Figure 5.
APC/CCdc20 activation is sufficient to stabilize aMTs at anaphase onset. (A) Schematic representation of the experimental setup pertinent to this figure. (B) Schematic representation of the pathways explaining spindle collapse in esp1-1 cells. Spindle collapse/disassembly is triggered by CDK inactivation. In yeast, CDK inactivation requires the activity of the MEN, a Ras-like GTPase signaling pathway localized on the SPB. MEN activation requires SPB movement into the bud, where the inactive cascade meets its activator, asymmetrically localized there. For this step, wild-type cells relies on spindle elongation. (C and D) On the contrary, esp1-1 cells may activate the MEN through a Dyn1-dependent pulling of the spindle, hence nucleus, into the bud before cohesin cleavage. esp-1 (Ry9490), esp1-1 dyn1∆ (Ry9516), esp1-1 cdc15-as1 (Ry9512), esp1-1 cdc5-as1 (Ry9134), esp1-1 cdc14-1 (Ry9131), esp1-1 cdc14-1 cdc5-as1 (Ry9128), and cdc14-1 cdc5-as1 (Ry1602) cells of the indicated phenotype were synchronized in G1 and released into restrictive conditions. Cells were scored at their terminal arrest. (C) Representative images of the indicated mutants at their terminal arrest are shown (scale bar = 5 µm). (D) For the mutants of interest, aMT length is shown in dot plots while aMT number is shown in bar charts (for aMT length, n = 100 aMTs; for aMT number, n = 100 cells). aMTs of esp1 mutants were significantly longer and more numerous than aMTs of cdc20 cells, resembling the ones of cdc5 cdc14 cells (with an average length of 4, 4.2, 4, 4.5, and 4.2 µm, and an average number of 3.8, 4.4, 4.1, 4.7, and 4.3 aMT/cell in esp1 cdc15, esp1 cdc5, esp1 cdc14 esp1, cdc5 cdc14, and esp1 dyn1 cells, respectively; compared to cdc20 and cdc14 cdc5 cells that showed an average aMT length of 2.6 and 4 µm, and an average number of 3.2 aMT/cell and 4.3 aMT/cell, respectively). ** = P < 0.01; *** = P < 0.001; **** = 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.

APC/CCdc20 activation is sufficient to stabilize aMTs at anaphase onset. (A) Schematic representation of the experimental setup pertinent to this figure. (B) Schematic representation of the pathways explaining spindle collapse in esp1-1 cells. Spindle collapse/disassembly is triggered by CDK inactivation. In yeast, CDK inactivation requires the activity of the MEN, a Ras-like GTPase signaling pathway localized on the SPB. MEN activation requires SPB movement into the bud, where the inactive cascade meets its activator, asymmetrically localized there. For this step, wild-type cells relies on spindle elongation. (C and D) On the contrary, esp1-1 cells may activate the MEN through a Dyn1-dependent pulling of the spindle, hence nucleus, into the bud before cohesin cleavage. esp-1 (Ry9490), esp1-1 dyn1∆ (Ry9516), esp1-1 cdc15-as1 (Ry9512), esp1-1 cdc5-as1 (Ry9134), esp1-1 cdc14-1 (Ry9131), esp1-1 cdc14-1 cdc5-as1 (Ry9128), and cdc14-1 cdc5-as1 (Ry1602) cells of the indicated phenotype were synchronized in G1 and released into restrictive conditions. Cells were scored at their terminal arrest. (C) Representative images of the indicated mutants at their terminal arrest are shown (scale bar = 5 µm). (D) For the mutants of interest, aMT length is shown in dot plots while aMT number is shown in bar charts (for aMT length, n = 100 aMTs; for aMT number, n = 100 cells). aMTs of esp1 mutants were significantly longer and more numerous than aMTs of cdc20 cells, resembling the ones of cdc5 cdc14 cells (with an average length of 4, 4.2, 4, 4.5, and 4.2 µm, and an average number of 3.8, 4.4, 4.1, 4.7, and 4.3 aMT/cell in esp1 cdc15, esp1 cdc5, esp1 cdc14 esp1, cdc5 cdc14, and esp1 dyn1 cells, respectively; compared to cdc20 and cdc14 cdc5 cells that showed an average aMT length of 2.6 and 4 µm, and an average number of 3.2 aMT/cell and 4.3 aMT/cell, respectively). ** = P < 0.01; *** = P < 0.001; **** = 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.

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