Cyclin A is destroyed in prometaphase and can delay chromosome alignment and anaphase - PubMed (original) (raw)
Cyclin A is destroyed in prometaphase and can delay chromosome alignment and anaphase
N den Elzen et al. J Cell Biol. 2001.
Abstract
Mitosis is controlled by the specific and timely degradation of key regulatory proteins, notably the mitotic cyclins that bind and activate the cyclin-dependent kinases (Cdks). In animal cells, cyclin A is always degraded before cyclin B, but the exact timing and the mechanism underlying this are not known. Here we use live cell imaging to show that cyclin A begins to be degraded just after nuclear envelope breakdown. This degradation requires the 26S proteasome, but is not affected by the spindle checkpoint. Neither deletion of its destruction box nor disrupting Cdk binding prevents cyclin A proteolysis, but Cdk binding is necessary for degradation at the correct time. We also show that increasing the levels of cyclin A delays chromosome alignment and sister chromatid segregation. This delay depends on the proteolysis of cyclin A and is not caused by a lag in the bipolar attachment of chromosomes to the mitotic spindle, nor is it mediated via the spindle checkpoint. Thus, proteolysis that is not under the control of the spindle checkpoint is required for chromosome alignment and anaphase.
Figures
Figure 1
Cyclin A–GFP is an appropriate marker for endogenous cyclin A. (A) Cyclin A and cyclin A–GFP have similar subcellular localizations. HeLa cells were synchronized in S phase or late G2 phase, fixed, and stained with anticyclin A antibodies, together with either anti-CTR453 antibodies to visualize centrosomes or anti–β-tubulin antibodies to visualize the mitotic spindle. DNA was visualized using TOTO-3 iodide. Confocal microscopy was used to compile a series of z-sections through cells. To analyze cyclin A–GFP localization, HeLa cells were microinjected in the nucleus in S phase (0–4 h after release from thymidine/aphidicolin block) or late G2 phase (9–11 h after release from thymidine/aphidicolin block) with cyclin A–GFP cDNA (0.1 mg/ml) or protein (9 mg/ml). The localization of cyclin A–GFP was assayed by time-lapse fluorescence and DIC microscopy. Note that the fluorescence levels in the metaphase cells have been enhanced relative to cells at earlier stages. (B) Cyclin A and cyclin A–GFP are degraded in mitosis. Examples of G2 phase and anaphase HeLa cells treated as in A and stained for endogenous cyclin A (top) or expressing cyclin A–GFP (bottom). Bars, 10 μm.
Figure 1
Cyclin A–GFP is an appropriate marker for endogenous cyclin A. (A) Cyclin A and cyclin A–GFP have similar subcellular localizations. HeLa cells were synchronized in S phase or late G2 phase, fixed, and stained with anticyclin A antibodies, together with either anti-CTR453 antibodies to visualize centrosomes or anti–β-tubulin antibodies to visualize the mitotic spindle. DNA was visualized using TOTO-3 iodide. Confocal microscopy was used to compile a series of z-sections through cells. To analyze cyclin A–GFP localization, HeLa cells were microinjected in the nucleus in S phase (0–4 h after release from thymidine/aphidicolin block) or late G2 phase (9–11 h after release from thymidine/aphidicolin block) with cyclin A–GFP cDNA (0.1 mg/ml) or protein (9 mg/ml). The localization of cyclin A–GFP was assayed by time-lapse fluorescence and DIC microscopy. Note that the fluorescence levels in the metaphase cells have been enhanced relative to cells at earlier stages. (B) Cyclin A and cyclin A–GFP are degraded in mitosis. Examples of G2 phase and anaphase HeLa cells treated as in A and stained for endogenous cyclin A (top) or expressing cyclin A–GFP (bottom). Bars, 10 μm.
Figure 2
Cyclin A is degraded from early prometaphase by a 26S proteasome-dependent pathway and is not inhibited by the spindle checkpoint. (A) Cyclin A begins to be degraded in prometaphase. HeLa cells synchronized in late G2 phase were microinjected in the nucleus with cyclin A–GFP protein (9 mg/ml), and followed by time-lapse fluorescence and DIC microscopy. Images (200-ms exposure) were taken at 3-min intervals. The total cell fluorescence minus background was quantified for each cell in successive images of a time series and plotted over time. A graph of a single cell (▪), representative of 42 cells analyzed, is shown. The start and finish of NEBD are marked and an arrow shows the time at which fluorescence levels began to decrease. The stages of mitosis are indicated at the top of the figure. Because metaphase is relatively short and variable in HeLa cells (9.9 ± 9.6 min in uninjected cells, n = 42) and images were only taken at 3-min intervals, in some cells (including the cell shown here) metaphase was not observed. Another cell was similarly injected and analyzed, but was treated with the 26S proteasome inhibitor MG-132 (100 μM; Calbiochem) during prometaphase. This cell remained in prometaphase for the duration of the experiment (data not shown). The graph shown (○) is representative of three cells analyzed. P, prophase; P/M, prometaphase; A, anaphase; T, telophase; G1, G1 phase. (B) Cyclin A begins to be degraded just after NEBD in PtK1 cells. Prophase PtK1 cells were microinjected with cyclin A–GFP protein (9 mg/ml) and analyzed as in A. A graph of a single cell, representative of nine cells analyzed, is shown. The initiation and completion of NEBD are marked and an arrow shows the time at which fluorescence levels began to decrease. The white arrowhead shows the localization of cyclin A–GFP between condensing chromosomes in prophase, and an asterisk indicates the original position of the nucleolus. M, metaphase. (C) Cyclin A–GFP is still degraded in the presence of taxol. HeLa cells synchronized in late G2 phase were injected with cyclin A–GFP protein and analyzed as above. Once in prometaphase, cells were treated with 10 μM taxol (Sigma-Aldrich). The degradation profile of a single cell, representative of four cells analyzed, is shown. DIC and fluorescence images show that cyclin A–GFP was completely degraded and that the cell remained in prometaphase for the duration of the experiment. Bars, 10 μm.
Figure 2
Cyclin A is degraded from early prometaphase by a 26S proteasome-dependent pathway and is not inhibited by the spindle checkpoint. (A) Cyclin A begins to be degraded in prometaphase. HeLa cells synchronized in late G2 phase were microinjected in the nucleus with cyclin A–GFP protein (9 mg/ml), and followed by time-lapse fluorescence and DIC microscopy. Images (200-ms exposure) were taken at 3-min intervals. The total cell fluorescence minus background was quantified for each cell in successive images of a time series and plotted over time. A graph of a single cell (▪), representative of 42 cells analyzed, is shown. The start and finish of NEBD are marked and an arrow shows the time at which fluorescence levels began to decrease. The stages of mitosis are indicated at the top of the figure. Because metaphase is relatively short and variable in HeLa cells (9.9 ± 9.6 min in uninjected cells, n = 42) and images were only taken at 3-min intervals, in some cells (including the cell shown here) metaphase was not observed. Another cell was similarly injected and analyzed, but was treated with the 26S proteasome inhibitor MG-132 (100 μM; Calbiochem) during prometaphase. This cell remained in prometaphase for the duration of the experiment (data not shown). The graph shown (○) is representative of three cells analyzed. P, prophase; P/M, prometaphase; A, anaphase; T, telophase; G1, G1 phase. (B) Cyclin A begins to be degraded just after NEBD in PtK1 cells. Prophase PtK1 cells were microinjected with cyclin A–GFP protein (9 mg/ml) and analyzed as in A. A graph of a single cell, representative of nine cells analyzed, is shown. The initiation and completion of NEBD are marked and an arrow shows the time at which fluorescence levels began to decrease. The white arrowhead shows the localization of cyclin A–GFP between condensing chromosomes in prophase, and an asterisk indicates the original position of the nucleolus. M, metaphase. (C) Cyclin A–GFP is still degraded in the presence of taxol. HeLa cells synchronized in late G2 phase were injected with cyclin A–GFP protein and analyzed as above. Once in prometaphase, cells were treated with 10 μM taxol (Sigma-Aldrich). The degradation profile of a single cell, representative of four cells analyzed, is shown. DIC and fluorescence images show that cyclin A–GFP was completely degraded and that the cell remained in prometaphase for the duration of the experiment. Bars, 10 μm.
Figure 2
Cyclin A is degraded from early prometaphase by a 26S proteasome-dependent pathway and is not inhibited by the spindle checkpoint. (A) Cyclin A begins to be degraded in prometaphase. HeLa cells synchronized in late G2 phase were microinjected in the nucleus with cyclin A–GFP protein (9 mg/ml), and followed by time-lapse fluorescence and DIC microscopy. Images (200-ms exposure) were taken at 3-min intervals. The total cell fluorescence minus background was quantified for each cell in successive images of a time series and plotted over time. A graph of a single cell (▪), representative of 42 cells analyzed, is shown. The start and finish of NEBD are marked and an arrow shows the time at which fluorescence levels began to decrease. The stages of mitosis are indicated at the top of the figure. Because metaphase is relatively short and variable in HeLa cells (9.9 ± 9.6 min in uninjected cells, n = 42) and images were only taken at 3-min intervals, in some cells (including the cell shown here) metaphase was not observed. Another cell was similarly injected and analyzed, but was treated with the 26S proteasome inhibitor MG-132 (100 μM; Calbiochem) during prometaphase. This cell remained in prometaphase for the duration of the experiment (data not shown). The graph shown (○) is representative of three cells analyzed. P, prophase; P/M, prometaphase; A, anaphase; T, telophase; G1, G1 phase. (B) Cyclin A begins to be degraded just after NEBD in PtK1 cells. Prophase PtK1 cells were microinjected with cyclin A–GFP protein (9 mg/ml) and analyzed as in A. A graph of a single cell, representative of nine cells analyzed, is shown. The initiation and completion of NEBD are marked and an arrow shows the time at which fluorescence levels began to decrease. The white arrowhead shows the localization of cyclin A–GFP between condensing chromosomes in prophase, and an asterisk indicates the original position of the nucleolus. M, metaphase. (C) Cyclin A–GFP is still degraded in the presence of taxol. HeLa cells synchronized in late G2 phase were injected with cyclin A–GFP protein and analyzed as above. Once in prometaphase, cells were treated with 10 μM taxol (Sigma-Aldrich). The degradation profile of a single cell, representative of four cells analyzed, is shown. DIC and fluorescence images show that cyclin A–GFP was completely degraded and that the cell remained in prometaphase for the duration of the experiment. Bars, 10 μm.
Figure 3
Cyclin A–GFP degradation is D-box independent and temporally regulated by Cdk binding. (A) Schematic diagram of cyclin A–GFP constructs. The D-box and cyclin box of human cyclin A and GFP are indicated. In the cyclin B1 D-box construct, the D-box of cyclin A has been replaced with that of cyclin B1. The R47A and ΔD-box constructs do not contain a functional D-box. MAAIL and 1–98 constructs are non-Cdk binding forms of cyclin A–GFP. The ΔN97 protein begins with a methionine, followed by amino acid 98 of cyclin A. (B–G) Degradation profiles of the cyclin A–GFP constructs in A. HeLa cells synchronized in late G2 phase were microinjected with cytomegalovirus promotor-driven cDNAs (0.1 mg/ml) and followed by time-lapse fluorescence and DIC microscopy at 3 min intervals. The total cell fluorescence minus background was quantified for each cell and plotted over time. A representative graph, together with the total number of cells analyzed, is shown for each construct. The stages of mitosis are indicated at the top of each figure. In G the stages of mitosis for the two different constructs are shown. For some cells, no obvious metaphase was observed. NEBD initiation and completion or chromosome segregation are marked. Arrows indicate the times at which fluorescence levels began to decrease.
Figure 3
Cyclin A–GFP degradation is D-box independent and temporally regulated by Cdk binding. (A) Schematic diagram of cyclin A–GFP constructs. The D-box and cyclin box of human cyclin A and GFP are indicated. In the cyclin B1 D-box construct, the D-box of cyclin A has been replaced with that of cyclin B1. The R47A and ΔD-box constructs do not contain a functional D-box. MAAIL and 1–98 constructs are non-Cdk binding forms of cyclin A–GFP. The ΔN97 protein begins with a methionine, followed by amino acid 98 of cyclin A. (B–G) Degradation profiles of the cyclin A–GFP constructs in A. HeLa cells synchronized in late G2 phase were microinjected with cytomegalovirus promotor-driven cDNAs (0.1 mg/ml) and followed by time-lapse fluorescence and DIC microscopy at 3 min intervals. The total cell fluorescence minus background was quantified for each cell and plotted over time. A representative graph, together with the total number of cells analyzed, is shown for each construct. The stages of mitosis are indicated at the top of each figure. In G the stages of mitosis for the two different constructs are shown. For some cells, no obvious metaphase was observed. NEBD initiation and completion or chromosome segregation are marked. Arrows indicate the times at which fluorescence levels began to decrease.
Figure 4
Cyclin A delays chromosome alignment and anaphase in a concentration-dependent manner. (A) Overexpression of wild-type cyclin A delays chromosome alignment. Late G2 phase HeLa cells were microinjected with cyclin A and GFP expression constructs. DIC images were taken at 3-min intervals. A cell in which the period of time from NEBD completion to chromosome alignment was prolonged (117 min) compared with uninjected cells (26 ± 9.5 min, n = 37) is shown and is representative of 5 out of 15 cells analyzed. The duration of each mitotic phase is indicated. (B) Concentration dependence of the cyclin A–induced delay. HeLa cells were microinjected in late G2 phase with cyclin A–GFP protein and followed by time-lapse fluorescence and DIC microscopy at 3 min intervals. Identical microscope and camera settings were used in all experiments and the time from the completion of NEBD to chromosome alignment was plotted against the total cell fluorescence minus background at NEBD (before cyclin A–GFP degradation) for each cell. For cells where stable chromosome alignment was not observed, the duration from NEBD to sister chromatid segregation was measured. The average period from completion of NEBD to chromosome alignment in uninjected HeLa cells (26 ± 9.5 min, n = 37) is indicated by a black square with error bars. Prometaphase was statistically prolonged compared with uninjected cells (significance level = 99%) in those cells with periods from NEBD completion to chromosome alignment >50 min (indicated by a dashed line). The amount of cyclin A–GFP is also given in units (x) equivalent to the amount of endogenous cyclin A in a late G2 cell (see Materials and Methods). (C) Chromosome alignment is delayed by MG-132. HeLa cells were treated in prophase (time 0) with 100 μM MG-132 (Calbiochem) to inhibit 26S proteasome-mediated degradation, and DIC images were taken at 3 min intervals. 1 cell, representative of 10 cells analyzed, is shown. NEBD was complete within 7 min, but chromosomes remained misaligned 160 min later. DMSO alone had no effect (data not shown). Bars, 10 μm.
Figure 4
Cyclin A delays chromosome alignment and anaphase in a concentration-dependent manner. (A) Overexpression of wild-type cyclin A delays chromosome alignment. Late G2 phase HeLa cells were microinjected with cyclin A and GFP expression constructs. DIC images were taken at 3-min intervals. A cell in which the period of time from NEBD completion to chromosome alignment was prolonged (117 min) compared with uninjected cells (26 ± 9.5 min, n = 37) is shown and is representative of 5 out of 15 cells analyzed. The duration of each mitotic phase is indicated. (B) Concentration dependence of the cyclin A–induced delay. HeLa cells were microinjected in late G2 phase with cyclin A–GFP protein and followed by time-lapse fluorescence and DIC microscopy at 3 min intervals. Identical microscope and camera settings were used in all experiments and the time from the completion of NEBD to chromosome alignment was plotted against the total cell fluorescence minus background at NEBD (before cyclin A–GFP degradation) for each cell. For cells where stable chromosome alignment was not observed, the duration from NEBD to sister chromatid segregation was measured. The average period from completion of NEBD to chromosome alignment in uninjected HeLa cells (26 ± 9.5 min, n = 37) is indicated by a black square with error bars. Prometaphase was statistically prolonged compared with uninjected cells (significance level = 99%) in those cells with periods from NEBD completion to chromosome alignment >50 min (indicated by a dashed line). The amount of cyclin A–GFP is also given in units (x) equivalent to the amount of endogenous cyclin A in a late G2 cell (see Materials and Methods). (C) Chromosome alignment is delayed by MG-132. HeLa cells were treated in prophase (time 0) with 100 μM MG-132 (Calbiochem) to inhibit 26S proteasome-mediated degradation, and DIC images were taken at 3 min intervals. 1 cell, representative of 10 cells analyzed, is shown. NEBD was complete within 7 min, but chromosomes remained misaligned 160 min later. DMSO alone had no effect (data not shown). Bars, 10 μm.
Figure 4
Cyclin A delays chromosome alignment and anaphase in a concentration-dependent manner. (A) Overexpression of wild-type cyclin A delays chromosome alignment. Late G2 phase HeLa cells were microinjected with cyclin A and GFP expression constructs. DIC images were taken at 3-min intervals. A cell in which the period of time from NEBD completion to chromosome alignment was prolonged (117 min) compared with uninjected cells (26 ± 9.5 min, n = 37) is shown and is representative of 5 out of 15 cells analyzed. The duration of each mitotic phase is indicated. (B) Concentration dependence of the cyclin A–induced delay. HeLa cells were microinjected in late G2 phase with cyclin A–GFP protein and followed by time-lapse fluorescence and DIC microscopy at 3 min intervals. Identical microscope and camera settings were used in all experiments and the time from the completion of NEBD to chromosome alignment was plotted against the total cell fluorescence minus background at NEBD (before cyclin A–GFP degradation) for each cell. For cells where stable chromosome alignment was not observed, the duration from NEBD to sister chromatid segregation was measured. The average period from completion of NEBD to chromosome alignment in uninjected HeLa cells (26 ± 9.5 min, n = 37) is indicated by a black square with error bars. Prometaphase was statistically prolonged compared with uninjected cells (significance level = 99%) in those cells with periods from NEBD completion to chromosome alignment >50 min (indicated by a dashed line). The amount of cyclin A–GFP is also given in units (x) equivalent to the amount of endogenous cyclin A in a late G2 cell (see Materials and Methods). (C) Chromosome alignment is delayed by MG-132. HeLa cells were treated in prophase (time 0) with 100 μM MG-132 (Calbiochem) to inhibit 26S proteasome-mediated degradation, and DIC images were taken at 3 min intervals. 1 cell, representative of 10 cells analyzed, is shown. NEBD was complete within 7 min, but chromosomes remained misaligned 160 min later. DMSO alone had no effect (data not shown). Bars, 10 μm.
Figure 5
The delay in chromosome alignment and anaphase does not correlate with an active spindle checkpoint. (A) Mad2 is not on kinetochores in cells delayed by cyclin A–GFP. HeLa cells expressing cyclin A–GFP cDNA were followed by time-lapse microscopy. Cells that had not aligned their chromosomes >50 min after NEBD were scored as having a cyclin A–GFP-induced delay, and were fixed and stained with anti-Mad2 antibodies and TOTO-3 iodide. DIC images were taken before fixation. Fluorescence images were compiled from a series of z sections through cells. 1 cell, representative of 11 cells analyzed, is shown. (B) Cyclin B1–YFP is degraded in cells delayed by cyclin A–CFP. HeLa cells synchronized in late G2 phase were coinjected with cyclin A–CFP and cyclin B1–YFP expression vectors. Cells were followed by time-lapse microscopy at 3-min intervals using custom-designed filter sets to discriminate CFP fluorescence from YFP fluorescence. The total cell fluorescence minus background was quantified independently for cyclin A–CFP and cyclin B1–YFP and plotted over time. Graphs from a cell with a cyclin A–CFP-induced delay in chromosome alignment and anaphase onset, representative of seven delayed cells analyzed, are shown. Initiation and completion of NEBD and chromosome segregation are marked. Chromosomes did not segregate until 187 min, 172 min after the completion of NEBD. Arrows indicate the time points at which cyclin A–CFP and cyclin B1–YFP fluorescence levels began to decrease (30 and 63 min, respectively). (C) Dominant negative Bub1 does not abrogate the cyclin A–induced delay. HeLa cells synchronized in late G2 phase were coinjected with cDNAs encoding dominant negative Bub1 and either GFP or cyclin A–GFP, and followed by time-lapse fluorescence and DIC microscopy. NEBD completion to chromosome segregation took 36 ± 8.9 min in uninjected cells (n = 46), but significantly less time (<13 min, significance level = 99%) in cells expressing Bub1 DN and GFP at a fluorescence ≥106 (_n_ = 11). In five out of nine cells coexpressing cyclin A–GFP and Bub1 DN at levels >106 (the level of cyclin A–GFP required to cause a delay in chromosome alignment in Fig. 4 B), NEBD to chromosome segregation was significantly prolonged compared with uninjected cells (>59 min). (D) Taxol does not stabilize cyclin B1 in cells with increased amounts of cyclin A. HeLa cells synchronized in late G2 phase were injected with cyclin B1–YFP alone or coinjected with cyclin A–CFP and cyclin B1–YFP expression constructs and followed as in B. Cells expressing cyclin B1–YFP cDNA alone were treated with taxol after cyclin B1–YFP degradation had commenced. The graph shown is representative of four such cells. Arrows indicate the timing of taxol addition and the stabilization of cyclin B1–YFP. Cells coexpressing cyclin A–CFP and B1–YFP were treated with 10 μM taxol if they had not aligned their chromosomes within 50 min of NEBD. Graphs of a single cell, representative of seven cells analyzed, are shown. The cell remained in prometaphase for the duration of the experiment. Bar, 5 μm.
Figure 5
The delay in chromosome alignment and anaphase does not correlate with an active spindle checkpoint. (A) Mad2 is not on kinetochores in cells delayed by cyclin A–GFP. HeLa cells expressing cyclin A–GFP cDNA were followed by time-lapse microscopy. Cells that had not aligned their chromosomes >50 min after NEBD were scored as having a cyclin A–GFP-induced delay, and were fixed and stained with anti-Mad2 antibodies and TOTO-3 iodide. DIC images were taken before fixation. Fluorescence images were compiled from a series of z sections through cells. 1 cell, representative of 11 cells analyzed, is shown. (B) Cyclin B1–YFP is degraded in cells delayed by cyclin A–CFP. HeLa cells synchronized in late G2 phase were coinjected with cyclin A–CFP and cyclin B1–YFP expression vectors. Cells were followed by time-lapse microscopy at 3-min intervals using custom-designed filter sets to discriminate CFP fluorescence from YFP fluorescence. The total cell fluorescence minus background was quantified independently for cyclin A–CFP and cyclin B1–YFP and plotted over time. Graphs from a cell with a cyclin A–CFP-induced delay in chromosome alignment and anaphase onset, representative of seven delayed cells analyzed, are shown. Initiation and completion of NEBD and chromosome segregation are marked. Chromosomes did not segregate until 187 min, 172 min after the completion of NEBD. Arrows indicate the time points at which cyclin A–CFP and cyclin B1–YFP fluorescence levels began to decrease (30 and 63 min, respectively). (C) Dominant negative Bub1 does not abrogate the cyclin A–induced delay. HeLa cells synchronized in late G2 phase were coinjected with cDNAs encoding dominant negative Bub1 and either GFP or cyclin A–GFP, and followed by time-lapse fluorescence and DIC microscopy. NEBD completion to chromosome segregation took 36 ± 8.9 min in uninjected cells (n = 46), but significantly less time (<13 min, significance level = 99%) in cells expressing Bub1 DN and GFP at a fluorescence ≥106 (_n_ = 11). In five out of nine cells coexpressing cyclin A–GFP and Bub1 DN at levels >106 (the level of cyclin A–GFP required to cause a delay in chromosome alignment in Fig. 4 B), NEBD to chromosome segregation was significantly prolonged compared with uninjected cells (>59 min). (D) Taxol does not stabilize cyclin B1 in cells with increased amounts of cyclin A. HeLa cells synchronized in late G2 phase were injected with cyclin B1–YFP alone or coinjected with cyclin A–CFP and cyclin B1–YFP expression constructs and followed as in B. Cells expressing cyclin B1–YFP cDNA alone were treated with taxol after cyclin B1–YFP degradation had commenced. The graph shown is representative of four such cells. Arrows indicate the timing of taxol addition and the stabilization of cyclin B1–YFP. Cells coexpressing cyclin A–CFP and B1–YFP were treated with 10 μM taxol if they had not aligned their chromosomes within 50 min of NEBD. Graphs of a single cell, representative of seven cells analyzed, are shown. The cell remained in prometaphase for the duration of the experiment. Bar, 5 μm.
Figure 5
The delay in chromosome alignment and anaphase does not correlate with an active spindle checkpoint. (A) Mad2 is not on kinetochores in cells delayed by cyclin A–GFP. HeLa cells expressing cyclin A–GFP cDNA were followed by time-lapse microscopy. Cells that had not aligned their chromosomes >50 min after NEBD were scored as having a cyclin A–GFP-induced delay, and were fixed and stained with anti-Mad2 antibodies and TOTO-3 iodide. DIC images were taken before fixation. Fluorescence images were compiled from a series of z sections through cells. 1 cell, representative of 11 cells analyzed, is shown. (B) Cyclin B1–YFP is degraded in cells delayed by cyclin A–CFP. HeLa cells synchronized in late G2 phase were coinjected with cyclin A–CFP and cyclin B1–YFP expression vectors. Cells were followed by time-lapse microscopy at 3-min intervals using custom-designed filter sets to discriminate CFP fluorescence from YFP fluorescence. The total cell fluorescence minus background was quantified independently for cyclin A–CFP and cyclin B1–YFP and plotted over time. Graphs from a cell with a cyclin A–CFP-induced delay in chromosome alignment and anaphase onset, representative of seven delayed cells analyzed, are shown. Initiation and completion of NEBD and chromosome segregation are marked. Chromosomes did not segregate until 187 min, 172 min after the completion of NEBD. Arrows indicate the time points at which cyclin A–CFP and cyclin B1–YFP fluorescence levels began to decrease (30 and 63 min, respectively). (C) Dominant negative Bub1 does not abrogate the cyclin A–induced delay. HeLa cells synchronized in late G2 phase were coinjected with cDNAs encoding dominant negative Bub1 and either GFP or cyclin A–GFP, and followed by time-lapse fluorescence and DIC microscopy. NEBD completion to chromosome segregation took 36 ± 8.9 min in uninjected cells (n = 46), but significantly less time (<13 min, significance level = 99%) in cells expressing Bub1 DN and GFP at a fluorescence ≥106 (_n_ = 11). In five out of nine cells coexpressing cyclin A–GFP and Bub1 DN at levels >106 (the level of cyclin A–GFP required to cause a delay in chromosome alignment in Fig. 4 B), NEBD to chromosome segregation was significantly prolonged compared with uninjected cells (>59 min). (D) Taxol does not stabilize cyclin B1 in cells with increased amounts of cyclin A. HeLa cells synchronized in late G2 phase were injected with cyclin B1–YFP alone or coinjected with cyclin A–CFP and cyclin B1–YFP expression constructs and followed as in B. Cells expressing cyclin B1–YFP cDNA alone were treated with taxol after cyclin B1–YFP degradation had commenced. The graph shown is representative of four such cells. Arrows indicate the timing of taxol addition and the stabilization of cyclin B1–YFP. Cells coexpressing cyclin A–CFP and B1–YFP were treated with 10 μM taxol if they had not aligned their chromosomes within 50 min of NEBD. Graphs of a single cell, representative of seven cells analyzed, are shown. The cell remained in prometaphase for the duration of the experiment. Bar, 5 μm.
Figure 5
The delay in chromosome alignment and anaphase does not correlate with an active spindle checkpoint. (A) Mad2 is not on kinetochores in cells delayed by cyclin A–GFP. HeLa cells expressing cyclin A–GFP cDNA were followed by time-lapse microscopy. Cells that had not aligned their chromosomes >50 min after NEBD were scored as having a cyclin A–GFP-induced delay, and were fixed and stained with anti-Mad2 antibodies and TOTO-3 iodide. DIC images were taken before fixation. Fluorescence images were compiled from a series of z sections through cells. 1 cell, representative of 11 cells analyzed, is shown. (B) Cyclin B1–YFP is degraded in cells delayed by cyclin A–CFP. HeLa cells synchronized in late G2 phase were coinjected with cyclin A–CFP and cyclin B1–YFP expression vectors. Cells were followed by time-lapse microscopy at 3-min intervals using custom-designed filter sets to discriminate CFP fluorescence from YFP fluorescence. The total cell fluorescence minus background was quantified independently for cyclin A–CFP and cyclin B1–YFP and plotted over time. Graphs from a cell with a cyclin A–CFP-induced delay in chromosome alignment and anaphase onset, representative of seven delayed cells analyzed, are shown. Initiation and completion of NEBD and chromosome segregation are marked. Chromosomes did not segregate until 187 min, 172 min after the completion of NEBD. Arrows indicate the time points at which cyclin A–CFP and cyclin B1–YFP fluorescence levels began to decrease (30 and 63 min, respectively). (C) Dominant negative Bub1 does not abrogate the cyclin A–induced delay. HeLa cells synchronized in late G2 phase were coinjected with cDNAs encoding dominant negative Bub1 and either GFP or cyclin A–GFP, and followed by time-lapse fluorescence and DIC microscopy. NEBD completion to chromosome segregation took 36 ± 8.9 min in uninjected cells (n = 46), but significantly less time (<13 min, significance level = 99%) in cells expressing Bub1 DN and GFP at a fluorescence ≥106 (_n_ = 11). In five out of nine cells coexpressing cyclin A–GFP and Bub1 DN at levels >106 (the level of cyclin A–GFP required to cause a delay in chromosome alignment in Fig. 4 B), NEBD to chromosome segregation was significantly prolonged compared with uninjected cells (>59 min). (D) Taxol does not stabilize cyclin B1 in cells with increased amounts of cyclin A. HeLa cells synchronized in late G2 phase were injected with cyclin B1–YFP alone or coinjected with cyclin A–CFP and cyclin B1–YFP expression constructs and followed as in B. Cells expressing cyclin B1–YFP cDNA alone were treated with taxol after cyclin B1–YFP degradation had commenced. The graph shown is representative of four such cells. Arrows indicate the timing of taxol addition and the stabilization of cyclin B1–YFP. Cells coexpressing cyclin A–CFP and B1–YFP were treated with 10 μM taxol if they had not aligned their chromosomes within 50 min of NEBD. Graphs of a single cell, representative of seven cells analyzed, are shown. The cell remained in prometaphase for the duration of the experiment. Bar, 5 μm.
Figure 6
Nondegradable cyclin A–GFP does not prevent chromosome alignment, but arrests cells in anaphase or telophase. Late G2 phase HeLa cells were microinjected with a ΔN97 cyclin A–GFP expression vector and analyzed by time-lapse microscopy. Cells were scored as arrested if they remained in the same phase of mitosis for >3 h. Images of cells arrested in anaphase (A) or telophase (B) are shown. The DNA in cell B was stained by adding 1 μg/ml Hoechst 33342 (Sigma-Aldrich). Data are representative of 23 cells analyzed: 12 arrested in anaphase, 7 arrested in telophase and 4 cells with low levels of ΔN97 cyclin A–GFP did not arrest. Bars, 10 μm.
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