DNA endoreduplication does not appear to affect this overall growth rate but may be required to sustain it beyond a critical cell size, giving rise to the robust continued growth of optoBem1 cells. It has been shown in other organisms, for example, that DNA endoreduplication enables large increases in cell size. One possibility by which our findings can be reconciled with prior observations of exponential growth in wild type budding yeast is that cells become surface area-limited at sizes just above that of wild type cells, thereby inducing a shift from volume proportional growth to surface area-proportional growth.Cell size control pathways exist to correct for deviations from a set-point size, yet most previously-identified size control pathways specifically operate on cells that are born too small, delaying cell cycle progression to enable further growth to occur. Because the light and temperature-shift stimuli with which we prepared ‘giant’ yeast are fully reversible, we reasoned that we could monitor the return to a steady-state size distribution after releasing giant cells from their block. We prepared giant optoBem1 cells by incubating them in red light for 8 h and monitored them by live-cell microscopy after releasing them into infrared light. Strikingly, we found that cell populations rapidly returned to their unperturbed state , with individual daughter cells reaching the set-point volume in as few as three rounds of division .Return to the set-point size is not driven by cell shrinking, as giant mothers maintained their maximum volume over multiple rounds of budding . Instead, the giant mothers are eventually diluted out as successive generations are born, an effect that is especially prominent in cell populations at least 10 h post-Bem1 release .

In these populations,hydroponic bucket size distributions have a single mode near the set-point volume but exhibit long tails towards larger volumes . Our observation that cell size recovers after only a few generations strongly supports the existence of size control acting on large cells and demonstrates that size homeostasis across a cell population is robust even to extreme increases in cell volume.Quantitatively monitoring cell growth in yeast—as well bacterial, archaeal, and mammalian cells—has shown that the behavior of many organisms is consistent with an adder that monitors size across an entire cell cycle to correct for deviations in cell size and maintain size homeostasis in the population. However, a recent study argued that in budding yeast, the adder behavior could arise from independent regulation of pre- and post-Start events, without a cell needing to keep track of its added volume across all cell cycle phases, and may fail under various perturbations. To test whether adder-based mechanisms could account for size control in giant yeast, we quantified inter division volume change in successive cell division cycles after releasing optoBem1 cells into infrared light. For this experiment we prepared optoBem1 cells that also expressed fluorescently-labeled septin rings, which enabled us to time both bud emergence and cytokinesis and thus separate pre-Start and post Start size regulation . The ‘adder’ model predicts that the cell volume at division should be proportional to cell volume at birth with a slope of 1. Indeed, for unperturbed cells, we found that cell volume at division was linearly related to volume at birth with a slope of 1.19 . However, we found that the adder model poorly explained the cell size relationships in our giant cells, where the volume at division was related to volume at birth with a slope of 1.73 . This relationship was also evident when individual cells were tracked over time: the interdivision volume change, Δ, was positively correlated with the volume at birth . This size-dependent volume change occurred entirely during S/G2/M phase, as cells added a minimal volume during G1 that did not vary with cell size . We also performed analogous experiments in cdk1-ts giant cells that were shifted back to the permissive temperature. These experiments revealed a similar relationship: large cells grew more than small cells, exhibiting a linear relationship between volume at division and volume at birth with a slope of 1.70 .

These results are broadly consistent with recent work showing that although size control in unperturbed cells resembles an adder-based mechanism, no mechanistic adder regulates volume addition across the entire cell cycle. Our data also suggest that any size regulation limiting the growth of large cells is likely a consequence of regulation in S/G2/M, as growth during G1 is negligible.If an adder is unable to explain size homeostasis in giant yeast, what regulatory mechanisms or growth laws might operate on the daughters of giant cells during S/G2/M? Two possibilities include a bud ‘sizer’, where bud growth would be restricted after reaching a critical size; and a bud ‘timer’ in which cytokinesis would occur at a fixed duration following the beginning of S/G2/M . Such ‘sizers’ and ‘timers’ have been proposed to operate in a variety of biological systems. To distinguish between these possibilities, we tracked the timing of bud emergence and cytokinesis by septin ring appearance and disappearance, respectively, following reactivation of Bem1 in giant optoBem1 cells . Daughter volume strongly correlated with mother volume , inconsistent with a bud sizer mechanism. Our prior observation that the inter division volume change scales positively with cell birth size further argues against a bud sizer for cell volume control. In contrast, our data were consistent with a timer specifying the duration of S/G2/M: the time from bud emergence to cytokinesis did not vary as a function of mother cell volume and took average 95 min across cells of all volumes .Similar experiments performed using cdk1-ts cells were consistent with our observations in optoBem1 cells, revealing a size-independent duration of budding. However, we observed one notable difference: the duration of the size-invariant bud timer in giant cdk1-ts cells was substantially longer than that of giant optoBem1 cells . As Cdk1 is a key driver of mitosis in eukaryotes, the increased duration of the bud timer in cdk1-ts cells may arise from the need to refold or synthesize new Cdk1 molecules to complete S/G2/M following a shift from the restrictive to permissive temperature. Furthermore, even when grown at the permissive temperature, the doubling time of cdk1-ts cells is longer than an isogenic wild type strain , suggesting that cdk1-ts may not be able to fully complement CDK1. In summary, we find that a timer specifying a constant budding duration describes how a cell population founded by ‘giant’ cells returns to their set-point volume.

Although mother and daughter sizes are correlated across a broad size range, daughters are always born smaller than mother cells. After cytokinesis, daughter cells remaining larger than the set-point volume exhibit a G1 phase with virtually no growth and bud rapidly, leading to a geometric shrinking in successive generations . Indeed, a back-of-the-envelope calculation demonstrates that if newly-budded daughters are each 50% smaller than their mothers, a 32-fold decrease in cell volume can be achieved in 5 generations . Assuming a 100 min doubling time , a return to the set-point size would take ~8 h. A fixed budding time, even in the absence of active molecular size sensors in S/G2/M, is sufficient to buffer against persistence of abnormally large cell sizes in the population. We also note that the bud duration timer we describe is quite complementary to G1-phase size sensors such as Whi5, which compensate for a small size at birth by elongating G1 phase.Our conclusions are derived from cells prepared using two independent perturbations: optogenetic inactivation of the Bem1 polarity factor and a temperature-sensitive cdk1 allele. Importantly,stackable planters each of these perturbations targets distinct cellular processes and thus produces distinct physiological defects. Cells lacking Bem1 activity exhibit weakened cells walls and undergo successive rounds of DNA endoreduplication following their initial arrest in G1 . In contrast, loss of Cdk1 does not produce such defects but its disruption requires incubating cells at 37˚C, which may broadly activate environmental stress response pathways. Furthermore, cdk1-ts may not fully complement CDK1, even at the permissive temperature . That each of these perturbations reveals similar mother-daughter size correlations as well as a size-invariant bud timer strongly supports the generality of our conclusions. The bud timer we describe here is consistent with prior work suggesting that the duration of budding tends to be invariant to changes in growth rates. However, such a timer need not be a dedicated biochemical circuit to sense budding duration, compare it to a set-point, and dictate the transition to cytokinesis. Its existence could simply arise due to the time required by independent cellular processes that coincide with bud growth, such as the combined duration of S-phase or mitosis. Nevertheless, one observation suggests more complex regulation: the duration of the size-invariant bud timer is markedly longer in enlarged cdk1-ts vs. optoBem1 cells , yet mother-daughter sizes are nearly identical in these two backgrounds . These data suggest that the duration of the bud timer may be inter-related to Cdk1 activity and cells’ growth rate during S/G2/M. Recent work has found that mitosis and bud growth rate are closely coordinated and that cells may extend the duration of mitosis to compensate for slow growth that occurs under poor nutrient conditions. Dissecting the dependencies between growth rate, Cdk1 activity and the duration of post-Start events presents a promising direction for future study.All yeast strains used are isogenic to an ‘optoBem1’ strain which was created in the w303 genetic background and contained exogenous PhyB-mCherry-Tom7 with endogenous Bem1 C-terminally tagged with mCherry-PIF, as previously described. The cdc28-13strain was a kind gift from David Morgan. A pACT1-CDC10-eGFP expression vector was created by Gibson assembly, with the CDC10 expression cassette inserted between the NotI and XmaI sites of the pRS316 vector.

For the experiments described in Figs 3 and 4; Fig D, E, F, and G in S1 Fig; and S2 Fig; the indicated vector was transformed into our optoBem1 or cdk1-ts strain and selection was maintained by growing yeast in synthetic complete media lacking uracil . For all other experiments, yeast were cultured in synthetic complete media .Preparation of yeast prior to optogenetic experiments was performed, in general, as previously described. Yeast undergoing exponential growth in synthetic media were treated with 31.25 μM phycocyanobilin and incubated in foil-wrapped tubes at 30˚C for a minimum of 2 h. For all microscopy experiments, yeast were spun onto glass-bottom 96-well plates coated with Concanavalin A and washed once with fresh PCB-containing media to remove floating cells. Cells remained approximately spherical following this procedure, as assessed by Concanavalin A staining .Imaging was performed atroom temperature. For experiments where isotropic growth was measured , yeast were plated and imaged immediately following PCB treatment. For experiments where growth following Bem1 reactivation was examined , PCB-treated yeast were first placed in clear culture tubes and incubated at room temperature for >6 h while undergoing constant illumination with a red LED panel . Cells were then plated and imaged. For experiments involving the cdk1-ts strain, cells were maintained in liquid cultures of synthetic complete media at 25˚C for at least 24 h and plated as described for the optoBem1 strain. Imaging was performed at 37˚C for experiments where isotropic growth during G1 was measured . For experiments where size control was assessed , cells were incubated at 37˚C for 8 hr, then shifted to 25˚C 30 min prior to imaging.For isotropic growth experiments, samples were imaged on a Nikon Eclipse Ti inverted microscope equipped with a motorized stage , a Lamba XL Broad Spectrum Light Source , a 60x 1.4 NA Plan Apo objective , and a Clara interline CCD camera . Samples were imaged by bright-field microscopy every 10 min for 12 h. Throughout experiments involving optoBem1 cells, a red LED panel was carefully balanced against the motorized stage and microscope body to provide oblique illumination to the cells and ensure that Bem1 remained deactivated. Generous amounts of lab tape were applied to the LED panel and scope to prevent slippage during image acquisition and stage movement. For the remaining experiments, samples were imaged on one of two spinning disk confocal microscopes, both of which were Nikon Eclipse Ti inverted microscopes with motorized stages . The first microscope was equipped with a Diskovery 50-μm pinhole spinning disk , a laser merge module with 405, 440, 488, 514, and 561-nm laser lines, a 60x 1.49 NA TIRF Apo objective , and a Zyla sCMOS camera . The second microscope was equipped with CSU-X1 spinning disk , a MLC400B monolithic laser combiner with 405, 488, 561, and 640-nm laser lines, a 60x 1.4 NA Plan Apo objective , and a Clara interline CCD camera.