S. japonicus scales its geometry to changes in cell volume

Advancing S. pombe cells into mitosis by inhibition of the tyrosine kinase Wee1 is thought to provide a straightforward way to decrease cellular length-to-width aspect ratio30. We decided to use this genetic approach to generate shorter S. japonicus cells. To this end, we engineered an ATP analog-sensitive allele of S. japonicus, wee1-as8, based on an established S. pombe version35. After treatment of asynchronous wee1-as8 populations with 20 μM ATP analog 3-BrB-PP1, cells first divided medially, albeit at a shorter length. As these short cells entered the next mitosis, their daughters assumed asymmetric pattern of growth. Whereas most of the cell cortex underwent transient isotropic growth, one of the cell tips hyperpolarized and grew out at a smaller diameter (Fig. 1a, b; see time-lapse images in Supplementary Fig. 1a). The next division typically occurred close to the neck of the pear-shaped cell. Following cytokinesis, the asymmetrically dividing cell produced a thinner daughter with scaled geometry that resumed symmetric divisions and a wider one that usually underwent another round of hyperpolarization and asymmetric division. The accuracy of division site positioning in terms of pole-to-pole distance in asymmetrically dividing cells remained comparable to control (Supplementary Fig. 1b). After a few cell cycles, the population of exponentially dividing wee1-as8 S. japonicus cells reset cellular length-to-width aspect ratio, with cells dividing at both smaller length and width (Fig. 1a, c). Upon reaching steady state, 3-BrB-PP1-treated wee1-as8 cells divided at 72% volume as compared to the solvent control (284.4 μm3 ± 42.5 μm3 in 3-BrB-PP1-treated wee1-as8, 393.8 μm3 ± 30.73 μm3 in control, n = 18).

Fig. 1 S. japonicus maintains cellular aspect ratio over a range of volumes. a S. japonicus analog-sensitive wee1-as8 cells incubated with methanol (solvent control) or 20 μM ATP analog 3-BrB-PP1. Note the morphological transition in 3-BrB-PP1-treated cells occurring at a 4-h time point. b Wee1 inhibition initially causes differences in the diameters of two daughter cells (orange circles indicate cells treated with 3-BrB-PP1 for 2 h; gray circles represent solvent control). Shown are scatter plots, where either “thinner” (top left) or “thicker” (top right) daughter cell diameter measurements are on x-axis, and the ratios between diameters of the daughters are on y-axis. Graph (bottom right) compares cell diameter changes of the two daughters between control and 3-BrB-PP1-treated wee1-as8 and wild type populations. c Quantifications of cell length, width and aspect ratio at division of wee1-as8 cells shown in (a) and similarly treated wild type cells shown in Supplementary Fig. 1c. d Wee1-inhibited cells recover their original dimensions following the removal of the ATP analog from the growth medium. Shown are wee1-as8 cells treated with 20 μM 3-BrB-PP1 for 7 h, following the washout of the drug for 2 and 6 h. e Quantifications of cell length, width and aspect ratio at division of cells shown in (d). Methanol-treated wee1-as8 cells washed with growth medium (5th column) were used to control for cell number increase and washing procedure. f wee1-G788E mutant cells cultured at 24 °C (left) and 30 °C (right) overnight. g Measurements of cellular length, width and aspect ratio of the wild type as compared to wee1-G788E cells shown in (f). h S. japonicus cells after the shift from YE to EMM for 0, 4, 7 h and overnight. i Quantifications of cell length, width and aspect ratio at division before and after shift from YE to EMM overnight, for cells shown in (h). a, d, f, h Shown are single z-plane bright-field micrographs of live cells; scale bars represent 5 μm. b, c, e, g, i Quantifications presented as box plots with whiskers calculated by the Tukey method; n indicated in figures; p values derived from Kolmogorov–Smirnov test Full size image

The reduction of cell width in Wee1-inhibited cells was fully reversible. When 3-BrB-PP1 was washed out, small wee1-as8 cells reverted to wild type size, by extending cell length at division and concomitantly increasing cell diameter, likely through partial depolarization of growth machinery (Fig. 1d, e).

We noted that 3-BrB-PP1 treatment also promoted hyperpolarization of growth in wild type S. japonicus, although to a lesser extent as compared to wee1-as8 cells (p < 0.0001 for all measured parameters, Kolmogorov–Smirnov tests), indicating off-target effects (Fig. 1b, c and Supplementary Fig. 1c, d, see also ref. 36). This did not affect division plane positioning (Supplementary Fig. 1e). To confirm that specific downregulation of Wee1 kinase activity contributed to the morphological changes in 3-BrB-PP1-treated wee1-as8 cells, we constructed an S. japonicus version of wee1-50 S. pombe allele (G788E amino acid substitution), where Wee1 is functional at 24 °C but loses activity upon temperature up-shift30. Of note, S. japonicus cells harboring the wee1-G788E mutation divided at both reduced length and width when grown already at 30 °C. The extent of cell width reduction was lower as compared to 3-BrB-PP1 treatment of wee1-as8 cells, resulting in a slight deviation from the wild-type aspect ratio (Fig. 1f, g, see wild type images in Supplementary Fig. 1f). S. japonicus cells advanced into mitosis due to conditional G146D mutation in Cdc2 (cdc2-1w) that renders CDK1 insensitive to Wee1 inhibition31,32,33, exhibited reduced cellular length and width already at 24 °C, with cell width decreasing further after a prolonged incubation at 36 °C (Supplementary Fig. 1h, i). In these cells, cellular length-to-width ratio was not maintained perfectly but decreased to approximately 2.5 (Supplementary Fig. 1i). Upon shift to the restrictive temperature, cells of both genotypes settled on new geometry parameters through an asymmetrically dividing stage (Supplementary Fig. 1g, h; 3-h time points).

Suggesting that the cellular aspect ratio scaling is a physiological response, S. japonicus prototrophic wild type cells underwent similar morphological transition upon shift from the nutrition-rich medium (3% glucose YE) to the chemically defined minimal medium (EMM) (Fig. 1h, i). Similarly, shifting cells to low glucose medium (0.2% glucose YE) also lead to decrease in cell length and width. The lower aspect ratio in cells grown in 0.2% glucose was likely due to glucose exhaustion in batch cultures, resulting in high phenotypic variability (Supplementary Fig. 1j, k). Overall, our results suggested that forcing S. japonicus cells to divide at smaller volume either by manipulating Cdk1 activation status or nutritional availability leads to re-scaling of cellular geometry. Presumably, maintenance of the length-to-width aspect ratio may allow S. japonicus to adjust the spatial patterning of division site positioning determinants across a range of cellular volumes.

A Cdc42 GAP Rga4 is required for cellular geometry scaling

Reasoning that such a transition in growth patterns may require dynamic regulation of the small GTPase Cdc42 that is known to control polarized growth in both budding and fission yeasts37,38, we used CRIB-3xGFP as an established fluorescent marker for Cdc42 activation39. Time-lapse sequences of CRIB-3xGFP-expressing interphase S. japonicus cells revealed relatively weak and highly dynamic localization of active Cdc42 to the cell tips (Fig. 2a, b). This was in contrast to an extremely polarized distribution of active Cdc42 in S. pombe (Fig. 2a, b and ref. 40). Estimations of the full width of the CRIB-3xGFP domain at half maximum (FWHM) in both species using Gaussian curve fit41 and normalizing this values to cell tip radii showed that this marker of Cdc42 activity explores a broader area around the cell tips in S. japonicus as compared to its sister species (Fig. 2b, bottom panel). CRIB-3xGFP did not show higher enrichment at cell tips in S. japonicus cells that stabilized their geometry following long-term Wee1 inhibition or growth in the minimal medium (Supplementary Fig. 2a).

Fig. 2 Activation zones of the small GTPase Cdc42 rescale during morphological transition. a Time-lapse montages of interphase S. japonicus (top) and S. pombe (bottom) cells expressing a Cdc42 activity reporter CRIB-3xGFP. Shown are maximum intensity z-projections of spinning-disk confocal images. b Representative kymographs of CRIB-3xGFP at cell periphery in both species (top). 16-Color calibration bars indicate fluorescence intensities in arbitrary units. A diagram showing FWHM fit (full width of the domain at half maximum, bottom left), normalized to cell tip radii (right). c Single plane spinning-disk confocal micrographs of cells expressing indicated fluorescent proteins in the wild type (wt) and wee1-as8 genetic backgrounds. Wild type cells were treated either with solvent control or 20 μM 3-BrB-PP1 for 2 h. wee1-as8 cells were treated with 20 μM 3-BrB-PP1 for 2 h. d Graphs show cellular tip-to-tip ratio in fluorescent intensities of markers shown in (c). Note that CRIB-3xGFP, Scd2 and Gef1 become enriched at the hyperpolarized cell tip upon Wee1 inhibition, whereas Rga4 is largely excluded from these growing cell protrusions. a–c Scale bars represent 5 μm. b, d Quantifications presented as 1D-scatter plots; black bars represent sample median with error bars indicating 95% confidence intervals; n indicated in figures; p values derived from Kolmogorov–Smirnov test Full size image

Of note, when S. japonicus cells initiated hyperpolarized growth soon after Wee1 inactivation, CRIB-3xGFP became more enriched at the growing cell tips (Fig. 2c, d; see relative tip intensity values in Supplementary Fig. 2). At this time point, the Cdc42 scaffold protein Scd2-mNeonGreen was also enriched at the hyperpolarized cell tips in Wee1-inhibited cells (Fig. 2c, d and Supplementary Fig. 2). Although 3-BrB-PP1 treatment also caused hyperpolarization in wild type cells, we did not detect enrichment of these markers of Cdc42 activity under these conditions (Fig. 2c, d and Supplementary Fig. 2d). Consistently, the density of actin cytoskeletal assemblies required for polarized cell growth, including actin cables and endocytic actin patches, also increased upon Wee1 inhibition (Supplementary Fig. 2b), and the cell polarity protein Tea4 became enriched at the hyperpolarized cell tips (Supplementary Fig. 2c).

The mNeonGreen-tagged Cdc42 guanine nucleotide exchange factor (GEF) Gef1 exhibited weak accumulation at the cell tips in control cells. The intensity of Gef1 increased slightly upon 3-BrB-PP1 treatment in wild type cells, rising further at the growing cell tips during morphological transition in Wee1-inhibited cells (Fig. 2c, d and Supplementary Fig. 2d). We did not detect any cortical enrichment of another Cdc42 GEF, Scd1-mNeonGreen, in either of these conditions (Fig. 2c, d and Supplementary Fig. 2d). Curiously, the Cdc42 GTPase activating protein (GAP) Rga4 covered virtually an entire cortex of Wee1-inhibited small cells with an exception of the narrow growing tip (Fig. 2c, d). The second Cdc42 GAP Rga6 (ref. 42 and Supplementary Fig. 2e) was spread broadly throughout the cortex in both control and Wee1-inhibited S. japonicus cells (Fig. 2c, d).

We concluded that whereas both fission yeast species maintain comparable rod-shaped morphology, the Cdc42-dependent growth machinery at steady state is considerably less polarized in S. japonicus as compared to S. pombe. Yet, the former species can potentially modify its polarity apparatus when scaling cellular aspect ratio with changes in the cell volume.

We wondered whether interfering with Cdc42 regulation could prevent cellular geometry scaling in a population and, if so, what would be functional consequences of such a failure. To this end, we analyzed the physiological response of 3-BrB-PP1-treated wee1-as8 cells lacking Cdc42 GEF and GAP activities. Loss of Gef1 did not prevent hyperpolarization of growth at one of the cell tips (Fig. 3a, d). We were unable to assess the role of Scd1 during morphological transition upon Wee1 inhibition, as single scd1Δ mutant cells were virtually spherical and did not grow in standard liquid media (Supplementary Fig. 3a), suggesting that Scd1 was essential for normal polarity maintenance in S. japonicus.

Fig. 3 Rga4-dependent rescaling of cellular geometry is essential for medial division plane positioning. a–c Single z-plane bright-field micrographs of S. japonicus cells of indicated genotypes incubated with 20 μM 3-BrB-PP1 (right) for 2 h. Note that Rga4 deficiency prevents hyperpolarization of growth upon Wee1 inhibition. d Quantifications of cell diameters in the “thinner” (top) and “thicker” daughter (bottom) cell at cytokinesis, when 3-BrB-PP1 treatment is combined with genetic disruption of Cdc42 module regulators shown in (a–c). e wee1-as8 rga4Δ cells incubated with 20 μM 3-BrB-PP1 (right) for 4 h show severe division site positioning defects. Shown are bright-field micrographs. f Quantifications of cellular aspect ratio at division of cells shown in (e). g Single z-plane spinning-disk confocal micrographs of Pom1-GFP-expressing rga4Δ and wee1-as8 rga4Δ cells treated with 20 μM 3-BrB-PP1 for 2 h. h A plot summarizing the accuracy of division plane positioning in cells shown in (e). Deviation from the geometric center of the cell is indicated on y-axis. Black bars represent sample median. i Time-lapse montage of maximum intensity z-projected spinning-disk confocal micrographs of 3-BrB-PP1-treated wee1-as8 rga4Δ S. japonicus cells expressing Rlc1-GFP and the nucleoplasmic protein Nhp6-mCherry. Cell boundaries are outlined by white dashed lines. Wee1-inhibited S. japonicus cells lacking Rga4 fail to anchor the actomyosin ring medially following the semi-open mitosis, as indicated by the dispersal and nuclear re-import of Nhp6. a–c, e, g, i Scale bars represent 5 μm. d, f, h n indicated in figures; p values derived using Kolmogorov–Smirnov test Full size image

The lack of the Cdc42 GAP Rga6 produced a noisy response to Wee1 inhibition—some cells polarized normally whereas others failed (Fig. 3b, d, top plot). Strikingly, although S. japonicus cells lacking the primary Cdc42 GAP Rga4 were able to maintain cylindrical morphology when grown in normal conditions, they failed to hyperpolarize upon Wee1 inhibition (Fig. 3c, d, top plot). They also failed to reduce cell width in response to 3-BrB-PP1 treatment (Supplementary Fig. 3b). Thus, negative regulators of Cdc42 may spatially constrain its activation to promote hyperpolarized growth.

As S. japonicus mutants lacking Rga4 cannot initiate hyperpolarized growth adjusting cellular aspect ratio to a smaller volume (Fig. 3e, f), the 3-BrB-PP1-treated rga4Δ wee1-as8 cells eventually became virtually spherical, with Pom1 kinase spreading throughout the cellular cortex (Fig. 3g). As both Pom1 and the growth machinery contribute to medial division site positioning in S. japonicus29, Wee1-inhibited rga4Δ cells with decreased aspect ratio failed to anchor the actomyosin rings at cell equator, leading to profoundly asymmetric cytokinesis (Fig. 3h, i; see Supplementary Fig. 3c for control time-lapse sequence). When rga4Δ cells were shifted from the rich YE to the poorer, chemically defined EMM medium, they were not able to scale their aspect ratio and exhibited severe division site mis-positioning resulting in the generation of multinucleated cells (Supplementary Fig. 3d–f, compare with Fig. 1h, i). Taken together, these data indicate that spatial regulation of Cdc42 activity by Rga4 is critical for volume-dependent cellular geometry scaling in S. japonicus and contributes to proper patterning of cortical domains.

Cellular geometry correction rescues positioning errors

Our data suggested that failure to adapt cellular aspect ratio to cell volume prevents proper division site positioning. We set out to test this hypothesis directly by developing a microfluidics-based experimental setup, where wee1 mutant S. japonicus cells lacking Rga4 could be constrained physically to ensure that they remained cylindrical upon Wee1 inactivation. The overall device design was based on the previously published model43, but modified to fabricate microchannels of 7 or 10 μm width that could accommodate S. japonicus cells. These experiments were performed with cells encoding the temperature-sensitive allele of wee1, wee1-G788E (Fig. 1f, g), since the non-hydrolysable ATP analogs are known to be absorbed by polydimethylsiloxane (PDMS)44, used to manufacture microfluidics chambers. The nucleoplasmic protein Nhp6-mCherry was used to assess division site positioning failure resulting in multinucleation. As expected, wee1-G788E rga4Δ cells grown in batch cultures were not able to scale upon the temperature shift from 24 °C to 30 °C, and failed in division site positioning (Fig. 4a, d). However, when such a temperature shift was performed in the 7 μm microdevice, the daughter cells generated following Wee1 inactivation were forced to grow in a linear pattern. Although the aspect ratio was not fully corrected, most cells remained cylindrical and divided close to cell middle (Fig. 4b, d, e). Quantification of these results suggested that, at least in this system, the minimal length-to-width aspect ratio allowing accurate division site positioning is above 1.5 (Fig. 4b, e). As a control, we used cells grown in broader 10 μm microchannels, which were not able to provide mechanical constraint (Fig. 4c). In this case, most cells failed to remain cylindrical and failed in division site positioning, similarly to batch cultures (Fig. 4d). We concluded that controlling cellular aspect ratio is important for proper division site positioning in S. japonicus.

Fig. 4 Cellular geometry dictates division site positioning in S. japonicus. a Nhp6-mCherry-expressing wee1-G788E rga4Δ cells grown in batch cultures at indicated temperatures. Shown are the pseudocolored epifluorescence images overlaid with bright-field. Note that these cells fail in cytokinesis plane positioning after incubation at 30 °C. b, c Nhp6-mCherry-expressing wee1-G788E rga4Δ cells grown in 7 μm (b) and 10 μm (c) channels, respectively, at 30 °C. Shown are the pseudocolored z-projected spinning-disk microscope images overlaid with bright-field. Time labels indicate minutes elapsed after cells grown at 24 °C in batch culture were loaded in channels at 30 °C. Gray dotted line in (c) indicates the border of two merged images. d Graph summarizing the accuracy of division plane positioning of wee1-G788E rga4Δ cells after 4-h incubations shown in (a–c). e A plot showing deviation of division septa from the geometric center of the cell (y-axis) vs. cellular aspect ratio (x-axis) of individual cells after 4-h incubation at 30 °C in 7 μm channel. a–c Scale bars represent 5 μm. Quantifications presented as scatter plots. Black bars represent sample median with error bars indicating 95% confidence intervals; n indicated in figures; p values derived from Kolmogorov–Smirnov test Full size image

Aspect ratio control aids cytokinesis positioning in S. pombe

It is broadly accepted in the field that the sister species of S. japonicus, S. pombe, modulates cell length rather than width when forced to divide at smaller volume. This notion has stemmed from the phenotype exhibited by wee1 temperature-sensitive mutant cells30. Indeed, upon the shift from the permissive temperature of 24 °C to the restrictive temperature of 36 °C, wee1-50 cells shorten significantly, losing their normal length-to-width aspect ratio (ref. 30 and Fig. 5a, b). Yet, careful examination showed that these mutants in fact became wider (Fig. 5a, b), similarly to the wild type cells that underwent the same treatment (Supplementary Fig. 4a and 4b). We have previously shown that such a temperature shift-up is sufficient to trigger a heat-stress response in S. pombe, resulting in transient depolarization of growth and an increase in the cell diameter45. In line with cellular polarity defects, wee1-50 mutants shifted to 36 °C exhibited frequent division site mis-positioning (Fig. 5a, c, see also ref. 34).

Fig. 5 Maintaining cellular aspect ratio in S. pombe upon reduction in cell volume ensures the fidelity of medial division plane positioning. a S. pombe temperature-sensitive wee1-50 haploid cells grown at 24 °C overnight (gray), shifted to 36 °C for 5 h (red) or grown at 30 °C overnight (orange). b Quantifications of cell length, width and aspect ratio at division shown in (a). c A plot summarizing the accuracy of division plane positioning in wee1-50 S. pombe haploid cells grown at indicated temperatures. d S. pombe wee1-50 diploid cells grown at 24 °C overnight (gray), shifted to 36 °C for 5 h (red) or to 30 °C for 5 h (orange). e Quantifications of cell length, width and aspect ratio shown in (d). f A plot summarizing the accuracy of division plane positioning in wee1-50 S. pombe diploid cells grown at indicated temperatures. g Plots showing the rates of longitudinal growth and cellular volume increase in haploid and diploid S. pombe cells, compared with these parameters obtained for haploid S. japonicus cells. h Wild type S. pombe grown overnight in EMM with glutamate as a nitrogen source shifted to glutamate- or proline-based EMM for indicated time. A diagram (bottom) illustrates the workflow of the nitrogen shift experiment. i Quantifications of cell length, width and aspect ratio of cell populations represented in (h). j Calcofluor White-stained live S. pombe wee1-50 tea1Δ haploids (top) and diploids (bottom) at indicated temperatures. k A plot summarizing the accuracy of division plane positioning of cells shown in (j). a, d, h Shown as single z-plane bright-field micrographs. Scale bars represent 5 μm. b, e, g, i Presented are box-and-whiskers plots using the Tukey method. c, f, k Shown as 1D-scatter plots. Deviation of septa from the geometric center of the cell is indicated on y-axis. Black bars represent sample median with error bars indicating 95% confidence intervals. n indicated in figures. p values derived using Kolmogorov–Smirnov test Full size image

However, wee1-50 cells exhibited a reduction in cell length at division already at the temperature of 30 °C (Fig. 5a, b). Interestingly, under these non-stressed conditions, wee1-50 mutants showed reduced cell diameter, resulting in partial correction of cellular aspect ratio, although the response across the population was quite noisy (Fig. 5a, b). Consistently, we detected occasional division site placement abnormalities although this phenotype was more pronounced in the conditions of heat stress (Fig. 5a, c). This was in contrast to S. japonicus wee1 mutants that were able to scale more efficiently (Fig. 1). Curiously, diploid wee1-50 S. pombe cells exhibited a more coherent scaling of cellular morphology across population after the temperature shift to 30 °C, as compared to haploids (Fig. 5d, e; see Supplementary Fig. 4c, d for wild type diploid data). In line with these data, the diploid wee1-50 mutant cells exhibited virtually normal division site positioning at 30 °C (Fig. 5d, f). One possible explanation for the variance in scaling efficiency between S. pombe haploids and diploids could be higher rates of longitudinal growth and cellular volume increase in diploid cells (Fig. 5g). Arguably, faster polarized growth may allow cells to reach a suitable aspect ratio for optimal division site positioning before the onset of mitosis. In line with this, S. japonicus, which is able to scale very efficiently, grows substantially faster as compared to S. pombe (Fig. 5g).

We also investigated the behavior of S. pombe wee1-as8 cells35 upon chemical inhibition of Wee1. The wild type S. pombe exhibited hyperpolarization upon incubation with the ATP analog 3-BrB-PP1, consistent with off-target effect on polarity machinery. 3-BrB-PP1-treated cells became slightly thinner and accordingly, increased in length, resulting in higher cellular aspect ratio (Supplementary Fig. 5a, b). This increase did not affect division site positioning (Supplementary Fig. 5c). Unlike in S. japonicus, the extent of hyperpolarization in 3-BrB-PP1-treated S. pombe wee1-as8 cells did not differ significantly from the 3-BrB-PP1-treated wild type (compare with Fig. 1). This is perhaps not surprising since both values are quite low in S. pombe. However, the Wee1-inhibited cells divided at shorter length and were able to reset cellular aspect ratio after several divisions (Supplementary Fig. 5d, e, 7-h time-point). At earlier time-points of 2 and 4 h post-treatment the populations were considerably heterogeneous, with many cells entering mitosis with abnormal morphology (Supplementary Fig. 5f). Consistently, we observed mild defects in division site placement especially at the 2-h time-point (Supplementary Fig. 5g). Similar to the situation with wee1-50 mutants, diploid 3-BrB-PP1-treated wee1-as8 S. pombe cells exhibited more coherent and efficient scaling across the population (Supplementary Fig. 5h–j).

To test if scaling was a physiological phenomenon in S. pombe, we assessed cellular geometry in prototrophic cells grown in different nitrogen sources15. Cells transferred from rich glutamate- to poor proline-based minimal medium initially exhibited pronounced reduction of cell length at division, resulting in lower aspect ratio (Fig. 5h, i, 4-h time-point). Of note, the geometry of these cells eventually recovered, both by reduction in cell width and partial recovery of cell length at division (Fig. 5h, i, 8-h time-point). We concluded that S. pombe was able to scale its geometry, similarly to S. japonicus.

As shown above, the diploid S. pombe cells at steady state already exhibit a higher length-to-width aspect ratio and also are more efficient in geometry scaling as compared to haploids. We wondered if this could facilitate correct positioning of the division site and cell survival under conditions where the “tip inhibition” pathway is compromised and the dominant actomyosin anchor Mid1 invades cell tips34. The tip inhibition pathway depends on the function of the kelch repeat protein Tea134. Consistent with previously published observations34, we detected severe division site placement abnormalities in haploid Wee1-deficient cells lacking Tea1, which fail to inhibit Mid1-dependent actomyosin ring assembly close to cell tips (Fig. 5j, k; see Supplementary Fig. 5k, l for wee1-as8 data). This is likely due to the aspect ratios of individual cells transiently dropping below an optimal threshold during morphological transition. As cells enter mitosis with decreased aspect ratio, many will fail in division site positioning resulting in severely decreased population fitness. On the other hand, the diploid wee1 mutants without Tea1 continued positioning the division site comparatively normally throughout the process of cellular geometry rescaling (Fig. 5j, k; see Supplementary Fig. 5k, l for wee1-as8 data). Taken together, our results indicate that both S. pombe and S. japonicus control cellular aspect ratio in proliferating populations. In spite of the two sister species using different strategies to position the division site23, aspect ratio control is instrumental for the fidelity of cytokinesis in cells dividing at different volumes (Fig. 6).