High resolution mapping of the actin fusion focus reveals myosin V-dependent transport of formin for actin aster compaction

  1. Valentine Thomas
  2. Hikari Mase
  3. Laetitia Michon
  4. Andrea Picco
  5. Marko Kaksonen
  6. Sophie G Martin

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Abstract

Many cellular processes such as polarized growth and secretion require the formation of specific actin networks. In fungi, the morphogenetic process of cell-cell fusion requires cell wall digestion mediated by the local secretion of lytic enzymes at the site of cell-cell contact. In Schizosaccharomyces pombe , lytic enzyme-containing secretory vesicles are transported by the myosin V Myo52 on the actin fusion focus, an aster-like actin network assembled by the condensate-forming formin Fus1. The fusion focus also concentrates proteins regulating cell polarity, cell-cell communication, actin cytoskeleton, exocytosis and membrane merging, but their precise position from the time of focus formation to cell fusion is unknown. Here, using centroid tracking and averaging, we present a first spatiotemporal map of the fusion site with a precision of 8 nm. We show that the bulk of secretory vesicles remains at constant distance from the plasma membrane as the actin structure condenses. Notably, though necessary to transport vesicles, Myo52 detaches from the vesicle pool and colocalizes with Fus1 in a more membrane-proximal position. We show that Myo52 physically interacts with Fus1 and transports it along actin filaments, and that Myo52 and Fus1 actin assembly activity contribute to focus compaction. Thus, myosin V-driven transport of the formin Fus1 along actin filaments nucleated by other Fus1 molecules underlies a positive feedback mechanism for actin aster formation.

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    Reply to the reviewers

    We thank the reviewers for their careful assessment and enthusiastic appreciation of our work.

    __Reviewer #1 (Evidence, reproducibility and clarity (Required)): __In this article, Thomas et al. use a super-resolution approach in living cells to track proteins involved in the fusion event of sexual reproduction. They study the spatial organization and dynamics of the actin fusion focus, a key structure in cell-cell fusion in Schizosaccharomyces pombe. The researchers have adapted a high-precision centroid mapping method using three-color live-cell epifluorescence imaging to map the dynamic architecture of the fusion focus during yeast mating. The approach relies on tracking the centroid of fluorescence signals for proteins of interest, spatially referenced to Myo52-mScarlet-I (as a robust marker) and temporally referenced using a weakly fluorescent cytosolic protein (mRaspberry), which redistributes strongly upon fusion. The trajectories of five key proteins, including markers of polarity, cytoskeleton, exocytosis and membrane fusion, were compared to Myo52 over a 75-minute window spanning fusion. Their observations indicate that secretory vesicles maintain a constant distance from the plasma membrane whereas the actin network compacts. Most importantly, they discovered a positive feedback mechanism in which myosin V (Myo52) transports Fus1 formin along pre-existing actin filaments, thereby enhancing aster compaction.

    This article is well written, the arguments are convincing and the assertions are balanced. The centroid tracking method has been clearly and solidly controlled. Overall, this is a solid addition to our understanding of cytoskeletal organization in cell fusion.

    Major comments: No major comment.

    Minor comments: _ Page 8 authors wrote "Upon depletion of Myo52, Ypt3 did not accumulate at the fusion focus (Figure 3C). A thin, wide localization at the fusion site was occasionally observed (Figure 3C, Movies S3)" : Is there a quantification of this accumulation in the mutant?

    We will provide the requested quantification. The localization is very faint, so we are not sure that quantification will capture this faithfully, but we will try.

    _ The framerate of movies could be improved for reader comfort: For example, movie S6 lasts 0.5 sec.

    We agree that movies S3 and S6 frame rates could be improved. We will provide them with slower frame rate.

    Reviewer #1 (Significance (Required)):

    This study represents a conceptual and technical breakthrough in our understanding of cytoskeletal organization during cell-cell fusion. The authors introduce a high-precision, three-color live-cell centroid mapping method capable of resolving the spatio-temporal dynamics of protein complexes at the nanometer scale in living yeast cells. This methodological innovation enables systematic and quantitative mapping of the dynamic architecture of proteins at the cell fusion site, making it a powerful live-cell imaging approach. However, it is important to keep in mind that the increased precision achieved through averaging comes at the expense of overlooking atypical or outlier behaviors. The authors discovered a myosin V-dependent mechanism for the recruitment of formin that leads to actin aster compaction. The identification of Myo52 (myosin V) as a transporter of Fus1 (formin) to the fusion focus adds a new layer to our understanding of how polarized actin structures are generated and maintained during developmentally regulated processes such as mating.

    Previous studies have shown the importance of formins and myosins during fusion, but this paper provides a quantitative and dynamic mapping that demonstrates how Myo52 modulates Fus1 positioning in living cells. This provides a better understanding of actin organization, beyond what has been demonstrated by fixed-cell imaging or genetic perturbation.

    Audience: Cell biologists working on actin dynamics, cell-cell fusion and intracellular transport. Scientists involved in live-cell imaging, single particle tracking and cytoskeleton modeling.

    I have expertise in live-cell microscopy, image analysis, fungal growth machinery and actin organization.

    We thank the reviewer for their appreciation of our work.

    __Reviewer #2 (Evidence, reproducibility and clarity (Required)): __ A three-color imaging approach to use centroid tracking is employed to determine the high resolution position over time of tagged actin fusion focus proteins during mating in fission yeast. In particular, the position of different protein components (tagged in a 3rd color) were determined in relation to the position (and axis) of the molecular motor Myo52, which is tagged with two different colors in the mating cells. Furthermore, time is normalized by the rapid diffusion of a weak fluorescent protein probe (mRaspberry) from one cell to the other upon fusion pore opening. From this approach multiple important mechanistic insights were determined for the compaction of fusion focus proteins during mating, including the general compaction of different components as fusion proceeds with different proteins having specific stereotypical behaviors that indicate underlying molecular insights. For example, secretory vesicles remain a constant distance from the plasma membrane, whereas the formin Fus1 rapidly accumulates at the fusion focus in a Myo52-dependent manner.

    I have minor suggestions/points: (1) Figure 1, for clarity it would be helpful if the cells shown in B were in the same orientation as the cartoon cells shown in A. Similarly, it would be helpful to have the orientation shown in D the same as the data that is subsequently presented in the rest of the manuscript (such as Figure 2) where time is on the X axis and distance (position) is on the Y axis.

    We have turned each image in panel B by 180° to match the cartoon in A. For panel D, we are not sure what the reviewer would like. This panel shows the coordinates of each Myo52 position, whereas Figure 2 shows oriented distance (on the Y axis) over time (on the X axis). Perhaps the reviewer suggests that we should display panel D with a rotation onto the Y axis rather than the X axis. We feel that this would not bring more clarity and prefer to keep it as is.

    (2) Figure 2, for clarity useful to introduce how the position of Myo52 changes over time with respect to the fusion site (plasma membrane) earlier, and then come back to the positions of different proteins with respect to Myo52 shown in 2E. Currently the authors discuss this point after introducing Figure 2E, but better for the reader to have this in mind beforehand.

    We have added a sentence at the start of the section describing Figure 2, pointing out that the static appearance of Myo52 is due to it being used as reference, but that in reality, it moves relative to the plasma membrane: “Because Myo52 is the reference, its trace is flat, even though in reality Myo52 also moves relative to other proteins and the plasma membrane (see Figure 2E)”. This change is already in the text.

    (3) First sentence of page 8 "..., peaked at fusion time and sharply dropped post-fusion (Figure S3)." Figure S3 should be cited so that the reader knows where this data is presented.

    Thanks, we have added the missing figure reference to the text.

    (4) Figure 3D-H, why is Exo70 used as a marker for vesicles instead of Ypt3 for these experiments? Exo70 seems to have a more confusing localization than Ypt3 (3C vs 3D), which seems to complicate interpretations.

    There are two main reasons for this choice. First, the GFP-Ypt3 fluorescence intensity is lower than that of Exo70-GFP, which makes analysis more difficult and less reliable. Second, in contrast to Exo70-GFP where the endogenous gene is tagged at the native genomic locus, GFP-Ypt3 is expressed as additional copy in addition to endogenous untagged Ypt3. Although GFP-Ypt3 was reported to be fully functional as it can complement the lethality of a ypt3 temperature sensitive mutant (Cheng et al, MBoC 2002), its expression levels are non-native and we do not have a strain in which ypt3 is tagged at the 5’ end at the native genomic locus. For these reasons, we preferred to examine in detail the localization of Exo70. We do not think it complicates interpretations. Exo70 faithfully decorates vesicles and exhibits the same localization as Ypt3 in WT cells (see Figure 2D) and in myo52-AID (see Figure 3C-D). We realize that our text was a bit confusing as we opposed the localization of Exo70 and Ypt3, when all we wanted to state was that the Exo70-GFP signal is stronger. We have corrected this in the text.

    (5) Page 10, end of first paragraph, "We conclude...and promotes separation of Myo52 from the vesicles." This is an interesting hypothesis/interpretation that is consistent with the spatial-temporal organization of vesicles and the compacting fusion focus, but the underlying molecular mechanism has not be concluded.

    This is an interpretation that is in line with our data. Firm conclusion that the organization of the actin fusion focus imposes a steric barrier to bulk vesicle entry will require in vitro reconstitution of an actin aster driven by formin-myosin V feedback and addition of myosin V vesicle-like cargo, which can be a target for future studies. To make clear that it is an interpretation and not a definitive statement, we have added “likely” to the sentence, as in: “We conclude that the distal position of vesicles in WT cells is a likely steric consequence of the architecture of the fusion focus, which restricts space at the center of the actin aster and promotes separation of Myo52 from the vesicles”.

    (6) Figure 5F and 5G, the results are confusing and should be discussed further. Depletion of Myo52 decreases Fus1 long-range movements, indicating that Fus1 is being transported by Myo52 (5F). Similarly, the Fus1 actin assembly mutant greatly decreases Fus1 long-range movements and prevents Myo52 binding (5G), perhaps indicating that Fus1-mediated actin assembly is important. It seems the author's interpretations are oversimplified.

    We show that Myo52 is critical for Fus1 long-range movements, as stated by the reviewer. We also show that Fus1-mediated actin assembly is important. The question is in what way.

    One possibility is that FH2-mediated actin assembly powers the movement, which in this case represents the displacement of the formin due to actin monomer addition on the polymerizing filament. A second possibility is that actin filaments assembled by Fus1 somehow help Myo52 move Fus1. This could be for instance because Fus1-assembled actin filaments are preferred tracks for Myo52-mediated movements, or because they allow Myo52 to accumulate in the vicinity of Fus1, enhancing their chance encounter and thus the number of long-range movements (on any actin track). Based on the analysis of the K1112A point mutant in Fus1 FH2 domain, our data cannot discriminate between these three different options, which is why we concluded that the mutant allele does not allow us to make a firm conclusion. However, the Myo52-dependence clearly shows that a large fraction of the movements requires the myosin V. We have clarified the end of the paragraph in the following way: “Therefore, analysis of the K1112A mutant phenotype does not allow us to clearly distinguish between Fus1-powered from Myo52-powered movements. Future work will be required to test whether, in addition to myosin V-dependent transport, Fus1-mediated actin polymerization also directly contributes to Fus1 long-range movements.”

    (7) Figure 6, why not measure the fluorescence intensity of Fus1 as a proxy for the number of Fus1 molecules (rather than the width of the Fus1 signal), which seems to be the more straight-forward analysis?

    The aim of the measurement was to test whether Myo52 and Fus1 activity help focalize the formin at the fusion site, not whether these are required for localization in this region. This is why we are measuring the lateral spread of the signal (its width) rather than the fluorescence intensity of the signal. We know from previous work that Fus1 localizes to the shmoo tip independently of myosin V (Dudin et al, JCB 2015), and we also show this in Figure 6. However, the precise distribution of Fus1 is wider in absence of the myosins.

    We can and will measure intensities to test whether there is also a quantitative difference in the number of molecules at the shmoo tip.

    (8) Figure 7, the authors should note (and perhaps discuss) any evidence as to whether activation of Fus1 to facilitate actin assembly depends upon Fus1 dissociating from Myo52 or whether Fus1 can be activated while still associated with Myo52, as both circumstances are included in the figure.

    This is an interesting point. We have no experimental evidence for or against Fus1 dissociating from Myo52 to assemble actin. However, it is known that formins rotate along the actin filament double helix as they assemble it, a movement that seems poorly compatible with processive transport by myosin V. In Figure 7, we do not particularly want to imply that Myo52 associates with Fus1 linked or not with an actin filament. The figure serves to illustrate the focusing mechanism of myosin V transporting a formin, which is more evident when we draw the formin attached to a filament end. We have now added a sentence in the figure legend to clarify this point: “Note that it is unknown whether Myo52 transports Fus1 associated or not with an actin filament.”

    (9) Figure 7, the color of secretory vesicles should be the same in A and B.

    This is now corrected.

    Reviewer #2 (Significance (Required)):

    This is an impactful and high quality manuscript that describes an elegant experimental strategy with important insights determined. The experimental imaging strategy (and analysis), as well as the insight into the pombe mating fusion focus and its comparison to other cytoskeletal compaction events will be of broad scientific interest.

    We thank the reviewer for their appreciation of our work.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Summary:

    Fission yeast cell-cell fusion during mating is mediated by an actin-based structure called the 'fusion focus', which orchestrates actin polymerization by the mating-specific formin, Fus1, to direct polarized secretion towards the mating site. In the current study, Thomas and colleagues quantitatively map the spatial distribution of proteins mediating cell-cell fusion using a three-color fluorescence imaging methodology in the fission yeast Schizosaccharomyces pombe. Using Myo52 (Type V myosin) as a fluorescence reference point, the authors discover that proteins known to localize to the fusion focus have distinct spatial distributions and accumulation profiles at the mating site. Myo52 and Fus1 form a complex in vivo detected by co-immunoprecipitation and each contribute to directing secretory vesicles to the fusion focus. Previous work from this group has shown that the intrinsically disordered region (IDR) of Fus1 plays a critical role in forming the fusion focus. Here, the authors swap out the IDR of fission yeast Fus1 for the IDR of an unrelated mammalian protein, coincidentally called 'fused in sarcoma' (FUS). They express the Fus1∆IDR-FUSLC-27R chimera in mitotically dividing fission yeast cells, where Fus1 is not normally expressed, and discover that the Fus1∆IDR-FUSLC-27R chimera can travel with Myo52 on actively polymerizing actin cables. Additionally, they show that acute loss of Myo52 or Fus1 function, using Auxin-Inducible Degradation (AID) tags and point mutations, impair the normal compaction of the fusion focus, suggesting that direct interaction and coordination of Fus1 and Myo52 helps shape this structure.

    Major Comments:

    (1) In the Results section for Figure 2, the authors claim that actin filaments become shorter and more cross-linked they move away from the fusion site during mating, and suggest that this may be due to the presence of Myo51. However, the evidence to support this claim is not made clear. Is it supported by high-resolution electron microscopy of the actin filaments, or some other results? This needs to be clarified.

    Sorry if our text was unclear. The basis for the claim that actin filaments become shorter comes from our observation that the average position of tropomyosin and Myo51, both of which decorate actin filaments, is progressively closer to both Fus1 and the plasma membrane. Thus, the actin structure protrudes less into the cytosol as fusion progresses. The basis for claiming that Myo51 promotes actin filament crosslinking comes mainly from previously published papers, which had shown that 1) Myo51 forms complexes with the Rng8 and Rng9 proteins (Wang et al, JCB 2014), and 2) the Myo51-Rng8/9 not only binds actin through Myo51 head domain but also binds tropomyosin-decorated actin through the Rng8/9 moiety (Tang et al, JCB 2016; reference 27 in our manuscript). We had also previously shown that these proteins are necessary for compaction of the fusion focus (Dudin et al, PLoS Genetics 2017; reference 28 in our manuscript). Except for measuring the width of Fus1 distribution in myo51∆ mutants, which confirms previous findings, we did not re-investigate here the function of Myo51.

    We have now re-written this paragraph to present the previous data more clearly: “The distal localization of Myo51 was mirrored by that of tropomyosin Cdc8, which decorates linear actin filaments (Figure 2B) (Hatano et al, 2022). The distal position of the bulk of Myo51-decorated actin filaments was confirmed using Airyscan super-resolution microscopy (Figure 2B, right). Thus, the average position of actin filaments and decreasing distance to Myo52 indicates they initially extend a few hundred nanometers into the cytosol and become progressively shorter as fusion proceeds. Previous work had shown that Myo51 cross-links and slides Cdc8-decorated actin filaments relative to each other (Tang et al, 2016) and that both proteins contribute to compaction of the fusion focus in the lateral dimension along the cell-cell contact area (perpendicular to the fusion axis) (Dudin et al, 2017). We confirmed this function by measuring the lateral distribution of Fus1 along the cell-cell contact area (perpendicular to the fusion axis), which was indeed wider in myo51∆ than WT cells (see below Figure 6A-B).”

    (2) In Figure 4, the authors comment that disrupting Fus1 results in more disperse Myo52 spatial distribution at the fusion focus, raising the possibility that Myo52 normally becomes focused by moving on the actin filaments assembled by Fus1. This can be tested by asking whether latrunculin treatment phenocopies the 'more dispersed' Myo52 localization seen in fus1∆ cells? If Myo52 is focused instead by its direct interaction with Fus1, the latrunculin treatment should not cause the same phenotype.

    This is in principle a good idea, though it is technically challenging because pharmacological treatment of cell pairs in fusion is difficult to do without disturbing pheromone gradients which are critical throughout the fusion process (see Dudin et al, Genes and Dev 2016). We will try the experiment but are unsure about the likelihood of technical success.

    We note however that a similar experiment was done previously on Fus1 overexpressed in mitotic cells (Billault-Chaumartin et al, Curr Biol 2022; Fig 1D). Here, Fus1 also forms a focus and latrunculin A treatment leads to Myo52 dispersion while keeping the Fus1 focus, which is in line with our proposal that Myo52 becomes focused by moving on Fus1-assembled actin filaments. Similarly, we showed in Figure 5B that Latrunculin A treatment of mitotic cells expressing Fus1∆IDR-FUSLC-27R also results in Myo52, but not Fus1 dispersion.

    (3) The Fus1∆IDR-FUSLC-27R chimera used in Figure 5 is an interesting construct to examine actin-based transport of formins in cells. I was curious if the authors could provide the rates of movement for Myo52 and for Fus1∆IDR-FUSLC-27R, both before and after acute depletion of Myo52. It would be interesting to see if loss of Myo52 alters the rate of movement, or instead the movement stems from formin-mediated actin polymerization.

    We will measure these rates.

    (4) Also, Myo52 is known to interact with the mitotic formin For3. Does For3 colocalize with Myo52 and Fus1∆IDR-FUSLC-27R along actin cables?

    This is an interesting question for which we do not have an answer. For technical reasons, we do not have the tools to co-image For3 with Fus1∆IDR-FUSLC-27R because both are tagged with GFP. We feel that this question goes beyond the scope of this paper.

    (5) If Fus1∆IDR-FUSLC-27R is active, does having ectopic formin activity in mitotic cells affect actin cable architecture? This could be assessed by comparing phalloidin staining for wildtype and Fus1∆IDR-FUSLC-27R cells.

    We are not sure what the purpose of this experiment is, or how informative it would be. If it is to evaluate whether Fus1∆IDR-FUSLC-27R is active, our current data already demonstrates this. Indeed, Fus1∆IDR-FUSLC-27R recruits Myo52 in a F-actin and FH2 domain-dependent manner (shown in Figure 5B and 5G), which demonstrates that Fus1∆IDR-FUSLC-27R FH2 domain is active. Even though Fus1∆IDR-FUSLC-27R assembles actin, we predict that its effect on general actin organization will be weak. Indeed, it is expressed under endogenous fus1 promoter, leading to very low expression levels during mitotic growth, such that only a subset of cells exhibit a Fus1 focus. Furthermore, most of these Fus1 foci are at or close to cell poles, where linear actin cables are assembled by For3, such that they may not have a strong disturbing effect. Because analysis of actin cable organization by phalloidin staining is difficult (due to the more strongly staining actin patches), cells with clear change in organization predicted to be rare in the population, and the gain in knowledge not transformative, we are not keen to do this experiment.

    Minor Comments:

    Prior studies are referenced appropriately. Text and figures are clear and accurate. My only suggestion would be Figure 1E-H could be moved to the supplemental material, due to their extremely technical nature. I believe this would help the broad audience focus on the experimental design mapped out in Figure 1A-D.

    We are relatively neutral about this. If this suggestion is supported by the Editor, we can move these panels to supplement.

    Reviewer #3 (Significance (Required)):

    Significance: This study provides an improved imaging method for detecting the spatial distributions of proteins below 100 nm, providing new insights about how a relatively small cellular structure is organized. The use of three-color cell imaging to accurately measure accumulation rates of molecular components of the fusion focus provides new insight into the development of this structure and its roles in mating. This method could be applied to other multi-protein structures found in different cell types. This work uses rigorously genetic tools such as knockout, knockdown and point mutants to dissect the roles of the formin Fus1 and Type V myosin Myo52 in creating a proper fusion focus. The study could be improved by biochemical assays to test whether Myo52 and Fus1 directly interact, since the interaction is only shown by co-immunoprecipitation from extracts, which may reflect an indirect interaction.

    Indeed, future studies should dissect the Fus1-Myo52 interaction, to determine whether it is direct and identify mutants that impair it.

    I believe this work advances the cell-mating field by providing others with a spatial and temporal map of conserved factors arriving to the mating site. Additionally, they identified a way to study a mating specific protein in mitotically dividing cells, offering future questions to address.

    This study should appeal to a range of basic scientists interested in cell biology, the cytoskeleton, and model organisms. The three-colored quantitative imaging could be applied to defining the architecture of many other cellular structures in different systems. Myosin and actin scientists will be interested in how this work expands the interplay of these two fields.

    I am a cell biologist with expertise in live cell imaging, genetics and biochemistry.

    We thank the reviewer for their appreciation of our work.

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    Fission yeast cell-cell fusion during mating is mediated by an actin-based structure called the 'fusion focus', which orchestrates actin polymerization by the mating-specific formin, Fus1, to direct polarized secretion towards the mating site. In the current study, Thomas and colleagues quantitatively map the spatial distribution of proteins mediating cell-cell fusion using a three-color fluorescence imaging methodology in the fission yeast Schizosaccharomyces pombe. Using Myo52 (Type V myosin) as a fluorescence reference point, the authors discover that proteins known to localize to the fusion focus have distinct spatial distributions and accumulation profiles at the mating site. Myo52 and Fus1 form a complex in vivo detected by co-immunoprecipitation and each contribute to directing secretory vesicles to the fusion focus. Previous work from this group has shown that the intrinsically disordered region (IDR) of Fus1 plays a critical role in forming the fusion focus. Here, the authors swap out the IDR of fission yeast Fus1 for the IDR of an unrelated mammalian protein, coincidentally called 'fused in sarcoma' (FUS). They express the Fus1∆IDR-FUSLC-27R chimera in mitotically dividing fission yeast cells, where Fus1 is not normally expressed, and discover that the Fus1∆IDR-FUSLC-27R chimera can travel with Myo52 on actively polymerizing actin cables. Additionally, they show that acute loss of Myo52 or Fus1 function, using Auxin-Inducible Degradation (AID) tags and point mutations, impair the normal compaction of the fusion focus, suggesting that direct interaction and coordination of Fus1 and Myo52 helps shape this structure.

    Major Comments:

    • In the Results section for Figure 2, the authors claim that actin filaments become shorter and more cross-linked they move away from the fusion site during mating, and suggest that this may be due to the presence of Myo51. However, the evidence to support this claim is not made clear. Is it supported by high-resolution electron microscopy of the actin filaments, or some other results? This needs to be clarified.

    • In Figure 4, the authors comment that disrupting Fus1 results in more disperse Myo52 spatial distribution at the fusion focus, raising the possibility that Myo52 normally becomes focused by moving on the actin filaments assembled by Fus1. This can be tested by asking whether latrunculin treatment phenocopies the 'more dispersed' Myo52 localization seen in fus1∆ cells? If Myo52 is focused instead by its direct interaction with Fus1, the latrunculin treatment should not cause the same phenotype.

    • The Fus1∆IDR-FUSLC-27R chimera used in Figure 5 is an interesting construct to examine actin-based transport of formins in cells. I was curious if the authors could provide the rates of movement for Myo52 and for Fus1∆IDR-FUSLC-27R, both before and after acute depletion of Myo52. It would be interesting to see if loss of Myo52 alters the rate of movement, or instead the movement stems from formin-mediated actin polymerization.

    • Also, Myo52 is known to interact with the mitotic formin For3. Does For3 colocalize with Myo52 and Fus1∆IDR-FUSLC-27R along actin cables?

    • If Fus1∆IDR-FUSLC-27R is active, does having ectopic formin activity in mitotic cells affect actin cable architecture? This could be assessed by comparing phalloidin staining for wildtype and Fus1∆IDR-FUSLC-27R cells.

    Minor Comments:

    • Prior studies are referenced appropriately.

    • Text and figures are clear and accurate. My only suggestion would be Figure 1E-H could be moved to the supplemental material, due to their extremely technical nature. I believe this would help the broad audience focus on the experimental design mapped out in Figure 1A-D.

    Significance

    Significance: This study provides an improved imaging method for detecting the spatial distributions of proteins below 100 nm, providing new insights about how a relatively small cellular structure is organized. The use of three-color cell imaging to accurately measure accumulation rates of molecular components of the fusion focus provides new insight into the development of this structure and its roles in mating. This method could be applied to other multi-protein structures found in different cell types. This work uses rigorously genetic tools such as knockout, knockdown and point mutants to dissect the roles of the formin Fus1 and Type V myosin Myo52 in creating a proper fusion focus. The study could be improved by biochemical assays to test whether Myo52 and Fus1 directly interact, since the interaction is only shown by co-immunoprecipitation from extracts, which may reflect an indirect interaction.

    I believe this work advances the cell-mating field by providing others with a spatial and temporal map of conserved factors arriving to the mating site. Additionally, they identified a way to study a mating specific protein in mitotically dividing cells, offering future questions to address.

    This study should appeal to a range of basic scientists interested in cell biology, the cytoskeleton, and model organisms. The three-colored quantitative imaging could be applied to defining the architecture of many other cellular structures in different systems. Myosin and actin scientists will be interested in how this work expands the interplay of these two fields.

    I am a cell biologist with expertise in live cell imaging, genetics and biochemistry.

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    A three-color imaging approach to use centroid tracking is employed to determine the high resolution position over time of tagged actin fusion focus proteins during mating in fission yeast. In particular, the position of different protein components (tagged in a 3rd color) were determined in relation to the position (and axis) of the molecular motor Myo52, which is tagged with two different colors in the mating cells. Furthermore, time is normalized by the rapid diffusion of a weak fluorescent protein probe (mRaspberry) from one cell to the other upon fusion pore opening. From this approach multiple important mechanistic insights were determined for the compaction of fusion focus proteins during mating, including the general compaction of different components as fusion proceeds with different proteins having specific stereotypical behaviors that indicate underlying molecular insights. For example, secretory vesicles remain a constant distance from the plasma membrane, whereas the formin Fus1 rapidly accumulates at the fusion focus in a Myo52-dependent manner.

    I have minor suggestions/points:

    (1) Figure 1, for clarity it would be helpful if the cells shown in B were in the same orientation as the cartoon cells shown in A. Similarly, it would be helpful to have the orientation shown in D the same as the data that is subsequently presented in the rest of the manuscript (such as Figure 2) where time is on the X axis and distance (position) is on the Y axis.

    (2) Figure 2, for clarity useful to introduce how the position of Myo52 changes over time with respect to the fusion site (plasma membrane) earlier, and then come back to the positions of different proteins with respect to Myo52 shown in 2E. Currently the authors discuss this point after introducing Figure 2E, but better for the reader to have this in mind beforehand.

    (3) First sentence of page 8 "..., peaked at fusion time and sharply dropped post-fusion (Figure S3)." Figure S3 should be cited so that the reader knows where this data is presented.

    (4) Figure 3D-H, why is Exo70 used as a marker for vesicles instead of Ypt3 for these experiments? Exo70 seems to have a more confusing localization than Ypt3 (3C vs 3D), which seems to complicate interpretations.

    (5) Page 10, end of first paragraph, "We conclude...and promotes separation of Myo52 from the vesicles." This is an interesting hypothesis/interpretation that is consistent with the spatial-temporal organization of vesicles and the compacting fusion focus, but the underlying molecular mechanism has not be concluded.

    (6) Figure 5F and 5G, the results are confusing and should be discussed further. Depletion of Myo52 decreases Fus1 long-range movements, indicating that Fus1 is being transported by Myo52 (5F). Similarly, the Fus1 actin assembly mutant greatly decreases Fus1 long-range movements and prevents Myo52 binding (5G), perhaps indicating that Fus1-mediated actin assembly is important. It seems the author's interpretations are oversimplified.

    (7) Figure 6, why not measure the fluorescence intensity of Fus1 as a proxy for the number of Fus1 molecules (rather than the width of the Fus1 signal), which seems to be the more straight-forward analysis?

    (8) Figure 7, the authors should note (and perhaps discuss) any evidence as to whether activation of Fus1 to facilitate actin assembly depends upon Fus1 dissociating from Myo52 or whether Fus1 can be activated while still associated with Myo52, as both circumstances are included in the figure.

    (9) Figure 7, the color of secretory vesicles should be the same in A and B.

    Significance

    This is an impactful and high quality manuscript that describes an elegant experimental strategy with important insights determined. The experimental imaging strategy (and analysis), as well as the insight into the pombe mating fusion focus and its comparison to other cytoskeletal compaction events will be of broad scientific nterest.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    • In this article, Thomas et al. use a super-resolution approach in living cells to track proteins involved in the fusion event of sexual reproduction. They study the spatial organization and dynamics of the actin fusion focus, a key structure in cell-cell fusion in Schizosaccharomyces pombe. The researchers have adapted a high-precision centroid mapping method using three-color live-cell epifluorescence imaging to map the dynamic architecture of the fusion focus during yeast mating. The approach relies on tracking the centroid of fluorescence signals for proteins of interest, spatially referenced to Myo52-mScarlet-I (as a robust marker) and temporally referenced using a weakly fluorescent cytosolic protein (mRaspberry), which redistributes strongly upon fusion. The trajectories of five key proteins, including markers of polarity, cytoskeleton, exocytosis and membrane fusion, were compared to Myo52 over a 75-minute window spanning fusion. Their observations indicate that secretory vesicles maintain a constant distance from the plasma membrane whereas the actin network compacts. Most importantly, they discovered a positive feedback mechanism in which myosin V (Myo52) transports Fus1 formin along pre-existing actin filaments, thereby enhancing aster compaction.

    • This article is well written, the arguments are convincing and the assertions are balanced. The centroid tracking method has been clearly and solidly controlled. Overall, this is a solid addition to our understanding of cytoskeletal organization in cell fusion. Major comments: No major comment.

    Minor comments:

    • Page 8 authors wrote "Upon depletion of Myo52, Ypt3 did not accumulate at the fusion focus (Figure 3C). A thin, wide localization at the fusion site was occasionally observed (Figure 3C, Movies S3)" : Is there a quantification of this accumulation in the mutant?

    • The framerate of movies could be improved for reader comfort: For example, movie S6 lasts 0.5 sec.

    Significance

    This study represents a conceptual and technical breakthrough in our understanding of cytoskeletal organization during cell-cell fusion. The authors introduce a high-precision, three-color live-cell centroid mapping method capable of resolving the spatio-temporal dynamics of protein complexes at the nanometer scale in living yeast cells. This methodological innovation enables systematic and quantitative mapping of the dynamic architecture of proteins at the cell fusion site, making it a powerful live-cell imaging approach. However, it is important to keep in mind that the increased precision achieved through averaging comes at the expense of overlooking atypical or outlier behaviors. The authors discovered a myosin V-dependent mechanism for the recruitment of formin that leads to actin aster compaction. The identification of Myo52 (myosin V) as a transporter of Fus1 (formin) to the fusion focus adds a new layer to our understanding of how polarized actin structures are generated and maintained during developmentally regulated processes such as mating.

    Previous studies have shown the importance of formins and myosins during fusion, but this paper provides a quantitative and dynamic mapping that demonstrates how Myo52 modulates Fus1 positioning in living cells. This provides a better understanding of actin organization, beyond what has been demonstrated by fixed-cell imaging or genetic perturbation.

    Audience: Cell biologists working on actin dynamics, cell-cell fusion and intracellular transport. Scientists involved in live-cell imaging, single particle tracking and cytoskeleton modeling.

    I have expertise in live-cell microscopy, image analysis, fungal growth machinery and actin organization.

  5. Version published to 10.1101/2025.04.29.651207 on bioRxiv