Figure 6.
Systematic analysis of putative APC/CCdc20 substrates involved in anaphase aMT stabilization unveils the key role of B-Cyclin Clb4. (A and B) Wild-type (Ry1), clb1∆ (Ry5976), clb2∆ (Ry20), clb3∆ (Ry10927), clb4∆ (Ry10924), and clb5∆ (Ry445) cells were arrested in S-phase and analyzed at their terminal phenotype. (A) The graphs show aMT length and number of the indicated genotypes (for aMT length, n = 100 aMTs; for aMT number, n = 100 cells). Among the different clb mutants, only clb4 cells showed longer and more numerous aMTs than wild-type cells (from 1.6 µm and 2.1 aMT/cell in wild-type cells to 2.6 µm and 3.2 aMT/cell in clb4 cells). **** = P < 0.0001; asterisks denote significant differences according to ordinary One-Way ANOVA and Tukey’s multiple comparisons test). (B) Representative images of wild-type and clb4∆ arrested in S-phase are shown (scale bar = 5 µm). (C) Schematic representation of the experimental setup pertinent to panel D. (D)cdc20-AID (Ry7873), cdc20-AID clb4∆ (Ry10741), and esp1-1 cdc15-as1 (Ry9512) cells were released from a G1 block into restrictive conditions and analyzed at their terminal arrest (T180 minutes). Graphs for aMT length and number are shown. Similarly to the ones of esp1 cdc15 cells, aMTs of cdc20 clb4 cells resulted more stable than the aMTs of cdc20 cells (from 2.4 µm and 3.2 aMT/cell in cdc20 cells to 3.9 µm and 4.5 aMT/cell in cdc20 clb4 cells and 3.5 µm and 4.4 aMT/cell in esp1 cdc15 cells; **** = P < 0.0001; asterisks denote significant differences according to ordinary One-Way ANOVA and Tukey’s multiple comparisons test). Error bars in graphs equal SEM. (E)pGAL-3HA-CDC20 CLB4-13myc (Ry10889) cells were arrested in S-phase with HU (10 mg/ml) in YEP media supplemented with Raffinose. Upon reaching the arrest (around 180 min from HU addition), the culture was split in two. One half was maintained in the same conditions, whereas 2% galactose was added to the other half to induce the expression of Cdc20. Clb2 and Clb4 protein levels were probed by Western blot analyses at the indicated timepoints. Pgk1 protein was used as an internal loading control in immunoblots. Size markers on the sides of the gel blots indicate relative molecular mass. Source data are available for this figure: SourceData F6.

Systematic analysis of putative APC/CCdc20 substrates involved in anaphase aMT stabilization unveils the key role of B-Cyclin Clb4. (A and B) Wild-type (Ry1), clb1∆ (Ry5976), clb2∆ (Ry20), clb3∆ (Ry10927), clb4∆ (Ry10924), and clb5∆ (Ry445) cells were arrested in S-phase and analyzed at their terminal phenotype. (A) The graphs show aMT length and number of the indicated genotypes (for aMT length, n = 100 aMTs; for aMT number, n = 100 cells). Among the different clb mutants, only clb4 cells showed longer and more numerous aMTs than wild-type cells (from 1.6 µm and 2.1 aMT/cell in wild-type cells to 2.6 µm and 3.2 aMT/cell in clb4 cells). **** = P < 0.0001; asterisks denote significant differences according to ordinary One-Way ANOVA and Tukey’s multiple comparisons test). (B) Representative images of wild-type and clb4∆ arrested in S-phase are shown (scale bar = 5 µm). (C) Schematic representation of the experimental setup pertinent to panel D. (D)cdc20-AID (Ry7873), cdc20-AID clb4∆ (Ry10741), and esp1-1 cdc15-as1 (Ry9512) cells were released from a G1 block into restrictive conditions and analyzed at their terminal arrest (T180 minutes). Graphs for aMT length and number are shown. Similarly to the ones of esp1 cdc15 cells, aMTs of cdc20 clb4 cells resulted more stable than the aMTs of cdc20 cells (from 2.4 µm and 3.2 aMT/cell in cdc20 cells to 3.9 µm and 4.5 aMT/cell in cdc20 clb4 cells and 3.5 µm and 4.4 aMT/cell in esp1 cdc15 cells; **** = P < 0.0001; asterisks denote significant differences according to ordinary One-Way ANOVA and Tukey’s multiple comparisons test). Error bars in graphs equal SEM. (E)pGAL-3HA-CDC20 CLB4-13myc (Ry10889) cells were arrested in S-phase with HU (10 mg/ml) in YEP media supplemented with Raffinose. Upon reaching the arrest (around 180 min from HU addition), the culture was split in two. One half was maintained in the same conditions, whereas 2% galactose was added to the other half to induce the expression of Cdc20. Clb2 and Clb4 protein levels were probed by Western blot analyses at the indicated timepoints. Pgk1 protein was used as an internal loading control in immunoblots. Size markers on the sides of the gel blots indicate relative molecular mass. Source data are available for this figure: SourceData F6.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal