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. 2024 Apr 4;15(10):mjad061.
doi: 10.1093/jmcb/mjad061.

Sgo1 interacts with CENP-A to guide accurate chromosome segregation in mitosis

Fengge Wu  1 , Hameed Akbar  1 , Chunyue Wang  1 , Xiao Yuan  1   2 , Zhen Dou  1   2   3 , McKay Mullen  1   3 , Liwen Niu  1   2 , Liang Zhang  1 , Jianye Zang  1 , Zhikai Wang  1   2 , Xuebiao Yao  1   2 , Xiaoyu Song  1   2 , Xing Liu  1   2
Affiliations

Sgo1 interacts with CENP-A to guide accurate chromosome segregation in mitosis

Fengge Wu et al. J Mol Cell Biol. .

Abstract

Shugoshin-1 (Sgo1) is necessary for maintaining sister centromere cohesion and ensuring accurate chromosome segregation during mitosis. It has been reported that the localization of Sgo1 at the centromere is dependent on Bub1-mediated phosphorylation of histone H2A at T120. However, it remains uncertain whether other centromeric proteins play a role in regulating the localization and function of Sgo1 during mitosis. Here, we show that CENP-A interacts with Sgo1 and determines the localization of Sgo1 to the centromere during mitosis. Further biochemical characterization revealed that lysine and arginine residues in the C-terminal domain of Sgo1 are critical for binding CENP-A. Interestingly, the replacement of these basic amino acids with acidic amino acids perturbed the localization of Sgo1 and Aurora B to the centromere, resulting in aberrant chromosome segregation and premature chromatid separation. Taken together, these findings reveal a previously unrecognized but direct link between Sgo1 and CENP-A in centromere plasticity control and illustrate how the Sgo1-CENP-A interaction guides accurate cell division.

Keywords: Aurora B; CENP-A; Sgo1; centromere; kinetochore; mitosis.

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Figures

Figure 1
Figure 1
Sgo1 interacts with CENP-A in vitro and in cells. (A) Schematic diagram illustrating the LacI–LacO targeting assay used in this study. (B) Representative images of U2OS cells co-transfected with mCherry-LacI-Sgo1 (red) and GFP-tagged CENP-A, CENP-C, CENP-I, CENP-N, and histone H3.1 (green). DNA was stained with DAPI. Boxed regions were magnified to the right of the merged images. Scale bar, 10 µm. (C) Quantification of the GFP fluorescence intensities specifically enriched at LacO arrays in B. Thirty cells were examined in each category. (D) Representative immunofluorescence staining of CENP-A (red) in GFP-Sgo1-expressing HeLa cells from different mitotic stages. Prometa., prometaphase. Scale bar, 10 µm. (E) Co-immunoprecipitation assay was performed to determine the physical interaction between Sgo1 and CENP-A. HEK293T cells were transiently transfected with FLAG-Sgo1 and GFP-histone H3.1 or GFP-CENP-A (full-length or truncations) for 24 h. Afterward, cells were harvested, pelleted, lysed, and clarified. The clarified cell lysates were then incubated with FLAG-M2 agarose beads. The immunoprecipitates were resolved on SDS–PAGE, followed by western blotting analyses with the indicated antibodies.
Figure 2
Figure 2
Sgo1 interacts with CENP-A via its three C-terminal fragments. (A) Schematic representation of the full-length human Sgo1 protein and its truncations with different domains. (B) In vitro pull-down assay was performed to map the regions in Sgo1 that are responsible for CENP-A binding. GST-Sgo1 truncations expressed in bacteria were purified and used as affinity matrices to isolate FLAG-CENP-A in HEK293T cells. The binding fractions were analyzed by SDS–PAGE and probed with a FLAG antibody (top). Protein levels were shown by gel staining with Coomassie Brilliant Blue (CBB). An asterisk marks the band corresponding to the protein of interest when multiple bands are present. (C) HeLa cells were transfected with Sgo1 siRNA to suppress the endogenous Sgo1 protein and treated with doxycycline to induce the expression of GFP-tagged full-length Sgo1 and the deletion mutants (ΔCABD1, ΔCABD2, and ΔCABD3). Cells were arrested in mitosis by nocodazole. After fixation, cells were co-stained for ACA (red) and DNA (blue). Scale bar, 10 µm. (D) Quantification of the GFP fluorescence intensities (relative to ACA) at centromeres in C. The data represent mean ± standard error of the mean (SEM) from three independent experiments. At least 30 cells for each group were examined. The Kruskal–Wallis test, followed by Dunn's post hoc test, was used to determine statistical significance. ***P < 0.001.
Figure 3
Figure 3
Lysine and arginine residues determine the Sgo1–CENP-A interaction. (A) The schematic diagram indicates the amino acid positions that were mutated within the Sgo1 protein. The basic amino acids and their positions in the protein sequence were indicated in red. (B and D) In vitro pull-down assay. GST-Sgo1 fragments and mutants expressed in bacteria were purified and used as affinity matrices to isolate FLAG-CENP-A in HEK293T cells. The binding fractions were analyzed by SDS–PAGE and probed with a FLAG antibody (top). Protein levels were shown by gel staining with CBB. An asterisk marks the band corresponding to the protein of interest when multiple bands are present. The high-molecular weight polypeptides purified with the recombinant proteins were bacterial proteins bound to the affinity matrix. (C and E) HeLa cells were transfected with Sgo1 siRNA to suppress the endogenous Sgo1 protein and treated with doxycycline to induce the expression of GFP-tagged WT Sgo1 and mutated Sgo1. Cells were arrested in mitosis by nocodazole. After fixation, cells were co-stained for ACA (red) and DNA (blue). Scale bar, 10 µm.
Figure 4
Figure 4
Lysine and arginine residues of CABD3 mediate Sgo1 centromeric localization in mitosis. (A) HeLa cells were transfected with Sgo1 siRNA to suppress the endogenous Sgo1 protein and treated with doxycycline to induce the expression of GFP-tagged WT Sgo1 and mutated Sgo1. Cells were arrested in mitosis by nocodazole. After fixation, cells were co-stained for ACA (red) and DNA (blue). Scale bar, 10 µm. (B) Quantification of the GFP fluorescence intensities (relative to ACA) at centromeres in A. The data represent the mean ± SEM from three independent experiments. A total of 30 cells for each group were examined. The Kruskal–Wallis test, followed by Dunn's post hoc test, was used to determine statistical significance. ***P < 0.001; NS, not significant. (C) Representative real-time images of chromosome movements in Sgo1-suppressed HeLa cells expressing GFP-tagged WT Sgo1 and mutated Sgo1. Time shows hours and minutes (h:min) since nuclear envelope breakdown. Scale bar, 10 µm. (D) Quantification of the prometaphase arrest of live HeLa cells in C. The data represent the mean ± SEM from three independent experiments. At least 30 cells for each group were examined. **P < 0.01 by two-tailed unpaired Student's t-test.
Figure 5
Figure 5
The Sgo1–CENP-A interaction is required for the centromeric localization of Aurora B in mitosis. (A, C, and E) HeLa cells were transfected with Sgo1 siRNA to suppress the endogenous Sgo1 protein and treated with doxycycline to induce the expression of GFP-tagged WT Sgo1, CABD3-deleted Sgo1, and CABD3-mutated Sgo1. Cells were arrested in mitosis by nocodazole. After fixation, cells were co-stained for Aurora B (red), ACA (gray), and DNA (blue). Scale bar, 10 µm. (B, D, and F) Quantification of the Aurora B fluorescence intensities (relative to ACA) at centromeres in A, C, and E. The data represent the mean ± SEM from three independent experiments. At least 30 cells for each group were examined. Statistical significance was tested by two-tailed Mann–Whitney U-test; ***P < 0.001.
Figure 6
Figure 6
The Sgo1–CENP-A interaction affects the inter-kinetochore distance. (A, C, and E) HeLa cells were transfected with Sgo1 siRNA to suppress the endogenous Sgo1 protein and treated with doxycycline to induce the expression of GFP-tagged WT Sgo1, CABD3-deleted Sgo1, and CABD3-mutated Sgo1. At 8 h after release from thymidine block, the cells were treated with 10 µM MG132 for 1 h, and the chromosomes were then squashed onto coverslips, followed by fixation. After fixation, cells were co-stained for ACA (red) and DNA (blue). Scale bar, 10 µm. (B, D, and F) The inter-kinetochore distance was measured for over 300 chromosomes in 30 different metaphase cells in A, C, and E. The data represent the mean ± SEM from three independent experiments. The Kruskal–Wallis test, followed by Dunn's post hoc test, was used to determine statistical significance. ***P < 0.001; NS, not significant.
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