Actin-binding domain of Rng2 sparsely bound on F-actin strongly inhibits actin movement on myosin II

Rng2CHD, an actin-binding domain of an IQGAP, induces cooperative conformational changes in actin filaments and inhibits movement driven by myosin II.

Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authors We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. ***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** ---------------------------------------------------------------------------Reviewer #1 (Comments to the Authors (Required)): In this manuscript, the authors address the impact of the presence of the calponin homology domain of Rng2 (Rng2CHD) on the structure of the actin filaments and its interaction with myosin motors. The authors addressed these two questions using different experimental approaches. First they observe that filament gliding on human myoII surface is impaired by the presence of Rng2CHD at low saturating amounts. This behaviour is not observed for myosin V and Dicty myoII. Using HS-AFM they observe that the helical pitch is reduced by ~5% upon Rng2CHD binding. While the authors come with a lot of arguments and hypotheses to reach their conclusion, I find that some possibilities have not been ruled out fully that may explain what the authors are observing. While I am not convinced by all the interpretations of the results made by the authors, I still think that this work is of interest to the actin community. Though the quality of the manuscript can be substantially improved by discussing and/or addressing the following points : -The main striking point is that, if Rng2CHD has such a strong impact on filament structure, it is surprising that myoV and Dicty myoII are insensitive to it (figure 1).
-The GFP-Rng2CHD binds with similar affinity to actin filaments. Still, for the same binding coverage, its impact on gliding is lower, while one would expect that the addition of GFP would induce a stronger inhibition due to some steric hindrance. -In the gliding assay, can the authors rule out the possibility that what is observed is due to Rng2CHD directly binding to glassanchored myosins, as shown in supp. figure 5 ? -Similarly, can the authors rule out that some amount of Rng2CHD binds non specifically to the glass surface, with consequences on the filament gliding ? For example, glass-anchored Rng2CHD could transiently bind to the side of filament, decreasing filament gliding velocity, while not creating any buckling.
-I am not convinced by the authors claim (from AFM and negative EM images) that actin filaments can be untwisted due to Rng2CHD binding. If this was the case, should we expect to detect it in fluorescence microscopy ? Should it also lead to major filament breakage as we can expect untwisted proto-filaments to be very fragile ? -Can the authors assess the dynamics of binding/unbinding of Rng2CHD, for example using speckle microscopy with a low concentration of GFP-Rng2CHD ? The transient binding of Rng2CHD is hypothesized by the authors in the discussion as a way to induce some memory of a change in the filament conformation. It would be thus very informative to have more information on this point. at line 277, the authors claim that they observed transient binding, citing figure 4B, but this panel can not show any transient binding as it is a still image. This is more apparent in movie 6. -The authors should discuss more extensively the (putative) binding interface of both Rng2CHD and myoII. This discussion would be useful to the reader to more thoroughly assess the author's claims.
Reviewer #2 (Comments to the Authors (Required)): I am willing to recommend acceptance of the article titled "Actin binding domain of Rng2 sparsely bound on F-actin strongly inhibits actin movement on myosin II" on the condition that substantial revisions be made to the manuscript.
Authors use in vitro motility assays, binding assays, AFM, and EM of negatively stained samples to effectively show that lowdensity decoration of actin by the calponin homology domain of Rng2, a S. pombe IQGAP-related protein, changes the conformation of filamentous actin and inhibits striated muscle myosin gliding velocity. It is plausible that the change in actin conformation could be driving the effects on myosin. Overall, I think experiments are well-performed and there is novelty in the findings, but it is unclear why the authors chose the particular mishmash of protein orthologs versus those of one coherent system (S. pombe). As a result, it is confusing as to whether the authors intended to study the functional properties of Rng2 or to employ this protein as a tool to underscore a novel mode of actin-based regulation in vitro (Major comments 2).
Authors give satisfying evidence that the observed inhibition of filament sliding by muscle myosin-II is not due to interactions between Rng2CHD and the surface or myosin while simultaneously binding to actin based on the diffusive nature of the filaments in the presence of Rng2CHD for Dicty myo-II, disruption of HMM binding to anchored filaments (AFM and GFP), weak interaction of Rng2CHD with filamentous myosin in pelleting assay, and myosins where velocity is unaffected. Furthermore, they provide some evidence supporting their model that Rng2CHD changes the conformation of the actin helix in AFM and EM of negatively stained samples. This is bolstered by recent evidence that this domain confers the ability to bend muscle actin into rings (Palani et al., 2021). Yet, one further set of experiments would solidify the notion that the effect on velocity is directly due to weakened actomyosin interaction (Major comments 1).
Major comments 1) The relative affinity of actomyosin in motility assays becomes more apparent when the ionic strength is increased. To strengthen the argument that effects in motility assays are due to weakened actomyosin affinity, the motility assays could be performed at higher ionic strengths (~100-200 mM KCl; without methylcellulose). If the authors are correct, under these conditions they should see fewer or no filaments bound to the surface when Rng2 is present than in its absence (for the myosin-II species they tested). This would be a clear-cut result that can be obtained with relative ease (within a week).
2) The major detractor from this article is that the mix-and-match of proteins from different species makes the findings hard to interpret in the context of S. pombe cells where Rng2 regulates cytokinesis. In the discussion, authors explain that the physiological significance of Rng2CHD's inhibitory effect is unresolved, but authors should more strongly emphasize that this is directly because Rng2's effects on myosin were observed only with proteins from heterologous systems. 2a) Strikingly, the use of striated muscle orthologs of actin and myosin are in many ways inappropriate stand-ins for the native proteins of S. pombe that are cytoplasmic in nature. S. pombe actin differs from striated muscle actin enough to suggest that effects of Rng2CHD on the conformation of muscle actin does not automatically translate to the same effects on pombe actin (Ti and Pollard, 2011. J Biol Chem. Feb 18;286(7):5784-92). It is generally understood that striated muscle actin has been a standard reagent for in vitro studies for decades and is an important tool for establishing proof of molecular principles, but obtaining cytoplasmic actin isoforms from different species, including S. pombe, is rapidly becoming amenable and necessary for determining relevant molecular mechanisms. When it comes to studies that reach the level of molecular detail such as this one, species differences are especially likely to impact the results in meaningful ways. This study would be strengthened greatly by demonstrating that Rng2CHD also modulates the helical pitch of pombe actin, or at the very least another cytoplasmic actin, e.g. platelet actin or Dictyostelium actin. However, I am also inclined to overlook the usage of striated muscle actin as long as authors emphasize in their manuscript the caveats associated with using this actin versus pombe actin. 2b) Importantly, given the reported disparity between the tested striated muscle myosin-II and Dictyostelium cytoplasm myosin-II or mouse myosin-V in the ability of Rng2CHD to inhibit sliding, it suggests that the conformational changes do not universally inhibit myosin binding. Then, the critical question emerges: how would pombe myosins found in the contractile ring, Myo2, Myp2/Myo3, and Myo51, be regulated by the Rng2-actin interaction? The essential myosin-II, Myo2, has been shown to differ from Dicty. and muscle myosins by its apparent inability to assemble into minifilaments and how light chain phosphorylation inhibits actin binding (Friend et al., 2018. Cytoskeleton (Hoboken). Apr;75(4): 164-173;Pollard et al., 2017. PNAS. Aug 29;114(35):E7236-E7244), and thus it is reasonable to expect that the effects of Rng2 on Myo2 could also be different, despite belonging to the same class-II subfamily. Moreover, the contractile ring actin in pombe is decorated with tropomyosin Cdc8, which also would be expected to modify any of Rng2CHD's effects from the filaments' sides. Cdc8 is also an example of an ABP having opposite effects on the actin-affinity of Myo2 (enhancing) and striated myosin (inhibiting; Clayton et al. 2015. Cytoskeleton (Hoboken). Mar;72(3):131-45). One speculation is that Cdc8 decoration may even completely inhibit Rng2CHD binding, which would be consistent with the lack of cellular phenotype when the CHD is abolished (Tebbs and Pollard, 2013). There is no good substitute for using Rng2's cognate myosins for in vitro experiments to test properties related to its cellular role. Therefore, this study's value would be increased substantially if Rng2CHD could be shown to have any effect on any of the three relevant myosins of S. pombe (which have been purified previously). My understanding, however, is that this is not the authors' focus, but rather on the novelty of a long-range conformational regulation of actin that affects an activity of an actin binding protein (myosin) downstream. If this is true, it should be made clearer.
2c) The abstract and introduction should reflect the true focus/novelty of the findings of the study and be rewritten to clarify that the authors employed an entirely artificial in vitro system consisting of components from disparate systems to probe into the long-distance effects that actin binding proteins can have on one another. The way the manuscript is written, it is confusing to the general audience whether the authors are studying Rng2's role in cytokinesis, which is unlikely if not directly assaying pombe myosin activity, or characterizing a novel phenomenon that occurs when Rng2 and muscle actomyosin are used as molecular tools to study basic properties of actin regulation. I also recommend that the authors rewrite the discussion in their "Future Studies" section concerning Rng2's physiological role (last paragraph) to emphasize that in vitro studies, as they were performed here, need to be conducted employing Rng2CHD with pombe actin and myosin(s), and perhaps with Cdc8, to probe into how Rng2CHD functions in the cellular context. In sum, because of the discrepancy between the orthologs employed in this study and the ones employed in the native system, any conclusions based on this study (as it currently stands) about the potential role of Rng2's CHD in cytokinesis are highly attenuated. This issue can be addressed either experimentally or by rewriting portions of the manuscript appropriately.

Minor comment
The statement "contraction of the CR appears to be regulated in an inhibitory manner" (Lines 657-659) is unclear and I recommend removing or revising it; the reason the authors give is that Myo2 gliding velocity in vitro is faster than the rate of ring constriction, but this is perhaps an oversimplification given the fact that the myosin in the CR during constriction is under load, unlike in filament sliding assays. Cell wall synthesis likely sets the rate of constriction under normal conditions in S. pombe (Zhou et al. 2015. Mol Biol Cell. Jan 1;26(1):78-90); Proctor et al. 2012. Curr Biol. 201222(17):1601-8).

Reviewer #3 (Comments to the Authors (Required)):
Rng2 is an actin-binding protein that is involved in the formation and regulation of the actin-myosin II contractile ring in the fission yeast S. Pombe. However, the physiological role and molecular mechanisms of Rng2 in the contractile ring are unclear.
Here Hayakawa et al. report that the CH domain of Rng2 inhibits the motility of actin filaments on skeletal muscle myosin II in vitro in gliding filament assays even at a low ratio of bound Rng2CHD to actin protomers. The authors used a variety of techniques, including TIRF microscopy, negative stain EM, and HS-AFM to visualize the binding of HMM, Rng2CHD, and S1 fragment to actin filaments, providing direct evidence that partial decoration of Rng2CHD on F-actin changes its half helical pitch, and suggesting that sparsely bound Rng2CHD induces long-range or global structural changes of actin filaments, which reduce the affinity between actin and S1 in the ADP state, and thus inhibit the actin motility on myosin II. This work is largely performed carefully and is of interest to audience in the field of biophysics and experts on actin cytoskeleton and its binding proteins. Although it does not reveal the exact conformation of F-actin evoked by partial decoration of Rng2CHD, nor does it provide a detailed molecular mechanism of what and how global structural changes of actin filaments inhibit actin motility on myosin II, I understand that those are beyond the scope of this paper and deserve a separate study. Therefore, I recommend acceptance of this manuscript if the following points are addressed properly.
Major points: 1. The observation that Rng2CHD decorated actin filaments can separate into two individual protofilaments is surprising and interesting (Figure supplement 3, Video 7). Video 7 ends right after the two protofilaments separate, but what happens afterwards? Do the protofilaments disassemble? For a double-helical structure like F-actin, separating the two strands of the double helix requires continuous unwrapping and turning of one strand relative to the other, unless the double helix breaks along its length. If this is true, Rng2CHD may play a role in the disassembly of actin filaments. The authors should do an actin motility assay with TIRF microscopy using the same concentration of Rng2CHD as they have used in Figure supplement 3 to confirm that the separation of the two protofilaments is not an artifact from HS-AFM image processing.
2. Through co-sedimentation assay and TIRF microscopy, the authors have shown that Rng2CHD inhibits the steady-state binding of muscle S1 to actin filaments in the presence of ADP, not ATP, and that it decreases the binding of HMM to actin filaments in the presence of low-concentration ATP (0.5 M). However, the most potent inhibition of actin motility occurs in the presence of high-concentration ATP (1 mM, Fig. 1A). What is the mechanism of this strong motility inhibition at high ATP concentration?
Minor points: 1. A time-coded color bar is needed in Fig. 1C.
2. More details need to be provided on the HS-AMF image processing. E.g., in figure supplement 3, how were the raw images on the left converted to the images on the right? Why does applying the Laplacian and Gaussian filters enhance the helical feature of actin filaments? Would that also enhance the features of the separated protofilaments?

Reviewer #1
-The main striking point is that, if Rng2CHD has such a strong impact on filament structure, it is surprising that myoV and Dicty myoII are insensitive to it (figure 1).
Response: To be accurate, Dicty myosin II was also inhibited by Rng2CHD, although in a manner different from muscle myosin II. But yes, it is very intriguing that different myosins respond differently to Rng2CHD. As mentioned in the manuscript, we and others have experienced different response of different myosins to certain actin mutations. Moreover, as pointed out by Reviewer 2, tropomyosin inhibits myosin II but not myosin I. Therefore, for us, the current results are intriguing but not very surprising.
-The GFP-Rng2CHD binds with similar affinity to actin filaments. Still, for the same binding coverage, its impact on gliding is lower, while one would expect that the addition of GFP would induce a stronger inhibition due to some steric hindrance.
Response: In both Rng2CHD and GFP-Rng2CHD, the binding densities required for potent inhibition is quite low. For example, 80% reduction in speed on muscle HMM were achieved by binding densities of 3.0% and 9.7%, respectively (Table 1), and therefore additional steric hindrance due to the presence of GFP moiety would not be significant. Rather, the fact that binding affinity of GFP-Rng2CHD to actin (K d =3.4 µM) was >3-fold weaker than Rn2CHD (K d =0.92 µM) suggests that the structural impact on actin caused by one molecule of bound GFP-Rng2CHD may be smaller than that of Rng2CHD. We have no satisfactory explanation as to why GFP-Rng2CHD binds only weakly to actin, despite the presence of a Gly-based 16-residue linker between GFP and Rng2CHD moieties.

-In the gliding assay, can the authors rule out the possibility that what is observed is due to
Rng2CHD directly binding to glass-anchored myosins, as shown in supp. figure 5 ?
Response: If the concern is crosslinking of myosin motors to the glass surface by Rng2CHD, we can eliminate such possibility since a similar level of inhibition was observed with thick filaments of muscle myosin, in which myosin motors are elevated tens of nm above the glass surface. If the concern is structural impact of Rng2CHD binding to the myosin motor domain, K d between myosin filaments and Rng2CHD was 18.8 µM, and binding of Rng2CHD to myosin would be negligible in the presence of inhibitory concentrations of Rng2CHD (<200 nM), which is more than two orders of magnitude lower than the K d . Finally, if the concern is crosslinking of myosin motors to actin filaments, the same K d argument eliminates extensive crosslinking between actin filaments and HMM on the surface.
However, even a small number of such crosslinks may impose load to the moving actin filaments on muscle HMM-coated surface and contribute to the reduction in speed, as Response: Such possibility can be rejected from several independent lines of evidence: (a) a similar level of motility inhibition was observed with filaments of muscle myosin II (Video 3), (b) in the case of Dicty myosin II, actin filaments tended to diffuse away from the surface in the presence of Rng2CHD (Fig 1, Video 1), and (c) in the newly added actin landing assay, landing of actin filaments onto the surface did not occur in the presence of a high concentration of Rng2CHD, if the surface has no bound HMM (Fig S2).

-I am not convinced by the authors claim (from AFM and negative EM images) that actin
filaments can be untwisted due to Rng2CHD binding. If this was the case, should we expect to detect it in fluorescence microscopy? Should it also lead to major filament breakage as we can expect untwisted proto-filaments to be very fragile?
Response: This is a good point. During AFM real time imaging, we did see disappearance of protofilaments, suggestive of fragmentation, following the separation of the protofilaments.
We also saw frequent filament fragmentation in negative-stain EM images. However, we did not notice increased filament fragmentation during fluorescence microscopic observations in the presence of Rng2CHD. This is presumably because, unlike the case of AFM and EM, we used rhodamine-phalloidin-stabilized actin filaments for fluorescence microscopic observation. We would like to examine how phalloidin affects Rng2CHD-induced separation of the protofilaments in future experiments.
- At 25 mM KCl, Rng2CHD did not increase the amount of S1 that co-sedimented with actin in the presence of ATP (Fig 7), which is different from the above new results at 75 mM KCl. There can be a number of explanations for this apparent discrepancy, but we decided not to speculate on this issue.
We deeply thank this reviewer for suggesting this experiment, which guided our thinking to the correct direction. Response: Again, we would like to thank this reviewer for understanding the primary purpose of our current study. As I stated above, we plan to examine effects of Rng2CHD in a more native context, including the addition of tropomyosin and use of cytoplasmic actin and S. pombe myosins. For now, however, we explicitly mentioned that the physiological function of Rng2CHD cannot be properly evaluated using tropomyosin-free muscle actin and muscle or Dicty myosins, and a more native experimental setup is required (line Response: To avoid the confusion raised by this reviewer, we have extensively modified Abstract, Introduction and the final part of Discussion, as described above.

Minor comment
The statement "contraction of the CR appears to be regulated in an inhibitory manner"  Proctor et al. 2012. Curr Biol. 201222(17):1601-8).
Response: While we see the points raised by this reviewer, we still think it is possible that the contraction of CRs is negatively regulated in cells without cell walls, and it is worth point out the huge disparity present between in vitro motility speeds and contraction speeds of CRs. However, that myosin in CRs is regulated in an inhibitory manner is only a speculation, and it is not an important issue of this paper. Thus, we have deleted the relevant statement in Discussion and Supplementary Information 3.
Reviewer #3: Major points: 1 Partial separation of protofilaments was observed by negative stain EM (Fig 5), and therefore, we do not think separation of the protofilaments is an artifact of AFM.
Moreover, in the EM images, actin filaments in the presence of Rng2CHD had many breaks and kinks, suggesting that Rng2CHD indeed breaks protofilaments, the possibility pointed out by this reviewer. Clearly there are many experiments that should be done before this phenomenon is fully understood, but we would like to save them for future independent studies.
Regarding TIRF observation, we have actually performed motility assays in the presence of 5 and 8 µM Rng2CHD (Fig 3). These concentrations are comparable to or higher than that which caused separation of protofilaments in AFM. However, we did not notice anything indicative of protofilament separation, such as filament fragmentation or shrinking. We speculate that this is because TIRF observation used phalloidin-or rhodamine-phalloidin stabilized actin filaments, whereas AFM and EM observations used unstabilized actin filaments. Effect of phalloidin on Rng2CHD-induced protofilament separation and filament severing is another issue that need to be clarified in the future.  , Fig. 1A). What is the mechanism of this strong motility inhibition at high ATP concentration?
Response The purpose of applying the Laplacian and Gaussian filters is to enhance the spatial resolution of AFM images to clearly visualize two separated protofilaments. The working principle of the Laplacian and Gaussian filters are explained in the revised legend to Figure   S4 (line 1435-1444). We utilized the normal helical features of the control actin filaments to identify the abnormal features of Rng2CHD-bound filaments, particularly when two single actin protofilaments were separated. Thank you for submitting your revised manuscript entitled "Actin binding domain of Rng2 sparsely bound on F-actin strongly inhibits actin movement on myosin II". We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines. Please revise and format the manuscript and upload materials by Thursday.
Along with points mentioned below, please tend to the following: -please refer to the final comments from Reviewers 1 and 2 -please upload your main manuscript text as an editable doc file -please upload your main and supplementary figures as single files -we encourage you to introduce the panels in your figure legends in alphabetical order -please incorporate the Supplementary Materials into the main Materials and Methods section  Figure S2A and B If you are planning a press release on your work, please inform us immediately to allow informing our production team and scheduling a release date.
LSA now encourages authors to provide a 30-60 second video where the study is briefly explained. We will use these videos on social media to promote the published paper and the presenting author (for examples, see https://twitter.com/LSAjournal/timelines/1437405065917124608). Corresponding or first-authors are welcome to submit the video. Please submit only one video per manuscript. The video can be emailed to contact@life-science-alliance.org To upload the final version of your manuscript, please log in to your account: https://lsa.msubmit.net/cgi-bin/main.plex You will be guided to complete the submission of your revised manuscript and to fill in all necessary information. Please get in touch in case you do not know or remember your login name.
To avoid unnecessary delays in the acceptance and publication of your paper, please read the following information carefully.

A. FINAL FILES:
These items are required for acceptance.
--An editable version of the final text (.DOC or .DOCX) is needed for copyediting (no PDFs).
--High-resolution figure, supplementary figure and video files uploaded as individual files: See our detailed guidelines for preparing your production-ready images, https://www.life-science-alliance.org/authors --Summary blurb (enter in submission system): A short text summarizing in a single sentence the study (max. 200 characters including spaces). This text is used in conjunction with the titles of papers, hence should be informative and complementary to the title. It should describe the context and significance of the findings for a general readership; it should be written in the present tense and refer to the work in the third person. Author names should not be mentioned.