The mitotic exit network Mob1p-Dbf2p kinase complex localizes to the nucleus and regulates passenger protein localization

Abstract

The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved signaling network that coordinates CDK inactivation, cytokinesis and G1 gene transcription. The MEN Cdc14p phosphatase is sequestered in the nucleolus and transiently released in early anaphase and telophase. Cdc14p mediates mitotic exit by dephosphorylating Cdk1p substrates and promoting Cdk1p inactivation. Cdc14p also regulates the localization of chromosomal passenger proteins, which redistribute from kinetochores to the mitotic spindle during anaphase. Here we present evidence that the MEN protein kinase complex Mob1p-Dbf2p localizes to mitotic nuclei and partially colocalizes with Cdc14p and kinetochore proteins. Chromatin immunoprecipitation (ChIP) experiments reveal that Mob1p, Dbf2p, and Cdc14p associate with centromere DNA and require the centromere binding protein Ndc10p for this association. We establish that Mob1p is essential for maintaining the localization of Aurora, INCENP, and Survivin chromosomal passenger proteins on anaphase spindles, whereas Cdc14p and the Mob1p-Dbf2p-activating kinase Cdc15p are required for establishing passenger protein localization on the spindle. Moreover, Mob1p, but not Cdc15p, is required for dissociating Aurora from the kinetochore region. These findings reveal kinetochores as sites for MEN signaling and implicate MEN in coordinating chromosome segregation and/or spindle integrity with mitotic exit and cytokinesis via regulation of chromosome passenger proteins.

Figure 1.

Mob1p localizes to the nucleus. (A) Homozygous diploid cells expressing chromosomally tagged GFP-Mob1p (FLY331) were analyzed by DIC and fluorescence microscopy. Faint GFP fluorescence could be detected in nuclei of some anaphase cells (arrowheads). The bright fluorescence spots are GFP-Mob1p on SPBs. See corresponding Supplementary Movie 1. (B) Asynchronously growing haploid cells expressing GFP-Mob1Δ132 and Nic96p-RFP (FLY 2055) were analyzed by fluorescence microscopy. Mob1Δ132 was readily detectable within the RFP-marked nuclear envelope. (C) Asynchronously growing haploid GFP-Mob1 (FLY316), GFP-Mob1Δ78 (FLY1066) and GFP-Mob1Δ132 cells (FLY1147) were analyzed by fluorescence microscopy. Truncated GFP-Mob1p was readily detectable in the nuclei of large budded anaphase cells, in addition to previously established SPB and bud neck localizations. The arrowheads point to patches of nuclear Mob1p in anaphase cells. The asterisk (*) denotes a cell with Mob1Δ132 on what appears to be the anaphase spindle. The percentages of unbudded, small-, medium- (preanaphase), and large-budded cells with nuclear Mob1p were plotted from asynchronous populations of cells. n = 247, 191, and 161 for GFP-Mob1p, GFP-Mob1Δ78, and GFP-Mob1Δ132 cells, respectively.

Figure 2.

Dynamics of truncated Mob1p localization. (A) Mob1Δ78 and Mob1Δ132 localize to nuclei in metaphase-arrested cells. GFP-Mob1p, GFP-Mob1Δ78, and GFP-Mob1Δ132 were analyzed in nocodazole (NDZ)-treated wild-type cells (left panels) and conditional cdc16-1 cells (right panels). The cells were synchronized in metaphase, as described in the text. The strains used in the NDZ experiments were FLY316, FLY1066, and FLY1147. The cdc16-1 cells (FLY547) contained pRS314-GFP-Mob1, pRS314-GFP-Mob1Δ78, or pRS314-GFP-Mob1Δ132. We obtained similar results for cdc16-1 cells expressing integrated Mob1p (unpublished data). (B) Representative images of GFP-Mob1Δ132 in homozygous diploid cells (FLY1875) from various stages of the cell cycle. The arrowheads point to GFP-Mob1Δ132 on early mitotic SPBs. Note that the second SPB in the last image on the bottom panel is out of focal plane. The pattern of GFP-Mob1Δ78 fluorescence was similar to GFP-Mob1Δ132 (Supplementary Movies 2–4). See Results for detailed description. All images in B are merges of 6 × 0.2-μm Z-sections.

Figure 3.

Quantitative immunoblots of full-length and truncated Mob1p. (B) Quantitative immunoblots of full-length and truncated GFP-Mob1p were conducted as described in the Materials and Methods. The ratio of Mob1p to G6PDH levels (arbitrary units) from asynchronous, G1 and S phase-synchronized cells from three independent experiments were averaged and plotted. The SDs are denoted by error bars.

Figure 4.

Dbf2p localizes to the nucleus during anaphase. DIC and fluorescence microscopy of wild-type (top panels) and Mob1Δ132 cells (bottom panels) expressing chromosomally tagged Dbf2-GFP (FLY626, FLY1608, respectively). Faint GFP fluorescence is detectable in nuclei of some anaphase wild-type cells (arrowheads). Nuclear Dbf2-GFP fluorescence is more prominent in Mob1Δ132 cells than in wild-type cells.

Figure 5.

Mob1Δ132 colocalizes with Cdc14p. (A) Asynchronously growing cells expressing truncated GFP-Mob1Δ132 and CFP-tagged Cdc14p (FLY1206) were analyzed by fluorescence microscopy. GFP, CFP, and merged images are labeled accordingly. The arrowheads point to regions of significant colocalization. Note the colocalization of Mob1Δ132 and Cdc14p in the nucleolus of a preanaphase cell (*). Similar results were obtained for GFP-Mob1Δ78 (unpublished data). (B) Asynchronously growing cells expressing GFP-Mob1Δ132 and RFP-tagged nucleolar protein Sik1p (FLY2056) were analyzed by fluorescence microscopy. The arrowheads point to areas of colocalization in both preanaphase (top panels) and anaphase cells (bottom panels).

Figure 6.

Mob1Δ132 partially colocalizes with kinetochore proteins. Asynchronously growing cells expressing truncated GFP-Mob1Δ132 and CFP-tagged Slk19p (FLY1213) and Okp1p (FLY1219) were analyzed by fluorescence microscopy. GFP, CFP, and merged images are labeled accordingly. The arrowheads point to regions of significant colocalization. Similar results were obtained for GFP-Mob1Δ78 (unpublished data).

Figure 7.

Mob1p, Dbf2p, and Cdc14p precipitate centromere DNA. (A) Centromere chromatin immunoprecipitation (ChIP) was conducted, as described in Materials and Methods. Myc-tagged-Mob1p, Dbf2p, Cdc14p, and Chl4p were immunoprecipitated from asynchronous cells. Dilutions of immunoprecipitated material (IP) were used as template for PCR to test for coprecipitating centromere DNA (CEN1, CEN3). tot, total cell extract used as positive control for PCR template. (B) ChIP of Myc-tagged Mob1p, Cdc14p, and Chl4p from conditional ndc10-1 or ndc10-42 cells.

Figure 8.

Chromosome passenger proteins are not maintained on anaphase spindles in mob1-77, cdc15-2, or cdc14-1 cells at 37°C. (A) The patterns of Ipl1-GFP, Bir1-GFP, and Sli15p-GFP were analyzed in late anaphase-arrested mob1-77 cells. Asynchronously growing cells (FLY1884, FLY1842, FLY1846) were shifted to 37°C for 2–3 h and analyzed by DIC and fluorescence microscopy. Ipl1-GFP, Bir1-GFP, and Sli15p-GFP were not maintained on anaphase spindles in late anaphase-arrested mob1-77 cells. Wild-type cells (wt) are shown for comparison. The mob1-77 cells rapidly recruited all three chromosomal passenger proteins to anaphase spindles when returned to permissive temperature (22°). (B) Sli15p-GFP and Ipl1p-GFP were not present on anaphase spindles in arrested cdc15-2 cells (FLY1849 and FLY2004). When returned to permissive temperature (22°), the cells rapidly recruited Sli15p and Ipl1p to the spindles before mitotic exit. (C) Ipl1p-GFP failed to localize to anaphase spindles in cdc14-1 cells (FLY2069), as previously described (Pereira and Schiebel, 2003). When returned to permissive temperature (22°C), the cells rapidly recruited Ipl1p to the spindles before mitotic exit. We obtained similar data for Sli15p localization (unpublished data). (D) The percentage of arrested cdc15-2, mob1-77, and cdc14-1 cells with Ipl1p-GFP on the kinetochore region was plotted (n = 200 for each strain).

Figure 9.

Ipl1p transiently localizes to anaphase spindles in mob1-77 cells, but not in cdc15-2 or cdc14-1 cells. Ipl1p-GFP was analyzed in wild-type, mob1-77, cdc15-2, and cdc14-1 cells after release from NDZ-induced metaphase arrest, as described in the text. On release from metaphase block (t = 0), the percentage of cells that displayed Ipl1p-GFP on the anaphase spindle was scored over time. The presented data are from a representative experiment.