Live-cell microscopy and image analysis

Yeast cultures were propagated in synthetic complete medium (SC, Sunrise Science) prior to imaging. Cells were immobilized on cover slips that were pre-coated with concanavalin A (Sigma) before imaging on an inverted Nikon Ti2 fluorescence microscope with Perfect Focus System and a Nikon Plan Apo 60 × 1.4 NA oil-immersion objective lens. The stage temperature and humidity were controlled with a Tokai Hit stage top incubator set to 30°C. At least four stage positions were chosen for each strain, and all strains shown for a given experiment were imaged on the same slide during the same imaging session. Images were collected on a Hamamatsu Flash4.0 V2 +sCMOS camera using NIS-Elements Image Acquisition Software. For each stage position, images were taken at 9 z-heights, each separated by. 35 μm, and image stacks were collected at 8 min timepoints for at least 90 min (Ctf3-GFP) or 60 min (Ctf19-GFP). Illumination and frame times were kept constant between experiments.

To analyze Ctf3-GFP images, we first calculated maximum intensity projections in the z direction. Movies were then segmented separately in the mCherry and GFP channels using TrackMate for Fiji (Schindelin et al., 2012; Tinevez et al., 2017). Segmentation settings were established for the wild-type strain and were subsequently applied to all samples without adjustment. All segmentation results were visually inspected to avoid segmentation artifacts. Measurements for all spots were written to files which were subsequently parsed and plotted. Mtw1-mCherry spots separated from their nearest neighbor by greater than 10 μm were counted as ‘No spindle’ observations. Histograms showing distributions of measured spindle lengths are shown in Figure 3—figure supplement 2B. For this panel, ‘No spindle’ observations were assigned a 0 μm inter-kinetochore distance. Image statistics from all stage positions from a given strain and experiment were pooled, while statistics from distinct experiments (imaging sessions) were kept separate and compared. Error bars shown indicate standard deviations for measurements from three distinct experiments.