Anaphase/mitotic exit Brr6 regulates nuclear integrity. (A and B) Cell cycle progression of brr6⁺ (A) and brr6.ts8 (B) cells harboring the int::GFP-β-Gal NLS nuclear integrity marker and pREP81atb2.Cherry to mark nuclear integrity and microtubules, respectively. (C and D) The temperature was shifted from 25°C to 36°C 0.5 h before filming. Cell cycle progression of nda3.KM311 (β-tubulin mutation) cells harboring the sid4.Tom SPB marker (red) and the int::GFP-β-Gal NLS nuclear integrity marker (green) were arrested by incubation at 19°C for 9 h before the temperature was raised to 36°C to restore tubulin function. A–D show frames from Videos 1–4, respectively. The integrity of the nuclear envelope is retained during mitotic commitment (A) and anaphase B/mitotic exit of the wild-type (C) but not the mutant cells (B and D). Asterisks indicate nuclei for which the ability to retain the nuclear marker is severely compromised while the bars under the times series in C and D highlight anaphase B nuclei. (E) Cultures of the indicated strains were either shifted from 25°C to 36°C or had DMSO added at the same time as 20 µg/ml NBD-cholesterol. 1 h later, fluorescence and DIC images were captured. Bars, 10 µm.