Article activity feed

  1. Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons


    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity (Required)): Excellent quality of cell biology and biochemistry. the additional supports are needed for the claim of actin elongation using different formin variants.

    Reviewer #1 (Significance (Required)): Ingrid Billault-Chaumartin and co-authors described interesting research that provides insights on formin-isoform specific function in fission yeast and a new role of Fus1 FH2 domain in cell-cell fusion event. While three formin isoforms have different localization, research proposed an additional dissection in their functional differences by having different functions in C-terminus, including FH1 FH2 and formin C-terminus. The work also described additional factors that regulate cell fusions from autotrophy effect and formin expression level, in addition to the well-accepted formin biochemical activities. Here are my comments regarding the strengths of the work and improvements that could further strengthen the story.

    Major comments

    1. Fig.1 shows Cdc12C could recapitulate Fus1 function by ~80% if fused with Fus1C, whereas deletion of the C-terminal tail of Cdc12 following FH2 introduces drastic dysfunction. Together with Fig. 3, these results indicate Cdc12 Cter plays more important roles than Fus1 Cter for there respective functions. Such results suggested a Cter-mediated mechanism that differentiates the functions of three fission yeast formin isoforms. The authors examined contributions from the difference in FH1 (Figs 4,5) and FH2 residues (Fig. 6). Whereas the obvious phenotype of Cter was not further investigated and not much discussed. The Cter of budding yeast formins interacts with nucleation-promoting factors, Bud6 and Aip5. Although S. Pombe does not have orthologs of budding yeast Bud6 and Aip5, I wonder would the author discuss the potential contribution of Cter in differentiating S. Pombe formins.

    The reviewer is correct that the C-terminal tail region of Cdc12 beyond the FH1-FH2 domains has a strong influence on the ability of Cdc12C to replace Fus1C. This is one reason why we specifically investigated the possible role of Fus1 C-terminal tail, which is much shorter than that of Cdc12. We found that Fus1 C-terminal tail plays only very minor role in regulating Fus1 function, as described in Figure 3. We note that contrary to what the reviewer states, Bud6 exists in S. pombe and binds the C-terminal tail of the formin For3 (see Martin et al, MBoC 2007), but whether it binds Fus1 is unknown. We have expanded our discussion to include a paragraph on the role of formin C-termini.

    Because the manuscript is focused on the function of Fus1 formin, we did not explore further the role of the Cdc12 C-terminal tail. It was previously shown that this region of Cdc12 contains an oligomerization domain that promotes actin bundling (Bohnert et al, Genes and Dev 2013). It is thus likely that this helps Cdc12 FH1-FH2 perform well in replacement of Fus1. In fact, it is likely that oligomerization boosts formin function, as we have discovered that Fus1 N-terminus contains a disordered region that fulfils exactly this function. This is described in a distinct manuscript under review elsewhere and just deposited on BioRxiv (Billault-Chaumartin et al, BioRxiv 2022; DOI: 10.1101/2022.05.05.490810). We have now cited this point in the discussion.

    1. Here, the study focuses on the FH1 between Fus1 and Cdc12 to understand their different functions in actin polymerization. FH1 mediated actin elongation through its interaction with profilin via polyP. The transfer rate of G-actin from profilin and profilin sliding depends on the polyP patterns regarding the length of each polyp motif and their distance to FH2 (Naomi Courtemanche and Thomas D. Pollard, JBC, 2012). To better understand the mechanisms by which these engineered FH1 variants on both Fus1 and Cdc12 in Fig. 4, the author may want to list the sequence of these engineered FH1 domains, including the information of the number and length of polyp motifs, and discuss these patterns.

    This list and discussion were available in the initial paper that characterized each of the constructs in vitro (Scott et al, MBoC 2011). We have now re-drawn it in a supplemental figure for convenience (as also answered in response to minor point 2), which is already provided in the revised manuscript as Figure S1. (Previous supplementary figures are re-numbered S1>S2, S2>S3 and S3>S4).

    1. Figs.4,5 cell biology results do not directly support the point of specific elongation rate unless the LifeAct-labeled actin cable elongation speed could be followed and quantified. The fluorescent tagging of tropomyosin does not show the actin cable pattern, which makes it very difficult to be used to study actin cable dynamics, such as elongation. Therefore, I feel the data in current Fig. 4 and Fig. 5 could not claim the differences in actin elongation without a quantitative comparison of elongation rate. I suggest a CK666 treatment to increase the visibility of the actin cable pattern of LifeAct, used before in both fission and budding yeasts, which would allow the author to quantify the actin cable elongation rate. Another way is to use the TIRF assay used in this study, which would give a better quantitation of formin nucleation and profilin-aided elongation.

    We respectfully disagree with the reviewer on this point. All the constructs we use in vivo have been characterized in vitro and their elongation rate carefully measured (Scott et al, MBoC 2011). These values are thus known and can be directly compared to our results in vivo.

    Of course, it would be fantastic to be able to directly measure formin elongation rates in vivo, but we are not aware that this has been done in any system. The proxy experiments that the reviewer suggests would be good ones, but each faces technical challenges that make them impossible in our system. First, because the fusion focus is a structure that forms in response to cell-cell pheromonal communication, we cannot add CK-666 or any other drug during this phase, as this perturbs the pheromone signal. Indeed, we had shown that simple buffer wash leads to loss of the fusion focus (see Dudin et al, Genes and Dev 2016). Second, the fusion focus is at the contact site between partner cells, i-e somewhat distant (1-2µm) from the coverslip during imaging. It is thus impossible to use TIRF. Finally, the fusion focus is a tightly packed actin structure. This is the reason why (rather than use of the tropomyosin marker) we cannot image single actin filaments (or even bundles) of which we could follow the dynamics as has been done to measure the retrograde flow of actin cables in yeast.

    What we have done is to use a better tropomyosin tag, mNeonGreen-Cdc8, which was just described (Hatano et al, BioRxiv 2022; DOI: 10.1101/2022.05.19.492673) to quantify amounts of linear actin. Although this is not a measure of elongation rate, it would give some sense about amounts of polymer assembled. We have obtained images with mNeonGreen-Cdc8 of all experiments previously conducted with GFP-Cdc8 and have replaced them in Figure 4C, Figure 5E, Figure 6E and Figure S2B. We have also quantified the relevant strains. The relative intensities of mNeonGreen-Cdc8 at the fusion focus at fusion time reflect remarkably well the measured elongation rates of the various formin constructs characterized in vitro. These data are now provided as new panels Figure 4F and Figure 5F.

    1. I appreciated the detailed biochemical dissections of multiple aspects of WTFus1 and Fus1R1054E, although the biochemical assays could not identify the mechanism by which R1054E causes the cell fusion. In many cases, the formin functions are diverse in diverse biological processes and sophisticated that cannot be explained well only from its biochemical activities in actin polymerization, such as the bundling, nucleation, and elongation studied in this story regarding fusion. This exciting information allows us to think of more possibilities that might regulate formin function rather than a direct change of formin activities in actin polymerization. I think a discussion of different aspects of functional regulation of formin might inspire society to investigate new possibilities to solve the mysteries. For example, the changes in formin behaviors and functions could be regulated by stress-induced formin turnover by degradation, cell signaling-regulated formin clustering and complex assembly, and their potential relevance to recruit protein constituents for fusion progression.

    We have added a paragraph on the role of Fus1 C-terminus. If you feel we should expand more on the diverse modes of regulation of formins, we could, but we have so far kept the discussion centred around the points of investigation in this paper, whose aim was to probe how changes in nucleation and elongation rates, rather than other regulations, affect the in vivo function of Fus1.

    Minor comments.

    1. There are two types of "C", one includes FH1/FH2 and one following FH2, used in the manuscript, and it is a bit confusing. Better to differentiate them that allows an easy following. Fig. 1 uses Cdc12C-deltaC, Fig. 3 uses Fus1-delta Cter.

    We have updated the nomenclature to make this clearer: the C-terminal region beyond the FH1-FH2 domains is now called Cter throughout the manuscript.

    1. It's better to specify the amino acid position on the schematic of formins, such as panel A in many figures. It's always more informative to compare formin activities by considering the domain lengths, especially for the C-terminal tail that is variable in lengths and sequences. With similar thoughts, I suggest a supplementary figure that lists the sequence of all FH1 domains variants and Cter domains, such as the FH2 domain in Fig. S1.

    We have made a supplementary figure (new Figure S1) listing all constructs with specific aa positions as well as the FH1 domain variants and their sequences (see also answer to point 2 above). We have not added the sequence of the Cter domains in this figure, as these are extremely divergent and not particularly informative at this point.

    1. "n" for the statistic needs to be provided for Fig. S3.

    We have added the information to the legend of the figure (now Fig S4).

    1. The SDS-PAGE staining gel of the purified recombinant proteins for biochemical assays should be provided, particularly for these newly reported mutant variants.

    This is now provided as new panel S4C. We show the purified recombinant Cdc122FH1-Fus1FH2 proteins, which are the newly reported ones.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)): In this study, Billaut-Chaumartin and colleagues investigate the molecular specialization of the S. pombe formin, Fus1. The authors systematically modulate the actin filament elongation and nucleation activities of Fus1 by expressing chimeric constructs that contain Formin Homology 1 and 2 domains from two other formins with known polymerization activities. By characterizing the architecture of the fusion focus and the efficiency of cell fusion, they find that both the elongation and nucleation properties of Fus1 are specifically tailored for its cellular role. Comparison of formin constructs with similar elongation and nucleation activities also reveals that the Fus1 FH2 domain possesses a specific property that promotes efficient cell fusion. Using sequence alignment and homology modeling, the authors identify R1054 as the residue that confers this novel, fusion-specific activity to Fus1, despite producing no effect on its bundling or polymerization properties in vitro.

    Overall, this study is well motivated, and the results support the conclusions that are drawn. I have only minor suggestions, as described below.

    Minor comments: (1) The schematic diagrams of the chimeric formin constructs are very helpful. However, it is difficult to distinguish the colors from one another, especially in the case of the Cdc12FH1-Fus1FH2 variant, which requires discernment of the relatively small purple region within the dark blue molecule. Would it be possible to modify the colors to increase their contrast? Similarly, the blue and gray data sets in Figure 3B are very difficult to discern.

    We have changed the colours to improve contrasts.

    (2) The affinities (Kd) with which the formins bind the barbed ends as described in the second-to-last paragraph on page 8, in Figure Legend 7G, and in the "Analysis of pyrene data" section of the Materials and Methods should be defined as dissociation "constants", rather than dissociation "rates". Also, these affinities are lacking units in the following sentence on page 8.

    We have corrected this. The unit is nM.

    (3) When comparing the TIRF micrographs in Figure S3A, it looks as though both formins (but especially the R1054E variant) nucleate more filaments in the presence of profilin than in its absence. Is this a reproducible effect? If so, can the authors provide an explanation for this?

    There is strong variability in the filament numbers observed by TIRF in replicate experiments, which makes it difficult to use this technique to determine the nucleation efficiency. This may be due for instance to the stickiness of the glass, which may influence the number of observed filaments. We have measured the number of filaments after 130s of polymerization for each condition to test whether there are any significant differences between conditions despite overall variability. The measurements suggest that the addition of profilin increases the number of actin filaments. However, these results should be taken very carefully due to the experimental variations (very large error bars). Additionally, because Fus1-associated filaments are very short in absence of profilin, it is quite likely that this influences their crowding at the glass surface compared to longer filaments (in presence of profilin). Since in TIRF we can only observe the filaments at the glass surface, we may miss a portion of short Fus1-bound actin filaments in absence of profilin.

    For these reasons, and because the possible role of profilin in modulating nucleation efficiency by formins is not the object of the work here, would thus prefer not to include this graph in the manuscript.

    Reviewer #2 (Significance (Required)): This study contributes a key advancement towards understanding how the polymerization activities of formins are tailored to support diverse and specific cellular functions. The results in this study nicely complement and expand upon similar recent work that dissected the polymerization requirements of the formin Cdc12, which mediates cytokinetic ring assembly in S. pombe, and For2, which drives the assembly of apical networks that are necessary for polarized growth in Physcomitrella patens. As such, this work will likely be of significant interest to scientists who study mechanisms of actin dynamics regulation. The identification of R1054 as a residue that confers a novel regulatory activity to the FH2 domain of Fus1 will also likely be of great interest to biochemists and other scientists who study formins at the molecular level.

    My expertise is in the field of formins and actin polymerization.

    Read the original source
    Was this evaluation helpful?
  2. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons


    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    In this study, Billaut-Chaumartin and colleagues investigate the molecular specialization of the S. pombe formin, Fus1. The authors systematically modulate the actin filament elongation and nucleation activities of Fus1 by expressing chimeric constructs that contain Formin Homology 1 and 2 domains from two other formins with known polymerization activities. By characterizing the architecture of the fusion focus and the efficiency of cell fusion, they find that both the elongation and nucleation properties of Fus1 are specifically tailored for its cellular role. Comparison of formin constructs with similar elongation and nucleation activities also reveals that the Fus1 FH2 domain possesses a specific property that promotes efficient cell fusion. Using sequence alignment and homology modeling, the authors identify R1054 as the residue that confers this novel, fusion-specific activity to Fus1, despite producing no effect on its bundling or polymerization properties in vitro.

    Overall, this study is well motivated, and the results support the conclusions that are drawn. I have only minor suggestions, as described below.

    Minor comments:

    1. The schematic diagrams of the chimeric formin constructs are very helpful. However, it is difficult to distinguish the colors from one another, especially in the case of the Cdc12FH1-Fus1FH2 variant, which requires discernment of the relatively small purple region within the dark blue molecule. Would it be possible to modify the colors to increase their contrast? Similarly, the blue and gray data sets in Figure 3B are very difficult to discern.
    2. The affinities (Kd) with which the formins bind the barbed ends as described in the second-to-last paragraph on page 8, in Figure Legend 7G, and in the "Analysis of pyrene data" section of the Materials and Methods should be defined as dissociation "constants", rather than dissociation "rates". Also, these affinities are lacking units in the following sentence on page 8.
    3. When comparing the TIRF micrographs in Figure S3A, it looks as though both formins (but especially the R1054E variant) nucleate more filaments in the presence of profilin than in its absence. Is this a reproducible effect? If so, can the authors provide an explanation for this?

    Significance

    This study contributes a key advancement towards understanding how the polymerization activities of formins are tailored to support diverse and specific cellular functions. The results in this study nicely complement and expand upon similar recent work that dissected the polymerization requirements of the formin Cdc12, which mediates cytokinetic ring assembly in S. pombe, and For2, which drives the assembly of apical networks that are necessary for polarized growth in Physcomitrella patens. As such, this work will likely be of significant interest to scientists who study mechanisms of actin dynamics regulation. The identification of R1054 as a residue that confers a novel regulatory activity to the FH2 domain of Fus1 will also likely be of great interest to biochemists and other scientists who study formins at the molecular level.

    My expertise is in the field of formins and actin polymerization.

    Read the original source
    Was this evaluation helpful?
  3. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons


    Referee #1

    Evidence, reproducibility and clarity

    Excellent quality of cell biology and biochemistry. the additional supports are needed for the claim of actin elongation using different formin variants.

    Significance

    Ingrid Billault-Chaumartin and co-authors described interesting research that provides insights on formin-isoform specific function in fission yeast and a new role of Fus1 FH2 domain in cell-cell fusion event. While three formin isoforms have different localization, research proposed an additional dissection in their functional differences by having different functions in C-terminus, including FH1 FH2 and formin C-terminus. The work also described additional factors that regulate cell fusions from autotrophy effect and formin expression level, in addition to the well-accepted formin biochemical activities. Here are my comments regarding the strengths of the work and improvements that could further strengthen the story.

    Major comments

    1. Fig.1 shows Cdc12C could recapitulate Fus1 function by ~80% if fused with Fus1C, whereas deletion of the C-terminal tail of Cdc12 following FH2 introduces drastic dysfunction. Together with Fig. 3, these results indicate Cdc12 Cter plays more important roles than Fus1 Cter for there respective functions. Such results suggested a Cter-mediated mechanism that differentiates the functions of three fission yeast formin isoforms. The authors examined contributions from the difference in FH1 (Figs 4,5) and FH2 residues (Fig. 6). Whereas the obvious phenotype of Cter was not further investigated and not much discussed. The Cter of budding yeast formins interacts with nucleation-promoting factors, Bud6 and Aip5. Although S. Pombe does not have orthologs of budding yeast Bud6 and Aip5, I wonder would the author discuss the potential contribution of Cter in differentiating S. Pombe formins.
    2. Here, the study focuses on the FH1 between Fus1 and Cdc12 to understand their different functions in actin polymerization. FH1 mediated actin elongation through its interaction with profilin via polyP. The transfer rate of G-actin from profilin and profilin sliding depends on the polyP patterns regarding the length of each polyp motif and their distance to FH2 (Naomi Courtemanche and Thomas D. Pollard, JBC, 2012). To better understand the mechanisms by which these engineered FH1 variants on both Fus1 and Cdc12 in Fig. 4, the author may want to list the sequence of these engineered FH1 domains, including the information of the number and length of polyp motifs, and discuss these patterns.
    3. Figs.4,5 cell biology results do not directly support the point of specific elongation rate unless the LifeAct-labeled actin cable elongation speed could be followed and quantified. The fluorescent tagging of tropomyosin does not show the actin cable pattern, which makes it very difficult to be used to study actin cable dynamics, such as elongation. Therefore, I feel the data in current Fig. 4 and Fig. 5 could not claim the differences in actin elongation without a quantitative comparison of elongation rate. I suggest a CK666 treatment to increase the visibility of the actin cable pattern of LifeAct, used before in both fission and budding yeasts, which would allow the author to quantify the actin cable elongation rate. Another way is to use the TIRF assay used in this study, which would give a better quantitation of formin nucleation and profilin-aided elongation.
    4. I appreciated the detailed biochemical dissections of multiple aspects of WTFus1 and Fus1R1054E, although the biochemical assays could not identify the mechanism by which R1054E causes the cell fusion. In many cases, the formin functions are diverse in diverse biological processes and sophisticated that cannot be explained well only from its biochemical activities in actin polymerization, such as the bundling, nucleation, and elongation studied in this story regarding fusion. This exciting information allows us to think of more possibilities that might regulate formin function rather than a direct change of formin activities in actin polymerization. I think a discussion of different aspects of functional regulation of formin might inspire society to investigate new possibilities to solve the mysteries. For example, the changes in formin behaviors and functions could be regulated by stress-induced formin turnover by degradation, cell signaling-regulated formin clustering and complex assembly, and their potential relevance to recruit protein constituents for fusion progression.

    Minor comments.

    1. There are two types of "C", one includes FH1/FH2 and one following FH2, used in the manuscript, and it is a bit confusing. Better to differentiate them that allows an easy following. Fig. 1 uses Cdc12C-deltaC, Fig. 3 uses Fus1-delta Cter.
    2. It's better to specify the amino acid position on the schematic of formins, such as panel A in many figures. It's always more informative to compare formin activities by considering the domain lengths, especially for the C-terminal tail that is variable in lengths and sequences. With similar thoughts, I suggest a supplementary figure that lists the sequence of all FH1 domains variants and Cter domains, such as the FH2 domain in Fig. S1.
    3. "n" for the statistic needs to be provided for Fig. S3.
    4. The SDS-PAGE staining gel of the purified recombinant proteins for biochemical assays should be provided, particularly for these newly reported mutant variants.
    Read the original source
    Was this evaluation helpful?