Heavy Meromyosin Research Articles

Synthetic thick filaments: A new avenue for better understanding the myosin super-relaxed state in healthy, diseased, and mavacamten-treated cardiac systems

A hallmark feature of myosin-II is that it can spontaneously self-assemble into bipolar synthetic thick filaments (STFs) in low-ionic-strength buffers, thereby serving as a reconstituted in vitro model for muscle thick filaments. Although these STFs have been extensively used for structural characterization, their functional evaluation has been limited. In this report, we show that myosins in STFs mirror the more electrostatic and cooperative interactions that underlie the energy-sparing super-relaxed (SRX) state, which are not seen using shorter myosin subfragments, heavy meromyosin (HMM) and myosin subfragment 1 (S1). Using these STFs, we show several pathophysiological insults in hypertrophic cardiomyopathy, including the R403Q myosin mutation, phosphorylation of myosin light chains, and an increased ADP:ATP ratio, destabilize the SRX population. Furthermore, WT myosin containing STFs, but not S1, HMM, or STFs-containing R403Q myosin, recapitulated the ADP-induced destabilization of the SRX state. Studies involving a clinical-stage small-molecule inhibitor, mavacamten, showed that it is more effective in not only increasing myosin SRX population in STFs than in S1 or HMM but also in increasing myosin SRX population equally well in STFs made of healthy and disease-causing R403Q myosin. Importantly, we also found that pathophysiological perturbations such as elevated ADP concentration weakens mavacamten’s ability to increase the myosin SRX population, suggesting that mavacamten-bound myosin heads are not permanently protected in the SRX state but can be recruited into action. These findings collectively emphasize that STFs serve as a valuable tool to provide novel insights into the myosin SRX state in healthy, diseased, and therapeutic conditions.

Myosin and gelsolin cooperate in actin filament severing and actomyosin motor activity

Actin is a major intracellular protein with key functions in cellular motility, signaling, and structural rearrangements. Its dynamic behavior, such as polymerization and depolymerization of actin filaments in response to intracellular and extracellular cues, is regulated by an abundance of actin binding proteins. Out of these, gelsolin is one of the most potent for filament severing. However, myosin motor activity also fragments actin filaments through motor-induced forces, suggesting that these two proteins could cooperate to regulate filament dynamics and motility. To test this idea, we used an in vitro motility assay, where actin filaments are propelled by surface-adsorbed heavy meromyosin (HMM) motor fragments. This allows studies of both motility and filament dynamics using isolated proteins. Gelsolin, at both nanomolar and micromolar Ca2+ concentration, appreciably enhanced actin filament severing caused by HMM-induced forces at 1 mM MgATP, an effect that was increased at higher HMM motor density. This finding is consistent with cooperativity between actin filament severing by myosin-induced forces and by gelsolin. We also observed reduced sliding velocity of the HMM-propelled filaments in the presence of gelsolin, providing further support of myosin-gelsolin cooperativity. Total internal reflection fluorescence microscopy–based single molecule studies corroborated that the velocity reduction was a direct effect of gelsolin binding to the filament and revealed different filament severing pattern of stationary and HMM propelled filaments. Overall, the results corroborate cooperative effects between gelsolin-induced alterations in the actin filaments and changes due to myosin motor activity leading to enhanced F-actin severing of possible physiological relevance.

Millisecond Conformational Dynamics of Skeletal Myosin II Power Stroke Studied by High-Speed Atomic Force Microscopy.

Myosin-based molecular motors are responsible for a variety of functions in the cells. Myosin II is ultimately responsible for muscle contraction and can be affected by multiple mutations, that may lead to myopathies. Therefore, it is essential to understand the nanomechanical properties of myosin II. Due to the lack of technical capabilities to visualize rapid changes in nonprocessive molecular motors, there are several mechanistic details in the force-generating steps produced by myosin II that are poorly understood. In this study, high-speed atomic force microscopy was used to visualize the actin-myosin complex at high temporal and spatial resolutions, providing further details about the myosin mechanism of force generation. A two-step motion of the double-headed heavy meromyosin (HMM) lever arm, coupled to an 8.4 nm working stroke was observed in the presence of ATP. HMM heads attached to an actin filament worked independently, exhibiting different lever arm configurations in given time during experiments. A lever arm rotation was associated with several non-stereospecific long-lived and stereospecific short-lived (∼1 ms) HMM conformations. The presence of free Pi increased the short-lived stereospecific binding events in which the power stroke occurred, followed by release of Pi after the power stroke.

A Myosin II-Based Nanomachine Devised for the Study of Ca2+-Dependent Mechanisms of Muscle Regulation.

The emergent properties of the array arrangement of the molecular motor myosin II in the sarcomere of the striated muscle, the generation of steady force and shortening, can be studied in vitro with a synthetic nanomachine made of an ensemble of eight heavy-meromyosin (HMM) fragments of myosin from rabbit psoas muscle, carried on a piezoelectric nanopositioner and brought to interact with a properly oriented actin filament attached via gelsolin (a Ca2+-regulated actin binding protein) to a bead trapped by dual laser optical tweezers. However, the application of the original version of the nanomachine to investigate the Ca2+-dependent regulation mechanisms of the other sarcomeric (regulatory or cytoskeleton) proteins, adding them one at a time, was prevented by the impossibility to preserve [Ca2+] as a free parameter. Here, the nanomachine is implemented by assembling the bead-attached actin filament with the Ca2+-insensitive gelsolin fragment TL40. The performance of the nanomachine is determined both in the absence and in the presence of Ca2+ (0.1 mM, the concentration required for actin attachment to the bead with gelsolin). The nanomachine exhibits a maximum power output of 5.4 aW, independently of [Ca2+], opening the possibility for future studies of the Ca2+-dependent function/dysfunction of regulatory and cytoskeletal proteins.

Lysozyme-induced suppression of enzymatic and motile activities of actin-myosin: Impact of basic proteins

Electrostatic interactions between actin filaments and myosin molecules, which are ubiquitous proteins in eukaryotes, are crucial for their enzymatic activity and motility. Nonspecific electrostatic interactions between proteins are unavoidable in cells; therefore, it is worth exploring how ambient proteins, such as polyelectrolytes, affect actin-myosin functions. To understand the effect of counterionic proteins on actin-myosin, we examined ATPase activity and sliding velocity via actin-myosin interactions in the presence of the basic model protein hen egg lysozyme. In an in vitro motility assay with ATP, the sliding velocity of actin filaments on heavy meromyosin (HMM) decreased with increasing lysozyme concentrations. Actin filaments were completely stalled at a lysozyme concentration above 0.08 mg/mL. Lysozyme decreased the ATP hydrolysis rate of the actin-HMM complex but not that HMM alone. Co-sedimentation assays revealed that lysozyme enhanced the binding of HMM to actin filaments in the presence of ATP. Additionally, lysozyme could bind to actin and myosin filaments. The inhibitory effect of poly-l-lysine, histone mixture, and lactoferrin on the motility of actin-myosin was higher than that of lysozyme. Thus, nonspecific electrostatic interactions of basic proteins are involved in the bundling of actin filaments and modulation of essential functions of the actomyosin complex.

Plasma skeletal muscle myosin phenotypes identified by immunoblotting are associated with pulmonary embolism occurrence in young adults

BackgroundPurified skeletal muscle myosin (SkM) binds factor Xa and is procoagulant. The molecular forms of SkM in human plasma have not been characterized. MethodHuman plasma SkM heavy chain (HC) isoforms of different molecular weights were detected by a newly developed immunoblotting protocol. In this pilot study, the distribution of SkM HC antigen isoforms in plasmas of healthy subjects and young adult patients with venous thrombosis was analyzed. ResultsMultiple SkM HC antigen bands were detected in human plasmas, corresponding to full-length SkM HC, heavy meromyosin, or the S1 fragment. Plasma immunoblots of healthy subjects displayed three major phenotypes: Type I with two primary bands for full-length SkM and heavy meromyosin, and two lesser bands including S1 fragment (54%); Type II with bands primarily for full-length SkM HC (34%); and Type III with only a band for the S1 fragment (12%). Plasma SkM HC antigen Type II phenotype was associated with an increased occurrence of isolated pulmonary embolism in younger patients, respectively (≤50 years old). ConclusionsThree SkM HC antigen phenotypes were identified in human plasma by immunoblotting, and Type II phenotype was correlated with the occurrence of isolated pulmonary embolisms in younger patients.

Effects of Mavacamten and Blebbistatin on the Small-Angle X-ray Scattering Structure of Human β-cardiac Myosin

Cardiac myosin is a motor protein that converts chemical energy from ATP hydrolysis to mechanical work that keeps the heart beating. Full-length myosin and even the smaller sub-fragment, heavy meromyosin (HMM) are large and flexible molecules and have not been possible to crystallize and therefore has been unavailable for structural studies. These properties make myosin a good candidate for small-angle X-ray scattering (SAXS) studies. Here we use size exclusion chromatography coupled with SAXS to study structural changes of human β-cardiac 2-hep and 25-hep HMM constructs in relaxing conditions treated with two different myosin inhibitors: blebbistatin and mavacamten. Under resting conditions, the 25-hep HMM had a radius of gyration (Rg) of ∼ 118 Å (Guinier fit) to ∼ 137 Å (GNOM fit) and the maximum dimension of the molecule (Dmax) of 526 Å. The Rg of 25-hep HMM decreased by about 5-10% with either blebbistatin or mavacamten treatment. The Dmax of 25-hep HMM decreased ∼ 20 Å by blebbistatin treatment while it decreased ∼ 80 Å by mavacamten treatment. There were only moderate structural changes (< 2%) for the 2-hep HMM with either blebbistatin or mavacamten treatment. Our results indicate that most of the HMM molecules in relaxing conditions maintain an open configuration and that the myosin S2 region is essential for HMM to adopt the folded back closed configuration in presence of mavacamten and blebbistatin. Supported, in part, by NIH P41 GM103622.

Conformational and rheological changes of high-pressure processing treated rabbit myosin subfragments during heating

Rabbit skeletal (M. psoas) myosin subunits, heavy meromyosin (HMM) and light meromyosin (LMM), were purified and treated by high pressure processing (HPP). The HPP (100, 200 and 300 MPa)-modified myosin subunits were subjected to thermal treatment (25–70 °C), during which their conformational and rheological properties were explored. The turbidity of all HMM and LMM samples slightly improved from 25 to 55 °C, and then sharply increased from 55 °C to 70 °C (from). Pressure ≥200 MPa changed the structural properties of HMM, exhibited as more exposed hydrophobic and sulfhydryl patches, whereas the increase of hydrophobicity induced by heating decelerated afterwards. The LMM portion exhibited inferior heating stability. Upon heating, both pressurized and nontreated HMM showed similar trends of changes of secondary structure, which included the reduction of α-helices and the increase of β-structures. Only a slight change of secondary structure occurred on LMM after HPP, indicating a higher resistance of LMM on HPP than HMM. These findings suggested that a moderate denaturation generated by HPP would not affect the thermal behavior of myosin subfragments, while pressures of ≥200 MPa might lead to disadvantageous rheological properties by mediating the thermophysical properties of HMM.

High-speed AFM reveals subsecond dynamics of cardiac thin filaments upon Ca2+ activation and heavy meromyosin binding

High-speed atomic force microscopy (HS-AFM) can be used to study dynamic processes with real-time imaging of molecules within 1- to 5-nm spatial resolution. In the current study, we evaluated the 3-state model of activation of cardiac thin filaments (cTFs) isolated as a complex and deposited on a mica-supported lipid bilayer. We studied this complex for dynamic conformational changes 1) at low and high [Ca2+] (pCa 9.0 and 4.5), and 2) upon myosin binding to the cTF in the nucleotide-free state or in the presence of ATP. HS-AFM was used to directly visualize the tropomyosin-troponin complex and Ca2+-induced tropomyosin movements accompanied by structural transitions of actin monomers within cTFs. Our data show that cTFs at relaxing or activating conditions are not ultimately in a blocked or activated state, respectively, but rather the combination of states with a prevalence that is dependent on the [Ca2+] and the presence of weakly or strongly bound myosin. The weakly and strongly bound myosin induce similar changes in the structure of cTFs as confirmed by the local dynamical displacement of individual tropomyosin strands in the center of a regulatory unit of cTF at the relaxed and activation conditions. The displacement of tropomyosin at the relaxed conditions had never been visualized directly and explains the ability of myosin binding to TF at the relaxed conditions. Based on the ratios of nonactivated and activated segments within cTFs, we proposed a mechanism of tropomyosin switching from different states that includes both weakly and strongly bound myosin.

Myosin Orientation in a Muscle Fiber using Bifunctional Spin Labels with 4 Degrees Angular Resolution

We have measured the orientation of myosin light chain domain in a demembranated muscle fiber bundle using electron paramagnetic resonance (EPR) and a bifunctional spin label (BSL), with the angular resolution of 4 degrees in situ. A pair of (i,i+4) Cys residues was engineered on an alpha-helix of the regulatory light chain (RLC) on the N- and C- lobes. By exchanging endogenous RLC with mutant RLC labeled with BSL on oriented muscle fibers, we were able to resolve angular distributions in several biochemical states due to the stereospecific attachment of BSL's two disulfide bonds. In this setup, the accurate determination of BSL's angular coordinates allowed us to determine the orientation of individual structural elements with respect to the muscle fiber axis. The addition of ATP in the absence of Ca shifted the orientational distribution to a much more disordered one. By decorating demembranated fiber bundle with heavy meromyosin with RLC labeled with BSL, we observed different dynamics in the N- and C- lobe orientation. We compared these results with cardiac myosin. We also developed a new global fitting analysis to narrow the confidence intervals and enhance the sensitivity to azimuthal angle between pi orbital and magnetic field. While we find that the majority of the RLC helices are disordered even in the absence of ATP, we resolved an oriented population of myosin with lever arm being perpendicular to muscle fiber axis previously observed directly in insect flight muscle. This work was supported by NIH R01AR032961 and R37AG26160 to DDT. YS was supported by NIH T32AG29796 and University of Minnesota Interdisciplinary Doctoral Fellowship.

The Dietary Inclusion of Chickpea Seeds (Cicer Arietinum L.) Influences the Thermal Properties of Muscle Proteins, But Not the Texture of Drumstick Muscle in Broiler Chickens

ABSTRACT In this study, the effect of replacement of soybean meal with raw chickpea seeds as a primary protein source in the diet of broiler chicks on physical properties of drumstick muscle (m. gastrocnemius) was examined. One-day-old Ross 308 broilers (n=160) were fed soybean meal (n=80) or raw chickpea seeds (n=80) as a primary protein source for 42 days. Drumstick muscles, after dissection and storage for 24 h at 5°C, were subjected to texture profile analysis to determine changes in the structural and material integrity, and the thermal stability of muscle proteins were assessed on the basis of differential scanning calorimetry measurements. Light meromyosin, heavy meromyosin and sarcoplasmic proteins in the meat samples of broilers fed raw chickpea seeds were characterized by higher thermal stability as the higher denaturation temperatures were observed. In addition, the calorimetric enthalpy of denaturation of light meromyosin was significantly higher in the muscles of broiler fed raw chickpea seeds, while higher enthalpy of denaturation of actin was determined in those fed soybean meal. Muscle weight, hardness, and the other evaluated textural traits, did not differ between treatments. In summary, dietary protein source affected only thermal stability of muscle proteins; however, the physiological consequences of these alterations are yet to be determined. This study also showed that thermal analysis can be a useful tool for analyzing the effect of nutrition on the development and structural changes in the muscle tissues of poultry.

Comparative analysis of widely used methods to remove nonfunctional myosin heads for the in vitro motility assay

The in vitro motility assay allows studies of muscle contraction through observation of actin filament propulsion by surface-adsorbed myosin motors or motor fragments isolated from muscle. A possible problem is that motility may be compromised by nonfunctional, “dead”, motors, obtained in the isolation process. Here we investigate the effects on motile function of two approaches designed to eliminate the effects of these dead motors. We first tested the removal of heavy meromyosin (HMM) molecules with ATP-insensitive “dead” heads by pelleting them with actin filaments, using ultracentrifugation in the presence of 1 mM MgATP (“affinity purification”). Alternatively we incubated motility assay flow cells, after HMM surface adsorption, with non-fluorescent “blocking actin” (1 µM) to block the dead heads. Both affinity purification and use of blocking actin increased the fraction of motile filaments compared to control conditions. However, affinity purification significantly reduced the actin sliding speed in five out of seven experiments on silanized surfaces and in one out of four experiments on nitrocellulose surfaces. Similar effects on velocity were not observed with the use of blocking actin. However, a reduced speed was also seen (without affinity purification) if HMM or myosin subfragment 1 was mixed with 1 mM MgATP before and during surface adsorption. We conclude that affinity purification can produce unexpected effects that may complicate the interpretation of in vitro motility assays and other experiments with surface adsorbed HMM, e.g. single molecule mechanics experiments. The presence of MgATP during incubation with myosin motor fragments is critical for the complicating effects.

Cleavage of loops 1 and 2 in skeletal muscle heavy meromyosin (HMM) leads to a decreased function

BackgroundThe mechanical work and the actin-activated ATP kinetics in skeletal muscles are closely associated with two surface loops that are present in the myosin molecule: loop 1 and loop 2. They are located close to the ATP-loop (loop 1), and the actin binding domain (loop 2). In this study we investigated the roles of loops 1 and 2 in the regulation of the load-dependent velocity of actin sliding and ATPase activity. MethodsHeavy meromyosin (HMM) from rabbit skeletal muscle was subjected to limited tryptic proteolysis to obtain fragments containing different amounts of loops 1 and 2. The amino-acid sequences of these fragments were confirmed with quantitative mass-spectrometry. The velocity of actin motility propelled by the HMM fragments was measured using in-vitro motility assays, with varying loads induced by the addition of different concentrations of α-actinin. ResultsThe load-dependent velocity of the myosin-propelled actin motility, and the fraction of actin filaments motility, were decreased in close association with the depletion of loop 1 in the HMM. The ATPase activity was decreased in close association with depletion of loops 1 and 2. ConclusionsLoop 1 is responsible for regulating the load-dependent velocity of actin motility. General significanceMyosin-actin interaction is closely regulated by two flexible loops in the structure of myosin. The results of this study are important for the understanding of the molecular mechanisms of contraction, and therefore the most basic functions of life, such as locomotion, heart beating, and breathing.

Inorganic polyphosphate hydrolysis catalyzed by skeletal muscular actomyosin complexes is uncoupled with motility

Hydrolysis of the triphosphate moiety of ATP, catalyzed by myosin, induces alterations in the affinity of the myosin heads for actin filaments via conformational changes, thereby causing motility of the actomyosin complexes. To elucidate the contribution of the triphosphate group attached to adenosine, we examined the enzymatic activity of heavy meromyosin (HMM) with actin filaments for inorganic tripolyphosphate (3PP) using a Malachite green method and evaluated using fluorescence microscopy the effects of 3PP on actin filament motility on HMM-coated glass slides. In the presence of MgCl2, HMM hydrolyzed 3PP at a maximum rate of 0.016 s−1 HMM−1, which was four times lower than the hydrolysis rate of ATP. Tetrapolyphosphate (4PP) was hydrolyzed at a rate similar to that of 3PP hydrolysis. The hydrolysis rates of 3PP and 4PP were enhanced by roughly 10-fold in the presence of actin filaments. In motility assays, the presence of polyphosphates did not lead to the sliding movement of actin filaments. Moreover, in the presence of ATP at low concentrations, the sliding velocity of actin filaments decreased as the concentration of added polyphosphate increased, indicating a competitive binding of polyphosphate to myosin heads with ATP. These results suggested that the energy produced by standalone triphosphate hydrolysis did not induce the unidirectional motion of actomyosin and that the link between triphosphate and adenosine was crucial for motility.

Piperine, an alkaloid inhibiting the super-relaxed state of myosin, binds to the myosin regulatory light chain

Piperine, an alkaloid from black pepper, was found to inhibit the super-relaxed state (SRX) of myosin in fast-twitch skeletal muscle fibers. In this work we report that the piperine molecule binds heavy meromyosin (HMM), whereas it does not interact with the regulatory light chain (RLC)-free subfragment-1 (S1) or with control proteins from the same muscle molecular machinery, G-actin and tropomyosin. To further narrow down the location of piperine binding, we studied interactions between piperine and a fragment of skeletal myosin consisting of the full-length RLC and a fragment of the heavy chain (HCF). The sequence of HCF was designed to bind RLC and to dimerize via formation of a stable coiled coil, thus producing a well-folded isolated fragment of the myosin neck. Both chains were co-expressed in Escherichia coli, the RLC/HCF complex was purified and tested for stability, composition and binding to piperine. RLC and HCF chains formed a stable heterotetrameric complex (RLC/HCF)2 which was found to bind piperine. The piperine molecule was also found to bind isolated RLC. Piperine binding to RLC in (RLC/HCF)2 altered the compactness of the complex, suggesting that the mechanism of SRX inhibition by piperine is based on changing conformation of the myosin.

Controlled Surface Silanization for Actin-Myosin Based Nanodevices and Biocompatibility of New Polymer Resists.

Molecular motor-based nanodevices require organized cytoskeletal filament guiding along motility-promoting tracks, confined by motility-inhibiting walls. One way to enhance motility quality on the tracks, particularly in terms of filament velocity but also the fraction of motile filaments, is to optimize the surface hydrophobicity. We have investigated the potential to achieve this for the actin-myosin II motor system on trimethylchlorosilane (TMCS)-derivatized SiO2 surfaces to be used as channel floors in nanodevices. We have also investigated the ability to supress motility on two new polymer resists, TU7 (for nanoimprint lithography) and CSAR 62 (for electron beam and deep UV lithography), to be used as channel walls. We developed a chemical-vapor deposition tool for silanizing SiO2 surfaces in a controlled environment to achieve different surface hydrophobicities (measured by water contact angle). In contrast to previous work, we were able to fabricate a wide range of contact angles by varying the silanization time and chamber pressure using only one type of silane. This resulted in a significant improvement of the silanization procedure, producing a predictable contact angle on the surface and thereby predictable quality of the heavy meromyosin (HMM)-driven actin motility with regard to velocity. We observed a high degree of correlation between the filament sliding velocity and contact angle in the range 10-86°, expanding the previously studied range. We found that the sliding velocity on TU7 surfaces was superior to that on CSAR 62 surfaces despite similar contact angles. In addition, we were able to suppress the motility on both TU7 and CSAR 62 by plasma oxygen treatment before silanization. These results are discussed in relation to previously proposed surface adsorption mechanisms of HMM and their relationship to the water contact angle. Additionally, the results are considered for the development of actin-myosin based nanodevices with superior performance with respect to actin-myosin functionality.

Detection of the Super-Relaxed State in Cardiac Heavy Meromyosin

Detection of the Super-Relaxed State in Cardiac Heavy Meromyosin

Light-induced ATP driven self-assembly of actin and heavy-meromyosin in proteo-tubularsomes as a step toward artificial cells.

In this work, we studied the light induced self-assembly of F-actin and heavy meromyosin (HMM) in tubular vesicles or "tubularsomes" during initiation by ATP. To mimic nature, light-induced ATP synthesis was used for the F-actin/HMM self-assembly inside these vesicles created from a triblock copolymer reconstituted with the membrane protein bacteriorhodopsin (bR) and F1F0-ATPase along with F-actin and HMM in the core.

Cover Image, Volume 74, Issue 12

ON THE BACK COVER: The cover composite is based on the Research Articles Flow‐aligned, single‐shot fiber diffraction using a femtosecond X‐ray free‐electron laser by David Popp et al., DOI: 10.1002/cm.21378 and Unidirectional growth of heavy meromyosin clusters along actin filaments revealed by real‐time fluorescence microscopy by Rika Hirakawa et al., 10.1002/cm.21408. image

Unidirectional growth of heavy meromyosin clusters along actin filaments revealed by real-time fluorescence microscopy.

Heavy meromyosin (HMM) forms clusters along actin filaments under low ATP concentrations. Here, we observed the growth of HMM clusters under low concentrations of ATP in real time using fluorescence microscopy. When actin filaments were loosely immobilized on positively charged lipid bilayers, clusters of HMM-GFP were readily formed. Time-lapse observation revealed that the clusters grew unidirectionally. When we used a mixture of actin filaments and copolymers of actin and acto-S1dC, a chimeric protein of actin and the myosin motor domain, HMM-GFP preferentially formed clusters along the copolymers. We thus suggest that binding of myosin motors carrying ADP and Pi induces unidirectional conformational changes in actin filaments and allosterically recruits more myosin binding. In contrast, when actin filaments and copolymers were anchored to glass substrate via stable biotin-avidin linkage, higher concentrations of HMM-GFP were required to form clusters than on the lipid bilayer. Moreover, actin filaments and copolymers were not discriminated regarding preferential cluster formation. This is presumably because the myosin-induced cooperative conformational changes in actin filaments involve changes in the helical twist. Consistent with this, cofilin clusters, which supertwist the helix, were readily formed along loosely immobilized actin filaments, but not along those anchored via biotin-avidin linkage.