INCENP-Δ539-747 localizes Aurora-B to centromeres during (pro) metaphase. (a–d) U2OS cells grown on coverslips were transfected with indicated plasmids and H2B-GFP (depicted in blue). Cells were synchronized using thymidine. Fourteen hours after thymidine release, cells were fixed and immunostained with the indicated antibodies. Insets show magnified images of the indicated regions. (c and d) The percentage (±SEM) of transfected cells with centromeric Aurora-B staining (c) and Aurora-B-levels on individual centromeres (d; ±SEM) was quantified. See Materials and Methods for more detail on how Aurora-B levels on individual centromeres were determined.

Syntelic attachments can be resolved in cells expressing INCENP-Δ539-747. (a) Schematic representation of experimental setup. U2OS cells grown on coverslips were transfected with indicated plasmids and H2B-GFP (blue). Cells were synchronized using thymidine. Ten hours after thymidine release monastrol was added for 2 h, and during the second hour MG132 was added. Cells were washed and released into fresh medium containing MG132. Cells were fixed after 90 min and immunostained with the indicated antibodies. (b and c) Typical examples (b) and quantification (c; ±SEM) from each category. (d–f) U2OS cells grown on coverslips were transfected with indicated plasmids and H2B-GFP (blue). Cells were synchronized using thymidine, and 12 h after thymidine release taxol and MG132 were added for 3 h. Cells were fixed and immunostained with the indicated antibodies. (e) The percentage (±SEM) of transfected cells with CLIP-170 kinetochore staining was quantified. (f) Quantification of the number (±SEM) of CLIP-170 positive kinetochores per cell. (g and h) U2OS cells were transfected with the indicated plasmids and GFP-Spectrin. Cells were synchronized using thymidine. Eighteen hours after release into taxol or nocodazole, cells were harvested, and mitotic percentages were determined by MPM-2/PI staining and FACS analysis. (h) A fraction of the cells from panel f was lysed and analyzed by immunoblotting with the indicated antibodies. Asterisk denotes aspecific background signal that was used as loading control.

In vivo activation of Aurora-B is normal in cells expressing INCENP-Δ539-747. (a–d) U2OS cells grown on coverslips were transfected with indicated plasmids and H2B-GFP (blue). Cells were synchronized using thymidine. Fourteen hours after thymidine release, cells were fixed and immunostained with the indicated antibodies. (b–d) Quantification of the percentage (±SEM) of transfected cells with positive phospho-CENP-A staining (b) and phospho-CENP-A levels on individual centromeres (c and d; ±SEM). See Materials and Methods for more detail on how phospho-CENP-A levels on individual centromeres were determined. (d) Experiments similar to panel a, with the exception that 3 h before fixation, taxol (in combination with MG132) was added to the cells. (e) Experiments similar to panel a, with the exception that upon release from thymidine block, nocodazole was added to the cells.

A spindle checkpoint defect in cells expressing INCENP-Δ539-747. (a) U2OS cells were transfected with the indicated plasmids and GFP-Spectrin. Cells were synchronized using thymidine. Eighteen hours after release into taxol, cells were harvested and mitotic percentages (±SEM) were determined by MPM-2/PI staining and FACS analysis. (b) U2OS cells were transfected with the indicated plasmids and pBabe-puro. Cells were selected for puromycin-resistance for 36 h to enrich for transfected cells. Cells were lysed and analyzed by immunoblotting with the indicated antibodies. Alpha-tubulin was used as loading control. (c) and (d) U2OS cells were grown on glass-bottom culture dishes and cotransfected with H2B-GFP and the indicated plasmids. Cells were synchronized using thymidine and were followed by time-lapse imaging starting 10 h after release from thymidine. Time in the bottom right corner refers to the elapsed time (hours:minutes) from the time of nuclear envelope breakdown (NEB). Quantification (±SEM) of live cell imaging data are shown in d. (e) U2OS cells were transfected with the indicated plasmids and GFP-Spectrin. Cells were synchronized using thymidine. Eighteen hours after thymidine release, cells were harvested, and mitotic percentages (±SEM) were determined by MPM-2 staining and FACS analysis. (f) Western blotting analysis of panel e.

Normal mitotic progression in cells expressing INCENP-Δ539-747. (a and b) U2OS cells grown on coverslips were transfected with indicated plasmids and H2B-GFP (blue). Cells were synchronized using thymidine, and 12 h after thymidine release MG132 was added for the final 3 h. Cells were fixed and immunostained with the indicated antibodies. (a) Typical examples of cells found in different samples. (b) Quantification (±SEM) of the observed metaphase configurations. (c) Similar to a and b, with the exception that cells were treated with MG132 for 45 (black bars ■) or 90 min (white bars □). Percentage (±SEM) of the metaphase cells within the total mitotic population is shown. (d and e) U2OS cells were grown on glass-bottom culture dishes and cotransfected with H2B-GFP and the indicated plasmids. Cells were synchronized and were followed by time-lapse imaging starting 10 h after release from a thymidine block. Time in the bottom right corner refers to the elapsed time (hours:minutes) from the time of nuclear envelope breakdown (NEB). (e) Quantifications (±SEM) of alignment state upon anaphase onset.

Checkpoint function and chromosome alignment are equally sensitive to Aurora-B inhibition. (a and b) U2OS cells were synchronized using thymidine. Directly after release cells were treated with the indicated concentrations of ZM447392 or DMSO with or without taxol. Eighteen hours later, cells were harvested and mitotic percentages (right panel) and percentages (±SEM) of cells with 4n DNA content (left panel) were determined by MPM-2 and PI staining and analyzed by FACS. (b) Cells were treated with DMSO or the indicated concentrations of ZM447392 in combination with nocodazole for 15 h. Mitotic cells were obtained by mechanical detachment and analyzed by immunoblotting with the indicated antibodies. (c and d) U2OS cells grown on coverslips were synchronized by thymidine treatment. Directly after release cells were treated with the indicated concentrations of ZM447392 or DMSO. Cells were treated with MG132 for 3 h starting 12 h after release from thymidine. (d) After fixation, mitotic figures were quantified (±SEM).

BubR1 is recruited normally to unattached kinetochores in cells expressing INCENP-Δ539-747. (a and b) U2OS cells grown on coverslips were transfected with indicated plasmids and H2B-GFP (blue). Cells were synchronized using thymidine, and nocoda-zole was added immediately after release from thymidine. Fourteen hours later cells were fixed and immunostained with the indicated antibodies. Typical examples (a) and quantification (b; ±SEM) from each category are shown. (c) Model depicting the bifurcated effect of the CPC on spindle checkpoint function. On the presence of nonbipolar attachments, lack of tension is detected by the CPC (in a currently unknown way). The concurrent response to the lack of tension across the sister chromatids is twofold. First, the CPC promotes destabilization of the microtubule-kinetochore interactions that do not create tension and thereby creates unattached kinetochores that subsequently produce a spindle assembly checkpoint signal. Second, the CPC (with a specific requirement for the coiled-coil domain) is required to strengthen the signal emanating from the unattached kinetochores to produce efficient checkpoint signals that are required to prevent premature anaphase onset. This effect could either be dependent or independent of Aurora-B (possibly via a Survivin/INCENP subcomplex).