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. 2022 Feb 1;11:e74447. doi: 10.7554/eLife.74447

Figure 8. Critical Wpl1-dependent and independent roles of Eco1 in chromosome segregation during both meiosis I and II.

(A) Meiotic double-strand break formation arrests eco1-aa cells in meiotic prophase. Wild-type (AM24167), wpl1Δ (AM24168), eco1-aa (AM24184), eco1-aa wpl1Δ (AM24169), spo11Δ (AM27670) spo11Δ wpl1Δ (AM27673), spo11Δ eco1-aa (AM27672), and spo11Δ eco1-aa wpl1Δ (AM27671) anchor-away strains carrying heterozygous SPC42-tdTOMATO and CENV-GFP were induced to sporulate. The percentage of cells with more than one Spc42-tdTomato focus was determined from cell populations fixed at the indicated timepoints after resuspension in SPO medium containing rapamycin. At least 100 cells were scored per timepoint. (B–F) Live-cell imaging of strains as in (A) sporulated in the presence of rapamycin. Every cell that had one Spc42-tdTomato focus at the start of the movie was scored for the duration of the movie. A small number of mitotic/dead cells (<1% for each strain) were excluded from the analysis. (B) Deletion of SPO11 restores meiotic progression to eco1-aa cells. The percentage of cells that displayed two (meiosis I) or four (meiosis II) Spc42-tdTomato foci for each strain is shown. (C) Representative images of spo11Δ background cells undergoing the meiotic divisions. (D) Percentage of cells with one Spc42-tdTomato focus (prophase I) where two GFP foci were visible. Only cells that progressed to anaphase I were scored in this analysis. (E) Eco1 counteracts Wpl1 to allow the establishment of sister kinetochore mono-orientation. Segregation of CEN5-GFP foci to the same (reductional; dark gray) or opposite (equational; green) poles was scored in meiosis I (as two Spc42-tdTomato foci separate; binucleate cells). (F) Eco1 is required for pericentromeric cohesion, even in the absence of Wpl1. Segregation of CEN5-GFP foci to opposite (dark gray) or the same pole(s) (blue) was scored in meiosis II (as four Spc42-tdTomato foci separate; tetranucleate cells). Cells that had already segregated their sister CEN5-GFPs in meiosis I (GFP foci in two nuclei at the binucleate stage) were scored as a separate category (green).

Figure 8.

Figure 8—figure supplement 1. Sister kinetochore mono-orientation and pericentromeric cohesion defects in eco1-aa cells are not a consequence of SPO11 deletion.

Figure 8—figure supplement 1.

Representative images of wild-type (AM24167), wpl1Δ (AM24168), eco1-aa (AM24184), and eco1-aa wpl1Δ (AM24169) anchor-away cells carrying heterozygous CEN5-GFP and SPC42-tdTomato. Cells were imaged in the presence of rapamycin together with spo11Δ background strains in the experiment shown in Figure 8B–F.
Figure 8—figure supplement 2. Sister kinetochore mono-orientation and pericentromeric cohesion defects in eco1-aa cells are not caused by mislocalization of cohesin protector proteins Spo13 or Sgo1.

Figure 8—figure supplement 2.

(A–C) Cohesin protector Spo13 localization follows the profile of Rec8 in metaphase I eco1-aa and wpl1Δ single and double mutant cells. Diploid strains wpl1Δ (AM24263), eco1-aa (AM24262), and wpl1Δ eco1-aa (AM24261) carrying RPL13A-FKBP12, fpr1Δ, tor1-1, pCLB2-CDC20, REC8-3HA, and SPO13-3FLAG and wild-type anchor-away diploid strains carrying either REC8-3HA (AM24236) or SPO13-3FLAG (AM24235) were induced to sporulate in 1 μM rapamycin and harvested after 6 hr. (A, B) Chromatin-bound Rec8 and Spo13 are increased in wpl1Δ and eco1-aa wpl1Δ mutants. Mean α-HA (A; Rec8-3HA) or α-FLAG (B, Spo13-3FLAG) ChIP-qPCR for the indicated sites is shown from four repeats with error bars representing standard error. *p<0.05, paired Student’s t-test. (C) Western immunoblot using α-Smc3-K112,113-Ac, α-FLAG, α-HA, and α-Kar2 (loading control) confirming similar total levels of Rec8 and Spo13 in the expected strains. (D, E) Failure to localize pericentromeric Sgo1 cannot explain the segregation defect of wpl1Δ eco1-aa cells. Wild-type (AM24054), wpl1Δ (AM25103), eco1-aa (AM25104), and eco1-aa wpl1Δ (AM25102) strains carrying RPL13A-FKBP12, fpr1Δ, tor1-1, pCLB2-CDC20, SGO1-6HA, as well as a no tag strain (AM23700) without SGO1-6HA, were induced to sporulate in 1 μM rapamycin and harvested after 6 hr (metaphase I). (D) Mean α-HA ChIP-qPCR (Sgo1-6HA) is shown for four replicates at the indicated sites with error bars showing standard error. **p<0.01, *p<0.05, paired Student’s t-test. (E) Western immunoblot probed with α-Smc3-K112,113-Ac, α-HA (Sgo1-6HA), and α-Kar2 (loading control). Note that Sgo1 migration was reproducibly increased in eco1-aa cells, which is likely a result of reduced phosphorylation caused by impaired progression into metaphase I.
Figure 8—figure supplement 2—source data 1. Source images for the blot in Figure 8—figure supplement 2C.
Figure 8—figure supplement 2—source data 2. Source images for the blot in Figure 8—figure supplement 2C.
Figure 8—figure supplement 2—source data 3. Source images for the blot in Figure 8—figure supplement 2C.
Figure 8—figure supplement 2—source data 4. Source images for the blot in Figure 8—figure supplement 2C.
Figure 8—figure supplement 2—source data 5. Source images for the blot in Figure 8—figure supplement 2E.
Figure 8—figure supplement 2—source data 6. Source images for the blot in Figure 8—figure supplement 2E.
Figure 8—figure supplement 2—source data 7. Source images for the blot in Figure 8—figure supplement 2E.