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. 2023 Aug 17;34(10):ar97. doi: 10.1091/mbc.E23-02-0063

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© 2023 Sherwin et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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FIGURE 7: — Combined Sli15 dephosphorylation by Cdc14 and Fin1-PP1 has an additive effect on CPC translocation and checkpoint response to syntelic attachment. (A) Viability loss in strains with the combination of phospho-deficient sli15 mutants (sli15-6A and sli15-17A) with fin1-5A after CIK1-CC overexpression. WT (Y300), sli15-6A (Y3594), and sli15-17A (4053-4-1) cells containing fin1-5A (pMB7) plasmid with either vector control (V, p1218) or P_GALCIK1-CC (CC, pHL002) were grown to log-phase in raffinose media at 25°C. Galactose was added to a final concentration of 2% to induce CIK1-CC overexpression. Samples were taken at time 0 and after 6 h and spread on a YPD plate for overnight incubation at 25°C. The percentage of viable colonies was quantified. The experiments were repeated three times (N = 3) and 300 cells were counted for viability. The bars represent mean values ± SD. p values were obtained by using a two-tailed unpaired t test, with asterisks indicating **** p < 0.0001. (B) The combination of sli15-17A with fin1-5A does not have an additive effect on the sensitivity to syntelic attachment. The same protocol was to analyze the effect of the combination of sli1-17A with fin1-5A on the viability loss after syntelic attachment induction. Statistical analysis was performed as described in panel A. (C) The combination of sli15-6A with fin1-5A shows an additive effect on the sensitivity to syntelic attachment. The same method was used to determine the effect of the combination of sli15-6A with fin1-5A on the viability loss after syntelic attachment induction. The experiments were repeated three times (N = 3) and 300 cells were counted for each experiment. See above for statistical analysis. (D) The combination of sli15-6A with fin1-5A shows an additive effect on CPC dissociation from KTs in metaphase cells. cdc26∆ IPL1-3GFP NUF2-mCherry (4421-3-2, WT), sli15-17A cdc26∆ IPL1-3GFP NUF2-mCherry (4437-1-3), and sli15-6A cdc26∆ IPL1-3GFP NUF2-mCherry (4426-5-3) cells containing either FIN1 (pMB6) or phospho-deficient fin1-5A (pMB7) plasmids were grown to log-phase in YPD at 25°C then shifted to 36°C for 2 h to inactivate Cdc26 for metaphase arrest. Samples were collected for imaging. Ipl1 localization was categorized as no KT localization, KT localization, or spindle-like localization. Cells representing loss of Ipl1 KT localization are outlined with white dotted line. Scale bar, 5 μm. The pictures are representative of three experimental repeats (N = 3) where 100 cells were counted for each experiment. The bars represent mean values ± SD. p values were obtained by using a two-tailed unpaired t test, with asterisks indicating ****p < 0.0001. 17A: sli15-17A; 6A: sli15-6A; 5A: fin1-5A. (F) A schematic model describing two distinct pathways of Sli15 dephosphorylation by Cdc14 and Fin1-PP1 that promote CPC translocation in anaphase.