Deletion mutants containing SPC110-GFP and GAL-MPS1 were grown overnight on YP RAFF plates at room temperature, then struck out onto YP RAFF/GAL plates (time zero). Samples were taken at time zero and fixed for 15 minutes in 3.7% formaldehyde at 30° in a roller drum. Plates were then incubated at room temperature for six hours to induce mitotic arrest (>90% large buds) and samples were taken and fixed in formaldehyde as above for imaging. Fluorescence was quantified for in-focus SPBs and then normalized using the photosensor value of the microscope. The distribution of GAL-MPS1 arrested SPB fluorescence in each mutant strain was compared to the wild type distribution using a Kolmogorov-Smirnov test. ncs2Δ, pom152Δ, and nup60Δ arrested distributions were consistently different from wild type (p-value <1×10∧-5). Histograms of SPB fluorescence values from asynchronously growing (time zero) and metaphase-arrested (six hour time point) yeast from a representative experiment are shown for these strains. The data was normalized by population size and the best fit Gaussian curve was fit to each asynchronous distribution. Histogram bins that fell below the signal to noise cutoff chosen during image analysis were excluded when fitting the Gaussian curves. The wild type asynchronous best fit curve is overlaid on the mutant asynchronous distributions in red for comparison. The wild type arrested SPB fluorescence distribution is overlaid onto the arrested mutant distribution histograms in light grey for comparison.

(A) TEV protease is expressed. A control strain containing galactose-inducible TEV protease and wild type SPC110 (KGY321-3A) and an Spc110 cleavage strain (KGY57) were grown overnight in YP RAFF liquid media. 2% galactose was added at a cell density of 40 Klett units, and samples were taken for TCA precipitation at 0, 30, and 60 minutes after galactose addition. Protein samples were loaded on a 10% polyacrylamide gel and then analyzed by anti-Myc Western blot for TEV protease. (B) Spc110 is cleaved by TEV protease. A control strain containing cleavable Spc110 but no TEV protease (KGY53) and an Spc110 cleavage strain (KGY61) were grown overnight in YP RAFF liquid media. 2% galactose was added at a cell density of 25 Klett and samples were taken at intervals for TCA precipitation. Protein samples were loaded on 10% polyacrylamide gels and analyzed by anti-Spc110 Western blot. The anti-Spc110 antibody recognizes full length Spc110 and the large cleavage product, which are indicated by arrows. (C) Spc110 cleavage strains are viable on galactose media. Wild type (CRY1), a control strain containing cleavable Spc110 but no TEV protease (KGY54), and an Spc110 cleavage strain (KGY57) were grown on YP RAFF and YP RAFF/GAL plates to determine the growth phenotype of the Spc110 cleavage strain under induced conditions. (D) Spc110 cleavage strain growth is dependent on checkpoint proteins Mad1 and Mad2. Wild type (CRY1), Spc110 cleavage (KGY57), mad1Δ (TDY439-1B), Spc110 cleavage + mad1Δ (KGY133), mad2Δ (SFY127-1A), and Spc110 cleavage + mad2Δ (KGY139) strains were grown on YP RAFF and YP RAFF/GAL plates to determine whether the spindle checkpoint is necessary for growth of the Spc110 cleavage strain under induced conditions.

(A) ubc4Δ, but not ubc5Δ, has a synthetic growth defect when combined with Spc110 cleavage. Strains were grown on YP RAFF/GAL plates at room temperature. (B) ubc4Δ has a more pronounced growth defect when combined with assembly mutant spc110-220 than ubc5Δ. Strains were grown on YPD plates at 25° and 37°. (C) Asynchronously growing ubc4Δ, but not ubc5Δ, cells show a defect in SPB size regulation. Strains were grown overnight at 23° in YPD liquid media to a cell density of 25 Klett units. α-factor was added to a concentration of 7.56 µg/ml at time zero and samples were taken and fixed in formaldehyde for imaging of the asynchronous cultures. Strains were then incubated at 23° for 3.5 hours (1.5 generations) and samples were taken from the G1 arrested cells and fixed in formaldehyde for imaging. SPB fluorescence of asynchronously growing and G1 arrested cells was measured and plotted as described. Best fit Gaussian curves were fit to each distribution and the wild type fit is overlaid in red on the ubc4Δ and ubc5Δ histograms for comparison. SPBs in the asynchronous population that were part of metaphase pairs were isolated and their SPB fluorescence values were also plotted. The distribution of SPB fluorescence after α-factor arrest shows a similar shift in wild type, ubc4Δ, and ubc5Δ cells, indicating that the mutant strains are able to reduce SPB size in α-factor as well as wild type.