Screening for Nuclear-Cytoplasmic Ratio Mutants

The 3-stage screen was carried out as described above. Firstly, deletion mutants were incubated at 25°C for 12 to 20h in 300 μl of YE4S in 96-well plates then inoculated onto YE4S agar plates containing DiOC₆ at 10 μg/ml (Life technologies) using a pin tool (V & P Scientific, Inc) and incubated at 25°C for 12 to 20h. The N/C ratio of each mutant strain was estimated by comparison with the wild type strain growing in the same plate using a Zeiss Axioskop 40 microscope. 102 of the 2,969 deletion mutants failed to grow on plates so were excluded from the screen. 366 mutant strains were selected for a secondary visual screen in liquid medium. Cells were collected from individual exponentially growing cultures of the 366 candidate mutants, stained with DiOC₆ and visually screened to estimate the N/C ratio. The 97 strains selected for the tertiary screen were tagged with the nuclear envelope marker protein Cut11-GFP and the nuclear and cellular volumes measured to assess the N/C ratio [5 (link)]. Images were analysed using ImageJ (NIH) as previously described [5 (link)]. Nuclear volume of shape mutants was calculated using ImageJ.