Multiple polarity kinases inhibit phase separation of F-BAR protein Cdc15 and antagonize cytokinetic ring assembly in fission yeast
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eLife assessment
This is a well-designed study to show how phosphorylation of the intrinsically disordered regions can control their ability to undergo liquid-liquid phase separation and thus impact protein function. The authors report how regulation of the F-BAR-containing protein Cdc15 via phosphorylation impacts its ability to phase separate and promote cytokinesis. This paper is of interest to not just the field of cytokinesis, but also to the general field of protein chemistry which is interested in how phase separation controls protein function.
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Abstract
The F-BAR protein Cdc15 is essential for cytokinesis in Schizosaccharomyces pombe and plays a key role in attaching the cytokinetic ring (CR) to the plasma membrane (PM). Cdc15’s abilities to bind to the membrane and oligomerize via its F-BAR domain are inhibited by phosphorylation of its intrinsically disordered region (IDR). Multiple cell polarity kinases regulate Cdc15 IDR phosphostate, and of these the DYRK kinase Pom1 phosphorylation sites on Cdc15 have been shown in vivo to prevent CR formation at cell tips. Here, we compared the ability of Pom1 to control Cdc15 phosphostate and cortical localization to that of other Cdc15 kinases: Kin1, Pck1, and Shk1. We identified distinct but overlapping cohorts of Cdc15 phosphorylation sites targeted by each kinase, and the number of sites correlated with each kinases’ abilities to influence Cdc15 PM localization. Coarse-grained simulations predicted that cumulative IDR phosphorylation moves the IDRs of a dimer apart and toward the F-BAR tips. Further, simulations indicated that the overall negative charge of phosphorylation masks positively charged amino acids necessary for F-BAR oligomerization and membrane interaction. Finally, simulations suggested that dephosphorylated Cdc15 undergoes phase separation driven by IDR interactions. Indeed, dephosphorylated but not phosphorylated Cdc15 undergoes liquid–liquid phase separation to form droplets in vitro that recruit Cdc15 binding partners. In cells, Cdc15 phosphomutants also formed PM-bound condensates that recruit other CR components. Together, we propose that a threshold of Cdc15 phosphorylation by assorted kinases prevents Cdc15 condensation on the PM and antagonizes CR assembly.
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Author Response
Reviewer #1 (Public Review):
The authors found that the IDR in Cdc15 gets phosphorylated by multiple kinases, Pom1/Shk1/Pck1/Kin1, and the phosphorylation on IDR inhibits the phase separation of the Cdc15 protein. The phosphorylation was demonstrated in the cell as well as in vitro. Moreover, the phosphorylation sites were identified by mass spectrometry. The phospho-regulation of Cdc15 LLPS was demonstrated by in vitro assay using recombinant proteins. The significance of the phosphorylation on contractile actomyosin ring (CAR) was demonstrated by using a cdc15 mutant carrying 31 Ala-substitutions at the phosphorylation sites (cdc15 31A). The CAR assembled comparable to cdc15+, but maturation and contraction of the ring were faster in the cdc15 31A mutant, suggesting the contribution of the phosphorylation for …
Author Response
Reviewer #1 (Public Review):
The authors found that the IDR in Cdc15 gets phosphorylated by multiple kinases, Pom1/Shk1/Pck1/Kin1, and the phosphorylation on IDR inhibits the phase separation of the Cdc15 protein. The phosphorylation was demonstrated in the cell as well as in vitro. Moreover, the phosphorylation sites were identified by mass spectrometry. The phospho-regulation of Cdc15 LLPS was demonstrated by in vitro assay using recombinant proteins. The significance of the phosphorylation on contractile actomyosin ring (CAR) was demonstrated by using a cdc15 mutant carrying 31 Ala-substitutions at the phosphorylation sites (cdc15 31A). The CAR assembled comparable to cdc15+, but maturation and contraction of the ring were faster in the cdc15 31A mutant, suggesting the contribution of the phosphorylation for delaying cytokinesis. This could be one of the mechanisms to ensure the completion of chromosome segregation before the cytokinesis. In this paper, the authors showed over-accumulation of type-II myosin regulatory light chain Rlc1 on CAR in the cdc15 31A mutant during the CAR assembly and its contraction. In addition, the kinases for the Cdc15 IDR phosphorylation are identified as polarity kinases, which restrict the assembly of the CAR formation in the middle. Indeed, inhibition of the kinases increases the ratio of septa formation at the cell tip in the mid1 knockout mutant, which lacks a major positive polarity cue during the mitotic phase. However, in this manuscript, this phenotype is not solely explained by the phosphorylation of the cdc15 31A, because the authors did not show the tip septa formation using cdc15 31A.
Preventing Cdc15 phosphorylation does not on its own promote tip septa formation (Bhattacharjee et al., 2020). The polarity kinases have other substrates in the tip exclusion pathway that presumably also play a key role in septation. Also, cells must also be in the correct part of the cell cycle to form functional CRs and septa. We described the necessary roles of other polarity kinase substrates in our discussion.
Overall, the data supports their conclusion, Cdc15 forms LLPS, and the process is inhibited by the phosphorylation of amino acid residues in the IDR in Cdc15 by polarity kinases. It is still unclear whether LLPS formation is a reversible process regulated by the protein kinases. In vitro experiments showed condensate formation by dephosphorylation of Cdc15 IDR but not diffusion of the LLPS by phosphorylation. I wonder if incubation of the kinases and the Cdc15 IDR condensates induces demolition of the LLPS.
This is an interesting idea but technically challenging. The reactions performed in vitro are done by adding phosphatase to induce droplet formation and there is no way to remove the phosphatase. Therefore, addition of kinase will battle the phosphatase and clear results are unlikely. What we do know from work in vivo is that without the ability to rephosphoryate Cdc15 with the Alanine mutants, the protein remains bound to membrane in clusters so it seems clear that it is the phosphostate of Cdc15 that governs this property of the protein.
The transition of the Cdc15 IDR phosphorylation and LLPS formation through the cell cycle progression is unclear. In asynchronous cells (most of the cells may be in the G2 phase) and nda3 or cps1 mutants, Cdc15 was still highly phosphorylated. This indicates that the Cdc15 is phosphorylated and the LLPS formation is inhibited throughout the cell cycle. The transition of the phosphorylation status for individual residues could be the next challenge for this research.
The cell cycle changes in Cdc15 phosphostatus and their correlation with localization have been well-documented (e.g. Fankhauser et al., Cell, 1998; Clifford et al., JCB, 2008; Roberts-Galbriath et al., Mol. Cell, 2010). Upon bulk analysis, Cdc15 is never fully dephosphorylated during mitosis but it is not highly phosphorylated in cells blocked in mitosis with nda3 or in cps1 cells when some portion of it is in CRs (please see the references indicated previously). As shown in the simulations, the protein need not be fully phosphorylated or dephosphorylated in order to undergo a conformational change that would allow condensate formation. A major conclusion of our work is that no particular phosphorylation site or sites is important but rather the overall charge on the dimer is important and that some threshold of phosphorylation keeps the protein off from forming clusters on the membrane. We agree with the reviewer that what that threshold is will be of interest in the future.
In addition, currently, there is no approach to monitor the LLPS in wild-type cells. Therefore, it is still unclear if LLPS formation is the physiological mechanism regulating cell division in wild-type cells.
We agree that we have not monitored LLPS in live cells. However, Cdc15’s condensate formation in live cells and its phosphorylation state are highly correlated. This suggestive of LLPS in vivo.
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eLife assessment
This is a well-designed study to show how phosphorylation of the intrinsically disordered regions can control their ability to undergo liquid-liquid phase separation and thus impact protein function. The authors report how regulation of the F-BAR-containing protein Cdc15 via phosphorylation impacts its ability to phase separate and promote cytokinesis. This paper is of interest to not just the field of cytokinesis, but also to the general field of protein chemistry which is interested in how phase separation controls protein function.
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Reviewer #1 (Public Review):
The authors found that the IDR in Cdc15 gets phosphorylated by multiple kinases, Pom1/Shk1/Pck1/Kin1, and the phosphorylation on IDR inhibits the phase separation of the Cdc15 protein. The phosphorylation was demonstrated in the cell as well as in vitro. Moreover, the phosphorylation sites were identified by mass spectrometry. The phospho-regulation of Cdc15 LLPS was demonstrated by in vitro assay using recombinant proteins. The significance of the phosphorylation on contractile actomyosin ring (CAR) was demonstrated by using a cdc15 mutant carrying 31 Ala-substitutions at the phosphorylation sites (cdc15 31A). The CAR assembled comparable to cdc15+, but maturation and contraction of the ring were faster in the cdc15 31A mutant, suggesting the contribution of the phosphorylation for delaying cytokinesis. …
Reviewer #1 (Public Review):
The authors found that the IDR in Cdc15 gets phosphorylated by multiple kinases, Pom1/Shk1/Pck1/Kin1, and the phosphorylation on IDR inhibits the phase separation of the Cdc15 protein. The phosphorylation was demonstrated in the cell as well as in vitro. Moreover, the phosphorylation sites were identified by mass spectrometry. The phospho-regulation of Cdc15 LLPS was demonstrated by in vitro assay using recombinant proteins. The significance of the phosphorylation on contractile actomyosin ring (CAR) was demonstrated by using a cdc15 mutant carrying 31 Ala-substitutions at the phosphorylation sites (cdc15 31A). The CAR assembled comparable to cdc15+, but maturation and contraction of the ring were faster in the cdc15 31A mutant, suggesting the contribution of the phosphorylation for delaying cytokinesis. This could be one of the mechanisms to ensure the completion of chromosome segregation before the cytokinesis. In this paper, the authors showed over-accumulation of type-II myosin regulatory light chain Rlc1 on CAR in the cdc15 31A mutant during the CAR assembly and its contraction. In addition, the kinases for the Cdc15 IDR phosphorylation are identified as polarity kinases, which restrict the assembly of the CAR formation in the middle. Indeed, inhibition of the kinases increases the ratio of septa formation at the cell tip in the mid1 knockout mutant, which lacks a major positive polarity cue during the mitotic phase. However, in this manuscript, this phenotype is not solely explained by the phosphorylation of the cdc15 31A, because the authors did not show the tip septa formation using cdc15 31A.
Overall, the data supports their conclusion, Cdc15 forms LLPS, and the process is inhibited by the phosphorylation of amino acid residues in the IDR in Cdc15 by polarity kinases. It is still unclear whether LLPS formation is a reversible process regulated by the protein kinases. In vitro experiments showed condensate formation by dephosphorylation of Cdc15 IDR but not diffusion of the LLPS by phosphorylation. I wonder if incubation of the kinases and the Cdc15 IDR condensates induces demolition of the LLPS.
The transition of the Cdc15 IDR phosphorylation and LLPS formation through the cell cycle progression is unclear. In asynchronous cells (most of the cells may be in the G2 phase) and nda3 or cps1 mutants, Cdc15 was still highly phosphorylated. This indicates that the Cdc15 is phosphorylated and the LLPS formation is inhibited throughout the cell cycle. The transition of the phosphorylation status for individual residues could be the next challenge for this research. In addition, currently, there is no approach to monitor the LLPS in wild-type cells. Therefore, it is still unclear if LLPS formation is the physiological mechanism regulating cell division in wild-type cells.
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Reviewer #2 (Public Review):
This is a very interesting paper that described how multiple kinases regulate the phase separation of Cdc15 and thus impact its localization and function during cytokinesis. The authors build upon their prior research to show that Cdc15 is phosphorylated in its intrinsically disordered region at multiple sites by different kinases. Molecular simulations suggest that phosphorylation of Cdc15 impacts its F-BAR domain's ability to interact with the membrane. Indeed, the authors show that non-phosphorylatable Cdc15 mutants appear in larger dynamic clusters in the cells and also increase the recruitment of cytokinetic proteins to the actomyosin ring. Furthermore, the authors show that the purified Cdc15 intrinsically disordered region undergoes phase separation when treated with a phosphatase. Also, the …
Reviewer #2 (Public Review):
This is a very interesting paper that described how multiple kinases regulate the phase separation of Cdc15 and thus impact its localization and function during cytokinesis. The authors build upon their prior research to show that Cdc15 is phosphorylated in its intrinsically disordered region at multiple sites by different kinases. Molecular simulations suggest that phosphorylation of Cdc15 impacts its F-BAR domain's ability to interact with the membrane. Indeed, the authors show that non-phosphorylatable Cdc15 mutants appear in larger dynamic clusters in the cells and also increase the recruitment of cytokinetic proteins to the actomyosin ring. Furthermore, the authors show that the purified Cdc15 intrinsically disordered region undergoes phase separation when treated with a phosphatase. Also, the phase-separated region can recruit cytokinetic proteins that are known interacting partners of Cdc15. Overall, this is a very well-designed study that provides a deep mechanistic insight into how the Cdc15 scaffold conformation is regulated so that it can bind other proteins and interact with the plasma membrane to facilitate cytokinesis. However, the authors do not show if the sites identified here are specifically involved in phase separation. The authors provide evidence that Cdc15 undergoes phase separation when dephosphorylated by a phosphatase. However, it is not shown if dephosphorylation at the sites identified is indeed responsible for the phase separation. It would be helpful to show whether the purified cdc15-31A mutant protein also undergoes phase separation and increased interaction with cytokinetic proteins even in the absence of phosphatase treatment. This would provide strong evidence that indeed the kinases phosphorylate the identified sites to prevent phase separation.
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