Quantification of fluorescent signals

Fiji/ImageJ (Schindelin et al., 2012) was used to quantify nuclear and cellular concentrations of the protein analyzed. Nuclear and cellular region of interest (ROI) at each time point were defined using TetR-tdTomato- and brightfield-based thresholding, respectively. The mean background outside of cells was subtracted. The degradation kinetics of securin and Cdc13 were determined in Matlab (MathWorks). A cubic spline was fit to the degradation curve. The onset of degradation was defined as the point when the degradation rate first reached 10% of the maximal degradation rate; the end of degradation was defined as the point when the curve first became flat. Data were normalized to the intensities at these two points. For the normalized degradation rate, the region of the curve between 30 and 70% of degradation was used to determine the slope. Plo1 binding to spindle pole bodies was monitored by recording the maximum signal within the cellular ROI. The onset of Plo1 removal from spindle pole bodies was defined as the point when the loss in maximum signal first reached 20% of the maximal loss rate. The time point of sister chromatid separation (SCS) was determined by splitting of the dh1L<<tetO/TetR-tdTomato signal (Sakuno et al., 2009), and all fluorescence intensity curves were aligned to SCS. Spindle length was determined by manually measuring the distance between Plo1-GFP at spindle pole bodies in Fiji/ImageJ.