S3D cofilin binds actin filaments more weakly than WT cofilin but with higher cooperativity
|WT||9.7 ± 2.0||5.3 ± 1.1||0.35 ± 0.51||17.3 ± 3.5||4.6 ± 6.7|
|S3D||154.1 ± 20.3||47.3 ± 6.2||0.48 ± 0.15||4.2 ± 1.2||0|
WT and S3D cofilin binding to actin filaments is linked to cation release
|Cofilin||n (Ka)||n (ω)|
|S3D||2.3 ± 0.7 K+||∼0|
|0.9 ± 0.1 Mg2+||∼0|
|WT||1.7 ± 0.2 K+||∼0|
|0.7 ± 0.1 Mg2+||∼0|
|mm||oC||kJ mol−1||kJ mol−1 T−1||degree cm2 mol−1||degree cm2 mol−1|
S3D cofilin weakly severs actin filaments over a broad range of binding densities
|Filament bending persistence length Lp|
|Intersubunit torsional rigidity Csub|
|Intersubunit torsional constant α|
|μm||newtons m2 radians−1||newtons m2 radians−1|
|Bare||8.3 ± 0.4||1.09 ± 0.07 × 10−27||3.95 ± 0.26 × 10−19|
|WT||2.6 ± 0.3||0.21 ± 0.05 × 10−27||0.75 ± 0.02 × 10−19|
|S3D||6.3 ± 1.2||1.26 ± 0.17 × 10−27||4.60 ± 0.63 × 10−19|
S3D cofilin severs at boundaries more slowly than WT cofilin
where R = , Lavg,spon is average filament length of bare actin, and k′dc, k′sc, and k′iso are the rate constants (relative to the intrinsic severing rate constant of bare actin) for severing at cofilins bound in a doubly contiguous (i.e. within a bound cofilin cluster ), singly contiguous (at edge of bound cofilin cluster or ), or isolated (non-contiguous ) binding mode, respectively. The best fits of the WT and S3D cofilin data to Equation 1 (smooth lines through data in Fig. 4A) indicate that severing at boundaries between bare and S3D cofilin-decorated segments occurs >4-fold more slowly than at bare–WT cofilin boundaries (Table 1, ksc). Therefore, the observed severing deficiency of S3D cofilin originates from a slower severing rate constant at boundaries as well as a reduction in overall boundary density.
- Ngo K.X.
- Kodera N.
- Katayama E.
- Ando T.
- Uyeda T.Q.P.
Cofilin competitors promote filament severing by S3D cofilin
S3D cofilin weakly affects filament bending and twisting dynamics
|Anisotropy decay model-independent analysis: sum of exponentials|
Total phosphorescence anisotropy decays after a 5-μs dead time were fitted to a double exponential in the form r(t) = r1 e−t/φ1 + r2 e−t/φ2 + r∞ to obtain the initial r0 = r1 + r2 + r∞ and final rω anisotropy values at time infinity. The percentage in parenthesis is relative to the initial total anisotropy value of bare actin filaments.
|r0||0.094 ± 0.002 (100%)||0.079 ± 0.003 (84%)||0.078 ± 0.005 (83%)|
|r∞||0.038 (40%)||0.030 (32%)||0.036 ± 0.004 (38%)|
S3D cofilactin has a structure similar to that of WT cofilactin
Ser-3 modification repositions the cofilin N terminus away from actin
Effects of cofilin Ser-3 modification on actin filament-binding interactions
Role of the cofilin N terminus in modulating actin filament mechanical properties
Effects of Ser-3 modification reveal factors contributing to cofilin-severing efficiency
Materials and methods
Protein expression, purification, and labeling
Equilibrium binding assays
where F0 and F∞ are the bare and cofilin-decorated pyrene-actin filament fluorescence values. The cofilin binding density (ν) satisfies the following implicit equation for non-cooperative binding (i.e. ω = 1) and cooperative binding with nearest neighbor interactions (i.e. ω ≠ 1), respectively (
for ω = 1 or
where ; Kd is the equilibrium constant for binding to an isolated site (i.e. intrinsic affinity for binding with no neighbors); ω is a dimensionless cooperativity parameter; n is the binding stoichiometry (n = 1 cofilin per actin filament subunit); and Ctot and Atot are total cofilin and actin concentrations, respectively. The measured Ctot-dependent fluorescence data were fitted to Equation 2 and either Equation 3 or Equation 4 following a numerical procedure with parameters Kd and ω unconstrained. During fitting iterations, ν is calculated using Equation 3 if the testing parameter ω = 1 and using Equation 4 if ω ≠ 1. WT and S3D cofilin equilibrium binding with unlabeled, pyrene-labeled (supplemental Fig. S5), or Alexa-labeled actin filaments (
Boundary density calculation
where the terms f and b refer to free and bound cofilin, respectively (see “Appendix” for specific definitions of each term). These expressions have been used previously (
Fluorescence microscopy, bending mechanics, and severing assays
where s is the filament segment length and θ is the tangent angle along the filament (
Phosphorescence intensity and anisotropy decay measurement and analysis
where θobs is the observed molar ellipticity value; θn and θu are the molar ellipticities of native (folded) and unfolded species, respectively; ΔH0′u and ΔCp0′u are the standard enthalpy and heat capacity changes (at constant pressure) associated with the unfolding reaction; R is the gas constant (8.31 J K−1 mol−1); T is the scanning temperature in Kelvin; and Tm is the transition (melting) temperature of unfolding.
Electron cryomicroscopy and structure refinement
Molecular dynamics simulations
Interface model for cofilin severing actin filaments
where ksever and kanneal are the average, fundamental, filament-severing and -annealing rate constants, respectively, and dN is the change in total filament number. The term ksever represents a microscopic rate constant, such that the overall observed severing rate constant is equal to ksever times the number of potential severing sites.
where n is the total number of actin filament subunits. At equilibrium, dN/dt = 0, and Equation A2 can be solved in terms of the average filament number,
and the average filament length (average number of actin subunits per filament) (
where k and sİ are the severing rate constant and mole fraction of cofilactin–cofilactin (c-c), cofilactin–actin (c-a), or actin–actin (a-a) interfaces (indicated by subscripts), respectively, whereas k′c-c and k′c-a represent the corresponding severing rate constants relative to that at an actin–actin interface. Applying Equation A5 allows Equation A4 to be rewritten as follows,
represents the average filament length of bare actin (i.e. in the absence of regulatory proteins) defined by the intrinsic bare actin filament severing and annealing reactions.
or for cooperative binding
equals unity, indicating that there is no counting problem in the derived expressions.
Critical cluster size for severing >3 predicts an asymmetric cofilin binding density dependence of severing activity
- Ngo K.X.
- Kodera N.
- Katayama E.
- Ando T.
- Uyeda T.Q.P.
For completeness, using the same technique when formulizing Equations A8–A10, we provide expressions for the fractions of total bound cofilin, sİ (c ≥ cİcrit ≥ 1) = sİb(c ≥ cİcrit ≥ 1) + sİdc(c ≥ cİcrit ≥ 1) and cofilin bound doubly contiguously, sİdc(c ≥ cİcrit ≥ 1), for a given critical cluster size. Note that doubly contiguous cofilin exists only for clusters ≥3.
where the following relation is applied.
- Proteins of the ADF/cofilin family: essential regulators of actin dynamics.Annu. Rev. Cell. Dev. Biol. 1999; 15: 185-230
- Cellular motility driven by assembly and disassembly of actin filaments.Cell. 2003; 112: 453-465
- Cellular functions of the ADF/cofilin family at a glance.J. Cell Sci. 2016; 129: 3211-3218
- Cofilin binding to muscle and non-muscle actin filaments: isoform-dependent cooperative interactions.J. Mol. Biol. 2005; 346: 557-564
- How cofilin severs an actin filament.Biophys. Rev. 2009; 1: 51-59
- Cofilin tunes the nucleotide state of actin filaments and severs at bare and decorated segment boundaries.Curr. Biol. 2011; 21: 862-868
- Cofilin-linked changes in actin filament flexibility promote severing.Biophys. J. 2011; 101: 151-159
- Cofilin changes the twist of F-actin: implications for actin filament dynamics and cellular function.J. Cell Biol. 1997; 138: 771-781
- Actin depolymerizing factor stabilizes and existing state of F-actin and can change the tilt of F-actin subunits.J. Cell Biol. 2001; 153: 75-86
- Remodeling of actin filaments by ADF/cofilin proteins.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 20568-20572
- Cofilin increases the bending flexibility of actin filaments: Implications for severing and cell mechanics.J. Mol. Biol. 2008; 381: 550-558
- Cofilin increases the torsional flexibility and dynamics of actin filaments.J. Mol. Biol. 2005; 353: 990-1000
- Biophysics of actin filament severing by cofilin.FEBS Lett. 2013; 587: 1215-1219
- Site-specific cation release drives actin filament severing by vertebrate cofilin.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 17821-17826
- Actin mechanics and fragmentation.J. Biol. Chem. 2015; 290: 17137-17144
- Mechanical heterogeneity favors fragmentation of strained actin filaments.Biophys. J. 2015; 108: 2270-2281
- Actin filament strain promotes severing and cofilin dissociation.Biophys. J. 2017; 112: 2624-2633
- Reactivation of phosphorylated actin depolymerizing factor and identification of the regulatory site.J. Biol. Chem. 1995; 270: 17582-17587
- Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase.Nature. 1998; 393: 805-809
- Phosphorylation of Ser-3 of cofilin regulates its essential function on actin.Genes Cells. 1996; 1: 73-86
- Uncoupling actin filament fragmentation by cofilin from increased subunit turnover.J. Mol. Biol. 2000; 298: 649-661
- Kinetic analysis of the interaction of actin-depolymerizing factor (ADF)/cofilin with G- and F-actins: comparison of plant and human ADFs and effect of phosphorylation.J. Biol. Chem. 1998; 273: 20894-20902
- Instantaneous inactivation of cofilin reveals its function of F-actin disassembly in lamellipodia.Mol. Biol. Cell. 2013; 24: 2238-2247
- Phosphorylation of Acanthamoeba actophorin (ADF/cofilin) blocks interaction with actin without a change in atomic structure.J. Mol. Biol. 2000; 295: 203-211
- Cooperative and noncooperative binding of protein ligands to nucleic acid lattices: experimental approaches to the determination of thermodynamic parameters.Biochemistry. 1986; 25: 1226-1240
- Theoretical aspects of DNA-protein interactions: co-operative and non-co-operative binding of large ligands to a one-dimensional homogeneous lattice.J. Mol. Biol. 1974; 86: 469-489
- Energetics and kinetics of cooperative cofilin-actin filament interactions.J. Mol. Biol. 2006; 361: 257-267
- Regulation of actin by ion-linked equilibria.Biophys. J. 2013; 105: 2621-2628
- Single-molecule imaging and kinetic analysis of cooperative cofilin-actin filament interactions.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 9810-9815
- Identification of cation-binding sites on actin that drive polymerization and modulate bending stiffness.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 16923-16927
- Competitive displacement of cofilin can promote actin filament severing.Biochem. Biophys. Res. Commun. 2013; 438: 728-731
- ADF/cofilin accelerates actin dynamics by severing filaments and promoting their depolymerization at both ends.Curr. Biol. 2017; 27: 1956-1967.e7
- Cofilin-induced unidirectional cooperative conformational changes in actin filaments revealed by high-speed atomic force microscopy.eLife. 2015;
- Architecture dependence of actin filament network disassembly.Curr. Biol. 2015; 25: 1437-1447
- Mechanism of actin filament turnover by severing and nucleation at different concentrations of ADF/cofilin.Mol. Cell. 2006; 24: 13-23
- Actin filament severing by cofilin.J. Mol. Biol. 2007; 365: 1350-1358
- Kinetics and thermodynamics of phalloidin binding to actin filaments from three divergent species.Biochemistry. 1996; 35: 14054-14061
- Transient kinetic analysis of rhodamine phalloidin binding to actin filaments.Biochemistry. 1994; 33: 14387-14392
- Actin dynamics: tropomyosin provides stability.Curr. Biol. 2002; 12: R523-R525
- The effect of phalloidin and jasplakinolide on the flexibility and thermal stability of actin filaments.FEBS Lett. 2004; 565: 163-166
- Tropomyosin isoforms specify functionally distinct actin filament populations in vitro.Curr. Biol. 2017; 27: 705-713
- Multi-platform compatible software for analysis of polymer bending mechanics.PLoS One. 2014; 9: e94766
- Lakowicz J. Principles of Fluorescence Spectroscopy. Plenum Press, New York1983: 98-353
- Differences in structural dynamics of muscle and yeast actin accompany differences in functional interactions with myosin.Biochemistry. 1999; 38: 14860-14867
- Solution structures and dynamics of ADF/cofilins UNC-60A and UNC-60B from Caenorhabditis elegans.Biochem. J. 2015; 465: 63-78
- Solution structure of human cofilin: actin binding, pH sensitivity, and relationship to actin-depolymerizing factor.J. Biol. Chem. 2004; 279: 4840-4848
- Structural analysis of human cofilin 2/filamentous actin assemblies: atomic-resolution insights from magic angle spinning NMR spectroscopy.Sci. Rep. 2017; 7: 44506
- Essential functions and actin-binding surfaces of yeast cofilin revealed by systematic mutagenesis.EMBO J. 1997; 16: 5520-5530
- The kinetics of cooperative cofilin binding reveals two states of the cofilin-actin filament.Biophys. J. 2010; 98: 1893-1901
- A genetic dissection of Aip1p's interactions leads to a model for Aip1p-cofilin cooperative activities.Mol. Biol. Cell. 2006; 17: 1971-1984
- Xenopus actin-interacting protein 1 (XAip1) enhances cofilin fragmentation of filaments by capping filament ends.J. Biol. Chem. 2002; 277: 43011-43016
- Aip1p interacts with cofilin to disassemble actin filaments.J. Cell Biol. 1999; 145: 1251-1264
- Single-molecule imaging of a three component ordered actin disassembly mechanism.Nat. Commun. 2015; 6: 7202
- Calculation of protein extinction coefficients from amino acid sequence data.Anal. Biochem. 1989; 182: 319-326
- A systematic nomenclature for mammalian tropomyosin isoforms.J. Muscle Res. Cell Motil. 2015; 36: 147-153
- A small molecule inhibitor of tropomyosin dissociates actin binding from tropomyosin-directed regulation of actin dynamics.Sci. Rep. 2016; 6: 19816
- The effect of two actin depolymerizing factors (ADF/cofilins) on actin filament turnover: pH sensitivity of F-actin binding by human ADF, but not of Acanthamoeba actophorin.Eur. J. Biochem. 1998; 256: 388-397
- Torsion dynamics and depolarization of fluorescence of linear macromolecules I: theory and application to DNA.Chem. Phys. 1979; 41: 35-59
- Rotational diffusion of deformable macromolecules with mean local cylindrical symmetry.Chem. Phys. 1984; 84: 71-96
- Microsecond rotational dynamics of actin: spectroscopic detection and theoretical simulation.J. Mol. Biol. 1996; 255: 446-457
- Myosin isoform determines the conformational dynamics and cooperativity of actin filaments in the strongly bound actomyosin complex.J. Mol. Biol. 2010; 396: 501-509
- Using circular dichroism spectra to estimate protein secondary structure.Nat. Protoc. 2006; 1: 2876-2890
- Ligand binding affinity determined by temperature-dependent circular dichroism: cyclin-dependent kinase 2 inhibitors.Anal. Biochem. 2005; 345: 187-197
- MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy.Nat. Methods. 2017; 14: 331-332
- Gctf: real-time CTF determination and correction.J. Struct. Biol. 2016; 193: 1-12
- Helical reconstruction in RELION.J. Struct. Biol. 2017; 198: 163-176
- RELION: implementation of a Bayesian approach to cryo-EM structure determination.J. Struct. Biol. 2012; 180: 519-530
- UCSF Chimera: a visualization system for exploratory research and analysis.J. Comput. Chem. 2004; 25: 1605-1612
- Molecular origins of cofilin-linked changes in actin filament mechanics.J. Mol. Biol. 2013; 425: 1225-1240
- VMD: visual molecular dynamics.J. Mol. Graph. 1996; 14 (27-28): 33-38
- Extending the treatment of backbone energetics in protein force fields: limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations.J. Comput. Chem. 2004; 25: 1400-1415
- Scalable molecular dynamics with NAMD.J. Comput. Chem. 2005; 26: 1781-1802
- Cations stiffen actin filaments by adhering a key structural element to adjacent subunits.J. Phys. Chem. B. 2016; 120: 4558-4567
- GROMACS: a message-passing parallel molecular dynamics implementation.Comput. Phys. Commun. 1995; 91: 43-56
- Particle mesh Ewald: an N*log(N) method for Ewald sums in large systems.J. Chem. Phys. 1993; 98: 10089-10092
- Canonical sampling through velocity rescaling.J. Chem. Phys. 2007; 126 (014101)
This work was supported by National Institutes of Health R01 Grants GM097348 (to E. M. D. L. C.), GM110533001 (to C. V. S.), and AR032961 (to D. D. T.); American Cancer Society Grant IRG5801255 (to C. V. S.); and the Department of Defense Army Research Office through MURI Grant W911NF1410403 (on which G. A. V. and E. M. D. L. C. are co-investigators). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
This article contains supplemental Table S1, Figs. S1–S5, and Movies S1 and S2.
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