Flexibility of Actin Filaments Derived from Thermal Fluctuations

Isambert, Hervé; Venier, Pascal; Maggs, A. C.; Fattoum, Abdelatif; Kassab, Ridha; Pantaloni, Dominique; Carlier, Marie‐France

doi:10.1074/jbc.270.19.11437

Cited by 566 publications

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References 73 publications

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“…3 Cofilin lowers the persistence length of actin filaments ∼5-fold ( Table 1). The persistence length of native actin filaments (without phalloidin) under our experimental conditions is 9.8 ±0.1 µm (Table1); comparable to a previously reported value of 9.0±0.5 µm measured under slightly different buffer conditions, 19 and the value predicted from molecular dynamics simulations. [20][21][22] 23 and vimentin 24,25 ).…”

Section: Resultssupporting

confidence: 75%

“…contour length of a segment) over which correlation between the corresponding tangent angles (θ(s)) of a fluctuating semi-flexible polymer, such as an actin filament, are lost in three-dimensional space. 17,18 The average cosine correlation function (〈C(s)〉) defines the L p -dependence of tangent angle (θ) correlation of any segment (arc) length (s) along the contour length (L) of a filament constrained to two-dimensional motion within a plane according to 19 :…”

Section: Resultsmentioning

confidence: 99%

“…19 The stiffness of a tropomyosin-decorated actin filament can be accounted for 26 by the increase in filament mass (I); there is little or no effect on the filament Young's elastic modulus (E). Cofilin binding also increases the filament mass (∼40%) and second moment of inertia (Table 1), but differs from tropomyosin in that it dramatically lowers the Young's filament elastic modulus ( Table 1).…”

Section: Discussionmentioning

confidence: 99%

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Cofilin Increases the Bending Flexibility of Actin Filaments: Implications for Severing and Cell Mechanics

McCullough

Blanchoin

Martiel

et al. 2008

Journal of Molecular Biology

206

254

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We determined the flexural (bending) rigidities of actin and cofilactin filaments from a cosine correlation function analysis of their thermally driven, two-dimensional fluctuations in shape. The persistence length of actin filaments is 9.8 µm, corresponding to a flexural rigidity of 0.040 pN µm 2 . Cofilin binding lowers the persistence length ∼5-fold to a value of 2.2 µm and the filament flexural rigidity to 0.0091 pN µm 2 . That cofilin-decorated filaments are more flexible than native filaments despite an increased mass indicates that cofilin binding weakens and redistributes stabilizing subunit interactions of filaments. We favor a mechanism in which the increased flexibility of cofilin-decorated filaments results from the linked dissociation of filament-stabilizing ions and reorganization of actin subdomain 2 and as a consequence promotes severing due to a mechanical asymmetry. Knowledge of the effects of cofilin on actin filament bending mechanics, together with our previous analysis of torsional stiffness, provide a quantitative measure of the mechanical changes in actin filaments associated with cofilin binding, and suggest that the overall mechanical and forceproducing properties of cells can be modulated by cofilin activity.

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Section: Resultssupporting

confidence: 75%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Cofilin Increases the Bending Flexibility of Actin Filaments: Implications for Severing and Cell Mechanics

McCullough

Blanchoin

Martiel

et al. 2008

Journal of Molecular Biology

206

254

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“…To achieve this aim, we first characterized the effect of phalloidin and nucleotide analogues separately. Our experiments showed that the binding of ADP.BeF x , ADP.AlF 4 , or phalloidin stabilized the structure of actin filaments, in agreement with previous studies (37,38). In the presence of 3 mM BeSO 4 and 10 mM NaF, the T m was greater (84.8 °C) than in the absence of them (64.1 °C) (Figure 2A Figure 2B).…”

supporting

confidence: 81%

Noncooperative Stabilization Effect of Phalloidin on ADP.BeF_x- and ADP.AlF₄-Actin Filaments

et al. 2008

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Actin plays important roles in eukaryotic cell motility. During actin polymerization, the actinbound ATP is hydrolyzed to ADP and P i . We carried out differential scanning calorimetry experiments to characterize the cooperativity of the stabilizing effect of phalloidin on actin filaments in their ADP.P i state. The ADP.P i state was mimicked by using ADP.BeF x or ADP.AlF 4 . The results showed that the binding of the nucleotide analogues or phalloidin stabilized the actin filaments to a similar extent when added separately. Phalloidin binding to ADP.BeF x -or ADP.AlF 4 -actin filaments further stabilized them, indicating that the mechanism by which phalloidin and the nucleotide analogues affect the filament structure was different. The results also showed that the stabilization effect of phalloidin binding to ADP.BeF x or ADP.AlF 4 -bound actin filaments was not cooperative. Since the effect of phalloidin binding was cooperative in the absence of these nucleotide analogues, these results suggest that the binding of ADP.BeF x or ADP.AlF 4 to the actin modified the protomer-protomer interactions along the actin filaments.Actin is one of the main components of the cytoskeleton and plays important roles in the motility of eukaryotic cells (1-7). The actin monomer can bind a nucleotide in complex with a divalent cation in the cleft between the two main domains of the protein (Figure 1) (8). During polymerization, the ATP is hydrolyzed to ADP and P i 1 (9-16). The ADP.P i state is transient as the inorganic phosphate product is released from actin after polymerization. For † This study was supported by grants from the Hungarian Scientific Research Fund [OTKA Grants K60186 and K60968 (M.Ny.)]. The SETARAM Micro DSC-II instrument was purchased with a grant MATERIALS AND METHODS ChemicalsKCl, MgCl 2 , CaCl 2 , MOPS, EGTA, AlCl 3 , and NaF were purchased from SIGMA-Aldrich (Budapest, Hungary). ATP, ADP, and β-mercaptoethanol were obtained from MERCK (Darmstadt, Germany). NaN 3 and BeSO 4 were purchased from Fluka (St. Gallen, Switzerland). Protein PreparationSkeletal actin was prepared from acetone powder obtained from rabbit muscle (12,34). After purification, the calcium-bound actin monomers were stored in a 2 mM MOPS buffer (pH 7.3) with 0.2 mM ATP, 0.1 mM CaCl 2 , 0.1 mM β-mercaptoethanol, and 0.005% NaN 3 .The actin monomer concentration was determined by absorption photometry using 0.63 mg −1 mL cm −1 as the extinction coefficient at 290 nm (35). Actin-bound calcium was exchanged for magnesium by incubating the samples with 0.2 mM EGTA and 0.1 mM MgCl 2 for 5 min (36). Actin polymerization was initiated by the addition of 2 mM MgCl 2 and 100 mM KCl. ADP.BeF x -actin filaments were prepared in a similar way as described by Levitsky and colleagues (37); BeSO 4 (0.03-3 mM) and NaF (0.1-10 mM) were added to the samples, and actin was polymerized at room temperature for 3 h. Note that since the presence of ADP, BeSO 4 , and NaF in the solution leads to formation of either ADP.BeF 2 (OH) -•H 2 O or ADP.BeF 3 -...

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“…However, this does not exclude the possibility that cooperativity occurs across rather than along the actin filament. To test this, we added phalloidin, which binds near the thin filament axis with an orientation that is invariant with thin filament conformation (22) and both decreases thin filament flexibility and alters strand-strand interactions (23)(24)(25). Any cooperativity that was dependent upon such interactions might be changed by the addition of phalloidin.…”

Section: Relationship Between Ca 2ϩ Binding To the Thin Filament Regumentioning

confidence: 99%

Cooperative Effect of Calcium Binding to Adjacent Troponin Molecules on the Thin Filament-Myosin Subfragment 1 MgATPase Rate

Butters

Tobacman

1997

Journal of Biological Chemistry

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The myosin subfragment 1 (S1) MgATPase rate was measured using thin filaments with known extents of Ca 2؉ binding controlled by varying the ratio of native cardiac troponin versus an inhibitory troponin with a mutation in the sole regulatory Ca 2؉ binding site of troponin C. Fractional MgATPase activation was less than the fraction of troponins that bound Ca 2؉ , implying a cooperative effect of bound Ca 2؉ on cross-bridge cycling. Addition of phalloidin did not alter cooperative effects between bound Ca 2؉ molecules in the presence or absence of myosin S1. When the myosin S1 concentration was raised sufficiently to introduce cooperative myosin-myosin effects, lower Ca 2؉ concentrations were needed to activate the MgATPase rate. MgATPase activation remained less than Ca 2؉ binding, implying a true, not just an apparent, increase in Ca 2؉ affinity. MgATPase activation by Ca 2؉ was more cooperative than could be explained by cooperativeness of overall Ca 2؉ binding, the discrepancy between Ca 2؉ binding and MgATPase activation, or interactions between myosins. The results suggest the thin filament-myosin S1 MgATPase cycle requires calcium binding to adjacent troponin molecules and that this binding is cooperatively promoted by a single cycling cross-bridge. This mechanism is a potential explanation for Ca 2؉ -mediated regulation of crossbridge kinetics in muscle fibers.Just as isometric tension is cooperatively activated by Ca 2ϩ , so is the cardiac thin filament-myosin S1 1 MgATPase rate, even under conditions where there is no cooperativity in myosin S1 binding (1, 2). A possible explanation for this behavior is that ATPase activation is proportional to Ca 2ϩ binding to the many TnCs on each thin filament and that this calcium binding is cooperative (3). We tested the idea that Ca 2ϩ binding and MgATPase activation are proportional and found to the contrary that they are not. Instead, fractional MgATPase activation was considerably less than fractional Ca 2ϩ binding and more closely paralleled the number of pairs of adjacent troponins with Ca 2ϩ bound to both.To accomplish the above experiment, we employ a constitutively inhibitory form of cardiac troponin containing an inactivating mutation of the sole regulatory site of TnC, site II (4). This troponin, designated CBMII-Tn, results in a low thin filament-myosin S1 MgATPase rate that is not increased by the addition of Ca 2ϩ , analogous to previous results in which a similar TnC mutant was exchanged into myofibrils or muscle fibers (5-7). CBMII-Tn binds to actin-tropomyosin with an affinity identical to that of normal troponin in the absence of Ca 2ϩ . This binding, which is very tight for both normal troponin and for 8,9), permits the present report in which thin filaments are assembled with defined mixtures of normal troponin and CBMII-Tn. In the presence of saturating Ca 2ϩ concentrations, such thin filaments exhibit a fractional saturation of the TnC regulatory sites that is experimentally controllable by varying the relative concentrations of the two forms ...

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Flexibility of Actin Filaments Derived from Thermal Fluctuations

Cited by 566 publications

References 73 publications

Cofilin Increases the Bending Flexibility of Actin Filaments: Implications for Severing and Cell Mechanics

Cofilin Increases the Bending Flexibility of Actin Filaments: Implications for Severing and Cell Mechanics

Noncooperative Stabilization Effect of Phalloidin on ADP.BeF_x- and ADP.AlF₄-Actin Filaments

Cooperative Effect of Calcium Binding to Adjacent Troponin Molecules on the Thin Filament-Myosin Subfragment 1 MgATPase Rate

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Flexibility of Actin Filaments Derived from Thermal Fluctuations

Cited by 566 publications

References 73 publications

Cofilin Increases the Bending Flexibility of Actin Filaments: Implications for Severing and Cell Mechanics

Cofilin Increases the Bending Flexibility of Actin Filaments: Implications for Severing and Cell Mechanics

Noncooperative Stabilization Effect of Phalloidin on ADP.BeFx- and ADP.AlF4-Actin Filaments

Cooperative Effect of Calcium Binding to Adjacent Troponin Molecules on the Thin Filament-Myosin Subfragment 1 MgATPase Rate

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Noncooperative Stabilization Effect of Phalloidin on ADP.BeF_x- and ADP.AlF₄-Actin Filaments