Kin4 Kinase Delays Mitotic Exit in Response to Spindle Alignment Defects  Gislene Pereira, Elmar Schiebel  Molecular Cell  Volume 19, Issue 2, Pages 209-221 (July 2005) DOI: 10.1016/j.molcel.2005.05.030 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Kin4 Inhibits Mitotic Exit in Response to Spindle Misalignment (A) The indicated cells with GFP-TUB1 pregrown at 30°C were incubated for 4, 8, and 20 hr at 14°C. Fixed cells were stained with DAPI and analyzed for the number and localization of DAPI staining regions. Asterisks indicate cells with two metaphase-like spindles in the mother cell body. The arrows highlight cells with multiple monopolar G1 spindles. Bar, 5 μm. (B) Quantification of (A). n > 100 cells per time point. (C) Wild-type (wt) KAR9 KIN4, kin4Δ, kar9Δ, and kar9Δ kin4Δ cells were first grown at 23°C and then incubated for 3 hr at 30°C. The DNA of fixed cells was stained with DAPI. Arrows point toward cells with greater than two DAPI staining regions. The asterisk highlights an anucleated cell. Bar, 5 μm. (D) Quantification of (C). n > 100. (E) Viability of wt, kar9Δ, kar9Δ bub2Δ, and kar9Δ kin4Δ cells. Cells were pregrown at 23°C and then incubated for 60, 120, 240, and 360 min at 37°C. Shown is the viability as a percentage. kin4Δ and bub2Δ cells behave as wt cells (data not shown). Molecular Cell 2005 19, 209-221DOI: (10.1016/j.molcel.2005.05.030) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 kin4Δ Cells Arrest in Metaphase in Response to Microtubule Depolymerization (A) Wt, bub2Δ, and kin4Δ cells with SPC42-eqFP CDC14-GFP were synchronized by α factor block. Cells were released into medium containing 10 μg/ml nocodazole at 30°C. Release of nucleolar Cdc14-GFP and the budding of fixed cells were determined. n > 100. (B) Cells of (A) were analyzed for Clb2 levels by immunoblotting. Anti-Tub2 antibodies were used as loading control. Molecular Cell 2005 19, 209-221DOI: (10.1016/j.molcel.2005.05.030) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Kin4 Is an Inhibitor of Mitotic Exit (A and B) Deletion of KIN4 suppresses the mitotic exit defect of lte1Δ cells. The indicated cells with CDC14-GFP SPC42-eqFP were synchronized for 2 hr with α factor at 30°C followed by an incubation for 30 min at 14°C. Cells were washed with precooled YPAD and incubated at 14°C (t = 0). Samples were fixed and stained with DAPI. Anaphase cells (A) and partial and full release of Cdc14-GFP from the nucleolus (B) were determined. n > 80 cells per time point. (C) Deletion of KIN4 allows growth of lte1Δ ste20Δ cells. The indicated cell types all containing LTE1 on the URA3 centromere-based plasmid pRS316 were grown on 5-FOA and YPAD plates (data not shown) for 3 days at 30°C. All strains grew equally well on YPDA plates (not shown). Only cells that can lose the URA3-based pRS316-LTE1 plasmid will grow on 5-FOA plates. (D) In cells with a misaligned anaphase spindle, KIN4 prevents the release of Cdc14 from the nucleolus. The indicated cell types expressing CDC14-GFP were grown for 3 hr at 30°C. Cells were fixed, and DNA was stained with DAPI. The arrows mark kar9Δ cells with two DAPI staining regions in the mother cell body and Cdc14-GFP entrapped in the nucleolus. Asterisks highlight kar9Δ bub2Δ and kar9Δ kin4Δ cells with two DAPI staining regions and Cdc14 released from the nucleolus. Bar, 5 μm. (E) Quantification of (D). Cells with two DAPI staining regions in the mother cell were analyzed for the release of nucleolar Cdc14-GFP. n > 50. Molecular Cell 2005 19, 209-221DOI: (10.1016/j.molcel.2005.05.030) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Kin4 Associates with the Cell Cortex, the SPB in the Mother Cell, and the Bud Neck (A) KIN4-GFP is functional. The indicated cell types all containing pRS316-LTE1 were tested for growth on 5-FOA plates for 3 days at 30°C. All cells grew equally well on YPD plates at 30°C (data not shown). In this test, KIN4-GFP behaves as KIN4 but distinctly from kin4Δ. Thus, KIN4-GFP is functional. (B) Kin4 associates in midanaphase with the mother SPB. KIN4-GFP SPC42-eqFP cells were grown at 30°C in YPDA. Localization of Kin4-GFP of unfixed cells was determined by fluorescence microscopy. The arrowheads point toward Kin4-GFP at the SPB in the mother cell. Bar, 5 μm. (C) KIN4-GFP SPC42-eqFP cells were synchronized with α factor (t = 0). Samples were immediately analyzed by fluorescence microscopy without fixation. Kin4-GFP localization with SPBs, Kin4-GFP at the bud neck, and anaphase cells were determined over time. Between 100 and 150 cells were counted per time point. Molecular Cell 2005 19, 209-221DOI: (10.1016/j.molcel.2005.05.030) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Kin4 Binding to SPBs Is Dependent on Nud1 and Cdc5 (A) Binding of Kin4 to SPBs is impaired in nud1-2 cells. NUD1 and nud1-2 cells with KIN4-GFP SPC42-eqFP were synchronized with α factor at 23°C (t = 0). After washing, cells were shifted to 37°C and analyzed by fluorescence microscopy for Kin4-GFP localization at SPBs (arrowhead in the NUD1 cell) and strong Kin4 association at the bud neck (asterisk). Shown are nud1-2 cells in anaphase with a correctly aligned (upper) and a misaligned (lower) anaphase spindle. The cell in row 1 was analyzed after 60 min and cells in rows 2–4 after 90 min. (B) Quantification of (A). n > 50 per time point. (C) Kin4 fails to bind to SPBs in cells depleted of Cdc5. CDC5 and Gal1-CDC5 cells with KIN4-GFP SPC42-eqFP grown in YP raffinose-galactose medium were synchronized with α factor at 30°C (t = 0). Cells were washed with YPAD to repress Gal1-CDC5 expression and to remove α factor. Cells were further incubated at 30°C and then analyzed by fluorescence microscopy. The arrowhead highlights SPB localization of Kin4 in a CDC5 wt cell, and the asterisk highlights strong bud-neck localization of Kin4. The cell in row 1 was analyzed after 60 min and cells in rows 2 and 3 after 90 min. Bars, 5 μm. (D) Quantification of (C). n > 50 per time point. Molecular Cell 2005 19, 209-221DOI: (10.1016/j.molcel.2005.05.030) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Kin4 Binds to Both SPBs in Cells with a Misaligned Anaphase Spindle and in Response to Microtubule Depolymerization (A) Localization of Bub2-GFP in kar9Δ kin4Δ cells. kar9Δ and kar9Δ kin4Δ cells with BUB2-GFP SPC42-eqFP were analyzed for Bub2-GFP localization by fluorescence microscopy. Shown are cells with a misaligned anaphase spindle in the mother cell body. Bar, 5 μm. (B) SPB localization of Kin4-GFP in kar9Δ cells. Kin4-GFP localization was studied in kar9Δ KIN4-GFP SPC42-eqFP cells grown in YPAD at 30°C. Shown is a cell with a misaligned anaphase spindle located in the mother cell body (top, arrow) and a kar9Δ cell with correctly positioned anaphase spindle (bottom, asterisk). Bar, 5 μm. (C) Quantification of (B). kar9Δ cells with misaligned and correctly aligned anaphase spindles were analyzed for Kin4 SPB localization. n > 50. (D) Kin4 binds to both SPBs in response to microtubule depolymerization. α factor-synchronized KIN4-GFP SPC42-eqFP cells were incubated at 30°C for 2 hr with 10 μg/ml nocodazole until >95% of cells arrested with a large bud. Kin4-GFP localization was determined by fluorescence microscopy. The two SPBs remained close together in response to microtubule depolymerization. Enlargement of the two SPBs (see inlet) revealed that Kin4 was at both SPBs (n = 15). Bar, 5 μm. (E) Metaphase arrest of cells is insufficient to target Kin4 to both SPBs. KIN4-GFP SPC42-eqFP Gal1-CDC20 cells arrested in metaphase upon repression of the Gal1 promoter by the addition of glucose. Kin4-GFP SPB localization was analyzed by fluorescence microscopy. n = 75. Bar, 5 μm. Molecular Cell 2005 19, 209-221DOI: (10.1016/j.molcel.2005.05.030) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 Evidence for a Function of Kin4 Upstream of Bub2-Bfa1 (A) Models for the inhibition of the MEN by Kin4. (a) Kin4 could function upstream of Cdc5 and inhibit Cdc5, (b) counteract the inhibitory activity of Cdc5, or (c) function parallel to Bub2-Bfa1 by inhibiting the activity of a MEN component. The line connecting Kin4 and Cdc5 indicates that SPB localization of Kin4 is dependent on Cdc5 (Figure 5C). (B) Deletion of KIN4 does not suppress the growth defect of cdc5-10 cells. The indicated cell types were grown on YPD plates at 37°C and 23°C for 3 and 5 days, respectively. (C) BFA1-11A inhibits mitotic exit of kar9Δ kin4Δ cells. kar9Δ kin4Δ cells were transformed with plasmid pRS315 (lane 1 and 6), pRS315-KAR9 (lanes 2 and 7), pRS315-KIN4 (lanes 3 and 8), pRS315-BFA1-11A (lanes 4 and 9), and pRS315-BFA1 (lanes 5 and 10). Cells were grown in selective medium at 23°C and then incubated for 1 hr at 37°C. DNA of fixed cells was stained with DAPI. Indicated cell types were determined. n > 100. (D) Kin4 inhibits Bfa1 phosphorylation. KIN4 BFA1-3HA (lanes 1 and 3), and kin4Δ BFA1-3HA cells (lanes 2 and 4) were incubated for 2 hr at 30°C with 10 μg/ml nocodazole to arrest cells in metaphase (+noco) (lanes 3 and 4). Cells before nocodazole treatment (−noco) were taken as controls (lanes 1 and 2). Protein extracts were analyzed by immunoblotting with anti-HA (12CA5) and anti-Tub2 antibodies (loading control). The asterisk in lane 4 (kin4Δ BFA1-3HA cells) indicates a phospho-form of Bfa1-3HA. (E) Bfa1-11A is not phosphorylated in kin4Δ cells. kin4Δ cells carrying pRS315-BFA1-3HA (lane 1) or pRS315-BFA1-11A-3HA (lane 2) were incubated with nocodazole (see Supplemental Data). Bfa1-3HA and Bfa1-11A-3HA were analyzed by immunoblotting. (F) Bfa1 phosphorylation of kin4Δ cells requires Cdc5. BFA1-3HA kin4Δ (lane 1) and Gal1-CDC5 BFA1-3HA kin4Δ cells (lane 2) were incubated in glucose-containing medium for 2 hr with nocodazole. Bfa1-3HA, Cdc5 and Tub2 (loading control) were analyzed by immunoblotting. Molecular Cell 2005 19, 209-221DOI: (10.1016/j.molcel.2005.05.030) Copyright © 2005 Elsevier Inc. Terms and Conditions