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*Reviewer #1 (Evidence, reproducibility and clarity (Required): *
*Using genetics and microscopy approaches, Cabral et al. investigate how fission yeast regulates its length and width in response to osmotic, oxidative, or low glucose stress. Miller et al. have recently found that the cell cycle regulators Cdc25, Cdc13 and Cdr2 integrate information about cell volume, time and cell surface area into the cellular decision when to divide. Cabral now build on this work and test how disruption of these regulators affects cell size adaptation. They find that each stress condition shows a distinct dependence on the individual regulators, suggesting that the complex size control network enables optimized size adaptation for each condition. Overall, the manuscript is clear and the detailed methods ensure that the experiments can be replicated.
Major comments:
1.) It would be much easier to follow the authors' conclusions, if in addition to surface area to volume ratio, length and width, they would also plot cell volume at division in Figs. 1-4.*
AUTHOR RESPONSE: Due to space constraints in the main (and supplemental) figures, we focused on SA:Vol ratio together with cell length and width, which directly define cell geometry in rod-shaped fission yeast. Surface area and volume are derived from these measurements and can be misleading when considered alone, as similar surface area or volume values can arise from distinct combinations of length and width. The SA:Vol ratio therefore serves as a robust integrative metric for capturing coordinated changes in length and width that reshape cell geometry. We would be happy to include individual surface area and volume plots if requested.
2.) To me, it seems that maybe even more than upon osmotic stress, the cdc13-2x strain differs qualitatively from WT in low glucose conditions, where the increased SA-V ratio is almost completely abolished.
AUTHOR RESPONSE: We agree with the reviewer and have revised the manuscript text to point out this difference. The newly added text states: “Under low glucose, cdc13-2x cells also showed a WT-like response, decreasing length and increasing in SA:Vol ratio (Figures 3B-D). However, this SA:Vol increase was reduced compared to WT (1% vs 8.5%; Figures 1D and 3B), suggesting impaired geometric remodeling under glucose limitation.”
3.) It is not entirely clear to me why two copies of Cdc13 would qualitatively affect the responses. Shouldn't the extra copy behave similarly to the endogenous one and therefore only lead to quantitative changes? Maybe the authors can discuss this more clearly or even test a strain in which Cdc13 function is qualitatively disrupted.
AUTHOR RESPONSE: Increased Cdc13 protein concentration in cdc13-2x cells disrupts the typical time-scaling of Cdc13 protein. Consistent with this, cdc13-2x cells enter mitosis at a smaller cell size. We have modified the text to clarify this point. The new text states: “To access the role of the Cdc13 time-sensing pathway, we disrupted Cdc13 protein abundance by creating a cdc13-2x strain carrying an additional copy of cdc13 integrated at an exogenous locus. cdc13-2x cells divided at a smaller size than WT, reflecting accelerated mitotic entry upon disruption of typical time-scaling of Cdc13 protein (Figure S1A).”
4.) I don't see why the authors come to the conclusion that under osmotic stress cells would maximize cell volume. It leads to a decreased cell length, doesn't it?
AUTHOR RESPONSE: WT cells under osmotic stress do decrease in length, but this is accompanied by an increase in cell width. Because width contributes disproportionately to cell volume in rod-shaped cells, this change results in a modest but reproducible reduction in the SA:Vol ratio relative to WT cells in control medium (Figure 1D). We note that the degree of this change under osmotic stress is small (-0.4%), although statistically significant (p *
Likewise, in Figure 2B, they interpret tiny changes in the SA/V. By my estimation, the difference between control and osmotic stress is only 2% (1.195/1.17), less that the wild-type case, which appears to be twice that (which is still pretty modest). The small amplitude of these changes is obscured by the fact that the graphs do not have a baseline at zero, which, as a matter of good data-presentation practice, they should.
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AUTHOR RESPONSE: We appreciate the reviewer’s distinction between statistical and biological significance and agree that this is an important point to clarify. We now note in the revised text that changes in SA:Vol ratio under osmotic stress are numerically small and should not be overinterpreted. Our revised text now states: “Under oxidative and osmotic stress, the SA:Vol ratio decreased, indicating greater cell volume expansion relative to surface area (Figure 1D). However, we note that the reduction in SA:Vol under osmotic stress, while statistically significant, was modest in magnitude (−0.4%).”
Although small in absolute terms, even subtle geometric changes can be biologically meaningful in fission yeast due to the small size of these cells, where minor shifts in length or width translate into measurable differences in membrane area relative to cytoplasmic volume. Importantly, in Figure 2B, the key observation is not the magnitude of the change but its direction: cdc25-degron-DaMP cells exhibit a ~2% increase in SA:Vol ratio under osmotic stress, in contrast to the decrease observed in WT cells under the same condition. This opposite response reflects altered cell geometry and is supported by corresponding changes in cell length and width. We have revised the Results text to emphasize both the modest magnitude and the directional nature of these effects: “Under osmotic stress, cdc25-degron-DaMP cells exhibited a ~2% increase in SA:Vol ratio, opposite to the modest decrease observed in WT cells. This increase arose from increased cell length and reduced width (Figures 2B-D).”
Regarding data presentation, because SA:Vol ratios vary over a narrow numerical range, setting the y-axis minimum to zero would compress the data and obscure all detectable differences. Instead, we have modifed our SA:Vol ratio graphs in Fig. 1-4 to have consistent axis scaling across panels to accurately convey relative changes while maintaining visual clarity. We are happy to provide full data tables and statistical outputs upon request.
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I am also concerned about the use of manual measurement of width at a single point along the cell. This approach is very sensitive to the choice of width point and to non-cylindrical geometries, several of which are evident in the images presented. MATLAB will return the ??? as well as the length from a mask, but even better, one can more accurately calculate the surface area and volume by assuming rotational symmetry of the mask. Given that surface area and volume calculation need to be redone anyway, as discussed below, I encourage the authors to calculate them directly from the mask, instead of using the cylindrical assumption.*
AUTHOR RESPONSE: In initial experiments to calculate surface area and volume of fission yeast cells for prior work (Miller et al., 2023, Current Biology) we found that automated width measurements by MATLAB or ImageJ were inaccurate for a subset of cells leading to noisy cell surface area and volume values. Measuring cell width by hand and assuming that each cell in a given strain had the same cell radius (average of population) for calculation of cell surface area and volume gave more consistent results and recapitulated established conclusions regarding size control mechanisms.
In this previous work and the current study, abnormally skinny or wide regions of a cell were avoided when drawing a line to measure the cell width by hand. For each strain and condition, an average cell width was determined per independent experiment and used for surface area and volume calculations. Additionally, previous analysis demonstrated that this approach yields results consistent with a rotation method derived directly from cell masks, which does not assume a cylindrical cell shape (Facchetti et al., 2019, Current Biology; Miller et al., 2023, Current Biology).
To test the validity of our size measurements and confirm the robustness of our results in this study we compared the surface area and volume of cells by this rotation method. We have added this additional information to our revised methods section and also added SA:Vol ratio graphs generated from the rotation size measurement to our revised Figure S1 E-J. Importantly, both approaches used to measure cell size gave consistent results and supported the same conclusions.*
The authors also need to be more careful about their claims about size-dependent scaling. The concentration of both Cdc13 and Cdc25 scale with size (perhaps indirectly, in the case of Cdc13), but Cdr2 does not. Cdr2 activity has been proposed to scale with size, and its density at cortical nodes has been reported to scale with size, although that claim has been challenged .*
AUTHOR RESPONSE: We have modified text in the Introduction and Results to address this point. Our revised text in the introduction states: “Recent work has shown that Cdk1 activation integrates size- and time-dependent inputs: the Wee1-inhibitory kinase Cdr2 cortical node density scales with cell surface area (Pan et al., 2014; Facchetti et al., 2019); Cdc25 nuclear accumulation scales with cell volume; and cyclin Cdc13 accumulates over time in the nucleus (Miller et al., 2023) (Figure 1B).” Our revised text in the results section states: “Cdr2 functions as a cortical scaffold that regulates Wee1 activity in relation to cell size, with Cdr2 nodal density reported to scale with cell surface area, enforcing a surface area threshold for mitotic entry (Pan et al., 2014; Allard et al., 2018; Facchetti et al., 2019; Sayyad and Pollard, 2022).”*
Even taking the authors approach at face value, there are observations that do not seem to make sense, which led me to realize that the wrong formulae were used to calculate surface area and volume.
In Figure 1E,F, the KCl-treated cells get shorter and wider; surely, that should result in a lower SA/V ratio. However, as noted above, in Figure 1D, they are shown to have a similar ratio. As a sanity check, I eye-balled the numbers off of the figure (control: 14 µm x 3.6 µm and KCl: 11 µm x 3.8 µm) and calculated their surface area and volume using the formula for a capsule (i.e., a cylinder with hemispheric ends).
SA = the surface area of the two hemispheres + the surface are of the cylinder in between = 4*pi*(width/2)^2 + pi*width*(length-width), the length-width term calculates the side length of the capsule (length without the hemispheres) from the full length of the capsule (length including the hemispheres)
V = the volume of the two hemispheres + the volume of the cylinder in between = 4/3*pi*(width/2)^3 + pi*(width/2)^2*(length-width).
I got SA/V ratios of around 2, which are way off from what is presented in Figure 1D, but my calculated ratio goes down in KCl, as expected, but not as reported.
To make sure I was not doing something wrong, I was going to repeat my calculations with the formulae in Table 1, which made me realize both are incorrect. The stated formula for the cell surface area-2*pi*RL-only represents to surface area of the cylindrical side of the cells, not its hemispherical ends. And it is not even the correct formula for the surface area of the side, because that calls for L to be the length of the side (without the hemispherical ends) not the length of the cell (which includes the hemispherical ends). L here is stated to be cell length (which is what is normally measured in the field, and which is consistent with the reported length of control cells in Figure 1E being 14 µm). The formula for the volume of a capsule in the form use in Table 1 (volume of a cylinder of length L - the volume excluded from the hemispherical ends) is pi*R^2*L - (8-(4/3*pi))*R^3.
Given these problems, I think I spent too much time thinking about the rest of the paper, because all of the calculations, and perhaps their interpretations, need to be redone.*
AUTHOR RESPONSE: The surface area and volume equations for a cylinder with hemispherical ends used in our study and listed in our table are correct and widely used in other work with fission yeast cells (Navarro and Nurse, 2012; Pan et al., 2014; Facchetti et al., 2019; BayBay et al., 2020; and Miller et al., 2023). We write our equations with variables for cell length and radius because these are biologically relevant and measured parameters for fission yeast cells. Cell length (L) refers to the total tip-to-tip length of the cell, including the hemispherical ends, and radius (R) refers to half the measured cell width. We have revised the Methods section to clarify this definition and avoid ambiguity (Please see methods section “Cell geometry measurements”)
Additionally, SA or Vol calculations were performed using the length of each individual cell and the average cell radius of the population. We did not use mean cell length of the population for our calculations like the reviewer assumed in their “sanity check” above. Please see methods section “Cell geometry measurements”. We hope that these clarifications and text revisions improve transparency and reproducibility.
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Minor Points:
Strains should be identified by strain number is the text and figure legends.*
AUTHOR RESPONSE: For clarity and readability, we refer to strains by genotype in the main text and figure legends, which we believe is more informative for readers than strain numbers. All strain numbers corresponding to each genotype are provided in Table S1, ensuring traceability and reproducibility without compromising clarity in data presentation.*
In the Introduction, "Most cell control their size" should be "Most eukaryotic cell control their size".*
AUTHOR RESPONSE: The text has been corrected as suggested.*
Reviewer #2 (Significance (Required)):
Nothing to add.*
*Reviewer #3 (Evidence, reproducibility and clarity (Required)):
Summary
This manuscript reports that fission yeast cells exhibit distinct cell size and geometry when exposed to osmotic, oxidative, or low-glucose stress. Based on quantitative measurements of cell length and width, the authors propose that different stress conditions trigger specific 'geometric adaptation' patterns, suggesting that cell size homeostasis is flexibly modulated depending on environmental cues. The study provides phenotypic evidence that multiple environmental stresses lead to distinct outcomes in the balance between cell surface area and volume, which the authors interpret as stress-specific modes of size control.
Major comments
1) The authors define the 48-hour time point as the 'long-term response', but no justification is provided for why 48 hours represents a physiologically relevant adaptation phase. It is unclear whether the size-control mode has stabilized by that time, or whether it may continue to change afterward. At minimum, the authors should provide a rationale (e.g., growth recovery dynamics, transcriptional adaptation plateau, or pilot time-course observations) to demonstrate that 48 hours corresponds to the steady-state adaptive phase rather than an arbitrarily selected time point.*
AUTHOR RESPONSE: We thank the reviewer for this important point and agree that the definition of the long-term response should be clarified. We have addressed this with new experiments and revised text. We now incorporate growth curve data and doubling time analyses for all yeast strains grown under control and stress conditions (See new Figure S3). These analyses show that following an initial transient stress-induced cell cycle delay, growth rates stabilize well before 48 hours. Notably, the slowest growth rate observed was in 1M KCl, with a doubling time of ~4 hours across all yeast strains tested. Thus, by 48 hours, cells in this condition have undergone more than 12 generations of growth, while cells in all other conditions with shorter doubling times have undergone even more divisions. So by allowing cells to grow for 48 hours prior to imaging, we are capturing cells that have resumed sustained cell cycle progression following transient stress-induced cell cycle delays. Because cell size control is tightly linked to the cell cycle, we define 48 hours as a physiologically relevant time point where cells have adapted to stress conditions.
Our revised methods now states: “Cultures were incubated at 25°C while shaking at 180 rpm for 48 h prior to imaging. This time point was chosen to ensure that cells had progressed beyond the initial transient stress response and reached a stable, condition-specific growth state, as confirmed by growth curve and doubling time analyses showing stabilization well before 48 h (Figure S3), including in the slowest growing condition (1 M KCl; doubling time ~4 h).”
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2*）Related to the above comment, the authors propose that different stresses lead to distinct cell size adaptations, yet the rationale for the chosen stress intensities and exposure times is insufficiently described. It remains unclear whether the osmotic, oxidative, and low-glucose conditions used here induce comparable levels of cellular stress. Dose-response and time-course analyses would greatly strengthen the conclusions. Without such analyses, it is difficult to support the interpretation that geometry modulation represents a direct adaptive response.
AUTHOR RESPONSE: * *We selected the specific stress conditions based on previously published work showing that these doses elicit robust responses while preserving overall cell viability and the capacity for recovery. We note that osmotic, oxidative, and low glucose conditions perturb fundamentally different cellular systems (turgor pressure and cell wall mechanics, redox balance, and metabolism etc.) and therefore do not generate directly comparable levels of cellular stress in a quantitative sense. Our goal was not to equalize stress intensity across conditions, but to examine how cells change their geometry in response to distinct classes of stressors.
We have clarified the rationale for specific stress conditions in the revised methods: “These stress intensities were selected based on prior studies demonstrating robust cellular responses while preserving cell viability and the capacity for recovery (Fantes and Nurse, 1977, Shiozaki and Russell, 1995, Degols, et al., 1996; López-Avilés et al., 2008; Sansó et al., 2008; Satioh et al., 2015, Salat-Canela et al., 2021, Bertaux et al., 2023).”
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3) The authors describe stress-induced size changes as an 'adaptive' response. While this is an appealing hypothesis, the presented data do not demonstrate that the change in cell size itself confers a fitness advantage. Evidence showing that blocking the size change reduces stress survival-or that the altered size improves growth recovery- would be required to support this claim. Without such data, the use of the term 'geometric adaptation' seems overstated.*
AUTHOR RESPONSE: We have revised the text to remove the term “adaptive” and now describe stress-induced size changes in descriptive terms. As discussed further in response to Comment 4, new growth curve and doubling time analyses show that defects in surface area or volume expansion do not uniformly impair growth or survival over the stress exposure examined here, reinforcing the decision to avoid fitness-based language.*
4) The authors conclude that mutants exhibit no major defects in growth or viability during 48-hour stress exposure based on comparable septation index values (Fig. S2). However, septation index alone does not fully capture growth performance or cell-cycle progression and is not sufficient to support claims regarding fitness or robustness of proliferation. If the authors intend to make statements about 'growth', 'viability', or 'cell-cycle progression', additional quantitative measures (e.g., growth curves, doubling time, colony-forming units, or microcolony growth measurements) would be necessary. Alternatively, the claims should be toned down to align with the measurements currently provided.*
AUTHOR RESPONSE: We have addressed this concern with new experiments and revised text. In addition to septation index measurements (now analyzed using chi-square tests of proportions; Figure S2), we performed growth curve experiments and doubling time analyses for all genotypes under control and stress conditions (new Figure S3). These additional data show that growth rates are largely comparable across genotypes in control, oxidative, and low-glucose conditions, with more pronounced genotype-dependent differences emerging under osmotic stress. Defects in surface area or volume expansion did not uniformly correspond to impaired population growth, indicating that geometric remodeling is not strictly required for proliferation over the 48-hour stress exposure examined here. We have refined our conclusion to emphasize that defects in surface area or volume expansion do not uniformly impair growth or survival. See revised Results text under the heading “Defects in surface area or volume expansion do not uniformly compromise growth or survival”.*
5) Related to the above comment, the manuscript does not adequately rule out the possibility that the decreased division size simply results from slower growth or delayed cell-cycle progression rather than a shift in the size-control mechanism. Measurements and normalizations of growth rate are required; without them, the interpretation remains speculative.*
AUTHOR RESPONSE: We agree that changes in growth rate or altered cell cycle timing are important to consider. We have revised our text: “Changes in growth rate or cell cycle progression under stress may influence division size by altering mitotic regulator accumulation. Future studies measuring mitotic regulator dynamics alongside growth rates will be needed to distinguish direct changes in size control mechanisms from growth- or timing-dependent effects.”
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6) Regarding the phenotypes of wee1-2x cells, it is interesting that they increase the SA:Vol ratio under all stress conditions and show phenotypes distinct from cdr2Δ cells. From these observations, the authors claims that Cdr2 and Wee1 function as a surface-area-sensing module that complements the volume-sensing and time-sensing pathways to maintain geometric homeostasis. To support this interpretation, the authors could consider additional experiments, such as analyzing cdr2Δ + wee1-2x cells under the same stress conditions. Such data would test whether increased Wee1 can rescue or modify the cdr2Δ phenotype, providing functional evidence for the proposed Cdr2-Wee1-Cdk1 regulatory relationship. Measurements of cell length, width, SA:Vol ratio, and, if feasible, Cdk1 activity markers in the strain would greatly strengthen the mechanistic claims.*
AUTHOR RESPONSE: We thank the reviewer for this insightful suggestion. While analysis of a cdr2Δ wee1-2x strain could provide additional mechanistic detail, such experiments address a distinct question beyond the scope of our current study, which focuses on how cell geometry changes under different stress conditions in cells with perturbed surface area-, volume-, or time-sensing pathways. Our conclusions regarding a surface area-sensing role for Cdr2-Wee1 signaling are based on previous studies (Pan et al., 2014; Facchetti et al., 2019; Miller et al., 2023) and the cell geometry phenotypes we observe of cdr2Δ and wee1-2x cells under stress conditions. *
Minor comments
1) The manuscript focuses on adaptation through changes in the surface-to-volume ratio; however, only the ratio is shown. Presenting the underlying values of surface area and volume would clarify which geometric parameter primary contributes to the observed changes.*
AUTHOR RESPONSE: Please see our response to Reviewer 1 major comment 1.*
*2) Statistical analysis for Fig.S2 should be provided.
AUTHOR RESPONSE: We have completed this. See revised Figure S2 and methods.*
3) The paper by Kellog and Levin 2022 is missing from the reference list.*
AUTHOR RESPONSE: Thank you for catching this. This reference has now been added. *
**Referees cross-commenting**
After reading the other reviewer's reports, I recognize that focal points differ, but they appear sequential rather than contradictory.
Reviewer 2 raises concerns regarding the surface area/volume calculations, which-if incorrect-would influence many of the quantitative conclusions. I agree that confirming the validity of these calculations (and recalculating if necessary) should be the top priority before evaluating the biological interpretations.
Reviewer 1 raises more mechanistic biological questions. These are certainly important, but in my view they depend on the robustness of the quantitative analysis highlighted by Reviewer 2.
Therefore, I regard the reports as complementary rather than conflicting. Once the analytical issue pointed out by Reviewer 2 is resolved, the field will be in a better position to assess the significance of the mechanistic points raised by Reviewer 1 (as well as those in my own report).
Reviewer #3 (Significance (Required)):
General assessment
One of the major strengths of this manuscript is its quantitative, side-by-side comparison of multiple environmental stresses under a unified experimental and analytical framework. The authors provide well-controlled morphometric measurements, allowing direct comparison of geometry changes that would otherwise be difficult to evaluate across studies. The observation that different stress types generate distinct geometric outcomes is particularly intriguing and has the potential to stimulate new conceptual thinking in the field of size control.
However, the strength of the conceptual conclusion is currently limited by several aspects of the experimental design and interpretation. In particular, it remains unclear whether the observed geometry changes represent active adaptive responses rather than non-specific consequences of prolonged or string stress exposure. Demonstrating whether geometry remodeling provides a fitness advantage, clarifying whether the changes reach a steady-state rather than reflecting slow drift over time, or identifying upstream stress pathways that govern the response would substantially strengthen the conceptual advance. Even if additional mechanistic or fitness-related data cannot be added, refining the interpretation so that it remains aligned with the present evidence will enhance the clarity, and impact of the study.
Advance
Previous study - including the 2023 publication by the James B. Moseley group - established that fission yeast integrates distinct size-control pathways related to surface area, volume, and time under normal growth conditions. The present manuscript extends this line of work to stressed environments and argues that each stress condition elicits a distinct size-control pattern. To our knowledge, a systematic comparison of cell geometry across multiple stress types in the context of size-control pathways has not been reported, and this represents a potentially valuable conceptual advance. The advance is primarily phenomenological and conceptual rather than mechanistic: the work presents new correlation between stress types and geometry but does not yet elucidate the pathways governing these responses or demonstrate a functional advantage. With additional evidence - or with qualifiers ensuring that claims match the current data - the study could make an important contribution to understanding how cells integrate environmental cues into size-control strategies.
Audience
Although the primary audience consists of researchers in the fields of cell growth, cell-cycle control, and stress responses in yeast, the conceptual contribution may interest broader fields such as growth homeostasis, metabolic adaptation, and pathological cell size changes in higher eukaryotes. Beyond yeast biology, the modular view of size regulation proposed here may inspire new investigations in stem cell biology, cancer research, and biotechnology where environmental adaptation and cell size are closely linked.
Expertise: nuclear morphology; cell morphology; cell growth; cell cycle; cytoskeleton*