1. Reviewer #3 (Public Review):

    In this article, Odermatt and colleagues report a detailed and quantitative description of the growth in dry mass and volume of single fission yeast cells. They first propose a new method for dry mass measurement and calibrate it. They then use this method to show that, while growth in dry mass shows a rather steady exponential trend, growth in volume changes with the cell cycle stage, depending on the rate of cell tip elongation, which results in changes in cell density. They then use various methods to arrest cells at various stages in the cell cycle, demonstrating that, for each cell cycle stage, the change in density is due to a specific growth rate which does not necessarily matches the growth in dry mass. All these experiments are very convincing. They close the article with two observations which are a bit harder to understand: first they show that there is an internal gradient of density, which corresponds to a difference in the rate of growth of both tips of the cell, and which is maintained on long timescales; second, they show that the differences in densities between the two ends of the cell lead, after the formation of the septum, to a difference in pressure which can be indirectly visualized from the bending of the septum - the denser side having a higher pressure, the septum is bent towards the lower density side.

    Overall, the article provides one of the very few available quantitative description of growth from single cell measurements, and shows for the first time that density variations can arise from an uncoupling between volume growth and dry mass growth, which does not seem to be compensated for. I think that this particular finding is significant enough to make this article broadly interesting for the cell biology community, as it concerns a very fundamental aspect of cell physiology.

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  2. Reviewer #2 (Public Review):

    Odermatt et al. apply quantitative phase imaging to fission yeast and show that the well-established cytokinetic growth pause is not accompanied by a parallel pause in biosynthesis and thus cellular density increases. Interestingly, cellular density does not quickly re-equilibrate after division. Instead, density slowly decrease throughout interphase, suggesting that cells can operate efficiently within at least a 20% range of cellular density.

    The work is of high technical quality. Comparisons with other measures of density and mass lend confidence to the robustness of the approach and the fact that it requires no custom hardware makes it accessibly to most workers in the field. However, the biological insight from the work is somewhat limited. The fact that density increases as growth pauses during cytokinesis is not surprising. Demonstrating it is an important contribution to the field, but it will not change the way people think about cell-cycle or growth control.

    To me, the most interesting result is that density gradients a stable within cells. This result must have important implications in cytosolic viscosity, which could have been discussed more explicitly. The discussion does claim that the "distributions of large organelles and total protein are not polarized", but it is not clear that the papers cited to support that claim would be able to detect the ~5% reported difference in density. The organelle paper contains no quantitation and the noise in the protein paper looks to be around 10%.

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  3. Reviewer #1 (Public Review):

    This paper provides quantitative detailed measurements of how dry mass density varies as a function of the cell cycle in fission yeast cells. They find that density decreases during G2 and increases during mitosis/cytokinesis. They also monitor the effects of cell cycle mutants and a drug that depolymerizes actin, and conclude that while dry mass increases continuously, cell cycle-regulated changes in volume growth in fission yeast create the density oscillations. This supports earlier less precise work in the field, and is therefore unsurprising. Overall, the quantitative data convincingly support these conclusions.

    More interesting is the discovery of intracellular density gradients in G2 cells, with lower density at faster growing ends. The basis for these gradients is not investigated. The gradients persist through mitosis and cytokinesis, giving birth to daughter cells whose mean densities differ. These findings would appear to suggest that density differences might accumulate with each generation, but this is clearly not the case as the density variation in a cell population is very small. This paper will be of interest to researchers working on fission yeast, and raises interesting questions for future investigation.

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  4. Evaluation Summary:

    This article contributes to the fundamental understanding of how a cell grows. It provides a broadly applicable method for dry mass measurement of single cells and, using it, it describe how cell density varies accross the cell division cycle. The key finding of this article is the fact that growth in mass and volume seem to be generally uncoupled, leading to significant density changes.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

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