Evolutionary divergence of anaphase spindle mechanics in nematode embryos constrained by antagonistic pulling and viscous forces

  1. Dhruv Khatri
  2. Thibault Brugière
  3. Chaitanya A. Athale
  4. Marie Delattre

  • Curated by eLife

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    Evaluation Summary:

    This work will be of interest to cell biologists studying the mechanism of asymmetric cell division and its diversity across species. Building on their earlier work, the authors show that that there is considerable variability in the mechanics of the spindle among six nematode species studied here. While the authors' main conclusion is plausible - that spindle oscillations require high force and low viscosity - stronger support by the data would be needed.

    (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.The reviewers remained anonymous to the authors.)

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Abstract

Little is known about the evolution of mechanical constraints in cells. We observed six nematode species for which the mitotic spindle is positioned asymmetrically in one-cell embryos, yet spindle motion varies between species. We asked which changes in biophysical parameters allow diversification in spindle motion but a constant output.

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  1. eLife

    Evaluation Summary:

    This work will be of interest to cell biologists studying the mechanism of asymmetric cell division and its diversity across species. Building on their earlier work, the authors show that that there is considerable variability in the mechanics of the spindle among six nematode species studied here. While the authors' main conclusion is plausible - that spindle oscillations require high force and low viscosity - stronger support by the data would be needed.

    (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.The reviewers remained anonymous to the authors.)

  2. eLife

    Reviewer #1 (Public Review):

    The authors look at a few different nematode species to compare the dynamics of anaphase. They find that in some species the spindle oscillates transversely in anaphase, and in other species it does not. They ask what accounts for this different behavior. To address this question, they use ablation of the central spindle, and conclude from the result, correctly, that after the ablation the centrosomes are pulled to the opposite poles of the cell in all species. However, the magnitude, half-time and initial velocity of the recoil differ.

    To understand what accounts for the quantitative difference, the authors

    1. use a simple viscoelastic model of a constant force, F, pulling against a spring (with constant stiffness k), while the object moves through the viscous medium.

    2. estimate the cytoplasmic viscosity from tracking yolk granules,

    3. estimate parameters F and k from fitting the exponential recoil curves. They find that the greatest correlation between having transverse oscillation or not is with lower or higher viscosity, not with magnitude of the force or stiffness of the spring.

    Two major problems with this study can be identified:

    1. Meaning and significance: It is not clear if the transverse oscillation have a functional significance. In fact, they are more likely than not simply a byproduct of complex nonlinear mechanics of the mitotic spindle. It is important to understand what we can learn about the spindle mechanics from these oscillations, but there may be no evolutionary significance here. If the authors were asking - how, in many different species, the spindle scales with the cell size in the same way (as was done in Farhadifar et al 2020, which the authors do not to cite) despite large parameter variations - that would be a different story. But asking which parameter change is responsible for the behavior change is less meaningful.

    2. The study is not convincing, mainly because the model used for the fit is overly simplistic. The force is not constant, the spring stiffness is not constant, the mechanics is not, etc. There are a few different, very complex models, of the anaphase spindle with transverse oscillations - comparing to simulations of these models would be more convincing. Also, I am not quite sure whether the volume fraction of yolk is a useful parameter. Does not measuring MSD give us the diffusion coefficient and viscosity directly? I think using the factor depending on the volume fraction artificially inflates the viscosity differences. Lastly, I do not understand the theoretical argument based on comparison with Nedelec's model: in that model, increasing viscosity only slowed the oscillations down, not abolished them.

    In short, much more thorough investigation would be needed to understand which differences between the species account for the presence or absence of the oscillations, and one may question whether the answer would have a deep impact on our understanding of spindle mechanics.

  3. eLife

    Reviewer #2 (Public Review):

    The authors ask the question of why the one-cell spindles of some nematode worms "rock" (i.e. undergo transverse oscillations with the anterior and posterior poles out of phase) whereas some do not. One limitation of the paper is that spindle oscillations may be an epiphenomenon with no adaptive function, as suggested by the fact that it is dispensable in C. elegans and not found in non-Caenorhabditis species.

    A strength of the paper is the use of laser-cutting and granule diffusion experiments to infer key mechanical parameters - force viscosity and stiffness (though the measurements are only indirect). It is interesting that there is a large range of parameters and that there are some similarities in parameter values in the two species that oscillate (C. elegans and C. remanei). However, a third species, O. tipulae, which does not oscillate, has parameters similar to C. remanei. Thus, the conclusion that oscillators depend on force and viscosity is not strongly supported by the data here. Given the number of parameters on which oscillation is thought to depend, 6 species might not be enough to draw definitive conclusions, especially if there is one exception.

  4. eLife

    Reviewer #3 (Public Review):

    The 1-cell stage embryo of the nematode, C. elegans, is a good model system for studying the mechanism of asymmetric division. The cell division at the 1-cell stage is asymmetric, in which fertilized P0 cells divide into large AB cells and small P1 cells. The different sizes of the daughter cells are caused by the asymmetric (off-center) localization of the mitotic spindle, and the asymmetry is caused by the asymmetry in the pulling forces from the anterior and posterior cell cortex. Therefore, characterization of the forces pulling the spindle from the cortex is important to understand the mechanism of asymmetric cell division.

    In some nematode species including C. elegans, an oscillatory behavior is observed upon the asymmetric localization of the spindle. In other species, the oscillation does not accompany the off-center motion of the spindle. The difference in the oscillation should be caused by the difference in either the magnitude of the pulling forces, or the magnitude of viscous drag (or both). It was not clear which is the case.

    This study solved this question by a nice collaboration between the Delattre group, who is an expert in comparative studies among the nematode species, and the Athale group, who developed image processing tools to characterize physical properties of the cytoplasm for non-labelled cells. Quantitative data on granule tracking and spindle laser ablation results are provided for 6 nematode species. The results will be a variable resource for future studies on diversity among species.

    The answer to the question on the difference behind the existence of the oscillation is provided in two ways, which are not mutually exclusive. One explanation is that, in non-oscillation species, the force is low (P. pacificus), or the viscosity of the cytoplasm is high (C. monodelphis), or both (D. sp. 1, and O. tipulae), compared to the oscillation species (C. elegans and C. remanei) (Fig. 5G). The second explanation is that the viscosity of the cytoplasm alone can explain the difference. It is known that, in theory, even a slight difference in viscosity can cause a large difference in oscillation. In the present study, the authors observed certain, but not always statistically significant, differences in viscosity that might account for the difference in oscillation (Fig. 5F).

    The data provided in this study is valuable, and the conclusions are overall reasonable. I find some aspect of force measurements and quantification of the parameters need to be clarified.

    #1. I wonder the force measurement, which is a critical part of this paper, is conducted appropriately. Previous studies on the C. elegans embryo observed the difference in cortical pulling forces between the anterior and posterior sides. This aspect was not reproduced in this study. The authors should explain the consistency of their measurements with the previous study, and evaluate whether the accuracy of their measurement of the forces is sufficient to draw the conclusion of this study.

    #2. Absolute values related to force, viscosity, and elasticity are quantified in this study. While the information is valuable, the values are obtained by incorporating several assumptions which is not so solid (for me). There would need to be careful discussion about the uncertainties associated with the assumptions. Such discussion is important for future research based on this study.

  5. Version published to 10.1091/mbc.e21-10-0532
  6. Version published to 10.1101/2021.08.10.455863v1 on bioRxiv