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. Author manuscript; available in PMC: 2015 May 19.
Published in final edited form as: Curr Biol. 2014 Apr 24;24(10):1101–1106. doi: 10.1016/j.cub.2014.03.040

Figure 4. Expression of the Ase1 short isoforms stabilizes the S-phase spindle.

Figure 4

(A) ase1-AA mutants are sensitive to HU. Saturated cell cultures with indicated genotypes were 10-fold serial diluted and spotted onto –TRP and –TRP + 100 mM HU plates and scanned after incubation at 30°C for 2 days. (B) ase1-AA cells show abnormal spindles after HU treatment. Cells with indicated genotypes were arrested in G1 and released to fresh selective media containing 200 mM HU for 3 hrs at 30°C. The spindle morphology (Tub1-GFP) in representative cells is shown in the bottom panel. The white arrow indicates a collapsed spindle. The percentage of cells with a normal bar-shaped spindle and an abnormal collapsed or dot-like spindle from three biological replicates is shown in the top panel (n > 100). Normal: WT 88% ± 2.3, ase1-AA 62.3% ± 8.2, ase1Δ 62.6% ± 3.6, ASE1-SF 57.9% ± 4.4, ase1-AA + ASE1-SF 83.7% ± 1.3, cin8Δ ase1-AA 73.2% ± 1.5. Abnormal: WT 12% ± 2.3, ase1-AA 37.7% ± 8.2, ase1Δ 38.4% ± 5.1, ASE1-SF 42.1% ± 4.4, ase1-AA + ASE1-SF 16.3% ± 1.3, cin8Δ ase1-A 26.8% ± 1.5. (C) Spindles are more dynamic and experience more collapse in ase1-AA mutants during HU treatment. Thirty min following G1 release, the cells with indicated genotypes were spotted onto the surface of a slide with an agarose medium pad containing 200 mM HU and subjected to live-cell microscopy. Five representative cells for each genotype were selected and spindle length was measured using Andor iQ2 software. Time zero was set to one image prior to the first separation of spindle poles and spindle length was plotted over time (bottom panel). Live-cell images of the spindle morphology in representative cells are shown in the top panel. Arrows indicate spindle collapse. See also Figure S4. (D) Ase1-SF reduces Ase1-FL spindle localization. G1-arrested cells with indicated genotypes were released to liquid media containing 200 mM HU for 1 hr, and then mounted to a slide as described in (C). The cells were subjected to live-cell microscopy for 30 min after G1 release for 2 hrs. The percentage of cells showing GFP localization to the spindle in several time points during the 30 min window is shown (n >100). See also Figure S4.