Multisizer 4 measurements
The system electrolyte was replaced with media closely matched to the cells' growth media. A 400 mL Multisizer beaker with pre-warmed growth media was equilibrated to a sample-appropriate temperature by a polyurethane heating tube connected to a temperature-controlled recirculating bath (NESLAB RTE-111) and integrated stirrer (speed for yeast: 15, L1210: 5). Cells were added to the prepared beaker for a final concentration of ~7500 mL−1 (5–7% coincidence correction). Back-to-back 150 s measurements with a 100 µm aperture tube and intermediate flushes were recorded for the duration of the timecourse. Occasionally, debris remained on the aperture after a flush, and an unblock operation was initiated by the user if a sharp increase in aperture resistance occurred during the first half of a measurement. For long timecourses, the sample beaker volume was replenished with pre-warmed (if necessary, pre-treated) growth medium to maintain sample temperature and sufficient volume for measurement. Alternatively, a basic pump system contained within the sample chamber may be implemented to continuously exchange media during the measurement.
Yeast strains, growth conditions and sample preparation
Cells were grown to OD600<0.7 in YEP supplemented with 2% glucose (YEPD) at room temperature (~21°C). For cdc28-as1, cells were arrested with 1-NM-PP1 (5 µM) at either room temperature or 30°C, and for cdc28-4, cells were arrested by temperature shift (34°C). After 1 h at arrest conditions, yeast were washed via vacuum filtration, resuspended in 0.2 µm filtered YEPD, sonicated, and transferred to a measurement beaker with identical arrest conditions (cdc28-as1: YEPD with 5 µM 1-NM-PP1 at 21°C or 30°C, cdc28-4: YEPD at 34°C). The Multisizer system electrolyte was 0.2 µm-filtered YEP. Additional pre-warmed growth medium was added after 2 h of measurement to better maintain sample temperature and increase sample volume. The first hour of arrest conditions was not measured because it mostly consists of the population collecting into the arrested phase and does not accurately portray an arrested population.
Cells were grown overnight in YEP +2% raffinose at 30°C, synchronized by centrifugal elutriation 
, and resuspended in YEP +2% glucose at 30°C. Aliquots from a parallel flask-grown culture were collected into 4% paraformaldehyde at indicated timepoints for quantifying the presence of buds. The Multisizer system was prepared and maintained as described above.
L1210 growth conditions and sample preparation
Cells were grown at 37°C in L-15 medium (Gibco/Invitrogen, Carlsbad, CA) supplemented with 0.4% (w/v) glucose, 10% (v/v) fetal bovine serum, 100 I.U. penicillin, and 100 µg/mL streptomycin. Cells were passaged every 2–3 days to maintain a cell concentration of ~100,000 mL−1. For measurement, cells were transferred to the Multisizer beaker with pre-warmed medium identical to growth medium and allowed to equilibrate for 30 min before measurement. After ~1 hour of measurement, cells were treated with 0.5 µM staurosporine or an equal volume of dimethyl sulfoxide (DMSO; mock-treated control) and measured for an additional hour. The Multisizer system electrolyte was L-15 medium.
Data extraction, interpolation, and analysis
Raw Multisizer files were batch-processed with custom MATLAB software (See File S1
and Figure S5
). This software is necessary for timecourses longer than a single file because combining files within the commercial software resets the start time of each file to zero and makes rate analysis for cell cycle progression or drug response impractical. Furthermore, data exported from the commercial software are limited to 5 010 pulses per file and files must be handled individually. The custom software batch-processes raw Multisizer data files to acquire time and volume data for all measurements and extend the time axis beyond that of a single file (). The method for volume data extraction is described in the main text. The corresponding time data are provided in two sections of the raw Multisizer file (*.#m4). Every 200 ms the instrument records the time passed (“[#TSms]”) and the number of measurements that have occurred since the measurement began ([#TSpulses]). Volume measurements are linearly distributed across each 200 ms time step such that each cell measurement has at least 200 ms time resolution.
For each file, the lowest 2% of data by volume were excluded from analysis as instrument noise. Bound pairs were determined for each file and data outside of a certain bound pair (exclusion bounds), typically the 10% bound pair, were excluded from rate analysis. Extreme size measurements skew the rate analysis and typically represent instrument noise and culture debris.
Large and instantaneous changes in the size distribution were interpreted as a partial occlusion of the instrument aperture or some other instrument error. The files identified as “debris” files contained a 25% bound pair that was greater than 1.025 times the corresponding bound pair in either of the neighboring files. These files were replaced with data interpolated from the two neighboring files and linearly spaced across the entire 150 s measurement. Interpolated data were written as a new Multisizer file and used for subsequent analysis.
Since data collected over an entire timecourse are usually too large to be imported in its entirety, files required for analysis on each averaging window are imported and cleared before analysis on subsequent averaging windows begin.