Mps1Mph1 Kinase Phosphorylates Mad3 to Inhibit Cdc20Slp1-APC/C and Maintain Spindle Checkpoint Arrests (original) (raw)
Fig 2
mad3 phospho-mutants are localised and assembled into MCC complexes effectively, but mad2 mad3 double phospho-mutants have severe defects in the maintenance of checkpoint arrests.
A. Mutant Mad3 proteins are efficiently targeted to kinetochores. GFP fusions were constructed for all three phospho-mutants and -KEN mutants. cdc25 strains were synchronised in G2 and then released into mitosis for GFP imaging. Images shown were taken 20 mins after release. DNA was stained with DAPI. The inset images are labelled with the % of cells having Mad3-GFP kinetochore foci. B. nuf2-3 strains containing mad3-N9A, mad3-C9A and mad3-18A alleles or the mad2 non-dimerising mutant (M2 dimer) were shifted to 36°C for 4 hrs. Cells were then fixed and microtubules were stained with anti-tubulin antibodies. The relative mitotic index (cells containing short metaphase spindles) is plotted after normalising the % of mitotic spindles in the nuf2-3 control strain (which has a functional checkpoint) to 100. >100 cells were scored for each sample and this experiment repeated 5 times (scatter dot plot: lines at mean with SD). C. cdc25 strains containing mad3-C9A, mad2-S92A, mad3-C9A and mad2-S92A, or mps1 mph1 -kd mutations were synchronised in G2, released in mitosis and then challenged to arrest in response to the anti-microtubule drug carbendazim (CBZ). Failure to maintain mitotic checkpoint arrest leads to septation and this was scored at 15 min time-intervals, by fixing cells and staining with calcofluor (see supp. data for images). This experiment was repeated twice and >100 cells scored for each strain at each time point (plotted as mean with SEM). D. nda3 strains containing mad3-C9A, mad2-S92A, or mad3-C9A and mad2-S92A mutations were grown to log phase and then shifted to 18°C to analyse their ability to metaphase-arrest for several hours in response to the absence of microtubules. Failure to maintain arrest results in septation. This experiment was repeated twice and 100 cells scored for each strain at each time point (plotted as mean with SEM).