Figure 4. Fzy is recruited on I-CreI– or laser-induced breaks in a BubR1 KEN box–dependent manner. (A) Time-lapse images of WT (also see Video 5), bubR1-KEN (also see Video 6), and fzy-DYY* neuroblasts expressing I-CreI. WT and bubR1-KEN mutant cells express GFP::Fzy, and fzy-DYY* mutant cells express GFP::Fzy-DYY*. The yellow arrows and cyan arrowheads indicate the localization of GFP::Fzy and GFP::Fzy-DYY* on the kinetochore and tether. The white arrowheads indicate the I-CreI–induced acentric chromatids. 100% of WT cells exhibited GFP::Fzy signal on the tether (n = 22). In contrast, no GFP::Fzy signal on the tether was detected in 44% of bubR1-KEN mutant cells (n = 16). GFP::Fzy-DYY* signal was not visible in 32% of cells (n = 25). (B) Time-lapse images of WT (also see Video 7), bubR1-KEN (also see Video 8), and fzy-DYY* mutant cells before and after laser ablation. The yellow arrows indicate the accumulation of GFP::Fzy and GFP::Fzy-DYY* on kinetochores. The yellow circles correspond to the zones of laser ablation. Time (given in minutes/seconds) 0:00 corresponds to the first acquisition immediately after laser ablation. The white arrowheads indicate laser-induced damage. The cyan arrowheads show the appearance of GFP::Fzy at the site of chromosome damage. For WT cells, the percentage represents the frequency of cells with GFP::Fzy signal on DNA breaks (n = 17). In the bubR1-KEN mutant, the percentage denotes the frequency of cells without GFP::Fzy signal on laser-induced damage (n = 11). Finally, for the fzy-DYY* mutant, the percentage indicates the frequency of cells with no detectable GFP::Fzy-DYY* signal on laser-induced breaks (n = 19). The cells are delineated with dotted lines. Bars, 10 µm. (C) Scatter dot plot of GFP::Fzy and GFP::Fzy-DYY* levels at the site of the I-CreI–induced tether during anaphase in WT, bubR1-KEN, and fzy-DYY* mutants. (D) Scatter dot plot of GFP::Fzy and GFP-Fzy-DYY* levels at the site of the laser-induced breaks during anaphase. (C and D) n = number of cells. The GFP level is normalized to the cytoplasmic GFP signal (see Materials and methods section Image analysis for quantification details). A Mann-Whitney nonparametric test was used to calculate p-values. Error bars represent mean ± 95% confidence interval. a.u., arbitrary units.
Figure 4.

Fzy is recruited on I-CreI– or laser-induced breaks in a BubR1 KEN box–dependent manner. (A) Time-lapse images of WT (also see Video 5), bubR1-KEN (also see Video 6), and fzy-DYY* neuroblasts expressing I-CreI. WT and bubR1-KEN mutant cells express GFP::Fzy, and fzy-DYY* mutant cells express GFP::Fzy-DYY*. The yellow arrows and cyan arrowheads indicate the localization of GFP::Fzy and GFP::Fzy-DYY* on the kinetochore and tether. The white arrowheads indicate the I-CreI–induced acentric chromatids. 100% of WT cells exhibited GFP::Fzy signal on the tether (n = 22). In contrast, no GFP::Fzy signal on the tether was detected in 44% of bubR1-KEN mutant cells (n = 16). GFP::Fzy-DYY* signal was not visible in 32% of cells (n = 25). (B) Time-lapse images of WT (also see Video 7), bubR1-KEN (also see Video 8), and fzy-DYY* mutant cells before and after laser ablation. The yellow arrows indicate the accumulation of GFP::Fzy and GFP::Fzy-DYY* on kinetochores. The yellow circles correspond to the zones of laser ablation. Time (given in minutes/seconds) 0:00 corresponds to the first acquisition immediately after laser ablation. The white arrowheads indicate laser-induced damage. The cyan arrowheads show the appearance of GFP::Fzy at the site of chromosome damage. For WT cells, the percentage represents the frequency of cells with GFP::Fzy signal on DNA breaks (n = 17). In the bubR1-KEN mutant, the percentage denotes the frequency of cells without GFP::Fzy signal on laser-induced damage (n = 11). Finally, for the fzy-DYY* mutant, the percentage indicates the frequency of cells with no detectable GFP::Fzy-DYY* signal on laser-induced breaks (n = 19). The cells are delineated with dotted lines. Bars, 10 µm. (C) Scatter dot plot of GFP::Fzy and GFP::Fzy-DYY* levels at the site of the I-CreI–induced tether during anaphase in WT, bubR1-KEN, and fzy-DYY* mutants. (D) Scatter dot plot of GFP::Fzy and GFP-Fzy-DYY* levels at the site of the laser-induced breaks during anaphase. (C and D) n = number of cells. The GFP level is normalized to the cytoplasmic GFP signal (see Materials and methods section Image analysis for quantification details). A Mann-Whitney nonparametric test was used to calculate p-values. Error bars represent mean ± 95% confidence interval. a.u., arbitrary units.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal