Shugoshin Protein Function in Chromosome Segregation
Figure 1
- a: Cells with Rec8 and Sgo1 alone can grow. When Sgo1 and Rec8 are expressed together, growth is impaired.
- b: A GFP dot marks centromere 2. In cells with Rec8 and Sgo1, the GFP marker migrates to the same side instead of separating, causing nondisjunction.
Nondisjunction: Sister chromatids fail to separate properly. - c: Quantifies nondisjunction seen in Rec8 and Sgo1, which causes a large increase in failed sister chromatid separation.
- d: Rec8-GFP normally disappears during anaphase. When Sgo1 is present, Rec8 remains at the centromere of most cells.
Figure 2
- a: Normal meiosis model where sister chromatid separation occurs only at meiosis II.
- b: In Sgo1Δ cells, meiosis I is mostly normal, but about half the cells show nondisjunction during meiosis II.
- c: In normal meiosis, the presence of two centromere-GFPs shows that homologous chromosomes separate, but in SgoΔ cells, more signals appear, showing that sister chromatids split.
- d: Wild-type cells retain Rec8-GFP in late anaphase I. SgoΔ cells show little centromeric Rec8, proving Sgo1 is needed to preserve Rec8 in M1.
- e: Before meiosis I, Rec8 localization is similar in wild-type and Sgo1Δ cells. Therefore, Sgo1 is not required initially to recruit Rec8, but is needed to protect Rec8 later during anaphase I.
Figure 3
- a: Uses mutant yeast (temperature-sensitive). Shifting the temperature forces cells to enter meiosis; monitoring Sgo1 expression shows Sgo1 appears in M1 and then disappears.
- b: Shows Sgo1 appears as centromeric dots during metaphase I, decreases during anaphase, and is gone in M2.
- c: Monitored Sgo1 and Mis6 location. Mis6 is known to localize at centromeres. Sgo1 was spotted near Mis6, confirming Sgo1 is also present at the centromeres.
- d: ChIP assay quantifying exactly where along the chromosome Sgo1 binds. It shows enrichment in pericentromeric regions, not chromosome arms, which is exactly where protected Rec8 is found.
- e: In wild-type cells, Sgo1 forms centromeric foci, whereas in bub1Δ cells, it is not present in the same area. Western blot shows Sgo1 is still present, indicating Bub1 controls Sgo1 localization.
- f: Rec8 is pulled down after Sgo1 is immunoprecipitated, showing that Sgo1 and Rec8 associate with each other in vivo.
- g: Model showing Bub1 helps localize Sgo1 to the centromere, Sgo1 protects Rec8 there, and Sgo1 is removed in anaphase I.
Figure 4
- a: Cells lacking Sgo2 are more sensitive to the spindle-destabilizing drug TBZ. Sgo2 helps maintain proper kinetochore and spindle function in mitosis.
- b: Combining Sgo2Δ and Swi6Δ causes a large growth defect. Sgo2 and heterochromatin cooperate for proper chromosome segregation.
- c: Wild-type cells show little chromosome nondisjunction. The Sgo2Δ/Swi6Δ mutant shows an increase, disrupting mitotic chromosome segregation.
- d: Sgo2-GFP localizes to centromeres during metaphase and disappears during anaphase. In bub1Δ cells, this localization is lost, showing that Bub1 is needed for Sgo2 localization.
- e: Sgo2 overlaps with the Mis6 protein, showing centromeric localization.
- f: ChIP assays show Sgo2 enrichment in the pericentromeric region.
Figure 5
- a: Yeast lacking ScSgo1 are sensitive to the spindle poison benomyl. ScSgo1 is important for kinetochore and spindle chromosome segregation.
- b: Wild-type cells have little chromosome loss. ScSgo1Δ cells have high chromosome loss, showing ScSgo1 is important for chromosome stability.
- c: In ScSgo1Δ cells, meiosis I occurs, but meiosis II segregation is abnormal, resembling Sgo1Δ in fission yeast.
- d: A tagged ScSgo1 strain shows 96% normal reductional segregation in M1, but substantial nondisjunction in M2. The primary defect stems from the loss of centromeric cohesion in M1.
- e: Wild-type sisters remain together in anaphase I. In ScSgo1-Myc, GFP dots frequently split, showing premature separation.
Figure 6
Compares shugoshin-like proteins from different organisms, showing conserved regions, especially the N- and C-terminals, which demonstrates that shugoshin is a conserved protein family.