Cells for microscopy were collected from liquid cultures, centrifuged at 5,000 rpm, and then washed into EMM5S for imaging. Live-cell microscopy was performed using a thin layer of EMM5S liquid medium with 20% gelatin (Sigma-Aldrich) and 0.1 mM n-propyl-gallate and observed at 23–25°C as previously described (Wu et al., 2006 (link); Coffman et al., 2009 (link)). To observe cells at 36°C, cells were spun down for 30 s at 7,000 rpm and placed on EMM5S + 2% agar pads prewarmed for 10 min at 36°C. All slides, coverslips, and cultures were kept at 36°C during preparations of samples to maintain cells at 36°C. An Objective Heater system (Bioptechs) was used to maintain the temperature at 36°C or other temperatures for microscopy of temperature-sensitive mutants. In general, fluorescence intensity was lower at 36°C than at 25°C, making it more difficult to observe nodes in some strains.
For imaging, we used a 100×/1.4 NA objective lens (Nikon) on a spinning-disk confocal microscope (UltraVIEW ERS; PerkinElmer) with a 440-nm solid-state laser, 488-, 514-, and 568-nm argon ion lasers, and a cooled charge-coupled device camera (ORCA-AG; Hamamatsu) with 2 × 2 binning or no binning (for distance measurement). Maximum intensity projections of color images, grayscale montages, and other image analyses were performed using ImageJ (National Institutes of Health). Images in figures are maximum intensity projections of z sections spaced at 0.2–0.8 µm except where noted. For genetic dependencies, the time intervals in time-lapse videos were as follows: 1 min for mYFP-Cdc4 and Rlc1-mYFP; 1.5–2 min for mYFP-Cdc15; and 2 min for Cdc12-3YFP, mYFP-Rng2, YFP-Myo2, and Mid1–monomeric enhanced Citrine (mECitrine).
For imaging, we used a 100×/1.4 NA objective lens (Nikon) on a spinning-disk confocal microscope (UltraVIEW ERS; PerkinElmer) with a 440-nm solid-state laser, 488-, 514-, and 568-nm argon ion lasers, and a cooled charge-coupled device camera (ORCA-AG; Hamamatsu) with 2 × 2 binning or no binning (for distance measurement). Maximum intensity projections of color images, grayscale montages, and other image analyses were performed using ImageJ (National Institutes of Health). Images in figures are maximum intensity projections of z sections spaced at 0.2–0.8 µm except where noted. For genetic dependencies, the time intervals in time-lapse videos were as follows: 1 min for mYFP-Cdc4 and Rlc1-mYFP; 1.5–2 min for mYFP-Cdc15; and 2 min for Cdc12-3YFP, mYFP-Rng2, YFP-Myo2, and Mid1–monomeric enhanced Citrine (mECitrine).
