The IQGAP family of proteins plays a key role in actin cytoskeleton regulation including the assembly and function of the contractile actomyosin ring in budding and fission yeasts (Briggs and Sacks, 2003; Eng et al., 1998; Epp and Chant, 1997; Lippincott and Li, 1998; Tebbs and Pollard, 2013). To study the mechanism and role of actin binding by the fission yeast IQGAP (encoded by the rng2 gene), we utilized a strategy to investigate its function when immobilized on supported lipid bilayers. We chose this approach, since during cytokinesis Rng2, which binds several actomyosin ring proteins, is tethered to the plasma membrane via Mid1, ensuring the formation and anchoring of the cytokinetic ring (Laplante et al., 2016; Laporte et al., 2011; Padmanabhan et al., 2011). We linked hexa-histidine tagged Rng2(1-189) to supported lipid bilayers containing nickel-chelating lipids (DOGS-NTA(Ni²⁺)) and observed the binding of fluorescently labeled actin filaments using live total internal reflection fluorescence (TIRF) microscopy as described earlier (Köster et al., 2016; Figure 1A; Figure 1—figure supplement 1A). Remarkably, actin filaments landing onto His₆-Rng2(1-189) decorated SLBs formed highly bent arcs and full rings with curvatures of C_curly = 1.7 ± 0.5 µm⁻¹ (Figure 1B,C; Figure 1—figure supplement 1D–G; Videos 1 and 2). Binding of other membrane tethering actin binding proteins in the same geometry, such as the CHD of α-actinin or the actin binding domain of Ezrin, did not appreciably bend actin filaments (Figure 1—figure supplement 2A–D). Membrane anchored fimbrin has also been shown not to bend actin (Murrell and Gardel, 2012). To our knowledge, the bending of individual actin filaments into rings is an unprecedented phenomenon among the known actin binding proteins and we will refer in the following to His6-Rng2(1-189) as ‘curly’.

Video 1

Download asset

Video 2

Download asset

Figure 1 with 2 supplements see all

Download asset Open asset

To understand the mechanism leading to actin filament bending and ring formation by curly, we tested the role of curly anchoring to lipid membranes and its orientation. Curly mobility within planar lipid membranes was important for actin bending as glass adsorbed, immobilized His₆-Curly led to reduced actin binding and bending (C_glass = 0.6 ± 0.3 µm⁻¹) (Figure 2A). However, placing the hexa-histidine tag to the C-terminus, Rng2(1-189)-His₆, did not affect actin filament bending (C_curly-his = 1.5 ± 0.5 µm⁻¹) (Figure 2B–D, Figure 2—figure supplement 1).

Figure 2 with 1 supplement see all

Download asset Open asset

Next, we studied whether actin bending by curly depended on the orientation of actin filaments by following the landing of already polymerized actin filaments decorated with labeled capping protein as a plus end marker (Bieling et al., 2016). We found that the bending was oriented anti-clockwise with respect to the plus end in all instances, wherein the plus end was clearly labeled to identify the orientation of filament bending (Figure 3A,B; Figure 3—figure supplement 1A,B). This was observed using both, the N-terminal and C-terminal hexa-histidine tagged curly, indicating that the internal sequence of the two actin binding sites within curly sets the chirality of actin bending and not the position of the membrane linker (Figure 3A,B; Figure 3—figure supplement 1A,B; Videos 3 and 4). Actin filaments appeared to bend concomitant with their landing on the supported lipid bilayer, which indicates that the bending did not require the full actin filament to be tethered to the SLB and underlined the earlier observation that the bending occurred locally.

Video 3

Download asset

Video 4

Download asset

Figure 3 with 5 supplements see all

Download asset Open asset

To decouple the actin filament bending from the landing of actin filaments, we induced the polymerization of new actin filaments on planar lipid membranes by membrane tethered formin (His₆-SpCdc12(FH1-FH2)), profilin-actin and ATP in the presence of membrane tethered His₆-Curly. Strikingly, actin filaments displayed characteristic bending shortly after the onset of polymerization (C_{formin, short} = 1.1 ± 0.3 µm⁻¹) and often grew into full rings (C_{formin rings} = 1.7 ± 0.4 µm⁻¹) (Figure 3C,D; Figure 3—figure supplement 2A–E; Video 5). By contrast, polymerization of actin filaments along SLBs decorated with His₁₀-SNAP-EzrinABD did not result in the formation of arcs or rings, establishing that actin filament bending was due to curly and not due to formin (Figure 3—figure supplement 2F,G). These observations show that actin bending occurs continuously due to the binding of membrane tethered curly and did not require the cross-linking of adjacent ends of the same filament as was observed with the actin cross-linker anillin (Kučera et al., 2020). Importantly, the uni-directional bending supports the hypothesis that the binding site of curly with actin filaments defines an orientation with the propagation of an established curved trajectory.

Video 5

Download asset

Actin filaments forming the cytokinetic ring in S. pombe are tightly associated with tropomyosin (Cdc8). By contrast, the actin cross-linker fimbrin is present outside the cytokinetic ring region in Arp2/3 generated actin patches and prevents tropomyosin binding to these patches (Skau and Kovar, 2010). To determine whether the actin bending effect of curly is conserved in tropomyosin-wrapped actin filaments, we incubated actin filaments with tropomyosin before adding them to His₆-Curly containing SLBs. Strikingly, addition of tropomyosin to actin filaments increased the frequency of actin ring formation without affecting actin filament curvature, while actin filaments incubated with the actin cross-linker fimbrin displayed reduced bending and ring formation (Figure 3—figure supplement 3; Video 6). Thus, the tropomyosin Cdc8 and curly cooperate to enhance actin filament bending and ring formation.

Video 6

Download asset

Interestingly, we could observe that long actin filaments coated with tropomyosin would trace consecutive rings around the same center while landing on curly decorated lipid membranes. Subtraction of the image after completion of the first round of actin filament landing into a ring from the image after the second round revealed that the second ring occupied the interior space of the first ring. In line with that, a comparison of the intensity profiles perpendicular to the actin filament of the first and second round of ring formation revealed a widening of the profile toward the ring’s interior (Figure 3—figure supplement 4A,B). A similar effect could be observed in examples of actin filaments polymerized by membrane tethered formin in the presence of membrane tethered curly (Figure 3—figure supplement 4C,D). This would suggest that curly can arrange long actin filaments into an inward-oriented spiral.

To test whether the curly induced actin rings can contract, we added rabbit skeletal muscle myosin II filaments and ATP to actin filaments bound to SLB tethered His₆-curly and followed actin filament dynamics over time. Shortly after myosin II addition, actin filaments (curved and straight ones) were propelled by myosin action leading to increased bending, rotation and finally to ring formation and contraction (Figure 3E–G, Figure 3—figure supplement 5A–D, Video 7). Interestingly, most actin rings displayed a counter-clockwise rotation (34/36 cases) and a slow contraction speed of 3 ± 2 nm s⁻¹ (Figure 3F; Video 8). The density of actin rings was strongly increased after myosin II addition (Figure 3—figure supplement 5E), indicating that actin sliding leads to more recruitment of curly. In line with this, actin filament rings displayed increased localization of fluorescently labeled curly after addition of myosin II filaments (Figure 3—figure supplement 5F–H). Despite reaching very high actin curvatures (up to 6.3 µm⁻¹) no breaking of actin filaments during the contraction process could be observed suggesting that binding of curly reduces the rigidity of actin filaments.

Video 7

Download asset

Video 8

Download asset

Since Rng2 belongs to the IQGAP protein family, we tested the N-terminal hexa-histidine tagged fragments of the IQGAP proteins Iqg1(1-330) (S. cerevisiae) and IQGAP1(1–678) (H. sapiens) and found that the bending of actin filaments was conserved (C_S.C. = 1.1 ± 0.4 µm⁻¹; C_H.S. = 1.0 ± 0.2 µm⁻¹) (Figure 4A–C; Figure 4—figure supplement 1A). Comparison of the available crystal structures of H. sapiens IQGAP1(28–190) with S. pombe Rng2(32-190) indicates high similarity between the two (Figure 4—figure supplement 1B).

Figure 4 with 5 supplements see all

Download asset Open asset

Video 9

Download asset

Finally, to test the effect of curly on the actin cortex in cells, we expressed GFP -tagged curly in the mammalian cell lines HEK293T and RPE-1, which resulted in striking changes of the actin cortex architecture with the prominent occurrence of curved and ring-shaped actin filaments and bundles with curvatures of C_HEK293T = 2.3 ± 0.4 µm⁻¹ and C_RPE-1 = 1.9 ± 0.6 µm⁻¹ (Figure 4D–F, Figure 4—figure supplement 2A–D, Figure 4—figure supplement 3). Co-expression with LifeAct-mCherry confirmed EGFP-Rng2(1-189) bound to actin filaments in cells (Figure 4—figure supplement 2A,B). To our surprise, addition of a CaaX domain to tether curly to cellular membranes did not enhance the actin bending effect but located curly mainly to structures resembling the endoplasmic reticulum (Figure 4—figure supplement 4A). The EGFP-Rng2(144-189) and EGFP-Rng2(144-189)-CaaX constructs showed only cytoplasmic localization (Figure 4—figure supplement 4B,C). To test whether the actin bending effect is conserved in the mammalian full-length protein, we imaged HEK293T cells expressing full-length EGFP-IQGAP1 and LifeAct-mCherry using lattice light sheet microscopy and indeed observed curved and ring-shaped actin structures inside the cell as well at its surface (Figure 4—figure supplement 5; Video 9). These experiments established that curly could instructively reorganize actin filaments into curved structures and rings and that this capacity is conserved in full-length IQGAP1.

Our results show that the N-terminal CHD of IQGAP proteins induces actin filament bending when tethered to lipid membranes, which constitutes a new type of actin binding protein and could be an important link between actin and membrane geometries (for a summary of results, see Supplementary file 1). This mechanism of actin ring formation stands out as it bends single actin filaments in contrast to other reported systems that generate rings made of bundles of actin filaments (Litschel et al., 2020; Mavrakis et al., 2014; Mishra et al., 2013; Miyazaki et al., 2015; Way et al., 1995). The actin-binding affinity of Rng2(1-189) is Kd = 0.9 μM (Hayakawa et al., 2020) and CHDs of other proteins, such as α-actinin (K_d = 4.7 μM; Wachsstock et al., 1993) or utrophin (Kd = 1.5 μM; Singh et al., 2017) do not show this behavior. Recently, Uyeda and colleagues reported that curly (Rng2(1-189)) in solution could induce kinks at random locations of the actin filament (Hayakawa et al., 2020). We do not know yet the precise mechanism how curly induces actin bending when constrained to a lipid membrane, but one possibility could be asymmetric binding of curly to actin filaments leading to a succession of kinks towards the same direction. With an estimated His₆-Curly surface density on SLBs of 5000 µm⁻² (Nye and Groves, 2008) the approximated curly to actin ratio would be 1:7 or higher. The mobility of curly on the SLB allowing accumulation under actin filaments is essential for actin filament bending as glass-immobilized curly failed to generate rings. Studies of the Goode laboratory on IQGAP1 indicate that the N-terminal part IQGAP1(1–522), which corresponds to Rng2(1-189), is monomeric and does not promote bundling of actin filaments (Hoeprich et al., 2021). The binding of curly to actin reduces the effective actin filament persistence length far below its typical value of 10 µm. This increased flexibility is particularly highlighted by the fact that addition of rabbit muscle myosin II filaments resulted in actin ring constriction without any evidence for filament breaking up to curvatures of 6.3 µm⁻¹ which is much higher than values reported for actin alone (C = 2.5 µm⁻¹) (Taylor et al., 2000). How this reduction of actin persistence length is achieved and whether it is related to the effect observed with cofilin (De La Cruz and Gardel, 2015; McCullough et al., 2008) remains to be deciphered in future work.

It was not evident that the addition of myosin II filaments would lead to actin ring constriction without having to add any actin cross-linkers. When considering that curly arranges actin filaments into an inward spiral, a possible explanation for actin ring constriction would be that the myosin II filament acts both as a cross-linker and motor protein: one end of the myosin II filament sits at the actin filament plus end while other myosin head domains of the same myosin II filament pull along the same actin filament to travel toward the plus end leading to constriction. This would result in sub-optimal myosin head orientations towards the actin filament, which could explain the observed slow constriction rates that were orders of magnitude slower than the reported values for actin propulsion by myosin II in motility assays (Toyoshima et al., 1990). Future works will provide more insights into this peculiar minimal ring constriction mechanism.

Highly bent actin filament structures are most likely important for many cellular structures such as axons (Vassilopoulos et al., 2019; Xu et al., 2013), mitochondrial actin cages (Kruppa et al., 2018), or secretion of the Weibel-Palade bodies (Nightingale et al., 2011), but the molecular mechanisms leading to their formation are still poorly understood. Future work on the role of curly in these processes could provide crucial insights into these mechanisms. In addition, our system of membrane-bound curly, actin filaments, and myosin II filaments constitutes a minimalistic system for actin ring formation and constriction and could be used in the future to design synthetic dividing vesicles and further exiting active membrane-cortex systems.

All data generated or analysed during the study are included in the manuscript and supporting files. Source data files for the actin curvature measurements (Figures 1-4) have been deposited and are freely available on Dryad (http://doi.org/10.5061/dryad.bvq83bk6f).

1. 1. Köster DV

   (2020) Dryad Digital Repository

   Calponin-Homology Domain mediated bending of membrane associated actin filaments.

   https://doi.org/10.5061/dryad.bvq83bk6f

1. 1. Bieling P
   2. Li TD
   3. Weichsel J
   4. McGorty R
   5. Jreij P
   6. Huang B
   7. Fletcher DA
   8. Mullins RD

   (2016) Force feedback controls motor activity and mechanical properties of Self-Assembling branched actin networks

   Cell 164:115–127.

   https://doi.org/10.1016/j.cell.2015.11.057

   • PubMed
   • Google Scholar
2. 1. Bombardier JP
   2. Eskin JA
   3. Jaiswal R
   4. Corrêa IR
   5. Xu MQ
   6. Goode BL
   7. Gelles J

   (2015) Single-molecule visualization of a formin-capping protein 'decision complex' at the actin filament barbed end

   Nature Communications 6:8707.

   https://doi.org/10.1038/ncomms9707

   • PubMed
   • Google Scholar
3. 1. Briggs MW
   2. Sacks DB

   (2003) IQGAP proteins are integral components of cytoskeletal regulation

   EMBO reports 4:571–574.

   https://doi.org/10.1038/sj.embor.embor867

   • PubMed
   • Google Scholar
4. 1. De La Cruz EM
   2. Gardel ML

   (2015) Actin Mechanics and Fragmentation

   Journal of Biological Chemistry 290:17137–17144.

   https://doi.org/10.1074/jbc.R115.636472

   • PubMed
   • Google Scholar
5. 1. Eng K
   2. Naqvi NI
   3. Wong KC
   4. Balasubramanian MK

   (1998) Rng2p, a protein required for cytokinesis in fission yeast, is a component of the actomyosin ring and the spindle pole body

   Current Biology 8:611–621.

   https://doi.org/10.1016/S0960-9822(98)70248-9

   • PubMed
   • Google Scholar
6. 1. Ennomani H
   2. Letort G
   3. Guérin C
   4. Martiel J-L
   5. Cao W
   6. Nédélec F
   7. De La Cruz EM
   8. Théry M
   9. Blanchoin L

   (2016) Architecture and connectivity govern actin network contractility

   Current Biology 26:616–626.

   https://doi.org/10.1016/j.cub.2015.12.069

   • Google Scholar
7. 1. Epp JA
   2. Chant J

   (1997) An IQGAP-related protein controls actin-ring formation and cytokinesis in yeast

   Current Biology 7:921–929.

   https://doi.org/10.1016/S0960-9822(06)00411-8

   • PubMed
   • Google Scholar
8. Preprint

   1. Hayakawa Y
   2. Takaine M
   3. Imai T
   4. Yamada M
   5. Hirose K
   6. Tokuraku K
   7. Ngo KX
   8. Kodera N
   9. Numata O
   10. Nakano K
   11. Uyeda TQP

   (2020) Actin binding domain of Rng2 strongly inhibits actin movement on myosin II HMM through structural changes of actin filaments

   bioarXiv.

   https://doi.org/10.1101/2020.04.14.041046

   • Google Scholar
9. Preprint

   1. Hoeprich GJ
   2. Shekhar S
   3. Goode BL

   (2021) Single-molecule imaging of IQGAP1 regulating actin filament dynamics in real time

   bioRxiv.

   https://doi.org/10.1101/2021.04.18.440338

   • Google Scholar
10. 1. Köster DV
    2. Husain K
    3. Iljazi E
    4. Bhat A
    5. Bieling P
    6. Mullins RD
    7. Rao M
    8. Mayor S

    (2016) Actomyosin dynamics drive local membrane component organization in an in vitro active composite layer

    PNAS 113:E1645–E1654.

    https://doi.org/10.1073/pnas.1514030113

    • Google Scholar
11. 1. Kruppa AJ
    2. Kishi-Itakura C
    3. Masters TA
    4. Rorbach JE
    5. Grice GL
    6. Kendrick-Jones J
    7. Nathan JA
    8. Minczuk M
    9. Buss F

    (2018) Myosin VI-Dependent Actin Cages Encapsulate Parkin-Positive Damaged Mitochondria

    Developmental Cell 44:484–499.

    https://doi.org/10.1016/j.devcel.2018.01.007

    • PubMed
    • Google Scholar
12. Preprint

    1. Kučera O
    2. Janda D
    3. Siahaan V
    4. Dijkstra SH
    5. Pilátová E
    6. Zatecka E
    7. Diez S
    8. Braun M
    9. Lansky Z

    (2020) Anillin propels myosin-independent constriction of actin rings

    bioRxiv.

    https://doi.org/10.1101/2020.01.22.915256

    • Google Scholar
13. 1. Laplante C
    2. Huang F
    3. Tebbs IR
    4. Bewersdorf J
    5. Pollard TD

    (2016) Molecular organization of cytokinesis nodes and contractile rings by super-resolution fluorescence microscopy of live fission yeast

    PNAS 113:E5876–E5885.

    https://doi.org/10.1073/pnas.1608252113

    • Google Scholar
14. 1. Laporte D
    2. Coffman VC
    3. Lee IJ
    4. Wu JQ

    (2011) Assembly and architecture of precursor nodes during fission yeast cytokinesis

    Journal of Cell Biology 192:1005–1021.

    https://doi.org/10.1083/jcb.201008171

    • PubMed
    • Google Scholar
15. 1. Lippincott J
    2. Li R

    (1998) Sequential assembly of myosin II, an IQGAP-like protein, and filamentous actin to a ring structure involved in budding yeast cytokinesis

    Journal of Cell Biology 140:355–366.

    https://doi.org/10.1083/jcb.140.2.355

    • PubMed
    • Google Scholar
16. Preprint

    1. Litschel T
    2. Kelley CF
    3. Holz D
    4. Koudehi MA
    5. Vogel SK
    6. Burbaum L
    7. Mizuno N
    8. Vavylonis D
    9. Schwille P

    (2020) Reconstitution of contractile actomyosin rings in vesicles

    bioRxiv.

    https://doi.org/10.1101/2020.06.30.180901

    • Google Scholar
17. 1. Mavrakis M
    2. Azou-Gros Y
    3. Tsai FC
    4. Alvarado J
    5. Bertin A
    6. Iv F
    7. Kress A
    8. Brasselet S
    9. Koenderink GH
    10. Lecuit T

    (2014) Septins promote F-actin ring formation by crosslinking actin filaments into curved bundles

    Nature Cell Biology 16:322–334.

    https://doi.org/10.1038/ncb2921

    • PubMed
    • Google Scholar
18. 1. McCullough BR
    2. Blanchoin L
    3. Martiel JL
    4. De la Cruz EM
    5. Cruz EMDL
    6. Haven N

    (2008) Cofilin increases the bending flexibility of actin filaments: implications for severing and cell mechanics

    Journal of Molecular Biology 381:550–558.

    https://doi.org/10.1016/j.jmb.2008.05.055

    • PubMed
    • Google Scholar
19. 1. Mishra M
    2. Kashiwazaki J
    3. Takagi T
    4. Srinivasan R
    5. Huang Y
    6. Balasubramanian MK
    7. Mabuchi I

    (2013) In vitro contraction of cytokinetic ring depends on myosin II but not on actin dynamics

    Nature Cell Biology 15:853–859.

    https://doi.org/10.1038/ncb2781

    • PubMed
    • Google Scholar
20. 1. Miyazaki M
    2. Chiba M
    3. Eguchi H
    4. Ohki T
    5. Ishiwata S

    (2015) Cell-sized spherical confinement induces the spontaneous formation of contractile actomyosin rings in vitro

    Nature Cell Biology 17:480–489.

    https://doi.org/10.1038/ncb3142

    • PubMed
    • Google Scholar
21. 1. Mosby LS
    2. Hundt N
    3. Young G
    4. Fineberg A
    5. Polin M
    6. Mayor S
    7. Kukura P
    8. Köster DV

    (2020) Myosin II Filament Dynamics in Actin Networks Revealed with Interferometric Scattering Microscopy

    Biophysical Journal 118:1946–1957.

    https://doi.org/10.1016/j.bpj.2020.02.025

    • PubMed
    • Google Scholar
22. 1. Murrell MP
    2. Gardel ML

    (2012) F-actin buckling coordinates contractility and severing in a biomimetic actomyosin cortex

    PNAS 109:20820–20825.

    https://doi.org/10.1073/pnas.1214753109

    • PubMed
    • Google Scholar
23. 1. Nightingale TD
    2. White IJ
    3. Doyle EL
    4. Turmaine M
    5. Harrison-Lavoie KJ
    6. Webb KF
    7. Cramer LP
    8. Cutler DF

    (2011) Actomyosin II contractility expels von Willebrand factor from Weibel–Palade bodies during exocytosis

    Journal of Cell Biology 194:613–629.

    https://doi.org/10.1083/jcb.201011119

    • Google Scholar
24. 1. Nye JA
    2. Groves JT

    (2008) Kinetic control of histidine-tagged protein surface density on supported lipid bilayers

    Langmuir 24:4145–4149.

    https://doi.org/10.1021/la703788h

    • PubMed
    • Google Scholar
25. 1. Padmanabhan A
    2. Bakka K
    3. Sevugan M
    4. Naqvi NI
    5. D'souza V
    6. Tang X
    7. Mishra M
    8. Balasubramanian MK

    (2011) IQGAP-related Rng2p organizes cortical nodes and ensures position of cell division in fission yeast

    Current Biology 21:467–472.

    https://doi.org/10.1016/j.cub.2011.01.059

    • PubMed
    • Google Scholar
26. 1. Palani S
    2. Köster DV
    3. Hatano T
    4. Kamnev A
    5. Kanamaru T
    6. Brooker HR
    7. Hernandez-Fernaud JR
    8. Jones AME
    9. Millar JBA
    10. Mulvihill DP
    11. Balasubramanian MK

    (2019) Phosphoregulation of tropomyosin is crucial for actin cable turnover and division site placement

    Journal of Cell Biology 218:3548–3559.

    https://doi.org/10.1083/jcb.201809089

    • PubMed
    • Google Scholar
27. 1. Pardee JD
    2. Spudich JA

    (1982) Purification of muscle actin

    Methods in Cell Biology 24:271–289.

    https://doi.org/10.1016/s0091-679x(08)60661-5

    • PubMed
    • Google Scholar
28. 1. Schmid B
    2. Tripal P
    3. Fraaß T
    4. Kersten C
    5. Ruder B
    6. Grüneboom A
    7. Huisken J
    8. Palmisano R

    (2019) 3Dscript: animating 3D/4D microscopy data using a natural-language-based syntax

    Nature Methods 16:278–280.

    https://doi.org/10.1038/s41592-019-0359-1

    • PubMed
    • Google Scholar
29. 1. Singh SM
    2. Bandi S
    3. Mallela KMG

    (2017) The N-Terminal Flanking Region Modulates the Actin Binding Affinity of the Utrophin Tandem Calponin-Homology Domain

    Biochemistry 56:2627–2636.

    https://doi.org/10.1021/acs.biochem.6b01117

    • PubMed
    • Google Scholar
30. 1. Skau CT
    2. Kovar DR

    (2010) Fimbrin and tropomyosin competition regulates endocytosis and cytokinesis kinetics in fission yeast

    Current Biology 20:1415–1422.

    https://doi.org/10.1016/j.cub.2010.06.020

    • PubMed
    • Google Scholar
31. 1. Skoumpla K
    2. Coulton AT
    3. Lehman W
    4. Geeves MA
    5. Mulvihill DP

    (2007) Acetylation regulates tropomyosin function in the fission yeast Schizosaccharomyces pombe

    Journal of Cell Science 120:1635–1645.

    https://doi.org/10.1242/jcs.001115

    • PubMed
    • Google Scholar
32. 1. Taylor KA
    2. Taylor DW
    3. Schachat F

    (2000) Isoforms of α-Actinin from Cardiac, Smooth, and Skeletal Muscle Form Polar Arrays of Actin Filaments

    Journal of Cell Biology 149:635–646.

    https://doi.org/10.1083/jcb.149.3.635

    • Google Scholar
33. 1. Tebbs IR
    2. Pollard TD

    (2013) Separate roles of IQGAP Rng2p in forming and constricting the Schizosaccharomyces pombe cytokinetic contractile ring

    Molecular Biology of the Cell 24:1904–1917.

    https://doi.org/10.1091/mbc.e12-10-0775

    • PubMed
    • Google Scholar
34. 1. Toyoshima YY
    2. Kron SJ
    3. Spudich JA

    (1990) The myosin step size: measurement of the unit displacement per ATP hydrolyzed in an in vitro assay (actin/crossbridge/muscle contraction)

    PNAS 87:7130–7134.

    https://doi.org/10.1073/pnas.87.18.7130

    • Google Scholar
35. 1. Vassilopoulos S
    2. Gibaud S
    3. Jimenez A
    4. Caillol G
    5. Leterrier C

    (2019) Ultrastructure of the axonal periodic scaffold reveals a braid-like organization of actin rings

    Nature Communications 10:636217.

    https://doi.org/10.1038/s41467-019-13835-6

    • PubMed
    • Google Scholar
36. 1. Wachsstock DH
    2. Schwartz WH
    3. Pollard TD

    (1993) Affinity of alpha-actinin for actin determines the structure and mechanical properties of actin filament gels

    Biophysical Journal 65:205–214.

    https://doi.org/10.1016/S0006-3495(93)81059-2

    • PubMed
    • Google Scholar
37. 1. Way M
    2. Sanders M
    3. Garcia C
    4. Sakai J
    5. Matsudaira P

    (1995) Sequence and domain organization of scruin, an actin-cross-linking protein in the acrosomal process of Limulus sperm

    Journal of Cell Biology 128:51–60.

    https://doi.org/10.1083/jcb.128.1.51

    • Google Scholar
38. 1. Xu K
    2. Zhong G
    3. Zhuang X

    (2013) Actin, Spectrin, and associated proteins form a periodic cytoskeletal structure in axons

    Science 339:452–456.

    https://doi.org/10.1126/science.1232251

    • PubMed
    • Google Scholar

Acceptance 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 the manuscript are important in the cytoskeleton, cell division and cell morphogenesis fields, and the authors have addressed the points raised by reviewers in the revised manuscript.

Decision letter after peer review:

Thank you for submitting your article "Calponin-Homology Domain mediated bending of membrane associated actin filaments" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Pekka Lappalainen as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Vivek Malhotra as the Senior Editor.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below additional data are required in order to move forward with the publication. We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

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, Howver, in some cases the results are somewhat preliminary and lack essential controls. Thus, additional experiments and data analysis are required to strengthen the study.

Essential revisions:

1. More thorough analysis of actin-interactions of Curly constructs should be done. The authors discuss (pages 1 -2) interactions of full-length Curly and its various deletion constructs with actin filaments. However, actin-binding was not properly tested for any of the constructs used in this study. Thus, the authors should carry out better actin filament co-sedimentation assays with all constructs. The assays should be performed with a constant concentration of Curly, and varying the actin concentration (form 0 μm to e.g. 8 uM) to obtain binding curves, and to be able to compare the affinities of different constructs for actin filaments. Given that Curly is proposed to contain two actin-binding sites, the authors should also test, or at minimum discuss, if this protein bundles filaments. Also, do multiple filaments ever become incorporated into the same ring? Finally, the authors should examine if various curly constructs used in the study are monomeric of dimeric, because this may shed light on the mechanism by which Curly induces curvature on actin filaments.

2. The cell biology data presented in Figure 4 and Figure 4 —figure supplement 2 are not particularly convincing. The authors should 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 in ring formation. Additionally, are the cellular rings a similar size to those observed in vitro? Because EGFP-Curly most likely does not associate with the plasma membrane in cells, it is somewhat confusing how it can still induce the formation of actin rings. 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. This should be, at minimum, discussed in the manuscript.

3. 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. It would 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 in all cases, 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). The authors should confirm that the "n" values have enough statistical power to support the conclusions.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your article "Calponin-Homology Domain mediated bending of membrane associated actin filaments" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Pekka Lappalainen as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Vivek Malhotra as the Senior Editor.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential Revisions:

The actin-binding experiments are not particularly convincing, and the current data do not provide strong support for authors' conclusions about two actin-binding sites in curly. Therefore, the authors should delete all co-sedimentation data and comments about different actin binding sites from the manuscript.

Reviewer #1:

This is a revised version of a manuscript providing evidence that, when anchored to the membrane, 'curly' region of IQGAP proteins can stabilize actin filaments in a curved geometry. The manuscript is improved, and the authors have addressed many of my previous concerns. However, there are still few issues that should be addressed to strengthen the manuscript.

1. Based on the data presented in Figure 1 – supplement 1A, the quality of many protein preps is not sufficient for reliable biochemical experiments. The His6-Rng2 fragments contain huge amount of impurities, and are thus not suitable for biochemcial experiments. Thus, it is strongly recommended that the authors would either delete all data obtained by using these proteins from the manuscript, or alternatively make much better quality protein preps and repeat the experiments with these.

2. The actin filament co-sedimentation assay presented in Figure 1 – supplement 1B and C is not very convincing. I do not understand why the authors carried out this assay with a constant actin concentration and varying the concentration of Rng2(1-189). This is because much more reliable way to carry out such assay would be to keep the concentration of Rng2(1-189) constant, and vary the concentration of actin. This way the possible spontaneous aggregation of Rng2(1-189) would not affect the interpretation of the results. Therefore, these inconclusive data should be deleted from the manuscript. Unless the authors can measure the affinities of various protein constructs for F-actin, they should remove all speculations about different actin-binding sites from the manuscript text (lines 51-54, 63-84, and 195-198).

3. I am still not fully convinced about the CP-SNAP647 experiments (presented in Figure 3A and Figure 3 – supplement 1). This is because, in addition to CP-dots at filament ends, also other CP-SNAP647 dots not associated with the filament ends are visible in the images. Moreover, the supplementary video examples are not particularly convincing/high quality. Thus, the authors may also consider removing these data from the manuscript.

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.

The authors have addressed all of the comments I made in the first round of reviews. I have a few questions remaining, as detailed below:

(1) I am confused about the y-axis scale on the graph of the amount of filamentous actin each Rng2 construct tethered to the bilayers that the authors included in their response to "Essential Revisions" Question 1. What does it mean for the value of Aactin/Atotal to be 100? Is this graph different from the graph included in Figure 2 Supplement 1 Part B?

(2) I find it curious that the authors did not perform a low-speed co-sedimentation assay with their curly construct to test for filament bundling activity. This would have provided a more quantitative basis for their discussion of whether multiple actin filaments might ever be incorporated into the same ring structure.

(3) In response to my question about a schematic of a myosin II-bound actin ring (originally in Figure 3 Supplement 4 Part E), the authors state that they have made modifications to the model. However, I am unable to locate this schematic in the revised manuscript. Did the authors remove it?

(4) In response to my question about the effects of the counterclockwise curvature on the structure of the actin filaments, the authors state: "However, data by XXX indicates that binding of curly can loosen/disrupt the helical structure of actin filaments…". Who is XXX?

(5) In response to my question about binding/structural cooperativity, the authors state that they do not have direct evidence to determine whether binding of curly to actin filaments is cooperative, or if filament curvature arises from an additive effect of multiple independent binding events. To reflect this fact, they indicate that they have rephrased the following (original) statement using more cautious language:

"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."

However, their revised statement still appears to suggest a cooperative model for binding/bending without acknowledging the possibility of other binding models.

(6) The authors mention that they have added a quantification of ring frequency in curly-expressing cells compared to cells expressing only Lifeact-mCherry. Perhaps I have misunderstood their graphs, but I was unable to locate this analysis (or any micrographs or analysis with either wild-type, "EGFP alone" or "Lifeact-mCherry alone" cells).

Reviewer #3:

The authors have addressed all my previous concerns (reviewer 3 in the rebuttal).

However, I think the authors have left a place holder in their response to reviewer 2 (However, data by XXX indicates that binding of curly can loosen/ disrupt the helical structure of actin filaments leading to kinks. This would indicate towards the 2nd suggested mechanisms), as I do not know who XXX is meant to indicate.

Essential revisions:

1. More thorough analysis of actin-interactions of Curly constructs should be done. The authors discuss (pages 1 -2) interactions of full-length Curly and its various deletion constructs with actin filaments. However, actin-binding was not properly tested for any of the constructs used in this study. Thus, the authors should carry out better actin filament co-sedimentation assays with all constructs. The assays should be performed with a constant concentration of Curly, and varying the actin concentration (form 0 μm to e.g. 8 uM) to obtain binding curves, and to be able to compare the affinities of different constructs for actin filaments. Given that Curly is proposed to contain two actin-binding sites, the authors should also test, or at minimum discuss, if this protein bundles filaments. Also, do multiple filaments ever become incorporated into the same ring? Finally, the authors should examine if various curly constructs used in the study are monomeric of dimeric, because this may shed light on the mechanism by which Curly induces curvature on actin filaments.

We have performed sedimentation assay with Rng2(1-189) and with Rng2(150-250) as these two constructs are the most important ones for the study. Full length, because it bends actin into rings and the Rng2(150-250) since it contains a previously unreported actin binding region. A problem was that inspection of Coomassie-stained gels of our truncations of Rng2(1-189) displayed low expression and multiple bands indicating protein instability and degradation. This made it impractical for us to quantify their actin binding affinity using the co-sedimentation assay (added to Figure 2-sup Figure 1).

However, we could find a binding constant of Kd = 1 μM for Rng2(1-189) similar to the value reported by Hayakawa et al., (2020). Unfortunately, the Rng2(150-250) construct aggregated and accumulated in the pellet which made it impossible to reliably estimate a binding coefficient for the construct (added to Figure 1-sup Figure 1).

As an alternative to the co-sedimentation assay, we included a quantification of the amount of F-actin that each construct tethered to SLBs to compare binding affinities of the different constructs. (added to Figure 2-sup Figure 2)

We do not have evidence that rng2(1-189) is a potent actin bundling protein when tethered to SLBs as most tethered actin filaments were single and did not bundle. However, any of the apparent actin bundles were straight and did not display high curvatures. Of all constructs studied here the construct with the highest propensity to induce actin bundling was S. cerevisiae -iqg1[1-330].

Regarding whether curly is a monomer or dimer, we revisited our data using fluorescently labelled curly, and did not find evidence for dimer formation as the majority of the labelled protein covered the SLB homogeneously and no bright puncta were observed. However, we did not have the means to perform fluorescence correlation spectroscopy to quantify the number of monomers vs. multimers on the SLB and would intend to do this in future studies.

It is a good question whether multiple actin filaments incorporate into the same ring structure. We observed that the same actin filaments can wind around the same ring multiple times. Given the low actin densities used in the study, it is very rare that an actin filament landed on top of a ring, but we would not rule out that multiple actin filaments form along the same ring. However, given limitations of fluorescence microscopy, we would not be able to say whether this is due to the binding of one actin filament to another one promoted by rng2(1-189). Future studies using e.g. EM would be very helpful in answering this question.

We added these aspects in the discussion.

2. The cell biology data presented in Figure 4 and Figure 4 —figure supplement 2 are not particularly convincing. The authors should 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 in ring formation. Additionally, are the cellular rings a similar size to those observed in vitro? Because EGFP-Curly most likely does not associate with the plasma membrane in cells, it is somewhat confusing how it can still induce the formation of actin rings. 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. This should be, at minimum, discussed in the manuscript.

We did only show a few cell images as we only wanted to show that Curly affects the cell cortex, and we agree that more data would be helpful. We have now imaged cells expressing lifeact-mCherry as an actin marker together with EGFP-Curly, EGFP alone, EGFP-rng2(150-250) as well as membrane targeted protein constructs using the CAAX-motif using lattice light sheet microscopy. As reported in the previous version of the manuscript, EGFP-Curly expression leads to a higher occurrence of highly bent actin filaments. A possible explanation could be that the additional curly enhances the effect of endogenous IQGAP1 in the cells and binds preferentially at highly curved actin filaments (Figure 4, Figure 4.-supplement Figure 2).

The EGFP-rng2(150-250) construct was mainly cytoplasmic. We also thought that membrane anchored curly would induce stronger actin ring formation in cells. However, adding the CAAX domain did result in high cell death and very little protein location at the plasma membrane. Interestingly, colleagues in the Mayor laboratory at NCBS have also observed similar problems when expressing e.g. the ezrin actin binding domain linked to a transmembrane protein pointing to a general problem of membrane tethered proteins with high affinity to actin. In future, we are planning to use e.g. the rapamycin inducible FRB-FKBP linker system to target curly to the plasma membrane in an efficient and controlled way, but this would go beyond the scope of this paper (this is now part of the discussion).

We quantified the actin curvatures in cells expressing EGFP-curly (Figure 4F in the previous manuscript) that turn out to be very similar to the ones found in vitro, we have added a quantification of ring frequency in curly expressing cells compared to cells expressing lifeact-mCherry only expressing ones.

3. 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. It would 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 in all cases, 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). The authors should confirm that the "n" values have enough statistical power to support the conclusions.

We thought to highlight the data on filament curvature because of which we plotted those as graphs. As suggested, we have now added accompanying panels in the figures for the other quantitations. To ease the reading flow, we have removed the stated values of the key parameters (curvature, number of rings, number of samples) from the main text and added a table summarising them. In addition, the relevant numbers are also listed in the figure legends.

Regarding the statistics, we are very sorry that the reviewers were of the impression that only a single FOV was analysed. We have analysed a minimum of 10 FOVs of two or more independent experiments. The low number of filaments is a result of low binding of the protein construct to actin and do not reflect a lower number of FOVs.

Since the difference in actin curvature is very striking when comparing His6-curly to its truncated counterparts was obvious we did not perform more experiments in the first run (e.g. in case of the Rng2(150-25) construct with linear filaments). We have added some more experiments with the Rng2(1-189)Δ(154-161) construct which confirmed the previous findings. A statistical comparison between the curvatures of the Rng2 truncations with the original Rng2(1-189) using one-way Anova test is included in Figure 2-Figure sup. 1 A. Here we compare the average values of independent experiments (three for each condition, measuring 10 FoV for each).

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Reviewer #1:

1. Based on the data presented in Figure 1 – supplement 1A, the quality of many protein preps is not sufficient for reliable biochemical experiments. This is because His6-Rng2(1-189)del(154-160), His6-Rng2(150-250), and His6-Rng2(41-147) samples appear to contain huge amount of impurities, and are thus not suitable for biochemical experiments. Thus, it is strongly recommended that the authors would either delete all data obtained by using these proteins from the manuscript, or alternatively make much better quality protein preps and repeat the experiments with these.

We appreciate your critical assessment of the biochemical data and agree that it would have been stronger if the protein purity/ stability would have been better. We agree fully with the reviewers that this data is not essential for the central message of the paper and have removed the data from this manuscript. We will reassess the protein expression and add further purification steps (e.g. size exclusion) to repeat the experiments which will form a future publication.

2. The actin filament co-sedimentation assay presented in Figure 1 – supplement 1B and C is not very convincing. I do not understand why the authors carried out this assay with a constant actin concentration and varying the concentration of Rng2(1-189). This is because much more reliable way to carry out such assay would be to keep the concentration of Rng2(1-189) constant, and vary the concentration of actin. This way the possible spontaneous aggregation of Rng2(1-189) would not affect the interpretation of the results. Therefore, these inconclusive data should be deleted from the manuscript. Unless the authors can measure the affinities of various protein constructs for F-actin, they should remove all speculations about different actin-binding sites from the manuscript text (lines 51-54, 63-84, and 195-198).

We are sorry to learn that reviewer#1 did not find the co-sedimentation data convincing. It is true that it could have been better, but we followed the actin-co-sedimentation assay as described in Heier et al., 2017, Jove (https://www.jove.com/t/55613/measuring-protein-binding-to-f-actin-by-co-sedimentation). And the assay was run similarly in (Hayakawa, Y. et al., Actin binding domain of Rng2 strongly inhibits actin movement on myosin II HMM through structural changes of actin filaments. bioRxiv (2020) doi:10.1101/2020.04.14.04104) who report an actin binding affinity of ~ 1 μM for the Rng2(1-189) like what we find.

We will remove the data from the paper though and will repeat the co-sedimentation as suggested by the reviewer for future work. This together with studies of the other protein constructs (after optimisation of the purification) will then be more insightful and might form a new publication.

3. I am still not fully convinced about the CP-SNAP647 experiments (presented in Figure 3A and Figure 3 – supplement 1). This is because, in addition to CP-dots at filament ends, also other CP-SNAP647 dots not associated with the filament ends are visible in the images. Moreover, the supplementary video examples are not particularly convincing/high quality. Thus, the authors may also consider removing these data from the manuscript.

We understand that it can be confusing to see non-actin associated fluorescent spots. Unfortunately, it seems that the BG647 fluorophore tends to associate with lipid bilayers. However, these spots can be still distinguished from actin associated dots (that move together with actin) and we did not want to alter the image background to suggest there wouldn’t be any unspecific binding of CP-SNAP647 to the lipid bilayer. Even though there were some actin filaments without CP and some CP without actin, we did not detect any actin filaments with CP that turned the other way, and all CP positive actin filaments, where CP localisation could be clearly associated to an actin filament end, were showing an anti-clockwise orientation. If we would have picked up some random co-localisation of CP and actin, one would expect a more equal distribution between clock- and anti-clockwise orientation.

In addition, our myosin data supports the observation that curly recognises the actin polarisation.

Reviewer #2:

(1) I am confused about the y-axis scale on the graph of the amount of filamentous actin each Rng2 construct tethered to the bilayers that the authors included in their response to "Essential Revisions" Question 1. What does it mean for the value of Aactin/Atotal to be 100? Is this graph different from the graph included in Figure 2 Supplement 1 Part B?

A_Actin/ A_total = 100 would mean that the entire field of view is covered by actin. We could have replaced the ‘total’ with ‘FoV’, but the data would be not shown.

(2) I find it curious that the authors did not perform a low-speed co-sedimentation assay with their curly construct to test for filament bundling activity. This would have provided a more quantitative basis for their discussion of whether multiple actin filaments might ever be incorporated into the same ring structure.

Yes, this will be an interesting aspect to look at, and we will do this in future.

(3) In response to my question about a schematic of a myosin II-bound actin ring (originally in Figure 3 Supplement 4 Part E), the authors state that they have made modifications to the model. However, I am unable to locate this schematic in the revised manuscript. Did the authors remove it?

Sorry for this confusion, but we finally decided to not put in any model as we did not have any strong data now to support it. So, we deemed it more sensible to leave it out.

(4) In response to my question about the effects of the counterclockwise curvature on the structure of the actin filaments, the authors state: "However, data by XXX indicates that binding of curly can loosen/disrupt the helical structure of actin filaments…". Who is XXX?

Our apologies for this omission, the paper referred to is Hayakawa, Y. et al., Actin binding domain of Rng2 strongly inhibits actin movement on myosin II HMM through structural changes of actin filaments. bioRxiv (2020) doi:10.1101/2020.04.14.04104

(5) In response to my question about binding/structural cooperativity, the authors state that they do not have direct evidence to determine whether binding of curly to actin filaments is cooperative, or if filament curvature arises from an additive effect of multiple independent binding events. To reflect this fact, they indicate that they have rephrased the following (original) statement using more cautious language:

"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."

However, their revised statement still appears to suggest a cooperative model for binding/bending without acknowledging the possibility of other binding models.

Our apologies, the statement was now altered by removing the mention of a cooperative process.

“Importantly, the uni-directional bending supports the hypothesis that the binding site of curly with actin filaments defines an orientation with the propagation of an established curved trajectory.”

(6) The authors mention that they have added a quantification of ring frequency in curly-expressing cells compared to cells expressing only Lifeact-mCherry. Perhaps I have misunderstood their graphs, but I was unable to locate this analysis (or any micrographs or analysis with either wild-type, "EGFP alone" or "Lifeact-mCherry alone" cells).

A box plot showing the number of rings per cell at the basal site for HEK293 cells transfected with EGFP-curly, EGFP-curly-CAAX and RPE-1 with EGFP-Curly was shown in Figure 4 – supplement figure 2. We have added now also the data for LifeAct-mCherry expressing cells that we forgot to add in the previous submission.

Reviewer #3:

The authors have addressed all my previous concerns (reviewer 3 in the rebuttal).

However, I think the authors have left a place holder in their response to reviewer 2 (However, data by XXX indicates that binding of curly can loosen/ disrupt the helical structure of actin filaments leading to kinks. This would indicate towards the 2nd suggested mechanisms), as I do not know who XXX is meant to indicate.

Our apologies for this omission, the paper referred to is Hayakawa, Y. et al., Actin binding domain of Rng2 strongly inhibits actin movement on myosin II HMM through structural changes of actin filaments. bioRxiv (2020) doi:10.1101/2020.04.14.04104

Author details

1. Saravanan Palani

   1. Centre for Mechanochemical Cell Biology and Warwick Medical School, Division of Biomedical Sciences, Coventry, United Kingdom
   2. Department of Biochemistry, Division of Biological Sciences, Indian Institute of Science, Bangalore, India

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1893-6777

2. Sayantika Ghosh

   Centre for Mechanochemical Cell Biology and Warwick Medical School, Division of Biomedical Sciences, Coventry, United Kingdom

   Contribution

   Investigation, Mammalian cell culture and transfections

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1399-1168

3. Esther Ivorra-Molla

   Centre for Mechanochemical Cell Biology and Warwick Medical School, Division of Biomedical Sciences, Coventry, United Kingdom

   Contribution

   Investigation, Mammalian cell culture and transfections

   Competing interests

   No competing interests declared

4. Scott Clarke

   Centre for Mechanochemical Cell Biology and Warwick Medical School, Division of Biomedical Sciences, Coventry, United Kingdom

   Contribution

   Investigation, Lattice-light-sheet microscopy

   Competing interests

   No competing interests declared

5. Andrejus Suchenko

   Centre for Mechanochemical Cell Biology and Warwick Medical School, Division of Biomedical Sciences, Coventry, United Kingdom

   Contribution

   Investigation, Help in protein purification

   Competing interests

   No competing interests declared

6. Mohan K Balasubramanian

   Centre for Mechanochemical Cell Biology and Warwick Medical School, Division of Biomedical Sciences, Coventry, United Kingdom

   Contribution

   Conceptualization, Supervision, Funding acquisition, Validation, Methodology, Writing - original draft, Project administration

   For correspondence

   m.k.balasubramanian@warwick.ac.uk

   Competing interests

   Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0002-1292-8602

7. Darius Vasco Köster

   Centre for Mechanochemical Cell Biology and Warwick Medical School, Division of Biomedical Sciences, Coventry, United Kingdom

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   D.Koester@warwick.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8530-5476

• 2,419

  Page views

• 342

  Downloads

• 11

  Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.