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1.

Figure 6. From: Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1.

Securin destruction in metaphase requires only the D-box not the KEN box. (A) Degradation of the securin KEN box mutant starts at metaphase. HeLa cells were injected with an expression vector for a securin KEN box mutant–YFP fusion protein and followed through mitosis by time-lapse microscopy. Fluorescence and DIC pictures were taken at 3-min intervals and analyzed as described in the legend to Fig 2. The times of the beginning of metaphase and chromosome segregation are indicated. The graph is representative of at least five cells analyzed. (B) HeLa cells were coinjected with cDNAs encoding a securin D-box deletion mutant–CFP and the securin wildtype–YFP fusion proteins. The cells were analyzed as described in the legend to Fig. 2. The times at which metaphase and anaphase B started are indicated. The degradation profile of a single cell is shown and is representative of at least five cells analyzed. All cells had a cut phenotype.

Anja Hagting, et al. J Cell Biol. 2002 June 24;157(7):1125-1137.
2.
Figure 5.

Figure 5. From: Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1.

Securin can be ubiquitinated by APC/CCdc20 and APC/CCdh1. (A) APC/Ccdh1 can ubiquitinate securin and securin–YFP with equal efficiency in vitro. (B) In vitro ubiquitination of securin–YFP by APC/CCDC20 is dependent on an intact D-box. (C) APC/CCDH1 ubiquitinates securin–YFP containing either an intact D-box or KEN box. In A–C, APC/C was immunoprecipitated from Xenopus egg extracts and activated with recombinant CDH1 or CDC20. 35S-labeled in vitro–translated securin or securin–YFP wild-type (wt), D-box mutant (ΔDB), KEN box mutant (ΔKB), or D-box/KEN box double mutants were used as substrates. (D and E) Quantification of the data shown in B and C. The amount of securin–YFP conjugated to ubiquitin is shown as the percent of the total amount of securin–YFP per reaction. Time points 0 (hatched bars) and 30 min (black bars) indicate, respectively, the start and end points of the reaction.

Anja Hagting, et al. J Cell Biol. 2002 June 24;157(7):1125-1137.
3.

Figure 1. From: Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1.

Securin–YFP is an appropriate marker for endogenous securin. (A) HeLa cells were microinjected in the nucleus with an expression construct for securin (left) or securin linked to YFP (right). After 3 h, the cells were fixed, and cells injected with the untagged securin were stained with antisecurin antibodies. Cells were analyzed by confocal fluorescence microscopy. A single z section is shown for each cell. (B) HeLa cells were transfected with an expression construct for securin tagged with a myc epitope (lanes 1 and 2) or an expression construct for securin–myc linked to YFP (lanes 3 and 4). Cells were arrested with nocodazole, and cell extracts were prepared and used to immunoprecipitate separase (S). An unrelated antibody was used as a control for immunoprecipitation (C). The precipitates were immunoblotted with specific antibodies against hseparase (top), hsecurin (middle), and YFP (bottom). Arrows indicate tagged forms of hsecurin; the arrowhead points to endogenous securin.

Anja Hagting, et al. J Cell Biol. 2002 June 24;157(7):1125-1137.
4.

Figure 7. From: Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1.

The degradation profile of a securin mutant dependent on a KEN box for destruction reveals when Cdh1 replaces Cdc20. (A) The securin D-box mutant is degraded in anaphase, and a D-box/KEN double box mutant is partially stabilized in mitosis. HeLa cells were coinjected with cDNAs encoding a securin D-box deletion mutant linked to CFP and a securin D-box/KEN box double mutant linked to YFP. Cells were followed through mitosis, and DIC and fluorescence pictures were taken at 3-min intervals and analyzed as in the legend to Fig 2. The times at which metaphase and anaphase B started are indicated. The graph of a single cell is shown and represents at least eight cells analyzed. (B) Coexpressing cyclin B1 enhances the degradation of the securin double mutant. HeLa cells were coinjected with expression constructs for a wild-type cyclin B1–CFP together with a securin D-box and KEN box double mutant linked to YFP. Cells were followed, and DIC and fluorescence pictures were taken every 3 min and analyzed as in the legend to Fig 2. The times at which metaphase and anaphase B started are indicated. The graph of a single cell is shown and represents at least five cells analyzed. (C) High cyclin B1/CDK1 activity prevents the degradation of a securin mutant that depends on a KEN box for its proteolysis. HeLa cells were coinjected with expression constructs for a nondegradable R42A–cyclin B1–CFP together with either securin–YFP (left) or a securin D-box mutant linked to YFP (right). Cells were followed, and DIC and fluorescence pictures were taken every 3 min and analyzed as in the legend to Fig 2. The times at which metaphase started are indicated. The graph of a single cell is shown and represents at least five cells analyzed for each securin mutant.

Anja Hagting, et al. J Cell Biol. 2002 June 24;157(7):1125-1137.
5.

Figure 4. From: Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1.

Nondegradable securin causes a cut phenotype. (A) Schematic diagram of securin constructs. The D-box (R61-N68) and KEN box (K9-N11) are indicated. (B and C) HeLa cells injected with an expression vector encoding a securin D-box deletion mutant–YFP fusion protein were followed through mitosis by time-lapse microscopy, and DIC pictures were taken every three min. These cells are representative of at least 18 cells analyzed. (D) Reversion of the sequence of anaphase A and B. HeLa cells were coinjected with cDNAs expressing cyclin B1–CFP and a securin D-box deletion mutant–YFP (top) or with cDNAs expressing securin D-box mutant–CFP and a securin D-box/KEN box double mutant–YFP (bottom). Cells were followed through mitosis by time-lapse microscopy, and DIC pictures were taken every three min. In a minority of cells, the sister chromatids separated after the cell elongated. (E) HeLa cells were coinjected with cDNAs expressing cyclin B1–CFP and a securin D-box deletion mutant–YFP. Cells were followed by time-lapse microscopy. Fluorescence and DIC pictures were taken at 3-min intervals and analyzed as described in the legend to Fig 2. The degradation profile of a single cell is shown and is representative of at least six cells analyzed. The times at which metaphase and anaphase B started are shown.

Anja Hagting, et al. J Cell Biol. 2002 June 24;157(7):1125-1137.
6.

Figure 3. From: Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1.

Cyclin B1 levels can affect sister chromatid separation. (A) Low levels of cyclin B1 at the end of metaphase cause a late anaphase arrest. PtK1 cells were injected with an expression construct for nondegradable cyclin B1–GFP (R42A mutation). Cells expressing low levels of R42A–cyclin B1–GFP (<106 pixels for GFP) were followed by time-lapse fluorescence and DIC microscopy at 3-min intervals. The cell shown is representative of more than four cells analyzed. (B and C) High levels of cyclin B1 at the end of metaphase prevent sister chromatid separation. PtK1 cells were injected with an expression construct for nondegradable cyclin B1–GFP as in A. Cells expressing moderate to high levels of R42A–cyclin B1–GFP (>106 pixels for GFP) were followed by time-lapse fluorescence and DIC microscopy at 3-min intervals. Once cells had been in metaphase for >1 h, they were fixed and stained with anti-CREST (B) or anti-CREST and phospho-histone H3 antibodies (C). Cells were analyzed by confocal laser scanning microscopy, and either a single z section (B) or a projection of several z sections is shown (B and C). The arrows in B indicate pairs of kinetochores. In the single z section, only one kinetochore of the pair is visible, and the chromosomes can be visualized by the negative stain for cyclin B. The cell shown is representative of more than four cells analyzed. The arrows in Fig. 3 C indicate unseparated sister chromatids. The cell shown is representative of more than three cells analyzed.

Anja Hagting, et al. J Cell Biol. 2002 June 24;157(7):1125-1137.
7.

Figure 2. From: Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1.

Securin is degraded in metaphase coincident with cyclin B1, and its proteolysis is controlled by the spindle checkpoint. (A) High levels of securin–FP extend metaphase. HeLa cells synchronized in late G2 phase were microinjected with an expression construct for securin–FP and followed by time-lapse fluorescence microscopy at 3-min intervals. The length of metaphase was determined and plotted against the level of securin–FP fluorescence at the beginning of metaphase. We defined metaphase as either the time between chromosome alignment and sister chromatid separation, or, in cells exhibiting chromosome nondisjunction, the time between chromosome alignment and cell elongation. The length of metaphase in uninjected cells was 9.9 ± 9.6 min (den Elzen and Pines, 2001). The dashed line indicates the maximum length of metaphase in control uninjected cells. (B) Securin degradation starts at metaphase. HeLa cells synchronized in late G2 phase were microinjected in the nucleus with an expression construct for securin–YFP and followed by time-lapse fluorescence and DIC microscopy 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. The degradation profile of a single cell, representative of 10 cells analyzed, is shown. The stages of mitosis are indicated at the bottom of the figure. (C and D) Securin degradation is proteasome- and spindle checkpoint–dependent. HeLa cells were injected and analyzed as described in A. To reimpose the checkpoint or to stop proteasome-dependent degradation, cells in metaphase were treated with 10 μM taxol (C) or 100 μM MG132 (D), respectively. The arrow indicates the point at which taxol or MG132 was added, and the arrowhead indicates the start of metaphase. Graphs are of single cells, representative of at least four cells analyzed for each chemical. (E) Securin degradation coincides with cyclin B1 destruction. HeLa cells synchronized in late G2 phase were coinjected with cDNAs encoding cyclin B1–CFP and securin–YFP. Cells were analyzed as in A. The degradation profile of a single cell, representative of at least five cells analyzed, is shown. (F) In the absence of the spindle checkpoint, securin degradation starts in prometaphase. HeLa cells were synchronized in late G2 phase and coinjected with cDNAs encoding securin–YFP and a dominant negative mutant of Bub1. Cells were followed by time-lapse microscopy and analyzed as in A. The times of the completion of nuclear envelope breakdown and the start of chromosome segregation are indicated. The degradation profile of a single cell, representative of at least four cells analyzed, is shown. (G) Cyclin B1 and securin do not exactly colocalize. HeLa cells were coinjected with cDNAs encoding cyclin B1–YFP and securin–CFP, and fluorescence and DIC pictures were taken at the beginning of metaphase.

Anja Hagting, et al. J Cell Biol. 2002 June 24;157(7):1125-1137.

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