Metamorph 7.7 imaging software

Time lapses of strains containing the 10-kb lacO/LacI-GFP array or containing the dicentric plasmid were performed at room temperature (25°C) using an Eclipse Ti wide-field inverted microscope (Nikon) with a 100× Apo TIRF 1.49 NA objective (Nikon) and Clara charge-coupled device camera (Andor) using Nikon NIS Elements imaging software (Nikon). Time lapses of strains containing the 10-kb lacO/LacI-GFP array were 10 min in duration with 30 s intervals. At each interval a seven-step Z-stack of 300-nm step size was acquired in the GFP, RFP, and Trans channels. Time lapses of strains containing the dicentric plasmid were the same as above but with a duration of 20 min.
Population images of the dicentric plasmid strains and of strains containing the 1.2-kb lacO/LacI-GFP array were imaged at room temperature (25°C) using an Eclipse E600FN microscope (Nikon) with a 100× Plan Apo TIRF 1.45 NA objective (Nikon) and ImagEM EM-CCD digital camera (Hamamatsu) with a custom Lumencor LED illumination system (Lumencor) using MetaMorph 7.7 imaging software (Molecular Devices). Each acquisition was a seven-step Z-stack with a 300-nm step size in the GFP, RFP, and Trans channels.

Budding yeast strains EMS219 (Mat alpha, his5 leu2–3,212 ura3–50 CAN1 asp5 gal2 (form I1 rDNA::leu2 URA3+)), intact rDNA and EMS60-UVR-12 (LEU2+, URA3+ CANS form I1 rDNA), translocated rDNA (22 (link)), were transformed with CDC14-GFP:KAN to label the nucleolus to generate DCY1021.1 and DCY1017.2, respectively. DCY1021.1 was transformed using pS01 plasmid to introduce the brn1–9 allele into strain AY1009. DCY1021.1 was transformed to knockout Fob1 and Hmo1 in strains DCY1055.1 and DCY1056.1, respectively. Cells were grown in YPD (1% Yeast extract, 2% Bacto-peptone, 2% Dextrose) with excess adenine. Strains were grown until mid-log phase prior to imaging. Images were acquired at room temperature for wild-type (WT), fob1Δ and hmo1Δ mutant strains (25°C). brn1–9 strains were shifted to 37°C 3 h prior to image analysis. G1 cells were found in the population by visual inspection of bud size. Images were acquired using a Ti-Eclipse inverted microscope (Nikon) with a 100 × Plan Apo 1.4 NA objective (Nikon) and Clara CCD digital camera (Andor) using MetaMorph 7.7 imaging software (Molecular Devices). Single stacks contained 7 Z-planes sections with 300 nm step-size. Image stacks were cropped and maximum intensity projections were created using ImageJ.