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The collapse of the spindle following ablation in S. pombe is mediated by microtubules and the motor protein dynein

Parsa Zareiesfandabadi, Mary Williard Elting

Posted on: 24 June 2021 , updated on: 16 August 2023

Preprint posted on 20 May 2021

Article now published in Biophysical Journal at https://doi.org/10.1016/j.bpj.2021.12.019

Ablating the microtubule spindle during cell division causes it to collapse. Zareiesfandabadi et. al. show how the minus-end directed motor protein dynein, plays a role in this collapse.

Selected by Leeba Ann Chacko

Categories: cell biology, microbiology

Background:

Schematic depicting the collapse of the spindle after laser ablation which is aided partially by dynein.

Unlike many of their mammalian counterparts, fission yeasts are unicellular eukaryotes that undergo ‘closed mitosis’, where the nuclear envelope does not undergo breakdown during cell division. Instead, at the onset of mitosis, cytoplasmic microtubules are reorganized to form the mitotic spindle within the closed nucleus1. This spindle elongates inside the nucleus causing it to expand and in the process, the DNA from the mother cell is equally distributed into identical daughter cells.

Scientists have wondered whether the nucleus and/or its contents can exert an opposing force to the elongating spindle. Previous work has shown that ablating the mitotic spindle with a laser causes the spindle to collapse2,3.  This collapse was suggested to occur due to passive viscoelastic relaxation of the nucleus and/or mechanical relaxation of stretched chromosomes. However, in this preprint, Zareiesfandabadi et. al. show that active forces driven by dynein contribute to the collapse of the spindle.

Dynein is known to aid chromosome biorientation to enable its proper segregation in fission yeast4,5. Here, Zareiesfandabadi et. al. demonstrate dynein’s role in ensuring mechanical force balance in the spindle through its ability to slide antiparallel microtubules towards each other as the spindle elongates in the opposite direction.

Key results:

Zareiesfandabadi et. al. used the technique of laser ablation to split the spindle in the middle to identify the forces being exerted on the broken, collapsing spindles.

Upon ablating the spindle, they showed that the collapse observed post-laser ablation follows an exponential relaxation response. While this observation is consistent with viscoelastic relaxation, it did not explain the observed spindle rotation within the nucleus. To test whether active forces are at play, the authors examined how much passive nuclear and chromosomal relaxation forces contribute to spindle collapse.

The authors found that upon ablating the early-stage, short spindle, the separated spindles collapsed towards each other while the chromosomes remained largely stationary. Contrastingly, when ablating the later-stage, long spindle, the chromosomes moved along with the collapsing spindle. Interestingly, the authors observed inward indentations in regions of the chromosome that were connected to the spindle ends. Similarly, there were inward indentations on the surface of the nuclear envelope that was in close proximity to the spindle ends. Based on these results, the authors concluded that neither mechanical relaxation of chromosomes nor viscoelastic relaxation of the nucleus contributed to the collapse of the spindle.

The authors determined that while actin plays no role in spindle collapse, microtubule dynamics are necessary for spindle collapse. They found that the minus end-directed motor protein, dynein, aids spindle collapse.  In the absence of dynein, the spindle opts for a prolonged rotational diffusion instead of exhibiting a relaxation response. From this, the authors concluded that the observed collapse of the spindle post-ablation is partially mediated by active forces from dynein and microtubule polymerization.

What I liked about this preprint:

What stood out most for me was the elegance and simplicity of the experiments that were used to test the hypothesis. The authors were able to uncover substantial molecular dynamics using the well-known technique of laser ablation. These experiments reiterate the importance of devoting time towards analyzing data extensively so that one does not miss out on discoveries like the inward indentations the authors observed.

References:

1. Mehta, K., et al., Association of mitochondria with microtubules inhibits mitochondrial fission by precluding assembly of the fission protein Dnm1. J Biol Chem, 2019. 294(10): p. 3385-3396.

2. Khodjakov, A., S. La Terra, and F. Chang, Laser microsurgery in fission yeast; role of the mitotic spindle midzone in anaphase B. Curr Biol, 2004. 14(15): p. 1330-40.

3. Tolic-Norrelykke, I.M., et al., Positioning and elongation of the fission yeast spindle by microtubule-based pushing. Curr Biol, 2004. 14(13): p. 1181-6.

4. Grishchuk, E.L., I.S. Spiridonov, and J.R. McIntosh, Mitotic chromosome biorientation in fission yeast is enhanced by dynein and a minus-end-directed, kinesin-like protein. Mol Biol Cell, 2007. 18(6): p. 2216-25.

5. Courtheoux, T., et al., Dynein participates in chromosome segregation in fission yeast. Biol Cell, 2007. 99(11): p. 627-37.

6. Schreiner, S.M., et al., The tethering of chromatin to the nuclear envelope supports nuclear mechanics. Nat Commun, 2015. 6: p. 7159.

Tags: fission yeast, laser ablation, microtubule, mitosis, s.pombe, spindle

doi: https://doi.org/10.1242/prelights.29767

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Author's response

Mary Williard Elting and Parsa Zareiesfandabadi shared

1. To completely rule out the possibility of viscoelastic forces from nuclear relaxation, would it be useful to see whether spindle collapse increases when the nuclear rigidity is lowered? Mutants in which the chromatin is untethered from the inner nuclear membrane exhibit deformable nuclei6. Perhaps these mutants could be used to test this?

This is a good follow-up experiment. It is conceivable that if the collapse speed were unchanged when the rigidity of NE is lowered, one could rule out the contribution of relaxation of the NE during the collapse. A change in collapse speed would also provide information on the nuclear envelope relaxation. What we know so far is that, as Figure 3D of the papers indicates, we did not find a different collapse for smalls vs. large spindles in wild-type cells, which was surprising since the elongated NE is thought to be at higher tension than the spherical stage NE. In new data (not in the preprint), we’ve also begun to examine the collapse where the spindle pole bodies were not tethered to the NE using a pkl1 deletion strain and did not detect a different collapse. But we agree with you that untethering chromatin would also be a way to get at this question in more depth.

2. Do you expect to see an increase in spindle collapse upon over-expressing dynein in the cells?

Great questions, and while that is an appealing idea, we think it’s hard to predict whether overexpressing would necessarily increase the collapse amount or rate. The reason is that dynein overexpression would not necessarily ensure more force generation on the spindle fragment (since we don’t know how it’s recruited), and it’s also possible that dynein overexpression could cause crowding of the microtubules, acting to effectively increase friction and actually slowing collapse. Even though we cannot reliably predict the outcome of overexpression of dynein, we agree that it would certainly be an interesting follow-up experiment. It also would likely help us identify where the dynein is during the collapse, a question that is still open.

3. Would it be feasible to observe the dynein localization during spindle collapse? Perhaps this could provide further insight into the precise mechanism through which dynein is able to pull the spindle inward?

Good question! We have not localized dynein and so we don’t know for sure where it’s localized or exactly how it enables collapse. This is an experiment that we’re very excited to try in the future!

We presume that the two half-spindles need to connect to generate a shortening force, though we don’t know how yet. Our mental model has been that perhaps dynein localizes to the new microtubule minus-ends (of each side of the cut spindle) and then pulls the poles toward each other by dragging one fragment along the other. A similar response to ablation is seen by dynein in severed spindle fibers in mammalian (see Elting and Hueschen, JCB 2014). However, that’s very much speculation at this point, and something we need to follow up on.

4. While it is known that dynein plays various roles during fission yeast meiosis, do you think dynein provides mechanical force balance to the spindle during meiosis I and II as well?

That is an intriguing question and one we have thought of but have not had the time to conduct. As the question mentions, dynein is known to be important for meiotic prophase in S. pombe – specifically for nuclear oscillations during this phase, through its localization at SPBs and astral microtubules (Yamamoto et al, JCB 1999). In contrast, dynein deletion does not affect normal spindle dynamics in mitosis, although it does cause a small defect in chromosome biorientation4,5. It has been proposed that this function is achieved through spindle microtubule bundling and/or anchoring at SPBs, a role that dynein performs in many other cell types. The data in our preprint are certainly consistent with this mitotic interpretation, though we have not yet localized or precisely identified the population of dynein contributing to spindle collapse. As for meiosis – the geometry (and thus the mechanics) of the S. pombe meiotic spindle are somewhat different than the mitotic spindle, so it’s hard to say whether dynein is playing the same role there or not (in addition to its known role in meiotic nuclear alignment). That said, we agree that this would be an interesting follow-up to our work!

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