Skip to main content
. 2018 May 22;23(8):2308–2317. doi: 10.1016/j.celrep.2018.04.082

Figure 2.

Figure 2

SMC ATPase Controls Chromosome Condensation, Segregation, and Condensin Turnover

(A) Metaphase rDNA morphology of Smc4 ATPase mutants visualized by structured illumination microscopy of the rDNA binding protein Net1. Cells were synchronized in G1 with α factor and released into a metaphase arrest due to overexpression of a Mad2-Mad3 fusion protein. Scale bars represent 4 μm.

(B) Quantification of rDNA condensation, measured by the number of high-density volumetric pixels in the high-frequency data of structured illumination images. Error bars represent mean ± 95% confidence interval (n ≥ 90); asterisks denote p values with respect to the first column (NS, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ordinary one-way ANOVA, Dunnett’s multiple comparison test).

(C) rDNA segregation in Smc4 ATPase mutants visualized by structured illumination microscopy of the rDNA binding protein Net1. Cells were synchronized in G1 and released into a late anaphase arrest due to overexpression of Bfa1. Arrowheads indicate chromatin bridges. Scale bars represent 4 μm.

(D) Quantification of rDNA segregation as the ratio of Net1 fluorescence intensity in the two cell halves. A ratio of 1 indicates equal segregation. Error bars represent mean ± 95% confidence interval (n ≥ 106); asterisks denote p values as in (B).

(E) Fluorescence recovery half-times, following photobleaching, of the Brn1-3mNeonGreen signal in homozygous diploid wild-type and smc4-R210A cells, arrested in G1 by overexpression of Sic1 or metaphase by overexpression of Mad2-Mad3. Error bars represent mean ± SD (n ≥ 37) (ordinary one-way ANOVA, Sidak’s multiple comparison test).

(F) Examples of fluorescence recovery of the Brn1-3mNeonGreen signal in wild-type and smc4-R210A cells (outlined) in metaphase, with the area around the bleach spot (indicated by circles) magnified for clarity. Scale bars for whole-cell images represent 4 μm; those for the inset represent 1 μm.

See also Figure S4 for illustration of the high-density voxel chromosome condensation assay and confirmation of the cell-cycle arrests.