Figure 4.
Figure 4. Weakened SAC and congression failure in K243R/+ MEFs. (A) WT and K243R/+ MEFs were treated with 200 ng/ml nocodazole (Noc; left) or 2 µM paclitaxel (Taxol; right). The cells were collected at the indicated time points, stained with MPM-2 and 7-amino-actinomycid D, and analyzed by flow cytometry. The results and SDs are from three independent experiments. (B) WT, K243R/+, and BubR1+/− MEFs were treated with 3.3 µM Noc or 2 µM Taxol or left untreated. Cells were subjected to coimmunostaining with anti-Mad2 or -Mad1 antibody and FITC-conjugated anti–α-tubulin antibodies. Mad2 or Mad1 at the KTs with similar intensities were scored in each setting (Asyn; prometaphase cell in untreated sample). 10 unaligned/unattached KTs were analyzed per cell. The result is from three independent experiments of 35 different cells each. Relative value compared with the untreated prometaphase cell (Asyn) in WT is depicted by bar graphs (mean ± SEM; n > 350 KTs each). (C) The asynchronous and mitotic MEFs of WT and K243R/+, respectively, were subjected to IP and WB as indicated. For the mitotic extracts, the cells were serum starved for 26 h, released for 20 h, and treated with nocodazole for 7 h. In Noc + MG132-treated MEFs, Noc treatment was followed by MG132 treatment for 2 h before lysis. IP with anti-HA antibody was included as a negative control (Neg.). A sample representing 3% of the total cell lysate (TCL) was loaded as a control. (D) Assessment of BubR1 acetylation levels in WT and K243R/+ MEFs. The MEFs were treated with Noc and MG132 as in C and subjected to IP and WB as indicated. A sample representing 1% of the TCL was loaded as a control. The ratio of AcK243/total BubR1, when the level in +/+ cells is normalized to 1, was the same for both the anti-BubR1 and anti-AcK IPs and is marked below each lane. IP with a mixture of rabbit serum and 12CA5 anti-HA antibody is shown as a negative control for each panel (Neg.). Only a non-specific band migrating just below the band recognized by anti-AcK antibody (top left two lanes) was detected. Black lines in the top four panels indicate the removal of an intervening lane for presentation purposes. (E) Quantification of BubR1 at KTs in chromosome spreads after treatment with 200 ng/ml Noc. The level of BubR1, determined by anti-BubR1 immunofluorescence in Noc-treated prometaphase MEFs, was scored in WT, BubR1+/−, and K243R/+ MEFs. Each dot represents the mean BubR1 intensity calculated from 20 randomly picked KTs per cell. 40 chromosome spreads from each genotype were scored in two independent experiments. Mean value is indicated with a line (mean ± SEM; n > 800 KTs each). (F) Statistical mitotic timing from the NEBD to anaphase onset. The MEFs were subjected to time-lapse microscopy with or without treatment with 200 ng/ml Noc or 2 µM Taxol. Images were captured every 5 min, and the live images were processed for 36 h. Without a spindle poison, mitosis required an average of 25 min in the WT cells (n = 170) and ∼20 min in the K243R/+ MEFs (n = 153). After spindle poison exposure, the WT cells remained in mitosis for 331 min with Noc (n = 66) and 106 min with Taxol (n = 218). The K243R/+ MEFs exited mitosis (chromosome decondensation) within 117 min with Noc treatment (n = 144) and within 87 min with Taxol treatment (n = 191). The bars in the box represent the median values. The outliers (open circles) and suspected outliers (asterisks), as determined by statistical analysis, are marked. (G) Representative images captured at the indicated time points from NEBD without Noc treatment. Of the 20 K243R/+ MEFs, 17 displayed congression failure (second row; Video 2) and 3 exited mitosis without segregation (third row; Video 3). See Video 1 for the WT MEF control (first row). The timing of the onset of anaphase is marked. The white arrow indicates the lagging chromosome. NEBD: 00:00. Bar, 5 µm. (H) WT, BubR1+/−, and K243R/+ MEFs were treated with MG132 for 2 h and subjected to cold MT assay, followed by staining with anti–α-tubulin and CREST. Enlarged images of the insets show the properly attached MT in WT cells; syntelic attachment in BubR1+/− cells; and unattached (c), syntelic (a and b), and monotelic attachment (d) in K243R/+ cells. Green, α-tubulin; red, CREST immunostaining. Bars: (yellow) 5 µm; (white) 1 µm. (I) The congression defects were scored in cells from each mouse strain in the absence of MT poison, and data are shown as bar graphs (mean ± SEM). Number of cells scored: WT, n = 75; K243R/+, n = 102; BubR1+/−, n = 60.

Weakened SAC and congression failure in K243R/+ MEFs. (A) WT and K243R/+ MEFs were treated with 200 ng/ml nocodazole (Noc; left) or 2 µM paclitaxel (Taxol; right). The cells were collected at the indicated time points, stained with MPM-2 and 7-amino-actinomycid D, and analyzed by flow cytometry. The results and SDs are from three independent experiments. (B) WT, K243R/+, and BubR1+/− MEFs were treated with 3.3 µM Noc or 2 µM Taxol or left untreated. Cells were subjected to coimmunostaining with anti-Mad2 or -Mad1 antibody and FITC-conjugated anti–α-tubulin antibodies. Mad2 or Mad1 at the KTs with similar intensities were scored in each setting (Asyn; prometaphase cell in untreated sample). 10 unaligned/unattached KTs were analyzed per cell. The result is from three independent experiments of 35 different cells each. Relative value compared with the untreated prometaphase cell (Asyn) in WT is depicted by bar graphs (mean ± SEM; n > 350 KTs each). (C) The asynchronous and mitotic MEFs of WT and K243R/+, respectively, were subjected to IP and WB as indicated. For the mitotic extracts, the cells were serum starved for 26 h, released for 20 h, and treated with nocodazole for 7 h. In Noc + MG132-treated MEFs, Noc treatment was followed by MG132 treatment for 2 h before lysis. IP with anti-HA antibody was included as a negative control (Neg.). A sample representing 3% of the total cell lysate (TCL) was loaded as a control. (D) Assessment of BubR1 acetylation levels in WT and K243R/+ MEFs. The MEFs were treated with Noc and MG132 as in C and subjected to IP and WB as indicated. A sample representing 1% of the TCL was loaded as a control. The ratio of AcK243/total BubR1, when the level in +/+ cells is normalized to 1, was the same for both the anti-BubR1 and anti-AcK IPs and is marked below each lane. IP with a mixture of rabbit serum and 12CA5 anti-HA antibody is shown as a negative control for each panel (Neg.). Only a non-specific band migrating just below the band recognized by anti-AcK antibody (top left two lanes) was detected. Black lines in the top four panels indicate the removal of an intervening lane for presentation purposes. (E) Quantification of BubR1 at KTs in chromosome spreads after treatment with 200 ng/ml Noc. The level of BubR1, determined by anti-BubR1 immunofluorescence in Noc-treated prometaphase MEFs, was scored in WT, BubR1+/−, and K243R/+ MEFs. Each dot represents the mean BubR1 intensity calculated from 20 randomly picked KTs per cell. 40 chromosome spreads from each genotype were scored in two independent experiments. Mean value is indicated with a line (mean ± SEM; n > 800 KTs each). (F) Statistical mitotic timing from the NEBD to anaphase onset. The MEFs were subjected to time-lapse microscopy with or without treatment with 200 ng/ml Noc or 2 µM Taxol. Images were captured every 5 min, and the live images were processed for 36 h. Without a spindle poison, mitosis required an average of 25 min in the WT cells (n = 170) and ∼20 min in the K243R/+ MEFs (n = 153). After spindle poison exposure, the WT cells remained in mitosis for 331 min with Noc (n = 66) and 106 min with Taxol (n = 218). The K243R/+ MEFs exited mitosis (chromosome decondensation) within 117 min with Noc treatment (n = 144) and within 87 min with Taxol treatment (n = 191). The bars in the box represent the median values. The outliers (open circles) and suspected outliers (asterisks), as determined by statistical analysis, are marked. (G) Representative images captured at the indicated time points from NEBD without Noc treatment. Of the 20 K243R/+ MEFs, 17 displayed congression failure (second row; Video 2) and 3 exited mitosis without segregation (third row; Video 3). See Video 1 for the WT MEF control (first row). The timing of the onset of anaphase is marked. The white arrow indicates the lagging chromosome. NEBD: 00:00. Bar, 5 µm. (H) WT, BubR1+/−, and K243R/+ MEFs were treated with MG132 for 2 h and subjected to cold MT assay, followed by staining with anti–α-tubulin and CREST. Enlarged images of the insets show the properly attached MT in WT cells; syntelic attachment in BubR1+/− cells; and unattached (c), syntelic (a and b), and monotelic attachment (d) in K243R/+ cells. Green, α-tubulin; red, CREST immunostaining. Bars: (yellow) 5 µm; (white) 1 µm. (I) The congression defects were scored in cells from each mouse strain in the absence of MT poison, and data are shown as bar graphs (mean ± SEM). Number of cells scored: WT, n = 75; K243R/+, n = 102; BubR1+/−, n = 60.

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