Cortical microtubule pulling forces contribute to the union of the parental genomes in the Caenorhabditis elegans zygote
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Evaluation Summary:
This manuscript would be interesting for cell biologists and biophysicists studying nuclear organization and mechanics. The work provides new insights into how pulling forces from the cell cortex influence the dynamics of nuclear rupture during mitosis.
(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
Previously, we reported that the Polo-like kinase PLK-1 phosphorylates the single Caenorhabditis elegans lamin (LMN-1) to trigger lamina depolymerization during mitosis. We showed that this event is required to form a pronuclear envelope scission event that removes membranes on the juxtaposed oocyte and sperm pronuclear envelopes in the zygote, allowing the parental chromosomes to merge in a single nucleus after segregation (Velez-Aguilera et al., 2020). Here, we show that cortical microtubule pulling forces contribute to pronuclear envelopes scission by promoting mitotic spindle elongation, and conversely, nuclear envelopes remodeling facilitates spindle elongation. We also demonstrate that weakening the pronuclear envelopes via PLK-1-mediated lamina depolymerization, is a prerequisite for the astral microtubule pulling forces to trigger pronuclear membranes scission. Finally, we provide evidence that PLK-1 mainly acts via lamina depolymerization in this process. These observations thus indicate that temporal coordination between lamina depolymerization and mitotic spindle elongation facilitates pronuclear envelopes scission and parental genomes unification.
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Evaluation Summary:
This manuscript would be interesting for cell biologists and biophysicists studying nuclear organization and mechanics. The work provides new insights into how pulling forces from the cell cortex influence the dynamics of nuclear rupture during mitosis.
(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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Reviewer #1 (Public Review):
Velez-Aguilera use molecular genetics and live imaging after fertilization in C. elegans zygotes to document a novel role for spindle elongation in promoting dis-assembly of the nuclear lamina prior to the merging of the sperm and egg genomes into a shared space during mitosis. The authors take advantage of their previous work showing that a lamin transgene that converts 8 PLK-1 kinase target sites to alanine stabilizes the nuclear lamina and results in failure of genome union or mixing. Here they show that decreased cortical pulling forces on spindle microtubules that limit elongation enhance the 8A lamin transgene defects, and that the 8A lamin transgene expression that stabilizes the lamina opposes spindle elongation. In addition, the authors show that decreased cortical pulling forces can result in …
Reviewer #1 (Public Review):
Velez-Aguilera use molecular genetics and live imaging after fertilization in C. elegans zygotes to document a novel role for spindle elongation in promoting dis-assembly of the nuclear lamina prior to the merging of the sperm and egg genomes into a shared space during mitosis. The authors take advantage of their previous work showing that a lamin transgene that converts 8 PLK-1 kinase target sites to alanine stabilizes the nuclear lamina and results in failure of genome union or mixing. Here they show that decreased cortical pulling forces on spindle microtubules that limit elongation enhance the 8A lamin transgene defects, and that the 8A lamin transgene expression that stabilizes the lamina opposes spindle elongation. In addition, the authors show that decreased cortical pulling forces can result in failed lamina breakdown, and increased cortical pulling forces can promote early dis-assembly. However, while decreased cortical pulling forces can enhance the 8A lamin defects, increased cortical pulling forces (generated by klp-7 knockdown) do not rescue 8A lamin breakdown or the genome union defects, indicating that lamina breakdown is a "pre-requisite" for envelope scission. The authors' data are compelling and strongly support their conclusions, although some points require clarification.
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Reviewer #2 (Public Review):
Velez-Aguilera et al. investigated the role of cortical pulling forces on nuclear scission during first division in C. elegans embryos. Mitosis in C. elegans is semi-open, and the nuclear envelope partially breaks down during spindle elongation and reforms after division is finished. Previous studies found that nuclear rupture requires depolymerization of lamin, a supporting polymer network beneath the nuclear membrane controlled by PLK-1 activity. A particular mutation in lamin, LMN-1 8A, stabilizes it, prevents nuclear membrane scission, and causes nuclear abnormality in later divisions. The author investigated the role of cortical pulling forces in nuclear envelope breakdown by perturbing the GPR-1/2, a well-studied gene in C. elegans, to manipulate cortical pulling forces. They found that in LMN-1 8A, …
Reviewer #2 (Public Review):
Velez-Aguilera et al. investigated the role of cortical pulling forces on nuclear scission during first division in C. elegans embryos. Mitosis in C. elegans is semi-open, and the nuclear envelope partially breaks down during spindle elongation and reforms after division is finished. Previous studies found that nuclear rupture requires depolymerization of lamin, a supporting polymer network beneath the nuclear membrane controlled by PLK-1 activity. A particular mutation in lamin, LMN-1 8A, stabilizes it, prevents nuclear membrane scission, and causes nuclear abnormality in later divisions. The author investigated the role of cortical pulling forces in nuclear envelope breakdown by perturbing the GPR-1/2, a well-studied gene in C. elegans, to manipulate cortical pulling forces. They found that in LMN-1 8A, removing GPR-1/2 generated a significantly severe paired nuclei phenotype. They found that this is primarily due to the absence of nuclear membrane scission in those embryos. To test whether cortical pulling force contributes to nuclear membrane scission, they measured the timing of this event in efa-6 and klp-7(RNAi) embryos, where pulling forces are enhanced and found that under these conditions, embryos go under premature nuclear scission. It has been known that spindle length, measured as the distance between the two centrosomes, is regulated by cortical pulling forces. The authors found that spindles are shorter in LMN-1 8A, which indicates force-balance is perturbed in these embryos. Further genetic perturbation experiments concluded that cortical microtubule pulling forces and PLK-1 activity together contribute to nuclear membrane scission and proper chromosome segregation.
While the experiments are carefully done, I see a few confusing points and concerns about the conclusions of the paper:
1- It is unclear whether nuclear membrane scission is a phenomenon controlled by mechanical forces, cell cycle, or both. Some of the experiments in the manuscript suggest mechanical forces control it, but some are not very supportive of this. For example, gpr-1/2 (RNAi) in lmn-1 8A supports the hypothesis that mechanical forces are important for nuclear scission. However, the lack of scission in hcp-3 (RNAi) in lmn-1 WT, where chromosome alignment is prevented, suggests a cell-cycle dependence mechanism. It would be good if the authors could elaborate more on this issue.
2- The relation between membrane scission and paired nuclei phenotype is unclear. It seems that paired nuclei phenotype in daughter cells is a direct consequence of lack of membrane scission earlier during first mitosis. The authors showed that in gpr-1/2 (RNAi) in lmn-1 WT, 83% of embryos have no nuclear membrane scission. However, only 5% of gpr-1/2 (RNAi) in lmn-1 WT embryos showed paired nuclei phenotype (Fig 2A). Is this an inconsistency? If not, it would be good if the authors could explain this in more detail.
3- Similarly, why in klp-7 (RNAi) lmn-1 8A there is paired nuclei phenotype while the membrane scission happens (figure 5D), and in hcp-3 (RNAi) in lmn-1 WT, there is no paired nuclei phenotype, while membrane scission is completely prevented? It seems that one needs more knowledge/explanation about nuclear membrane scission and paired nuclei phenotype to understand these results. The short speculation at the top of page 11 does not seem enough.
4- The authors suggested an interesting mechanism for spindle length regulation in a one-cell stage embryo as a force balance between cortical pulling forces and membrane tension. To my knowledge, the role of the nuclear membrane had not been discussed for spindle length regulation. This could be interesting to highlight in conclusions.
5- The connection between plk-1ts experiments and the cortical pulling forces is not clear. The authors start with the proposal of testing the effect of pulling forces on membrane scission, but by this point in the manuscript, it seems that pulling forces have a secondary effect on scission. By the end, the authors argue that lamina depolymerization, chromosome alignment, and cortical pulling forces are all important for nuclear membrane scission. If so, it would be good if the authors could indicate which one is the primary factor and how the others are comparable to the primary factor.
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