Human Bub1 protects centromeric sister-chromatid cohesion through Shugoshin during mitosis

Abstract

Sister chromatids in mammalian cells remain attached mostly at their centromeres at metaphase because of the loss of cohesion along chromosome arms in prophase. Here, we report that Bub1 retains centromeric cohesion in mitosis of human cells. Depletion of Bub1 or Shugoshin (Sgo1) in HeLa cells by RNA interference causes massive missegregation of sister chromatids that originates at centromeres. Surprisingly, loss of chromatid cohesion in Bub1 and Sgo1 RNA-interference cells does not appear to require the full activation of separase but, instead, triggers a mitotic arrest that depends on Mad2 and Aurora B. Bub1 maintains the steady-state levels and centromeric localization of Sgo1. Therefore, Bub1 protects centromeric cohesion through Shugoshin in mitosis.

Fig. 1.

Loss of hBub1 or hSgo1 causes chromosome missegregation and mitotic arrest. (A) Log phase or nocodazole-treated control, hBub1, and hSgo1 RNAi HeLa tet-on cells were lysed with SDS sample buffer and blotted with the indicated antibodies. APC2 was used as the loading control. (B) Mitotic indices of control, hBub1 and hSgo1 RNAi cells 24 or 48 h after transfection. Live cells were stained with Hoechst 33342 and observed with an inverted fluorescence microscope. Mitotic cells were scored based on their cell shape and DNA morphology. Three separate fields with >100 cells each were counted. The average values and standard deviation are shown. (C) HeLa tet-on cells stably expressing H2B-GFP (green) were transfected with control, hBub1, or hSgo1 siRNA; fixed with paraformaldehyde; and stained with either anti-tubulin or CREST (red). The percentages of cells with a given phenotype and the number of examined cells are shown on the right.

Fig. 2.

Loss of centromeric cohesion in hBub1 and hSgo1 RNAi cells. (A) Mitotic chromosome spread of control, hBub1, and hSgo1 RNAi cells. The percentages of cells with a given phenotype and the number of cells examined are shown on the right. (Scale bars, 10 μm.) (B) Four major categories (I–IV) of phenotypes observed in mitotic chromosome spread of hBub1 and hSgo1 RNAi cells. Some of the sister chromatids that are separated only at centromeres are indicatedby arrows and magnified in the Insets.(C) Mitotic chromosome spread of hSgo1 RNAi cells. Chromosomes are shown in green, and the kinetochores stained with CREST are shown in red. Chromosomes that are separated at the centromeres but remain attached at one or both arms are indicated by arrows.

Fig. 3.

hBub1 maintains hSgo1 at centromeres. (A) HeLa tet-on cells were stained with anti-hSgo1 (red), CREST (green), and DAPI (blue). (B) HeLa tet-on cells were stained with anti-hSgo1 or anti-hBub1 (red), CREST (green), and DAPI (blue). The boxed areas are magnified and shown in Insets. (C) Control and hBub1 RNAi cells in mitosis were stained with anti-hSgo1 (red), CREST (green), and DAPI (blue).

Fig. 4.

Loss of hBub1 or hSgo1 triggers the Mad2- and Aurora B-dependent spindle checkpoint. (A) HeLa tet-on cells stably expressing H2B-GFP (green) were transfected with control or hBub1 siRNA and stained with anti-cyclin A2 or anti-securin (red). (B) Live-cell imaging of hSgo1 RNAi, hSgo1/Mad2 RNAi, and hSgo1/Aurora B RNAi HeLa tet-on cells stably expressing H2B-GFP (green). Note that some cells do not express H2B-GFP. DIC, differential-interference contrast. (C and D) Mitotic indices of H2B-GFP expressing HeLa tet-on cells transfected with the indicated siRNAs. Three separate fields with >100 cells each were counted. The average values and standard deviation are shown.

Fig. 5.

Depletion of separase by RNAi does not prevent sister-chromatid separation in hSgo1 RNAi cells. (A) Time course of the RNAi experiment shown in B.(B) Percentage of mitotic cells treated as described in A that exhibit the indicated phenotype. The number of analyzed cells analyzed is given in parentheses.