Myosin II regulatory light chain phosphorylation and formin availability modulate cytokinesis upon changes in carbohydrate metabolism
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eLife assessment
Force generation by the myosin motor plays an important role during cell division. Myosin activity is regulated by phosphorylation, which activates myosin in animals but was thought to inactivate it in yeast. In this valuable study, the authors use a combination of convincing approaches to show that under some growth conditions, dependent on the carbon source of the growth medium, phosphorylation becomes essential for myosin function.
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Abstract
Cytokinesis, the separation of daughter cells at the end of mitosis, relies in animal cells on a contractile actomyosin ring (CAR) composed of actin and class II myosins, whose activity is strongly influenced by regulatory light chain (RLC) phosphorylation. However, in simple eukaryotes such as the fission yeast Schizosaccharomyces pombe , RLC phosphorylation appears dispensable for regulating CAR dynamics. We found that redundant phosphorylation at Ser35 of the S. pombe RLC homolog Rlc1 by the p21-activated kinases Pak1 and Pak2, modulates myosin II Myo2 activity and becomes essential for cytokinesis and cell growth during respiration. Previously, we showed that the stress-activated protein kinase pathway (SAPK) MAPK Sty1 controls fission yeast CAR integrity by downregulating formin For3 levels (Gómez-Gil et al., 2020). Here, we report that the reduced availability of formin For3-nucleated actin filaments for the CAR is the main reason for the required control of myosin II contractile activity by RLC phosphorylation during respiration-induced oxidative stress. Thus, the restoration of For3 levels by antioxidants overrides the control of myosin II function regulated by RLC phosphorylation, allowing cytokinesis and cell proliferation during respiration. Therefore, fine-tuned interplay between myosin II function through Rlc1 phosphorylation and environmentally controlled actin filament availability is critical for a successful cytokinesis in response to a switch to a respiratory carbohydrate metabolism.
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Author Response
Reviewer #2 (Public Review):
- My main reservation is the presentation of the work. The writing style is conversational and expansive, which makes it challenging for the reader. Furthermore, long paragraphs shift from one topic to the next rather than using separate paragraphs with strong topic sentences to cover each topic. I suggested a few places to start new paragraphs, but many more paragraphs could be divided.
We have also made significant efforts to reduce the text of the manuscript in each section, with more compact phrasing (including the headlines for the different results sections), and more short paragraphs to make the paper more readable. This has resulted in an overall reduction in the total number of words in the manuscript from ~11.000 to 9.000 (including Abstract, Introduction, Results, Discussion, …
Author Response
Reviewer #2 (Public Review):
- My main reservation is the presentation of the work. The writing style is conversational and expansive, which makes it challenging for the reader. Furthermore, long paragraphs shift from one topic to the next rather than using separate paragraphs with strong topic sentences to cover each topic. I suggested a few places to start new paragraphs, but many more paragraphs could be divided.
We have also made significant efforts to reduce the text of the manuscript in each section, with more compact phrasing (including the headlines for the different results sections), and more short paragraphs to make the paper more readable. This has resulted in an overall reduction in the total number of words in the manuscript from ~11.000 to 9.000 (including Abstract, Introduction, Results, Discussion, Materials and Methods, and Figure legends sections), equivalent to approximately four pages of typed text.
- Most of the figures are also overly complicated. I did not attempt to edit one of them, but I am sure that findings will be much clearer with about half of the panels moved to supplemental materials, so the reader can concentrate on the most important data.
As recommended by the reviewer, we have significantly reduced the number of panels within the figures in the revised manuscript. Accordingly, the total number of panels in the modified figures compared to the original version is as follows: Figure 1 (7 vs 8); Figure 2 (8 vs 10); Figure 3 (7 vs 10); Figure 4 (7 vs 12); Figure 5 (6 vs 11); Figure 6 (4 vs 8).
The remaining panels, including quantitative data such as cable-to-patch ratios, or percentages of septated/multiseptated cells, among others, have been moved to existing and new supplementary figures. The total number of supplementary figures is now 9 versus 6 in the original version.
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eLife assessment
Force generation by the myosin motor plays an important role during cell division. Myosin activity is regulated by phosphorylation, which activates myosin in animals but was thought to inactivate it in yeast. In this valuable study, the authors use a combination of convincing approaches to show that under some growth conditions, dependent on the carbon source of the growth medium, phosphorylation becomes essential for myosin function.
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Reviewer #1 (Public Review):
The author's findings are as follows:
(A) S.pombe Rlc1 is highly phosphorylated at Ser35 during the mitotic phase under respiratory metabolism.
(B) This phosphorylation promotes the assembly and contraction of the contractile actomyosin ring (CAR).
(C) This mechanism sustains CAR assembly and contraction under the respiratory metabolism, generating ROS. The ROS activates SAPK, which inhibits F-actin formation by reducing For3 expression.They are important findings explaining the robustness of proliferative and regenerative activity of the eukaryotic cells.
The data presented in the paper support the author's model.Although there are controversial reports on Rlc1 phosphorylation, whether it activates or inactivates type II myosin in S.pombe, this paper does not terminate the debate. Inhibitory …
Reviewer #1 (Public Review):
The author's findings are as follows:
(A) S.pombe Rlc1 is highly phosphorylated at Ser35 during the mitotic phase under respiratory metabolism.
(B) This phosphorylation promotes the assembly and contraction of the contractile actomyosin ring (CAR).
(C) This mechanism sustains CAR assembly and contraction under the respiratory metabolism, generating ROS. The ROS activates SAPK, which inhibits F-actin formation by reducing For3 expression.They are important findings explaining the robustness of proliferative and regenerative activity of the eukaryotic cells.
The data presented in the paper support the author's model.Although there are controversial reports on Rlc1 phosphorylation, whether it activates or inactivates type II myosin in S.pombe, this paper does not terminate the debate. Inhibitory phosphorylation on Rlc1 was reported by Mohan Balasubramanian laboratory and Susan Lowey laboratory before, as the authors referred to in this paper. In contrast, the author's model showed that Rlc1 is phosphorylated to facilitate cell division. Since the molecular mechanism of the CAR assembly and contraction is still not defined well yet, the research field should welcome this study to facilitate the discussion in the future.
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Reviewer #2 (Public Review):
I agree with the authors (line 421) that their "findings provide a remarkable example of how carbohydrate metabolism dictates the relative importance of different sources of actin filaments for CAR dynamics during cellular division." The scope of the work is very broad, and the manuscript reports dozens of interesting phenotypes with high-quality experimental data. However, most points are not investigated in depth.
My main reservation is the presentation of the work. The writing style is conversational and expansive, which makes it challenging for the reader. Furthermore, long paragraphs shift from one topic to the next rather than using separate paragraphs with strong topic sentences to cover each topic. I suggested a few places to start new paragraphs, but many more paragraphs could be divided. I edited …
Reviewer #2 (Public Review):
I agree with the authors (line 421) that their "findings provide a remarkable example of how carbohydrate metabolism dictates the relative importance of different sources of actin filaments for CAR dynamics during cellular division." The scope of the work is very broad, and the manuscript reports dozens of interesting phenotypes with high-quality experimental data. However, most points are not investigated in depth.
My main reservation is the presentation of the work. The writing style is conversational and expansive, which makes it challenging for the reader. Furthermore, long paragraphs shift from one topic to the next rather than using separate paragraphs with strong topic sentences to cover each topic. I suggested a few places to start new paragraphs, but many more paragraphs could be divided. I edited one paragraph to illustrate how the text might be cut in half.
Most of the figures are also overly complicated. I did not attempt to edit one of them, but I am sure that findings will be much clearer with about half of the panels moved to supplemental materials, so the reader can concentrate on the most important data.
Line 873: Fig 1C and many other figures. The legend says the error bars are SD's but they include far less than 2/3 of the measurements, so something is wrong. In Fig 4A and other figures, three data points are insufficient to verify a normal distribution, a prerequisite for using the Student's T-test. Furthermore, the T-test requires equal SD's.
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Reviewer #3 (Public Review):
In this study, the authors provide the first molecular clue to the apparent dispensability of RLC phosphorylation at S35,S36 (equivalent of RLC T18,S19 in non-muscle myosin II) for cytokinesis in Schizosaccharomyces pombe. Using point-mutant alleles, they successfully demonstrated that the S35 residue of Rlc1 is phosphorylated during cytokinesis in cells growing on glucose and that a mutant expressing the Rlc1-S35A allele is inviable on glycerol. The mutant cells exhibit slow CAR constriction and disassembly, multi-septated phenotype, and occasional cell lysis.
Rlc1 phosphorylation at S35 increases glycerol, which requires either Pak1 or Pak2. Although the localization of endogenously tagged Pak2-GFP was not detectable, the authors showed that Pak2-GFP expressed from the pak1+ promoter can localize to the …
Reviewer #3 (Public Review):
In this study, the authors provide the first molecular clue to the apparent dispensability of RLC phosphorylation at S35,S36 (equivalent of RLC T18,S19 in non-muscle myosin II) for cytokinesis in Schizosaccharomyces pombe. Using point-mutant alleles, they successfully demonstrated that the S35 residue of Rlc1 is phosphorylated during cytokinesis in cells growing on glucose and that a mutant expressing the Rlc1-S35A allele is inviable on glycerol. The mutant cells exhibit slow CAR constriction and disassembly, multi-septated phenotype, and occasional cell lysis.
Rlc1 phosphorylation at S35 increases glycerol, which requires either Pak1 or Pak2. Although the localization of endogenously tagged Pak2-GFP was not detectable, the authors showed that Pak2-GFP expressed from the pak1+ promoter can localize to the division site in both glucose and glycerol conditions. Next, the authors elucidated the physiological significance of Rlc1 phosphorylation by looking at the regulation of formin For3. Previously, the authors showed that For3 is downregulated at the protein level (probably through degradation) in response to latrunculin A treatment in a Sty1-dependent manner. Similarly, the shift from glucose to glycerol caused phosphorylation of Sty1 and concomitant downregulation of For3 protein levels, which in turn caused a reduced actin cable-to-path ratio. Because expression of For3-DAD (a constitutively active allele) or a lack of For3 downregulation was sufficient to fully rescue mutants in which Rlc1-S35 phosphorylation is impaired in glycerol conditions, the authors concluded that this phosphorylation compensates for the reduced actin-cable nucleation.
Finally, the authors hypothesized that ROS production during respiratory growth is responsible for Sty1-dependent For3 downregulation, and showed that the addition of the antioxidant GSH was sufficient to rescue the reduction in For3 levels and (as expected) the inviability of mutants lacking Rlc1-S35 phosphorylation in glycerol.
This will be the first report on the cellular response in the regulation of cytokinesis to a shift from fermentative to respiratory growth. It provides a new and important context to the value of fission yeast as a model to study animal cytokinesis and the effects of oxidative stress during the process. Data are generally well presented and clear-cut, and the components of two molecular pathways involved (SAPK-For3 and PAK-Rlc1) appear to behave in manners consistent with the authors' conclusions.
Some areas of weakness are as follows:
(1) Lack of use of phosphomimetic Rlc1 alleles (e.g., Sladewski et al., MBoC 2009) to strengthen the author's conclusions.
(2) It is not very clear how the two pathways (SAPK-For3 and PAK-Rlc1) interact with each other. Fig. S6 suggests that the authors favor the model they are regulated independently under respiratory conditions. However, alternative models are possible and testable.
(3) The authors conclude that oxidative stress causes Sty1 phosphorylation and that this phosphorylation is ultimately responsible for For3 downregulation and dependency on phosphorylation at Rlc1-S35. However, it is formally possible that all of these are independent events, which could easily be tested by using the sty1∆ mutant that the authors have used in publication. -
