Figure 4. BRISC is required for chromosomal MT assembly. (A) Quantification of the number of MT asters in each mitotic cell fixed at the indicated times after NOC washout, as described in Fig. S3 B (n = 120 for each time point; means ± SD; three independent experiments). (B) Representative confocal images of K-fiber formation in control ABRO1/BRCC36–silenced cells. HeLa cells released from NOC for 6 min, as described in Fig. S3 B, were fixed and stained for Hec1 (green) and α-tubulin (red). K-fiber formation is significantly reduced in ABRO1/BRCC36-silenced cells compared with that in controls. Insets show K-fibers. (C) Representative confocal images of the kinetochore–MT attachment in control and BRCC36-silenced cells. Cells were treated with 10 µM MG132 for 4 h, fixed, and stained for Hec1 (green) and α-tubulin (red). End-on amphitelic attachment of MTs to kinetochores is observed in control cells. However, kinetochore–MT attachment was deficient or impaired in BRCC36-silenced cells, and many kinetochores were laterally bound to MT bundles. (D) Representative confocal images of the interkinetochore distance in control and BRCC36-silenced cells. Control or BRCC36-silenced HeLa cells were treated with 10 µM MG132 or 30 ng/ml NOC for 4 h, and then fixed and stained for the outer kinetochore marker Hec1 (red) and CREST (green). CREST sera are human autoantibodies against centromeric proteins. The images correspond to confocal microscopy maximum projections for NOC-treated cells and single confocal microscopy sections for 10 µM of MG132-treated cells. The white arrows show paired sister kinetochores. (E) Box-and-whiskers plots showing the quantification of the interkinetochore distance. Boxes show the upper and lower quartiles (25–75%) with a line at the median. Whiskers extend from the 10th to the 90th percentile, and dots correspond to outliers. Results are means ± SD. The mean interkinetochore distance is significantly lower in BRCC36-silenced cells than in the controls (1.10 vs. 1.51 µm). This effect is dependent on MT forces, as the interkinetochore distance in control and BRCC36-silenced cells is similar after NOC treatment (1.01 vs. 1.02 µm). ***, P < 0.001; Student’s t test. Bars, 5 µm. Error bars show mean ± SD.
Figure 4.

BRISC is required for chromosomal MT assembly. (A) Quantification of the number of MT asters in each mitotic cell fixed at the indicated times after NOC washout, as described in Fig. S3 B (n = 120 for each time point; means ± SD; three independent experiments). (B) Representative confocal images of K-fiber formation in control ABRO1/BRCC36–silenced cells. HeLa cells released from NOC for 6 min, as described in Fig. S3 B, were fixed and stained for Hec1 (green) and α-tubulin (red). K-fiber formation is significantly reduced in ABRO1/BRCC36-silenced cells compared with that in controls. Insets show K-fibers. (C) Representative confocal images of the kinetochore–MT attachment in control and BRCC36-silenced cells. Cells were treated with 10 µM MG132 for 4 h, fixed, and stained for Hec1 (green) and α-tubulin (red). End-on amphitelic attachment of MTs to kinetochores is observed in control cells. However, kinetochore–MT attachment was deficient or impaired in BRCC36-silenced cells, and many kinetochores were laterally bound to MT bundles. (D) Representative confocal images of the interkinetochore distance in control and BRCC36-silenced cells. Control or BRCC36-silenced HeLa cells were treated with 10 µM MG132 or 30 ng/ml NOC for 4 h, and then fixed and stained for the outer kinetochore marker Hec1 (red) and CREST (green). CREST sera are human autoantibodies against centromeric proteins. The images correspond to confocal microscopy maximum projections for NOC-treated cells and single confocal microscopy sections for 10 µM of MG132-treated cells. The white arrows show paired sister kinetochores. (E) Box-and-whiskers plots showing the quantification of the interkinetochore distance. Boxes show the upper and lower quartiles (25–75%) with a line at the median. Whiskers extend from the 10th to the 90th percentile, and dots correspond to outliers. Results are means ± SD. The mean interkinetochore distance is significantly lower in BRCC36-silenced cells than in the controls (1.10 vs. 1.51 µm). This effect is dependent on MT forces, as the interkinetochore distance in control and BRCC36-silenced cells is similar after NOC treatment (1.01 vs. 1.02 µm). ***, P < 0.001; Student’s t test. Bars, 5 µm. Error bars show mean ± SD.

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