Figure 7.
Figure 7. TopBP1 is required for timely resolution of anaphase bridges. In all experiments, quantification was performed on the basis of time-lapse microscopy with an imaging frequency of 2 min for 30 min. Exponentially growing cells were monitored from anaphase through telophase, and bridges were scored. The maximum number of bridges visible at one time point was noted as representative for the entire mitosis of a given cell. Asterisk indicates significant differences from the untreated (P < 0.05); error bars represent 95% confidence intervals. The number of cells analyzed is indicated (n). (A) TopBP1 and PICH colocalizing UFBs are induced by DNA replication stress but not topological stress. Cells expressing TopBP1-YFP-AID, PICH-TFP, OsTIR, and hH2B-mCherry (RTP151) were treated with 0.4 µM APH for 24 h, 0.5 µM ICRF-193 for 30 min, or 0.0125% DMSO (vol/vol, untreated) for 24 h before imaging. (B) A subset of TopBP1 bridges colocalizes with RPA. Cells (RTP156) express TopBP1-YFP, RPA1-CFP, and hH2B-mCherry. Yellow, blue, and green arrowheads indicate TopBP1, RPA1, and colocalizing bridges, respectively. (C) Colocalization of TopBP1 and RPA bridges is induced by both DNA replication stress and topological stress. Cells expressing TopBP1-YFP, RPA-CFP, and hH2B-mCherry (RTP156) were treated with 0.4 µM APH for 24 h, 0.5 µM ICRF-193 for 30 min, or 0.0125% DMSO (vol/vol, untreated) for 24 h before imaging. (D) Depletion of TopBP1 leads to a reduction of UFBs and induction of chromatin bridges. Cells expressing TopBP1-YFP-AID, PICH-TFP, and hH2B-mCherry with OsTIR present (RTP151) or absent (RTP177) were treated with 500 µM IAA or 0.2% ethanol (vol/vol, untreated) for 30 min before imaging. After incubation with IAA for 30 min, the level of TopBP1-YFP-AID fluorescence had decreased below detection in the majority of cells. (E) ATR inhibition induces chromatin bridges and colocalizing PICH- and TopBP1-coated UFBs. Cells expressing TopBP1-YFP-AID, PICH-TFP, OsTIR, and hH2B-mCherry (RTP151) were treated with 2 µM ATRi or 0.2% DMSO (vol/vol, untreated) for 30 min before imaging. (F–H) TopBP1 depletion, replication stress, and ATR inhibition affect the length of PICH UFBs. The length distribution of UFBs was quantified in anaphase/telophase cells from panels D (IAA), A (APH), and E (ATRi).

TopBP1 is required for timely resolution of anaphase bridges. In all experiments, quantification was performed on the basis of time-lapse microscopy with an imaging frequency of 2 min for 30 min. Exponentially growing cells were monitored from anaphase through telophase, and bridges were scored. The maximum number of bridges visible at one time point was noted as representative for the entire mitosis of a given cell. Asterisk indicates significant differences from the untreated (P < 0.05); error bars represent 95% confidence intervals. The number of cells analyzed is indicated (n). (A) TopBP1 and PICH colocalizing UFBs are induced by DNA replication stress but not topological stress. Cells expressing TopBP1-YFP-AID, PICH-TFP, OsTIR, and hH2B-mCherry (RTP151) were treated with 0.4 µM APH for 24 h, 0.5 µM ICRF-193 for 30 min, or 0.0125% DMSO (vol/vol, untreated) for 24 h before imaging. (B) A subset of TopBP1 bridges colocalizes with RPA. Cells (RTP156) express TopBP1-YFP, RPA1-CFP, and hH2B-mCherry. Yellow, blue, and green arrowheads indicate TopBP1, RPA1, and colocalizing bridges, respectively. (C) Colocalization of TopBP1 and RPA bridges is induced by both DNA replication stress and topological stress. Cells expressing TopBP1-YFP, RPA-CFP, and hH2B-mCherry (RTP156) were treated with 0.4 µM APH for 24 h, 0.5 µM ICRF-193 for 30 min, or 0.0125% DMSO (vol/vol, untreated) for 24 h before imaging. (D) Depletion of TopBP1 leads to a reduction of UFBs and induction of chromatin bridges. Cells expressing TopBP1-YFP-AID, PICH-TFP, and hH2B-mCherry with OsTIR present (RTP151) or absent (RTP177) were treated with 500 µM IAA or 0.2% ethanol (vol/vol, untreated) for 30 min before imaging. After incubation with IAA for 30 min, the level of TopBP1-YFP-AID fluorescence had decreased below detection in the majority of cells. (E) ATR inhibition induces chromatin bridges and colocalizing PICH- and TopBP1-coated UFBs. Cells expressing TopBP1-YFP-AID, PICH-TFP, OsTIR, and hH2B-mCherry (RTP151) were treated with 2 µM ATRi or 0.2% DMSO (vol/vol, untreated) for 30 min before imaging. (F–H) TopBP1 depletion, replication stress, and ATR inhibition affect the length of PICH UFBs. The length distribution of UFBs was quantified in anaphase/telophase cells from panels D (IAA), A (APH), and E (ATRi).

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