1. Volume 40, Issue 1, Pages 37-52 (January 2017)
Casein Kinase 1 Coordinates Cohesin Cleavage, Gametogenesis, and Exit from M Phase in Meiosis II 
Orlando Argüello-Miranda, Ievgeniia Zagoriy, Valentina Mengoli, Julie Rojas, Katarzyna Jonak, Tugce Oz, Peter Graf, Wolfgang Zachariae 
Developmental Cell 
Volume 40, Issue 1, Pages (January 2017)
DOI: /j.devcel
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2. Developmental Cell 2017 40, 37-52DOI: (10.1016/j.devcel.2016.11.021)
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3. Figure 1
Key Events of Meiosis II Require Hrr25 Activity after Anaphase I
(A) Inhibition of Hrr25 at prophase. Spindles (GFP-tubulin), nucleolar release of Cdc14-GFP, and nuclei (histone H2B-RFP) were imaged in HRR25 and hrr25-as cells treated with 1NM-PP1 (5 μM) at 2 hr in SPM. Top: time-lapse series. Bottom: scoring of cellular features. Results were similar with 20 μM 1NM-PP1.
(B–D) Inhibition of Hrr25 at anaphase I. CDC20-mAR control and hrr25-as cells were cultured in SPM for 8 hr, released from metaphase I with CuSO4 (t = 0), and treated with 1NM-PP1 at t = 40 min. (B) IF microscopy of DNA, tubulin, Pds1-myc18, and Cdc14. (C) Quantification of meiotic progression. The average yield is 78% ± 4% tetranucleates (HRR25) or 69% ± 6% binucleates (hrr25-as, n = 6 experiments). (D) Immunoblot detection of proteins.
See also Figure S1.
Developmental Cell  , 37-52DOI: ( /j.devcel )
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4. Figure 2
Removal of Centromeric Cohesin Requires Hrr25 and Separase Activity in Meiosis II
(A) Analysis of centromeric cohesin in CDC20-mAR control and hrr25-as cells treated with 1NM-PP1 at anaphase I (t = 40 min). Top: chromatin spreads stained for DNA, SPBs (γ-tubulin), kinetochores (Mtw1-myc9), and Rec8-ha3. Bottom: percentages of spreads with two SPBs (meiosis I) or four SPBs (meiosis II) and Rec8 on the whole chromatin or at centromeres.
(B) Imaging of Rec8-GFP and SPBs (Cnm67-RFP) in CDC20-mAR control and mutant cells treated to inactivate Hrr25-as, Esp1-2, or Cdc5-as at anaphase I (t = 40 min). Top: representative time-lapse series. Bottom: persistence of centromeric Rec8-GFP signals in control (gray) and mutant (red) cells. Blue lines denote mean values. Control and mutant distributions differ at p < or p < 0.01 (CDC5/cdc5-as).
See also Figure S2.
Developmental Cell  , 37-52DOI: ( /j.devcel )
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5. Figure 3
Cleavage of Rec8 Expressed in Meiosis II Depends on Hrr25 Activity
Immunoblot analysis of Ha3-Scc1 and Ha3-Rec8 expressed from the PEST promoter at metaphase II in CDC20-mAR cells. EST was added at t = 50 min. Dbf4 is degraded at anaphase I and Cdc5 at anaphase II, which requires Hrr25 activity. Cleavage products were quantified relative to the total lane signal.
(A) Ha3-Scc1 is cleaved in HRR25 and hrr25-as cells treated with 1NM-PP1 at anaphase I (arrows).
(B) Ha3-Rec8 is cleaved in HRR25 cells, while Ha3-Rec8-18D but not Ha3-Rec8 is cleaved in hrr25-as cells treated with 1NM-PP1 at anaphase I (arrows).
(C) Ha3-Rec8 but not Ha3-Rec8-24A is cleaved in HRR25 cells.
(D) Ha3-Rec8 is present in α-Myc immunoprecipitates from HRR25-myc9 but not from HRR25 cells.
See also Figure S3.
Developmental Cell  , 37-52DOI: ( /j.devcel )
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6. Figure 4
Rec8 Phosphorylation and PP2A Removal from Centromeres Are Required for the Meiosis II Division and Depend on Hrr25
(A and B) Detection of Sgo1-myc9 and Rts1-ha3 in CDC20-mAR control and hrr25-as cells treated with 1NM-PP1 at anaphase I. (A) Immunoblotting of protein extracts. (B) Percentages of chromatin spreads with two SPBs (γ-tubulin, meiosis I) or four SPBs (meiosis II) and foci of Sgo1 or Rts1.
(C) Anchor-away of Sgo1 before and after inhibition of Hrr25-as at anaphase I. CDC20-mAR hrr25-as cells carrying SGO1-FRB and ribosomal RPL13-FKBP12 were released from metaphase I (t = 0) and treated with 1NM-PP1 at t = 40 min. Rapamycin was omitted (left) or added either 180 min before release (middle) or 50 min after release (right). Shown are time-lapse series and scoring of nuclei (H2B-RFP) and Rts1-GFP foci.
(D) Anchoring Rts1 to centromeric Rec8 blocks the meiosis II division. Rapamycin was added at anaphase I to CDC20-mAR REC8-FKBP12 cells with RTS1 (control) or RTS1-FRB (anchoring to Rec8). Shown are time-lapse series and scoring of SPBs (Spc42-GFP) and nuclei (H2B-RFP).
See also Figure S4.
Developmental Cell  , 37-52DOI: ( /j.devcel )
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7. Figure 5
Sgo1 Degradation at Anaphase II Is Required for Proper Nuclear Division at Meiosis II
Analysis of CDC20-mAR SGO1-myc9 cells expressing Myc9-tagged Sgo1 proteins from the PEST promoter at metaphase II. EST was added at t = 15 min. Data with different green letters differ at p <
(A) Top: immunoblot detection of proteins. Bottom: kinetics of cells with one division (two nuclei), two divisions (more than two nuclei), cells in meiosis I (two SPBs), cells with meiosis I or II spindles, and spreads with Sgo1 foci.
(B) Percentages of chromatin spreads from anaphase II (four SPBs, two stretched DNA masses; n ≥ 72) showing Sgo1 foci.
(C) Percentages of anaphase II cells (two spindles, Cdc14 released; n = 200) having completed (four nuclei) or not completed (two nuclei) the meiosis II division.
(D) Percentages of post-anaphase II cells (t = 140 min, n ≥ 150) with two nuclei, four nuclei, or three to four nuclei of unequal size.
(E) Live-imaging analysis of the time from the meiosis I division (H2B-RFP) to the removal of centromeric Rts1-GFP foci. Red box, time from the meiosis I division (H2B-RFP) to meiosis II spindle disassembly (GFP-tubulin) in CDC20-mAR cells.
(F) Lifetimes of centromeric Rec8-GFP signals quantified from the imaging of Rec8-GFP and SPBs (Cnm67-RFP).
See also Figure S5.
Developmental Cell  , 37-52DOI: ( /j.devcel )
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8. Figure 6
Mps1 Degradation Contributes to Removal of Sgo1-PP2A from Centromeres
(A and B) Detection of Ha3-Mps1 and Bub1-myc18 in CDC20-mAR control and hrr25-as cells treated with 1NM-PP1 at anaphase I (arrows). (A) Immunoblotting of protein extracts. (B) Percentages of spreads with Bub1 foci at different stages of meiosis (n ≥ 128, p > 0.5).
(C–E) Analysis of CDC20-mAR SGO1-myc9 Ha3-MPS1 cells expressing Mps1-mD and Sgo1-mD or Sgo1-mD-3A from the PEST promoter at metaphase II. EST was added at t = 15 min. (C) Top: immunoblot detection of proteins. Bottom: kinetics of cells with one division (two nuclei), two divisions (more than two nuclei), cells in meiosis I (two SPBs), cells with meiosis I or II spindles, and spreads with Sgo1 foci.
(D) Live-imaging analysis of the time from the meiosis I division (H2B-RFP) to the removal of centromeric Rts1-GFP foci. Data with different green letters differ at p < 0.05 (a versus c) or p < (a, c versus b). Red box, time from the meiosis I division (H2B-RFP) to meiosis II spindle disassembly (GFP-tubulin) in CDC20-mAR cells.
(E) Lifetimes of centromeric Rec8-GFP signals quantified from the imaging of Rec8-GFP and SPBs (Cnm67-RFP). Data with different green letters differ at p <
See also Figure S6.
Developmental Cell  , 37-52DOI: ( /j.devcel )
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9. Figure 7 Analysis of Spindle Disassembly at Exit from Meiosis II
(A) Live-imaging analysis of the times from the onset of metaphase II to nuclear division (H2-RFP) or spindle disassembly (GFP-tubulin). CDC20-mAR strains were released from metaphase I at 23°C (t = 0), treated with EST (t = 10 min, to induce PEST-CLB1-mDK), and shifted to 34°C at metaphase II (t = 50 min, to inactivate Cdc14-3). Top: representative time-lapse series. Bottom: times to nuclear division (red) do not differ significantly (p = 0.13). Times to spindle disassembly (gray) with different green letters differ at p < 0.05 (a versus b) or p < (a, b versus c).
(B) Protein levels in extracts from CDC20-mAR control cells and cells expressing non-degradable Clb1 at metaphase II (PEST-CLB1-mDK) and/or low amounts of Ama1 (PDMC1-AMA1).
(C) Disassembly of anaphase II spindles upon inhibition of Cdc5. CDC20-mAR PDMC1-AMA1 PEST-CLB1-mDK cells carrying CDC5 or cdc5-as were released from metaphase I (t = 0) and treated with CMK at anaphase II (t = 100 min). Meiotic events were quantified in fixed cells.
See also Figure S7.
Developmental Cell  , 37-52DOI: ( /j.devcel )
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