Timing of EB1-scorable mitotic events.

Time-lapse movies at varying frame-rates (top row) were analyzed manually, and mean and standard deviation of time intervals is presented (all in minutes). ‘Pole sep’: separation of the anterior EB1 signal into two separate foci. ‘SP1, SP2, SP3’: full formation of bipolar spindle in 1st, 2nd, 3rd rounds of division. A spindle was scored when the EB1-NG signal was continuous across the midline of the cell, and the orientation of the signal was roughly perpendicular to the following cleavage furrow. ‘SP1B, SP2B, SP3B’: spindle breakdown 1st, 2nd, 3rd rounds of division. Spindle breakdown was scored when the EB1-NG signal was no longer perpendicular to the following cleavage furrow. Spindle formation and breakdown were highly synchronous in progeny within a single cell, although not all spindles were in focus in every division. To calculate the spindle duration, or SP to SPB, the frame number of the first visible spindle was subtracted from the frame number of the start of the spindle breakdown, and then multiplied by the frame frequency. In cases where a spindle was not visible in any frame it was given a value of 0 frames, but only if the preceding PS was clearly visible; in these cases, it was assumed that the spindle was likely missed because it was not present at the times the images were captured. If neither the PS nor the SP were seen, the values were not recorded. ‘CF1, CF2, CF3’: detectable cleavage furrow initiation in 1st, 2nd, 3rd rounds of division (for later divisions, furrow formation in any progeny cell was counted due to image complexity). A CF was scored when a visible indent associated with a dark line in brightfield, aligned with the upcoming plane of separation of the cells. The 10-sec frame-rate movie was at 26°C; the 20-sec and 3-min movies were at 33°C. Entries are in minutes: mean +/- standard deviation (number of cells). ND: not determined, for the following reasons: (1) 10 and 20 sec frame-rates were only usable for the first division, as cell viability dropped from light exposure; (2) cleavage furrow formation was scored from brightfield images that were captured only at 3 min resolution. Effective detection of pole separation was difficult at 3 min resolution because it was easiest to observe when moving density could be compared between adjacent frames. Only the later phases of the PS were easily seen once the poles were separated and very bright. Therefore, this was not scored in the 3 min frame-rate movies. See Figs 6 and 7 and S3 and S4 Videos for illustrative examples.