Calponin-homology domain mediated bending of membrane-associated actin filaments

  1. Saravanan Palani
  2. Sayantika Ghosh
  3. Esther Ivorra-Molla
  4. Scott Clarke
  5. Andrejus Suchenko
  6. Mohan K Balasubramanian
  7. Darius Vasco Köster

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Abstract

Actin filaments are central to numerous biological processes in all domains of life. Driven by the interplay with molecular motors, actin binding and actin modulating proteins, the actin cytoskeleton exhibits a variety of geometries. This includes structures with a curved geometry such as axon-stabilizing actin rings, actin cages around mitochondria and the cytokinetic actomyosin ring, which are generally assumed to be formed by short linear filaments held together by actin cross-linkers. However, whether individual actin filaments in these structures could be curved and how they may assume a curved geometry remains unknown. Here, we show that ‘curly’, a region from the IQGAP family of proteins from three different organisms, comprising the actin-binding calponin-homology domain and a C-terminal unstructured domain, stabilizes individual actin filaments in a curved geometry when anchored to lipid membranes. Although F-actin is semi-flexible with a persistence length of ~10 μm, binding of mobile curly within lipid membranes generates actin filament arcs and full rings of high curvature with radii below 1 μm. Higher rates of fully formed actin rings are observed in the presence of the actin-binding coiled-coil protein tropomyosin and when actin is directly polymerized on lipid membranes decorated with curly. Strikingly, curly induced actin filament rings contract upon the addition of muscle myosin II filaments and expression of curly in mammalian cells leads to highly curved actin structures in the cytoskeleton. Taken together, our work identifies a new mechanism to generate highly curved actin filaments, which opens a range of possibilities to control actin filament geometries, that can be used, for example, in designing synthetic cytoskeletal structures.

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  1. Version published to 10.7554/elife.61078 on eLife
  2. Version published to 10.1101/2020.07.10.197616v3 on bioRxiv
  4. eLife

    ###Reviewer #3:

    General assessment:

    The manuscript "Calponin-Homology Domain mediated bending of membrane associated actin filaments" by Palani et al investigates the role of truncated versions IQGAP1 (from yeast and humans) in forming ring-like structures on lipid supported bilayers in an in vitro TIRF assay. This reviewer is still confused by the mechanism that the "curly" truncation uses to bend actin filaments and context between this new "curved actin" generating mechanism with the mechanisms for generating actin rings in other contexts could help the reader understand this advance with more clarity. The authors mention several physiological contexts where the formation of actin rings might apply (associated with mitochondria, in axons, and during cell division in the actomyosin ring) however do not follow up with experiments addressing these specific ringed structures, rather non-specific cortical actin rings in several cell types. While this work has strong potential and is very intriguing additional support/clarification is required to back the claims made by the authors.

    Numbered summary of substantive concerns:

    1. The visual components of this work are striking. However, the accompanying quantification is somewhat confusing. Throughout the text mean values are listed for various parameters beyond those shown in the figures and it will improve the flow of the manuscript/aid the reader if these were represented as panels in each figure. Further, at least 3 FOVs should be analyzed for all analysis, from independent experiments, however it appears that a single FOV was measured in several figures (i.e. Figure 3 sup 1; Figure 3 sup 2). Other experiments also have relatively low "n" (i.e. 6 filaments measured for the analysis in Figure 2 sup 2). Do these N values have enough statistical power to support these conclusions?

    2. In the movies provided it looks like many of the "rings" are formed away from the coverslip and "fall" down into the TIRF field. Are these movies the most representative of ring formation for these versions of IQGAP? A comparison to actin filaments "alone" but with the lipids might ease this concern.

    3. Are the two IQGAP1 truncations dimers or monomers? Based on sequence alone it appears the dimerization domain is lacking from these constructs, but the SNAP-labeled images in Figure 2 have bright punctate and dimmer filament-like structures. The addition of a model or further clarification on how this arrangement of labeled IQGAP leads to ring formation would aid the reader.

    4. From the image presented in Figure 4 the "rings" from the human IQGAP1 truncation look substantially different than that from the yeast version - they are much larger (about 5x) and while "curvy" not exactly tight rings like I can see in the yeast examples. Yet the quantification as presented looks very similar. Is there a different optimal lipid content between mammalian or yeast lipids? Is the longer unstructured region in the mammalian isoform contributing to the difference?

    5. The authors should provide an explanation in the body of the manuscript of what "curly" constructs are being used in mammalian cells. From the methods it looks like the yeast truncations are being expressed. This should be compared to the mammalian version. Additionally, are the cellular rings a similar size to those observed in vitro (perhaps from the example in mammalian cells they are, but not for the yeast?). Additionally, this work would be really sing the in vitro rings were linked to a specific population(s) of cellular actin rings - what is the nature of the cortical rings analyzed by the authors? Are these actin associated mitochondria? Where is IQGAP1 during cell division?

  5. eLife

    ###Reviewer #2:

    In this manuscript, Palani and coworkers investigate the structural effects of binding of a fragment of the IQGAP family of proteins, called "curly", to actin filaments. When tethered to a supported lipid bilayer, curly induces curvature in actin filaments, ultimately giving rise to ring-shaped filament structures. Filament decoration by tropomyosin increases the propensity of ring formation, and introduction of myosin II filaments induces constriction.

    This manuscript presents novel and intriguing insights into the mechanisms that regulate the formation of cytoskeletal structures with curved geometries. The manuscript is well written, and the experiments are logically described. As such, this paper is sure to be of interest to a broad audience.

    Below are a few suggestions I would like to see addressed:

    1. What is the magnitude of curly's affinity for actin filaments? How does this compare to the binding affinity of the isolated CH domain?

    2. Given that curly is proposed to contain two actin-binding sites, has this protein ever been observed to bundle filaments? Also, do multiple filaments ever become incorporated into the same ring?

    3. How does the counter-clockwise direction of curvature of the actin rings compare to the helical pitch of the actin filament? In other words, are the actin subunits being wound tighter around the filament's long axis or are they being loosened?

    4. The authors compare the structural effects of curly binding to those produced by cofilin. Cofilin binding has been reported to alter the twist of actin filaments. Is this what is proposed to happen for curly-bound filaments as well?

    5. At the bottom of page 3, the authors state that: "Importantly, the uni-directional bending supports the hypothesis that the binding site of curly with actin filaments defines an orientation, and the propagation of a curved trajectory once established indicates a cooperative process."

    Cooperativity implies that a process becomes easier once it is started. Do the authors have evidence that it becomes easier to bend the filament along its length once the first binding/bending event occurs? Or is it possible that the additive effect of multiple filament bending events eventually generates a ring-like shape?

    1. It is unclear to me how the model of the myosin II-bound actin ring in Figure 3 Supplement 4 Part E illustrates a possible mechanism for myosin-induced constriction of the actin ring. If I am interpreting the schematic correctly, the authors indicate that ring constriction occurs via the application of force in the upward direction to the inner portion of the filament on the left side of the ring, and in the downward direction to both the inner and outer parts of the filament on the right side of the ring. However, it is my understanding that pulling simultaneously on the outer and the inner parts of the filament on the right side of the ring would not stimulate constriction. I believe one would have to pull on only one of those outer and inner segments at a time to slide them along each other and constrict the ring.

    If I am misunderstanding the schematic, can the authors correct me by expanding on their proposed mechanism?

    1. How constrained are the motions of Rng2 in S. pombe? Once Rng2 localizes to cytokinetic nodes, do the nodes move around enough to be mimicked by tethering curly to the supported lipid bilayer?

    2. The reference to the Tebbs and Pollard paper has an incorrect author listing in the References.

    3. The filament on the left in Figure 1A has a left-handed helical twist and should be corrected. The same is true for the filaments in Figure 3 Supplement 2, and Figure 3 Supplement 3.

  6. eLife

    ###Reviewer #1:

    The IQGAP family proteins interact with actin, and contribute e.g. to the formation of cytokinetic rings. Here, Palani et al. provide evidence that the N-terminal fragments of these proteins, composed of a CH domain and 'unstructured region', contain two separate actin-binding sites and can bend actin filaments into rings. This activity requires anchoring of the IQGAP fragment, which they named 'Curly', on the surface of a membrane. Moreover, they demonstrate that actin filament bending by Curly can be enhanced by addition of tropomyosin, and that myosin II can contract these actin rings.

    Major comments:

    1. The authors discuss on pages 1 -2 how full-length Curly and its various deletion constructs bind actin filaments. However, actin-binding was not properly tested for any of the constructs used in this study. Thus, the authors should carry out proper actin filament co-sedimentation assays for all constructs. The assays should be performed with a constant concentration of Curly, and varying the actin concentration (form 0 uM to e.g. 8 uM) to obtain binding curves, and to be hence able to compare the F-actin affinities of different constructs.

    2. The cell biology data presented in Fig. 4 and Fig. 4 - figure supplement 2 are not particularly convincing. The authors should thus perform a careful quantification of F-actin curvature and 'actin ring frequency' in cells transfected with plasmids expressing (i). EGFP, (ii). EGFP-Curly, and (iii). an EGFP-Curly mutant defective in ring formation. Because EGFP-Curly most likely does not associate with the plasma membrane in cells, it is somewhat confusing how it could still induce the formation of actin rings. Thus, the authors may observe much more robust actin ring formation in cells if they would use a membrane-anchored Curly-EGFP instead of soluble EGFP-Curly.

  7. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 2 of the manuscript.

    ###Summary:

    This manuscript reports the structural effects of a fragment of the IQGAP family proteins, called "curly", on actin filaments. When tethered to a supported lipid bilayer, Curly induces curvature in actin filaments, ultimately giving rise to ring-shaped filament structures. Moreover, this study demonstrates that filament decoration by tropomyosin increases the propensity of ring formation, and introduction of myosin II filaments induces constriction of actin rings.

    The findings presented in this manuscript are potentially very important. However, in some cases the results are somewhat preliminary and lack essential controls. Thus, additional experiments and data analysis are required to strengthen the study.

  8. Version published to 10.1101/2020.07.10.197616v2 on bioRxiv
  9. Version published to 10.1101/2020.07.10.197616v1 on bioRxiv