Surface area-proportional growth could arise if nutrient/waste exchange across the plasma membrane is a limiting factor for growth. We observed that red light-illuminated optoBem1 cells also exhibited a change in DNA content over time. While most cells maintained a ploidy of 2N or less during the first 3 h of Bem1 disruption, a population of 4N cells appeared following 6 h of arrest , consistent with prior reports suggesting that after Bem1 disruption, some arrested cells eventually leak through the cell cycle block and undergo DNA endoreduplication. To ensure that the surface area-proportional growth was not an artifact of increased ploidy, we set out to generate ‘giant yeast’ via a second, non-optogenetic method: disruption of Cdk1/Cdc28 using the temperature-sensitive allele cdc28-13 . Unlike optoBem1 cells, nearly all cdk1-ts cells at the restrictive temperature arrest in G1 without undergoing further DNA replication. We found that cdk1-ts cells grown at the restrictive temperature to induce arrest in G1 also exhibited a linear increase in cell diameter, consistent with growth proportional to surface area . However, cdk1-ts were unable to maintain this rate of growth over the entire 12-h time-course: After reaching a volume of 500–700 μm3 , cell growth stalled . Taken together, our results from both optoBem1 and cdk1-ts cells indicate that the isotropic growth rate during G1 is proportional to surface area over a wide range of cell sizes.
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,hydroponic nft system 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, 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 postStart size regulation . The ‘adder’ model predicts that the cell volume at division should be proportional to cell volume at birth with a slope of. 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,nft channel 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, 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 cdc28-13 strain 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 at room 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 .