Meiotic depletion of Smc5 or Nse4 leads to meiotic catastrophe.
(A) Western blot of depletion of 3HA-Smc5 (Y941) and 3HA-Nse4 (Y942) protein levels under the PCLB2 promoter. Mutants are referred to as smc5 and nse4 throughout. (B) FACS analysis of S-phase progression in wild type (Y940), smc5 (Y941) and nse4 (Y942) mutants. (C) Population kinetics of spindle pole body separation (n = 200 per time point). (D) Population kinetics of nuclear divisions (n = 200 per time point). (E) Montage of time series of nuclear divisions and spindle dynamics from representative time-lapse movies. H2B-mCherry and Tub1-GFP are pseudo-coloured in magenta and green, respectively. Maximum projections are shown. Bars: 4 µm. Full movies are available as Supplemental Movies S1 to S4. Arrows indicate examples of nuclear spikes and arrowheads show fragmentation/micronuclei. Strains: WT (Y3606), smc5 (Y3627), nse4 (Y3630). (F) DNA encapsulation failure in smc5 and nse4 mutants. Upper panel DIC, lower panel, DAPI (DNA). The boxed asci are shown with DNA (green) overlaid in the insets in the lower panel, bottom left. Note that the samples are taken from different time points in the various strains. Bars, 5 µm. (G) Proportion of cells completing meiosis and forming an ascus (di-tyrosine fluorescence). (H) Proportion of asci with encapsulated DNA (bottom). All data were collected after 24 hours in liquid sporulation medium. Three independent diploids were assessed for each genotype (standard deviations are shown). Strains: WT (Y1381), smc5 (Y2705), PCLB2-SMC5-AID (Y3252), nse4 (Y2704), smc5 nse4 (Y3185).
