Centrosomal Aki1 and cohesin function in separase-regulated centriole disengagement

Abstract

Sister chromatid separation at anaphase is triggered by cleavage of the cohesin subunit Scc1, which is mediated by separase. Centriole disengagement also requires separase. This dual role of separase permits concurrent control of these events for accurate metaphase to anaphase transition. Although the molecular mechanism underlying sister chromatid cohesion has been clarified, that of centriole cohesion is poorly understood. In this study, we show that Akt kinase-interacting protein 1 (Aki1) localizes to centrosomes and regulates centriole cohesion. Aki1 depletion causes formation of multipolar spindles accompanied by centriole splitting, which is separase dependent. We also show that cohesin subunits localize to centrosomes and that centrosomal Scc1 is cleaved by separase coincidentally with chromatin Scc1, suggesting a role of Scc1 as a connector of centrioles as well as sister chromatids. Interestingly, Scc1 depletion strongly induces centriole splitting. Furthermore, Aki1 interacts with cohesin in centrosomes, and this interaction is required for centriole cohesion. We demonstrate that centrosome-associated Aki1 and cohesin play pivotal roles in preventing premature cleavage in centriole cohesion.

Figure 1.

Aki1 localizes to centrosomes. (A) HeLa cells were costained for Aki1 (green) and centrin2 (red). Interphase or mitotic cells are shown. Arrows indicate the position of centrosomes, and insets show high magnification images of centrosomes. (B) HeLa cells stably expressing AcGFP-Aki1 (green) were counterstained for γ-tubulin (γ-tub; red). (C) Immunoblots were performed on cytoplasmic, nuclear, and sucrose gradient fractions (centrosome preparations) from HeLa cells with the indicated antibodies. Bars, 5 µm.

Figure 2.

Centrosomal Aki1 is essential for formation of bipolar spindles. (A) HeLa cells were transfected with control, Aki1-2, or Aki1-3 siRNA. Cell lysates were immunoblotted with the indicated antibodies. (B–D) HeLa cells were transfected with control or Aki1-2 siRNA and costained for Aki1 (green) and γ-tubulin (γ-tub; red), or for α-tubulin (α-tub; green) and γ-tubulin or pericentrin (red; C and D). (E) Percentage of multipolar cells of mitotic cells in control, Aki1-2, or Aki1-3 siRNA–treated cells. (F) Percentage of multipolar cells in control or Aki1-2 siRNA–treated cells in interphase or mitosis. (G) HeLa cells stably expressing AcGFP-WT or ΔC815-Aki1 (green) were counterstained for γ-tubulin (red). (H) Percentage of multipolar cells of mitotic cells in control or Aki1-2 siRNA–treated cells expressing AcGFP-mock, AcGFP-WT-rAki1, or AcGFP-ΔC815-rAki1. Error bars represent SD of triplicate experiments (150–200 total cells were scored per condition). Bars, 5 µm.

Figure 3.

Depletion of Aki1 induces spindle checkpoint arrest. (A) Percentage of mitotic cells in control or Aki1-2 siRNA–treated cells. Error bars represent SD of triplicate experiments (2,000–2,500 total cells were scored per condition). (B) HeLa cells were transfected with control or Aki1-2 siRNA. Cell lysates were immunoblotted with the indicated antibodies. (C–F) HeLa cells were transfected with control or Aki1-2 siRNA and costained for α-tubulin (α-tub; green) and BubR1, CENP-E, phospho-Ser¹⁰–histone (P-his), or cyclin B1 (red). (G) HeLa cells were transfected with control or Aki1-2 siRNA. Cell viability was measured using the cell viability assay. (H) HeLa cells were transfected with control or Aki1-2 siRNA. Cell lysates were immunoblotted with the indicated antibodies. Error bars represent SD of triplicate experiments. Bars, 5 µm.

Figure 4.

Formation of multipolar spindles induced by Aki1 depletion is dependent on separase. (A) HeLa cells were transfected with control or Aki1-2 siRNA and costained for α-tubulin (α-tub; green) and centrin2 (red). Centrosomes are shown enlarged on the left. (B) Quantitative analysis of centriole number at each spindle pole in control or Aki1-2 siRNA–treated cells (mean of three experiments; 100 total cells were scored per condition). (C) HeLa cells were transfected with the indicated siRNA. Cell lysates were immunoblotted with the indicated antibodies. (D) Percentage of multipolar cells of mitotic cells in indicated siRNA-treated cells. Error bars represent SD of triplicate experiments (150–200 total cells were scored per condition). (E) HeLa cells were transfected with control or Aki1-2 siRNA and harvested asynchronously (Asyn) or 0 or 2 h after release (Rel) from nocodazole (Noc) arrest. Cell lysates were immunoblotted with the indicated antibodies. Bars, 5 µm.

Figure 5.

Formation of a centrosomal Aki1–cohesin complex is required for centriole cohesion. (A) Immunoblots were performed on cytoplasmic, nuclear, and sucrose gradient fractions (centrosome preparations) from HeLa cells with the indicated antibodies. (B) HeLa cells were harvested 0 or 2 h after release from nocodazole arrest, and centrosomes were purified. Extracts obtained from indicated sucrose gradient fractions and chromatin fractions were immunoblotted with the indicated antibodies. Arrows indicate the full length (top) and C terminus (bottom) of Scc1. The top Scc1 blot shows a longer exposure of the bottom blot. (C) HeLa cells were transfected with control or Scc1 siRNA and costained for α-tubulin (α-tub; green) and centrin2 (red). Centrosomes are shown enlarged on the left. (D) Centrosomes were purified from thymidine (Thy)-arrested cells, nocodazole (Noc)-arrested cells, or cells released (Rel) from a nocodazole arrest for 2 h. Centrosomal proteins were immunoprecipitated with control rabbit IgG, anti-Aki1, or anti-SMC1 (for reciprocal immunoprecipitation [IP]) antibody. Immunoprecipitated proteins, nuclear/chromatin (Nu/ch) lysates, and centrosomal (Cen) lysates were immunoblotted with the indicated antibodies. (E) 293T cells were transfected with pFlag-CMV-2 vector encoding none (mock), WT-Aki1, or Aki1 mutants as indicated. Immunoprecipitated proteins and cell lysates were immunoblotted with antibodies to SMC1 or Flag. (F) Percentage of multipolar cells of mitotic cells in control or Aki1-2 siRNA–treated cells expressing AcGFP-mock, AcGFP-WT-rAki1, or AcGFP-ΔN415-rAki1. Error bars represent SD of triplicate experiments (150–200 total cells were scored per condition). (G) Centrosomes were purified from thymidine-arrested control cells, nocodazole-arrested control cells, or Aki1-depleted cells harvested by mitotic shake off. Cytoplasmic (C), nuclear (N), and centrosomal lysates (fractions 10–12) were immunoblotted with the indicated antibodies. Bars, 5 µm.