analysis of landmarks of mitotic progression. B) Indirect immunofluorescence of Cdc14-3Myc protein in red, spindle staining in green and DAPI-stained DNA in blue. At 90 minutes, an early anaphase wild-type cell undergoing FEAR (arrow) and two net1-6cdk cells that have not released Cdc14 (arrows); at 110 minutes, a wild-type cell undergoing MEN release of Cdc14 (arrow) and three net1-6cdk cells undergoing MEN release; at 130 minutes both wild-type and net1-6cdk cells with Cdc14 returned to the nucleolus.