Interaction Of Actin Filaments Research Articles

The non-muscle actinopathy-associated mutation E334Q in cytoskeletal γ-actin perturbs interaction of actin filaments with myosin and ADF/cofilin family proteins.

Various heterozygous cytoskeletal γ-actin mutations have been shown to cause Baraitser-Winter cerebrofrontofacial syndrome, non-syndromic hearing loss, or isolated eye coloboma. Here, we report the biochemical characterization of human cytoskeletal γ-actin carrying mutation E334Q, a mutation that leads to a hitherto unspecified non-muscle actinopathy. Following expression, purification, and removal of linker and thymosin β4 tag sequences, the p.E334Q monomers show normal integration into linear and branched actin filaments. The mutation does not affect thermal stability, actin filament nucleation, elongation, and turnover. Model building and normal mode analysis predict significant differences in the interaction of p.E334Q filaments with myosin motors and members of the ADF/cofilin family of actin-binding proteins. Assays probing the interactions of p.E334Q filaments with human class 2 and class 5 myosin motor constructs show significant reductions in sliding velocity and actin affinity. E334Q differentially affects cofilin-mediated actin dynamics by increasing the rate of cofilin-mediated de novo nucleation of actin filaments and decreasing the efficiency of cofilin-mediated filament severing. Thus, it is likely that p.E334Q-mediated changes in myosin motor activity, as well as filament turnover, contribute to the observed disease phenotype.

The non-muscle actinopathy-associated mutation E334Q in cytoskeletal γ-actin perturbs interaction of actin filaments with myosin and ADF/cofilin family proteins

Various heterozygous cytoskeletal γ-actin mutations have been shown to cause Baraitser–Winter cerebrofrontofacial syndrome, non-syndromic hearing loss, or isolated eye coloboma. Here, we report the biochemical characterization of human cytoskeletal γ-actin carrying mutation E334Q, a mutation that leads to a hitherto unspecified non-muscle actinopathy. Following expression, purification, and removal of linker and thymosin β4 tag sequences, the p.E334Q monomers show normal integration into linear and branched actin filaments. The mutation does not affect thermal stability, actin filament nucleation, elongation, and turnover. Model building and normal mode analysis predict significant differences in the interaction of p.E334Q filaments with myosin motors and members of the ADF/cofilin family of actin-binding proteins. Assays probing the interactions of p.E334Q filaments with human class 2 and class 5 myosin motor constructs show significant reductions in sliding velocity and actin affinity. E334Q differentially affects cofilin-mediated actin dynamics by increasing the rate of cofilin-mediated de novo nucleation of actin filaments and decreasing the efficiency of cofilin-mediated filament severing. Thus, it is likely that p.E334Q-mediated changes in myosin motor activity, as well as filament turnover, contribute to the observed disease phenotype.

Advances in Understanding the Molecular Mechanisms of Neuronal Polarity.

The establishment and maintenance of neuronal polarity are important for neural development and function. Abnormal neuronal polarity establishment commonly leads to a variety of neurodevelopmental disorders. Over the past three decades, with the continuous development and improvement of biological research methods and techniques, we have made tremendous progress in the understanding of the molecular mechanisms of neuronal polarity establishment. The activity of positive and negative feedback signals and actin waves are both essential in this process. They drive the directional transport and aggregation of key molecules of neuronal polarity, promote the spatiotemporalregulationof ordered and coordinated interactions of actin filaments and microtubules, stimulate the specialization and growth of axons, and inhibit the formation of multiple axons. In this review, we focus on recent advances in these areas, in particular the important findings about neuronal polarity in two classical models, in vitro primary hippocampal/cortical neurons and in vivo cortical pyramidal neurons, and discuss our current understanding of neuronal polarity.​.

Towards cell scale agent-based models for membrane cytoskeleton interactions.

The cytoskeleton is a dynamic far-from-equilibrium polymer network. It consumes energy to do mechanical work, and it interacts with complex chemical reaction networks in the cytoplasm and cell membrane to organize into various structures. We will discuss how the MEDYAN (medyan.org) model and software can simulate the chemical and mechanical interactions of actin filaments, crosslinks, myosin motor proteins, and membranes at the micrometer length scale and minute time scale. The next generation of MEDYAN is written in the Julia programming language (MEDYAN.jl). It has significant performance improvements including a 10x faster cylinder volume exclusion algorithm and a 10x faster Gillespie implementation for scheduling chemical reaction events. In addition, it includes an HDF5-based snapshot format that stores the full state of the simulation and enables restarts, and detailed linker and filament aging reactions that have reaction rates that adjust to changes to local monomer states, linker states, and linker mechanics. More complex reactions and reaction rates can be added using user-defined reaction callbacks. It also includes a deformable mesh-based membrane model. MEDYAN.jl is being used to study the role of ARP2/3 in the actin structure in the core of an axon growth cone. MEDYAN.jl is also being used to prototype models of membrane cytoskeleton chemical and mechanical interactions, including activation of ARP2/3.

Confinement Geometry Tunes Fascin-Actin Bundle Structures and Consequently the Shape of a Lipid Bilayer Vesicle.

Depending on the physical and biochemical properties of actin-binding proteins, actin networks form different types of membrane protrusions at the cell periphery. Actin crosslinkers, which facilitate the interaction of actin filaments with one another, are pivotal in determining the mechanical properties and protrusive behavior of actin networks. Short crosslinkers such as fascin bundle F-actin to form rigid spiky filopodial protrusions. By encapsulation of fascin and actin in giant unilamellar vesicles (GUVs), we show that fascin-actin bundles cause various GUV shape changes by forming bundle networks or straight single bundles depending on GUV size and fascin concentration. We also show that the presence of a long crosslinker, α-actinin, impacts fascin-induced GUV shape changes and significantly impairs the formation of filopodia-like protrusions. Actin bundle-induced GUV shape changes are confirmed by light-induced disassembly of actin bundles leading to the reversal of GUV shape. Our study contributes to advancing the design of shape-changing minimal cells for better characterization of the interaction between lipid bilayer membranes and actin cytoskeleton.

Abstract 5817: Anti-tropomyosin drugs prevent the rescue of vincristine-induced mitotic spindle defects

Abstract Drugs targeting a major component of the actin filaments of cancer cells, tropomyosin Tpm3.1, synergize with anti-microtubule drugs in neuroblastoma and lung cancer models both in vitro and in vivo and a wide range of other cancer types in vitro. We have determined the mechanism of synergy in HeLa cells to gain insight into the potential interaction of actin filaments and microtubules in the survival and proliferation of cancer cells. HeLa cells exhibit a strong synergistic response to the combined treatment of vincristine (VCR) and anti-Tpm3.1 compounds, marked by an enhanced reduction in cell viability, apoptosis induction and mitotic cell cycle arrest. Tpm3.1 localizes to the cell cortex during mitosis, potentially associating with the microtubule network, particularly the dynein/dynactin complexes responsible for mediating cortical pulling forces during spindle assembly. VCR alone causes supernumerary NuMA organized acentrosomal microtubule organizing centers upon nuclear envelope breakdown, which can be resolved via a clustering mechanism to achieve bipolar cell division. The addition of anti-Tpm3.1 compounds inhibits NuMA-associated clustering in VCR-treated cells, leading to irreparable defects during spindle assembly and thus a largely increased number of cells with multi-polar spindles undergoing mitotic delay and catastrophe. We conclude that actin/Tpm3.1 filaments contribute to the formation of the bipolar spindle and play a critical role in the clustering of acentrosomal microtubule asters. Citation Format: Peter Gunning, Yao Wang, Jeff H. Stear, Ashleigh Swain, Xing Xu, Nicole Bryce, Irina B. Alieva, Vera B. Dugina, Timothy Cripe, Justine Stehn, Edna C. Hardeman. Anti-tropomyosin drugs prevent the rescue of vincristine-induced mitotic spindle defects [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5817.

Collective and contractile filament motions in the myosin motility assay.

Cells require mechanical forces for their physiological functions. The forces are generated mainly from molecular interactions between actin filaments, cross-linking proteins, and myosin motors in the actin cytoskeleton. To better understand the molecular interactions, many studies employed myosin motility assays with actin filaments propelled by myosin heads fixed on a surface. Various interesting behaviors of actin filaments have been observed in the motility assay experiments. Despite the popularity of the motility assays, there were only a few computational models designed for simulating the motility assay systems. Most of the previous models have limitations which precluded full understanding of molecular origins for behaviors of actin filaments. In this study, we used an agent-based computational model based on Brownian dynamics for simulating the motility assay system. Our model rigorously describes the mechanics, dynamics, and interactions of actin filaments, cross-linking proteins, and molecular motors. Using the model, we first investigated how properties of actin filaments and motors affect gliding motions of actin filaments without volume-exclusion effects as a base study. We found that actin filaments can continuously glide at relative fast speed only when they are sufficiently longer than the average spacing between neighboring motors and that the gliding speed of F-actins shows a biphasic dependence on processivity of motors. Then, we showed that volume-exclusion effects between actin filaments can induce diverse collective movements and alignment of actin filaments, thus creating thick bundles and ring-like structures in the absence of cross-linking proteins. Lastly, we demonstrated that cross-linking proteins can lead to distinct contractile behaviors of actin networks depending on the density and kinetics of the cross-linking proteins. Results from our study show the ability of our model to simulate the motility assay system under various conditions and provide insights into understanding of different behaviors of actin filaments.

Special Issue: The Actin-Myosin Interaction in Muscle: Background and Overview.

Muscular contraction is a fundamental phenomenon in all animals; without it life as we know it would be impossible. The basic mechanism in muscle, including heart muscle, involves the interaction of the protein filaments myosin and actin. Motility in all cells is also partly based on similar interactions of actin filaments with non-muscle myosins. Early studies of muscle contraction have informed later studies of these cellular actin-myosin systems. In muscles, projections on the myosin filaments, the so-called myosin heads or cross-bridges, interact with the nearby actin filaments and, in a mechanism powered by ATP-hydrolysis, they move the actin filaments past them in a kind of cyclic rowing action to produce the macroscopic muscular movements of which we are all aware. In this special issue the papers and reviews address different aspects of the actin-myosin interaction in muscle as studied by a plethora of complementary techniques. The present overview provides a brief and elementary introduction to muscle structure and function and the techniques used to study it. It goes on to give more detailed descriptions of what is known about muscle components and the cross-bridge cycle using structural biology techniques, particularly protein crystallography, electron microscopy and X-ray diffraction. It then has a quick look at muscle mechanics and it summarises what can be learnt about how muscle works based on the other studies covered in the different papers in the special issue. A picture emerges of the main molecular steps involved in the force-producing process; steps that are also likely to be seen in non-muscle myosin interactions with cellular actin filaments. Finally, the remarkable advances made in studying the effects of mutations in the contractile assembly in causing specific muscle diseases, particularly those in heart muscle, are outlined and discussed.

Common and Specific Functions of Nonmuscle Myosin II Paralogs in Cells.

Various forms of cell motility critically depend on pushing, pulling, and resistance forces generated by the actin cytoskeleton. Whereas pushing forces largely depend on actin polymerization, pulling forces responsible for cell contractility and resistance forces maintaining the cell shape require interaction of actin filaments with the multivalent molecular motor myosin II. In contrast to muscle-specific myosin II paralogs, nonmuscle myosin II (NMII) functions in virtually all mammalian cells, where it executes numerous mechanical tasks. NMII is expressed in mammalian cells as a tissue-specific combination of three paralogs, NMIIA, NMIIB, and NMIIC. Despite overall similarity, these paralogs differ in their molecular properties, which allow them to play both unique and common roles. Importantly, the three paralogs can also cooperate with each other by mixing and matching their unique capabilities. Through specialization and cooperation, NMII paralogs together execute a great variety of tasks in many different cell types. Here, we focus on mammalian NMII paralogs and review novel aspects of their kinetics, regulation, and functions in cells from the perspective of how distinct features of the three myosin II paralogs adapt them to perform specialized and joint tasks in the cells.

The actin filament twist changes abruptly at boundaries between bare and cofilin-decorated segments

Cofilin/ADF proteins are actin-remodeling proteins, essential for actin disassembly in various cellular processes, including cell division, intracellular transport, and motility. Cofilins bind actin filaments cooperatively and sever them preferentially at boundaries between bare and cofilin-decorated (cofilactin) segments. The cooperative binding to actin has been proposed to originate from conformational changes that propagate allosterically from clusters of bound cofilin to bare actin segments. Estimates of the lengths over which these cooperative conformational changes propagate vary dramatically, ranging from 2 to >100 subunits. Here, we present a general, structure-based method for detecting from cryo-EM micrographs small variations in filament geometry (i.e. twist) with single-subunit precision. How these variations correlate with regulatory protein occupancy reveals how far allosteric, conformational changes propagate along filaments. We used this method to determine the effects of cofilin on the actin filament twist. Our results indicate that cofilin-induced changes in filament twist propagate only 1-2 subunits from the boundary into the bare actin segment, independently of the boundary polarity (i.e. irrespective of whether or not the bare actin segment flanks the pointed or barbed-end side of the boundary) and the pyrene fluorophore labeling of actin. These observations indicate that the filament twist changes abruptly at boundaries between bare and cofilin-decorated segments, thereby constraining mechanistic models of cooperative actin filament interactions and severing by cofilin. The methods presented here extend the capability of cryo-EM to analyze biologically relevant deviations from helical symmetry in actin as well as other classes of linear polymers.

Interaction of microtubules with the actin cytoskeleton via cross-talk of EB1-containing +TIPs and γ-actin in epithelial cells.

Actin microfilaments and microtubules are both highly dynamic cytoskeleton components implicated in a wide range of intracellular processes as well as cell-cell and cell-substrate interactions. The interactions of actin filaments with the microtubule system play an important role in the assembly and maintenance of 3D cell structure. Here we demonstrate that cytoplasmic actins are differentially distributed in relation to the microtubule system. LSM, 3D-SIM, proximity ligation assay (PLA) and co-immunoprecipitation methods applied in combination with selective depletion of β- or γ-cytoplasmic actins revealed a selective interaction between microtubules and γ-, but not β-cytoplasmic actin via the microtubule +TIPs protein EB1. EB1-positive comet distribution analysis and quantification have shown more effective microtubule growth in the absence of β-actin. Our data represent the first demonstration that microtubule +TIPs protein EB1 interacts mainly with γ-cytoplasmic actin in epithelial cells.

Differences in the spatial distribution of actin in the left and right ventricles of functioning rabbit hearts

We examined the effect of the differences in spatial arrangement of actin in contracting muscles from the left (LV) and right (RV) ventricles of rabbit heart. Spatial arrangement is a fundamental property of muscle because it reflects the interaction of individual actin molecules with myosin cross-bridges. Macroscopic parameters of contraction, such as maximum tension, speed of shortening or ATPase activity, are unlikely to reveal differences between the ventricles because they are made on whole organs containing trillions of actin and myosin molecules. Averaging the data collected from such a large assembly is likely to conceal small differences. To eliminate complications arising from the differences in the basic myocardial architecture and fiber structures of the ventricles, and to ensure that the molecular crowding influences contraction in the same way as under the in-vivo conditions, we collected data from isolated myofibrils ex-vivo. Control anisotropy experiments revealed that the orientation of actin reflected the orientation of myosin cross-bridges. The results showed that the changes in the distribution of actin induced by the interaction of actin filaments with myosin heads were demonstrated only in LVs, not in RVs. This suggests that both ventricles interact differently with myosin cross-bridges. We think that the stress induced in the thin filaments of LV causes loosening of bonds between actin monomers which leads to greater mobility of actin monomers.

Differences in the Spatial Distribution of Actin in the Left and Right Ventricles of Healthy Human Hearts

The pressure necessary to expel blood into the pulmonary circulation by the right ventricle is smaller than the pressure necessary to pump blood into the systemic circulation by the left ventricle. Nevertheless, there is little reason to believe that the left (LV) and right (RV) ventricular muscles are intrinsically different, because sarcomeric proteins of both ventricles are known to be expressed from the same genes. However, recent identification of disparities in their physiology and proteins has modified the classical outlook of their similarity and their respective cause for cardiovascular diseases. To decide whether ventricular muscles are functionally different we compared steady-state distribution of actin orientations in the left and the right ventricles at molecular levels. This distribution is a fundamental property of muscle because it reflects interaction of individual actin molecules with myosin cross-bridges. To eliminate complications arising from the differences in the basic fiber structures of the ventricles and to ensure that the molecular crowding influences contraction in the same way as it would in in-vivo conditions, we collected data from isolated myofibrils ex-vivo. To assure that each actin molecule acts independently, we labeled every 1,000 actin monomers with a fluorescent dye. The results showed that the changes in distribution of actin induced by the interaction of actin filaments with myosin heads were demonstrated only in LVs, not in RVs. This suggests that both ventricles interact differently with myosin cross-bridges. We think that the stress induced in the thin filaments of LV causes loosening of bonds between actin monomers and greater mobility of actin monomers.

Tropomyosin - master regulator of actin filament function in the cytoskeleton.

Tropomyosin (Tpm) isoforms are the master regulators of the functions of individual actin filaments in fungi and metazoans. Tpms are coiled-coil parallel dimers that form a head-to-tail polymer along the length of actin filaments. Yeast only has two Tpm isoforms, whereas mammals have over 40. Each cytoskeletal actin filament contains a homopolymer of Tpm homodimers, resulting in a filament of uniform Tpm composition along its length. Evidence for this 'master regulator' role is based on four core sets of observation. First, spatially and functionally distinct actin filaments contain different Tpm isoforms, and recent data suggest that members of the formin family of actin filament nucleators can specify which Tpm isoform is added to the growing actin filament. Second, Tpms regulate whole-organism physiology in terms of morphogenesis, cell proliferation, vesicle trafficking, biomechanics, glucose metabolism and organ size in an isoform-specific manner. Third, Tpms achieve these functional outputs by regulating the interaction of actin filaments with myosin motors and actin-binding proteins in an isoform-specific manner. Last, the assembly of complex structures, such as stress fibers and podosomes involves the collaboration of multiple types of actin filament specified by their Tpm composition. This allows the cell to specify actin filament function in time and space by simply specifying their Tpm isoform composition.

Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism

Myosin-binding protein C (MyBP-C) is an accessory protein of striated muscle thick filaments and a modulator of cardiac muscle contraction. Defects in the cardiac isoform, cMyBP-C, cause heart disease. cMyBP-C includes 11 Ig- and fibronectin-like domains and a cMyBP-C-specific motif. In vitro studies show that in addition to binding to the thick filament via its C-terminal region, cMyBP-C can also interact with actin via its N-terminal domains, modulating thin filament motility. Structural observations of F-actin decorated with N-terminal fragments of cMyBP-C suggest that cMyBP-C binds to actin close to the low Ca(2+) binding site of tropomyosin. This suggests that cMyBP-C might modulate thin filament activity by interfering with tropomyosin regulatory movements on actin. To determine directly whether cMyBP-C binding affects tropomyosin position, we have used electron microscopy and in vitro motility assays to study the structural and functional effects of N-terminal fragments binding to thin filaments. 3D reconstructions suggest that under low Ca(2+) conditions, cMyBP-C displaces tropomyosin toward its high Ca(2+) position, and that this movement corresponds to thin filament activation in the motility assay. At high Ca(2+), cMyBP-C had little effect on tropomyosin position and caused slowing of thin filament sliding. Unexpectedly, a shorter N-terminal fragment did not displace tropomyosin or activate the thin filament at low Ca(2+) but slowed thin filament sliding as much as the larger fragments. These results suggest that cMyBP-C may both modulate thin filament activity, by physically displacing tropomyosin from its low Ca(2+) position on actin, and govern contractile speed by an independent molecular mechanism.

Active Regulation of Cellular Membrane Tension

Cell membrane tension plays a critical rule in a variety of cellular activities, including membrane protein signaling, cell motility and cell blebbing. The specific mechanical environment of eukaryotic cells may result the spatially dependent membrane tension, which is crucial to actin filaments activity. In this work, we will start with simple fluid model to estimate lipid membrane tension in terms of active contractile interaction of actin filaments and polymerization/depolymerization rate. We includes the case of suspended cells and lamellipodium-membrane tension at cells resting on 2D substrates. The simple mechanical model will give us an overall scene of membrane tension distribution at specific order of magnitude, including the surface tension gradient, which cannot be measured by conventional experiment. Membrane tension also actively controls cell membrane blebbing. We also discuss the case of cell motility. We purpose the jet propulsion is the main mechanism for cell moving. The fluid field around the cell imposes an unique mechanical environment to the cell, which we will further demonstrate surface tension distribution and membrane net flow due to the spatially variant tension.

Formation and Ingression of Division Furrow Can Progress Under the Inhibitory Condition of Actin Polymerization in Ciliate Tetrahymena pyriformis

In eukaryotic cells that multiply by binary fission, the interaction of actin filaments with myosin II in the contractile ring is widely recognized to generate force for membrane ingression into the cleavage furrow; however, the expression of myosin II is restricted in animals, yeast, fungi, and amoeba (collectively, unikonts). No corresponding motor protein capable of forming mini-filaments that could exert sufficient tension to cleave the cell body is found in bikonts, consisting of planta, algae, and most protozoa; however, cells in some bikont lineages multiply by binary fission, as do animal cells. Of these, the ciliate Tetrahymena is known to form an actin ring beneath the division furrow in cytokinesis. Here, we investigated the role of filamentous actin in the cytokinesis of Tetrahymena pyriformis by treating synchronized dividing cells with an actin-inhibiting drug, Latrunculin-A. Video microscopic observation of live cells undergoing cytokinesis was performed, and contrary to expectation, we found that initiation of furrow ingression and its progress are not suppressed under the inhibitory condition of actin polymerization in Tetrahymena cells. We suggest that an actin filament-independent mechanism of binary fission may have been acquired during the evolution in this organism.

All-Atom Simulation and Coarse-Grained Analysis of the Rigor Actomyosin System

Interactions of actin filaments with myosin motor proteins are important for a variety of cellular functions. The myosin mechanochemical cycle is a complicated sequence of steps including ATP hydrolysis by myosin, binding and unbinding of myosin with actin, and phosphate release from myosin during the force-producing “powerstroke.” No high-resolution crystal structure of actomyosin exists, however low resolution data from cryo-EM has allowed a characterization of the rigor state (ATP free) of actomyosin at an atomic level of detail. Here we present the first simulation study at an all-atom level of detail of the myosin II S1 domain in the rigor state interacting with a fully periodic actin filament. Additionally, we study myosin II in the rigor and post-rigor states in the absence of actin. Through a combination of all-atom level and coarse-grained (CG) analysis, we are able to identify effects of myosin binding on the actin filament, the effects on myosin dynamics of being bound to actin, and differences in the collective motions between simulated myosin states. CG level analysis allows further characterization of the influence of myosin binding on the definitions of domains which exhibit collective motion for the actin monomers in the periodic filament, and the identification of CG domains of myosin which interact strongly with actin domains based on a heterogeneous elastic network model analysis.

Spatiotemporal Domains in Active Actin Gels

Using a standard actin filament gliding assay, rhodamine-phalloidin labeled actin filaments were visualized moving over a myosin-II coated surface by fluorescence microscopy and filament tracks were analyzed by computer. When micromolar amounts of non-fluorescent phalloidin-stabilised actin were added, filament motion became aligned into a common direction (Butt et al., 2010, J Biol Chem285:4964-4974). Experiments conducted at low filament densities show that, on average, there is a small angular deflection that tends to align the paths of individual colliding filaments. Path alignment is independent of relative track directionality and increases with collision incident angle. Filament elastic deformation during collision events indicates the collision bending energy is ∼20kBT. As increasing amounts of bulk actin are added, individual filament paths become straighter and start to move in a common direction. Spatial correlation analysis shows that increased alignment is due to incremental recruitment of filament tracks into one orientation rather than fusion of seed domains to larger ones. Experiments performed over a wide range of actin filament concentrations and HMM surface densities, show that ordering of filament motion peaks over a range of conditions and decreases at extreme values. This finding differs from theoretical prediction of a critical phase transition (Kraikivski et al., 2006, Phys Rev. Lett.96:258103). Loss of order at high bulk actin concentration might be explained if there is increased filament exchange between bulk, disordered, actin and surface bound material. Loss of order at high myosin surface densities might arise from increased probability of motor attachment that would reduce ability of actin to change its path following a collision. Although our results are broadly compatible with generation of long-range order from mechanics of individual actin filament interactions; other phenomena become important at high protein concentrations.

Electron Microscopy and 3D Reconstruction Reveals Filamin Ig Domain Binding to F-Actin

Filamin A (FLNa) is an actin-binding protein that cross-links F-actin into networks of orthogonally branched filaments. FLNa also directs the networks to integrins while responding to mechanochemical signaling pathways. Flexible, 160-nm-long FLNa molecules are tail-to-tail dimers, each subunit of which contains an N-terminal calponin homology (CH)/actin-binding domain connected by a series of 24 immunoglobulin (Ig) repeats to a dimerization site at their C-terminal end. Whereas the contribution of the CH domains to F-actin affinity is weak (apparent K(a)~10(5)), the binding of the intact protein to F-actin is strong (apparent K(a)~10(8)), suggesting involvement of additional parts of the molecule in this association. Indeed, previous results indicate that Ig repeats along FLNa contribute significantly to the strength of the actin filament interaction. In the current study, we used electron microscopy and three-dimensional reconstruction to elucidate the structural basis of the Ig repeat-F-actin binding. We find that FLNa density is clearly delineated in reconstructions of F-actin complexed either with a four-Ig-repeat segment of FLNa containing Ig repeat 10 or with immunoglobulin-like filamin A repeat (IgFLNa)10 alone. The mass attributable to IgFLNa10 lies peripherally along the actin helix over the N-terminus of actin subdomain 1. The IgFLNa10 interaction appears to be specific, since no other individual Ig repeat or fragment of the FLNa molecule examined, besides ones with IgFLNa10 or CH domains, decorated F-actin filaments or were detected in reconstructions. We conclude that the combined interactions of CH domains and the IgFLNa10 repeat provide the binding strength of the whole FLNa molecule and propose a model for the association of IgFLNa10 on actin filaments.