Microtubule reorganization during female meiosis in C. elegans
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Abstract
Most female meiotic spindles undergo striking morphological changes while transitioning from metaphase to anaphase. The ultra-structure of meiotic spindles, and how changes to this structure correlate with such dramatic spindle rearrangements remains largely unknown. To address this, we applied light microscopy, large-scale electron tomography and mathematical modeling of female meiotic Caenorhabditis elegans spindles. Combining these approaches, we find that meiotic spindles are dynamic arrays of short microtubules that turn over within seconds. The results show that the metaphase to anaphase transition correlates with an increase in microtubule numbers and a decrease in their average length. Detailed analysis of the tomographic data revealed that the microtubule length changes significantly during the metaphase-to-anaphase transition. This effect is most pronounced for microtubules located within 150 nm of the chromosome surface. To understand the mechanisms that drive this transition, we developed a mathematical model for the microtubule length distribution that considers microtubule growth, catastrophe, and severing. Using Bayesian inference to compare model predictions and data, we find that microtubule turn-over is the major driver of the spindle reorganizations. Our data suggest that in metaphase only a minor fraction of microtubules, those closest to the chromosomes, are severed. The large majority of microtubules, which are not in close contact with chromosomes, do not undergo severing. Instead, their length distribution is fully explained by growth and catastrophe. This suggests that the most prominent drivers of spindle rearrangements are changes in nucleation and catastrophe rate. In addition, we provide evidence that microtubule severing is dependent on katanin.
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###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on June 30, 2020, follows.
Summary
In this manuscript, findings from tomographic datasets of 10 C. elegans meiotic spindles from metaphase and anaphase (early, mid, and late) spindles (6 MI and 4 MII) are presented. The focus of the manuscript is on the observation that the transition from metaphase to anaphase involves a significant reorganization of the structure in which the number of MTs increases and the mean length decreases 2-fold. The authors develop a mathematical model to assess the relative contributions of 1) changes in MT dynamics, and 2) increased MT severing activity to the reorganization phenomenon. The model explains the data by a global change in MT dynamics and, in fact, indicates that MT severing makes hardly any …
###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on June 30, 2020, follows.
Summary
In this manuscript, findings from tomographic datasets of 10 C. elegans meiotic spindles from metaphase and anaphase (early, mid, and late) spindles (6 MI and 4 MII) are presented. The focus of the manuscript is on the observation that the transition from metaphase to anaphase involves a significant reorganization of the structure in which the number of MTs increases and the mean length decreases 2-fold. The authors develop a mathematical model to assess the relative contributions of 1) changes in MT dynamics, and 2) increased MT severing activity to the reorganization phenomenon. The model explains the data by a global change in MT dynamics and, in fact, indicates that MT severing makes hardly any contribution to the MT shortening observed in anaphase. The work is timely and the topic is of great interest; the quality of the EM data is excellent and these data can be expected to become a valuable resources in the field.
Essential Revisions
To compare the model with the data, the authors "average away" a large amount of detailed information present in the EM data and make additional simplifying assumptions that may be questioned. For example, it may be an oversimplification to assume mono-modal length distributions in the model that can be described by averages. In figure 1B the metaphase spindles look like there are two populations. The situation in anaphase looks even more complicated particularly if there is a surge of nucleation at the start of anaphase generating new short MT. The detailed 3D data sets are simplified down to a single spatial dimension (the spindle axis) and single length estimator (the average). The authors should provide some evidence/do some tests to validate their approach. How sensitive are the predictions of the model to the simplifying assumptions made and to the averaging out of detail?
A major weakness of the manuscript is considered to be the lack of experimental test of the prediction of the model which the authors present as their main conclusion. It should be possible to perform FRAP experiments to test the effect of katanin mutants on microtubule turnover to confirm or contradict the main conclusion that the authors derive from their model and that in part argues against pervious work. There is a well-characterized (and fast acting) ts allele of MEI-1 called mei-1(or642) (O'Rourke et al., PLoS One 2011 and McNally et al., MBoC, 2014) that could be used to test the effect of katanin on microtubule turnover by FRAP.
The authors should please be a bit clearer which EM data sets are new and which ones were re-used from previous work (for example including this information in Table 1). The expectation would be that the information from the new datasets is also used in the theoretical analysis presented in this manuscript. The context to previous work by others could be explained more clearly by being more specific when presenting background in the introduction so that it will be easier to understand what's new and different here compared to previous work (particularly compared to Yu et al. 2019 and Srayko et al. 2006).
Technical concerns: 4.1) FRAP analysis: To which extent does flux versus microtubule polymerization/depolymerization contribute to recovery. Is using a mono-exponential function to fit the recovery curves justified given that flux may contribute to recovery? How are the FRAP data used in the model? Is the contribution from flux to recovery considered separately from the contribution of polymerization/ depolymerization?
4.2) p.10, 2nd paragraph: Is the observed decrease in average microtubule length really independent of position? What is the factor of decrease as a function of position? Is the notion of global vs local change really fully supported by the data?
4.3) Model: Are microtubule minus ends considered stable after severing? Alpha is introduced, but the authors do not seem to come back to it later. What is it?
4.4) Model: Throughout, it would be useful to provide confidence intervals for the values that the authors extract from their model or provide some other statistical measure for the reliability of the prediction.
4.5) Does the model make the same predictions for meiosis II spindles or is turnover regulation different there?
4.6) Model: On page 9, lines 15-17 - the authors claim that if all the dynamic parameters except nucleation do not change then the length distribution should not change. However, if there is a change in nucleation, there will be a short-term increase in short MT, thereby shifting the length distribution.
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