Cross-link Actin Filaments Research Articles

Differential Regulation of Unconventional Fission Yeast Myosins via the Actin Track

Fission yeast possesses three unconventional myosins: Myo1p (a class I myosin that functions at endocytic actin patches) and Myo51p and Myo52p (class V myosins that function at contractile rings and actin cables, respectively). Here we used a combination of in vivo and in vitro approaches to investigate how changes in the actin track influence the motor activity and spatial regulation of these myosins. We optimized the isolation of Myo1p, Myo51p, and Myo52p. All three myosins exhibited robust motor activity in ATPase and actin filament gliding assays. However, decoration of actin with tropomyosin differentially regulates the activity of these motors. Tropomyosin inhibits Myo1p by blocking its ability to form productive associations with actin filaments, whereas tropomyosin increases the actin affinity and ATPase activity of Myo51p and Myo52p. The actin filament crosslinking protein fimbrin rescues Myo1p motor activity by displacing tropomyosin from actin filaments. Consistent with our in vitro findings, fimbrin and tropomyosin have opposing effects on Myo1p function at actin patches. Defects in tropomyosin function led to shorter Myo1p patch lifetimes, whereas loss of fimbrin extended Myo1p lifetimes. Furthermore, defects in tropomyosin function decreased the efficiency of Myo52p-directed motility along actin cables in the cell. Tropomyosin promotes myosin-V motility along actin cables. Accumulation of fimbrin at actin patches relieves Myo1p from tropomyosin-mediated inhibition, ensuring maximal myosin-I motor activity at these sites. Thus, spatial regulation of myosin motor function is in part controlled by specific changes in the composition of the actin track.

The effects of filament aging and annealing on a model lamellipodium undergoing disassembly by severing

We use numerical simulations to study the properties of a model lamellipodium as it disassembles by filament severing. The growing lamellipodium is modeled as a 2D or 3D periodic lattice of crosslinked actin filaments. At each time step a new layer of actin filaments is added at the membrane, existing filaments are severed stochastically and disconnected sections of the network are removed. Filament aging is modeled by including several different filament chemical states. Filament annealing is included by allowing existing filaments to grow new filaments. The properties of the model are studied as functions of the number of states and the severing and annealing rates. The network width is proportional to the sum of the average lifetimes of the states, and is well modeled by a simple kinetic theory. The length of the network scales linearly with actin concentration and has a finite width even at high severing protein concentrations. The edge of the growing network becomes sharper as either the number of states or the dimensionality is increased. Annealing increases the average length of the network, and the network length diverges at a critical annealing rate.

Proteomic analysis of ACTN4-interacting proteins reveals it’s a putative involvement in mRNA metabolism

Alpha-actinin 4 (ACTN4) is an actin-binding protein. In the cytoplasm, ACTN4 participates in structural organisation of the cytoskeleton via cross-linking of actin filaments. Nuclear localisation of ACTN4 has also been reported, but no clear role in the nucleus has been established. In this report, we describe the identification of proteins associated with ACTN4 in the nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and MALDI-TOF mass-spectrometry revealed a large number of ACTN4-bound proteins that are involved in various aspects of mRNA processing and transport. The association of ACTN4 with different ribonucleoproteins suggests that a major function of nuclear ACTN4 may be regulation of mRNA metabolism and signaling.

Biochemical Basis of the Interaction between Cystic Fibrosis Transmembrane Conductance Regulator and Immunoglobulin-like Repeats of Filamin

Mutations in the chloride channel cystic fibrosis transmembrane regulator (CFTR) cause cystic fibrosis, a genetic disorder characterized by defects in CFTR biosynthesis, localization to the cell surface, or activation by regulatory factors. It was discovered recently that surface localization of CFTR is stabilized by an interaction between the CFTR N terminus and the multidomain cytoskeletal protein filamin. The details of the CFTR-filamin interaction, however, are unclear. Using x-ray crystallography, we show how the CFTR N terminus binds to immunoglobulin-like repeat 21 of filamin A (FlnA-Ig21). CFTR binds to beta-strands C and D of FlnA-Ig21 using backbone-backbone hydrogen bonds, a linchpin serine residue, and hydrophobic side-chain packing. We use NMR to determine that the CFTR N terminus also binds to several other immunoglobulin-like repeats from filamin A in vitro. Our structural data explain why the cystic fibrosis-causing S13F mutation disrupts CFTR-filamin interaction. We show that FlnA-Ig repeats transfected into cultured Calu-3 cells disrupt CFTR-filamin interaction and reduce surface levels of CFTR. Our findings suggest that filamin A stabilizes surface CFTR by anchoring it to the actin cytoskeleton through interactions with multiple filamin Ig repeats. Such an interaction mode may allow filamins to cluster multiple CFTR molecules and to promote colocalization of CFTR and other filamin-binding proteins in the apical plasma membrane of epithelial cells.

Disruption of fear memory consolidation and reconsolidation by actin filament arrest in the basolateral amygdala

The dynamic re-arrangement of actin filaments is an essential process in the plasticity of synaptic connections during memory formation. In this study, we determined in mice effects of actin filament arrest in the basolateral complex of the amygdala (BLA) at different time points after memory acquisition and re-activation, using the fungal cytotoxin phalloidin. Our data show a selective disruption of auditory cued but not contextual fear memory, when phalloidin was injected 6 h after conditioning. In contrast, no effect was observed when phalloidin was applied after 24 h, ruling out an interference with the retrieval or expression of conditioned fear. A comparable result was obtained after memory re-activation, hence suggesting similar actin-dependent mechanisms to be active during consolidation and reconsolidation of auditory fear memory. Biochemical analysis showed that phalloidin-mediated filament arrest leads to a transient increase of highly cross-linked actin filaments in the BLA, evident 2 h after injection. Together, these observations indicate that dynamic re-arrangements of actin filaments in the BLA during a late phase of fear memory consolidation and reconsolidation are critical for fear memory storage.

Abstract 5116: p70 S6 kinase regulates the actin cytoskeleton in ovarian cancer cell motility by binding and cross-linking actin filaments

Abstract p70 S6 kinase (p70S6K) is a downstream effector of phosphatidylinositol 3-kinase and is frequently activated in human ovarian cancer. Here we show for the first time that p70S6K functions in actin cytoskeleton reorganization responsible for the acquisition of invasiveness during tumor progression. Ectopic expression of active p70S6K in ovarian cancer cells induced dramatic reorganization of the actin cytoskeleton and promoted cell migration. Inhibition of p70S6K by siRNA or small molecule inhibitor resulted in diminished cell migration and actin cytoskeleton reorganization, confirming that these effects were p70S6K specific. By using cosedimentation and differential sedimentation assays, p70S6K was found to bind actin filaments, and was more effective in the presence than in the absence of phosphorylation. Importantly, purified p70S6K could be cosedimented with purified filamentous actin, indicating a direct association between p70S6K and actin. The response to p70S6K could be blocked with cytochalasin D, indicating that the binding of p70S6K to the cytoskeleton is mediated by actin. In addition, our results suggest that this is a functional interaction, because p70S6K was capable of generating specialized, actin-based cell morphologies via direct cross-linking activity. This bundling activity was confirmed by light scattering and electron microscopy. These results, in conjunction with our prior evidence that p70S6K regulates Rac and Cdc42 GTPases, suggest that p70S6K serves as an important regulator of the actin cytoskeleton, highlighting a role for p70S6K in the regulation of tumor progression and metastasis in ovarian cancer (This work is supported by Hong Kong Research Grants Council Grant HKU7599/05M to A.S.T.W.). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5116.

Molecular mechanisms underlying nucleocytoplasmic shuttling of actinin-4

In addition to its well-known role as a crosslinker of actin filaments at focal-adhesion sites, actinin-4 is known to be localized to the nucleus. In this study, we reveal the molecular mechanism underlying nuclear localization of actinin-4 and its novel interactions with transcriptional regulators. We found that actinin-4 is imported into the nucleus through the nuclear pore complex in an importin-independent manner and is exported by the chromosome region maintenance-1 (CRM1)-dependent pathway. Nuclear actinin-4 levels were significantly increased in the late G2 phase of the cell cycle and were decreased in the G1 phase, suggesting that active release from the actin cytoskeleton was responsible for increased nuclear actinin-4 in late G2. Nuclear actinin-4 was found to interact with the INO80 chromatin-remodeling complex. It also directs the expression of a subset of cell-cycle-related genes and interacts with the upstream-binding factor (UBF)-dependent rRNA transcriptional machinery in the M phase. These findings provide molecular mechanisms for both nucleocytoplasmic shuttling of proteins that do not contain a nuclear-localization signal and cell-cycle-dependent gene regulation that reflects morphological changes in the cytoskeleton.

Force generated by actomyosin contraction builds bridges between adhesive contacts

Extracellular matrices in vivo are heterogeneous structures containing gaps that cells bridge with an actomyosin network. To understand the basis of bridging, we plated cells on surfaces patterned with fibronectin (FN)-coated stripes separated by non-adhesive regions. Bridges developed large tensions where concave cell edges were anchored to FN by adhesion sites. Actomyosin complexes assembled near those sites (both actin and myosin filaments) and moved towards the centre of the non-adhesive regions in a treadmilling network. Inhibition of myosin-II (MII) or Rho-kinase collapsed bridges, whereas extension continued over adhesive areas. Inhibition of actin polymerization (latrunculin-A, jasplakinolide) also collapsed the actomyosin network. We suggest that MII has distinct functions at different bridge regions: (1) at the concave edges of bridges, MIIA force stimulates actin filament assembly at adhesions and (2) in the body of bridges, myosin cross-links actin filaments and stimulates actomyosin network healing when breaks occur. Both activities ensure turnover of actin networks needed to maintain stable bridges from one adhesive region to another.

Measurement of Filamin a Torsion In-Singulo

Filamin A (FLNa) is one of the most ubiquitous actin binding proteins that cross-link actin filaments. The protein is a v-shaped homodimer. Each monomer consists of twenty-four β-sheet “rod-like” domains which separate the actin binding domain at the N-terminus from the C-terminus at the dimerization site. The rod-like domains are divided by two unstructured “hinge” sequences between repeats 15 and 16 and repeats 23 and 24. While it has been reported that these hinge sequences play a key role in determining the nonlinear elastic response of stressed actin networks, how the hinge segment elicits such changes in network elasticity remains unknown.

The regulatory action of α-actinin on actin filaments is enhanced by cofilin

We have used fluorescence recovery after photobleaching to study the effect of muscle alpha-actinin on the structure of actin filaments in dilute solutions. Unexpectedly we found that alpha-actinin partitioned filaments into two types: those with a high mobility and those with low mobility. We have determined that the high mobility (smaller sized) population is too large to be simple monomeric actin:alpha-actinin complexes. Although it is known that cofilin encourages the transformation of alpha-actinin:actin gels into large meshworks of inter-digitating actin filament bundles (Maciver et al. 1991), we have found that the presence of cofilin also increases the cross-linking of actin filaments by alpha-actinin and hypothesize that this is due to cofilin's ability to alter the filament twist. This effectively makes more potential alpha-actinin binding sites per unit of actin filament. As expected from previous work, this effect was more marked at pH 6.5 than at pH 8.0. Both effects are likely to operate in cells to deny other actin-binding proteins access to binding these particular filaments and may explain how very different actin cytoskeletal structures may co-exist in the same cell at the same time.

Filamin A Controls Embryonic Stem Cell Differentiation.

Abstract 4599Filamin A is a major non-muscle actin binding protein that plays an important role in cross-linking cortical actin filaments into three-dimensional networks. In addition to its role as a cytoskeletal scaffolding molecule, Filamin A is also known to bind more than 30 other proteins, regulating their subcellular location and coordinating their ability to signal. To analyze the role of filamin A in mouse embryonic stem (ES) cell maturation, we generated filamin ALow ES cells by introducing a micro-RNA that specifically downregulates filamin A expression under the control of a cytomegalovirus promoter. Filamin ALow ES cells exhibited a more rounded morphology than did their wild-type filamin ANormal counterparts, and expressed increased levels of the ES cell transcription factor Nanog. In contrast, non-transfected cells in the same culture dish retained normal expression of filamin A, expressed low levels of Nanog, and exhibited a more elongated and spread phenotype characteristic of differentiating cells. Further evidence for a role for filamin A in ES cell differentiation was provided by the observation that withdrawing leukemia inhibitory factor (LIF) to induce ES cell differentiation was accompanied by increased expression of filamin A, a concomitant loss of Nanog expression, and acquisition of a differentiated morphology. Filamin ALow ES cells were able to retain their undifferentiated phenotype, as evaluated by alkaline phosphatase (Alp) activity, in the presence of a 10-fold lower concentration of LIF than was permissive for filamin ANormal ES cells, or following exposure to the differentiating agent, bone morphogenic protein 4 (BMP4). LIF-induced phosphorylation of ERK was decreased in filamin ALow relative to filamin ANormal ES cells, as was BMP-induced phosphorylation of Smad1/5 - two signaling pathways that initiate ES cell differentiation. Finally, embryoid bodies comprised of filamin ALow ES cells were unable to differentiate into CD41+ hematopoietic progenitor cells. Taken together, these data demonstrate that filamin A plays a previously unrecognized, but critical, scaffolding function that support both the LIF - ERK and BMP4 - Smad1/5 signaling pathways leading to ES and hematopoietic cell differentiation. Manipulation of filamin levels might be useful in the future to modulate the differentiation requirements for a variety of clinically-and therapeutically-useful stem cells. Disclosures:Newman:Novo Nordisk: Consultancy; New York Blood Center: Membership on an entity's Board of Directors or advisory committees.

Phylogenetic analysis of gene structure and alternative splicing in alpha-actinins.

The alpha-actinins are an important family of actin-binding proteins with the ability to cross-link actin filaments when in dimer form. Members of the alpha-actinin family share a domain topology composed of highly conserved actin-binding and EF-hand domains separated by a rod domain composed of spectrin-like repeats. Functional diversity within this family has arisen through exon duplication and the formation of alternate splice isoforms as well as gene duplications during the evolution of vertebrates. In addition to the known functional domains, alpha-actinins also contain a consensus PDZ-binding site. The completed genome sequence of over 32 invertebrate species has allowed the analysis of gene structure and exon-gene duplication over a diverse range of phyla. Our analysis shows that relative to early branching metazoans, there has been considerable intron loss especially in arthropods with few cases of intron gains. The C-terminal PDZ-binding site is conserved in nearly all invertebrates but is missing in some nematodes and platyhelminths. Alternative splicing in the actin-binding domain is conserved in chordates, arthropods, and some nematodes and platyhelminths. In contrast, alternative splicing of the EF-hand domain is only observed in chordates. Finally, given the prevalence of exon duplications seen in the actin-binding domain, this may act as a significant mechanism in the modification of actin-binding properties.

In Silico Reconstitution of Actin-Based Symmetry Breaking and Motility

Author SummaryNetworks of actin filaments provide the force that drives eukaryotic cell movement. In a model system for this kind of force generation, a spherical bead coated with an actin nucleating protein builds and rockets around on an actin “comet tail,” much like the tails observed in some cellular systems. How does a spherically symmetric bead break the symmetry of the actin coat and begin to polymerize actin in a directional manner? A previous theoretical model successfully explained how symmetry breaks, but suggested that the subsequent motion was driven by actin squeezing the bead forwards—a prediction refuted by experiment. To understand how motility occurs, we created a parsimonious computer model that predicted novel experimental behaviors, then performed new experiments inspired by the model and confirmed these predictions. Our model demonstrates how the elastic properties of the actin network explain not only symmetry breaking, but also the details of subsequent motion and how the bead maintains direction.

Actin and myosin regulate cytoplasm stiffness in plant cells: a study using optical tweezers

Here, we produced cytoplasmic protrusions with optical tweezers in mature BY-2 suspension cultured cells to study the parameters involved in the movement of actin filaments during changes in cytoplasmic organization and to determine whether stiffness is an actin-related property of plant cytoplasm. Optical tweezers were used to create cytoplasmic protrusions resembling cytoplasmic strands. Simultaneously, the behavior of the actin cytoskeleton was imaged. After actin filament depolymerization, less force was needed to create cytoplasmic protrusions. During treatment with the myosin ATPase inhibitor 2,3-butanedione monoxime, more trapping force was needed to create and maintain cytoplasmic protrusions. Thus, the presence of actin filaments and, even more so, the deactivation of a 2,3-butanedione monoxime-sensitive factor, probably myosin, stiffens the cytoplasm. During 2,3-butanedione monoxime treatment, none of the tweezer-formed protrusions contained filamentous actin, showing that a 2,3-butanedione monoxime-sensitive factor, probably myosin, is responsible for the movement of actin filaments, and implying that myosin serves as a static cross-linker of actin filaments when its motor function is inhibited. The presence of actin filaments does not delay the collapse of cytoplasmic protrusions after tweezer release. Myosin-based reorganization of the existing actin cytoskeleton could be the basis for new cytoplasmic strand formation, and thus the production of an organized cytoarchitecture.

Dynamin2 GTPase and Cortactin Remodel Actin Filaments

The large GTPase dynamin, best known for its activities that remodel membranes during endocytosis, also regulates F-actin-rich structures, including podosomes, phagocytic cups, actin comet tails, subcortical ruffles, and stress fibers. The mechanisms by which dynamin regulates actin filaments are not known, but an emerging view is that dynamin influences F-actin via its interactions with proteins that interact directly or indirectly with actin filaments. We show here that dynamin2 GTPase activity remodels actin filaments in vitro via a mechanism that depends on the binding partner and F-actin-binding protein, cortactin. Tightly associated actin filaments cross-linked by dynamin2 and cortactin became loosely associated after GTP addition when viewed by transmission electron microscopy. Actin filaments were dynamically unraveled and fragmented after GTP addition when viewed in real time using total internal reflection fluorescence microscopy. Cortactin stimulated the intrinsic GTPase activity of dynamin2 and maintained a stable link between actin filaments and dynamin2, even in the presence of GTP. Filaments remodeled by dynamin2 GTPase in vitro exhibit enhanced sensitivity to severing by the actin depolymerizing factor, cofilin, suggesting that GTPase-dependent remodeling influences the interactions of actin regulatory proteins and F-actin. The global organization of the actomyosin cytoskeleton was perturbed in U2-OS cells depleted of dynamin2, implicating dynamin2 in remodeling actin filaments that comprise supramolecular F-actin arrays in vivo. We conclude that dynamin2 GTPase remodels actin filaments and plays a role in orchestrating the global actomyosin cytoskeleton.

Atomic Structures of Two Novel Immunoglobulin-like Domain Pairs in the Actin Cross-linking Protein Filamin

Filamins are actin filament cross-linking proteins composed of an N-terminal actin-binding domain and 24 immunoglobulin-like domains (IgFLNs). Filamins interact with numerous proteins, including the cytoplasmic domains of plasma membrane signaling and cell adhesion receptors. Thereby filamins mechanically and functionally link the cell membrane to the cytoskeleton. Most of the interactions have been mapped to the C-terminal IgFLNs 16-24. Similarly, as with the previously known compact domain pair of IgFLNa20-21, the two-domain fragments IgFLNa16-17 and IgFLNa18-19 were more compact in small angle x-ray scattering analysis than would be expected for two independent domains. Solution state NMR structures revealed that the domain packing in IgFLNa18-19 resembles the structure of IgFLNa20-21. In both domain pairs the integrin-binding site is masked, although the details of the domain-domain interaction are partly distinct. The structure of IgFLNa16-17 revealed a new domain packing mode where the adhesion receptor binding site of domain 17 is not masked. Sequence comparison suggests that similar packing of three tandem filamin domain pairs is present throughout the animal kingdom, and we propose that this packing is involved in the regulation of filamin interactions through a mechanosensor mechanism.

Adapter Protein SH2B1β Cross-Links Actin Filaments and Regulates Actin Cytoskeleton

The Src homology 2 (SH2) domain-containing adapter protein SH2B1beta plays a role in severe obesity, leptin and insulin resistance, and infertility. SH2B1beta was initially identified as a Janus tyrosine kinase 2 (JAK2) substrate, and it has been implicated in cell motility and regulation of the actin rearrangement in response to GH and platelet-derived growth factor. SH2B1beta is also required for maximal actin-based motility of Listeria. Here we have used a low-speed pelleting assay and electron microscopy to demonstrate that SH2B1beta has two actin-binding sites and that it cross-links actin filaments in vitro. Wild-type SH2B1beta localized to cell ruffles and along filopodia, but deletion of amino acids 150-200 (the first actin-binding site) led to mislocalization of the protein to filopodia tip complexes where it colocalized with vasodilator-stimulated phosphoprotein (VASP). Based on studies performed in VASP-deficient MVD7(-/-) cells, with or without green fluorescent protein-VASP reconstitution, we concluded that the proper intracellular localization of native SH2B1beta required the presence of the first SH2B1beta actin-binding site and VASP. Finally, we found that both SH2B1beta actin-binding domains were required for maximal GH- and prolactin-induced cell ruffling. Together, these results suggest that SH2B1beta functions as an adapter protein that cross-links actin filaments, leading to modulation of cellular responses in response to JAK2 activation.

Role of Alpha Actinin4 in Transcriptional Regulation

Alpha actinins (ACTNs) are known for their ability to modulate cytoskeletal organization and cell motility by crosslinking actin filaments. However, recently it has been shown that the ACTNs can also play an important role in transcriptional regulation. ACTN4 mutations including K228E, T232I and S235P are linked to familial focal segmental glomerulosclerosis (FSGS), a kidney disease characterized by proteinuria. The mechanisms by which these mutations in ACTN4 are associated with FSGS are not well defined. We have demonstrated here that ACTN4 potently activated transcription by nuclear receptors including the retinoid acid receptor (RAR), the vitamin D3 receptor (VDR) and peroxisome proliferator‐activated receptor (PPARγ). ACTN4 physically interacted with nuclear receptors in a hormone dependent manner and mutation in the nuclear receptor interacting motif disrupted its ability to stimulate transcription. Furthermore, FSGS‐linked ACTN4 mutants localized aberrantly and failed to potentiate transcriptional activation by nuclear hormone receptors. Our data demonstrate that the inability of FSGS to activate transcription by nuclear receptors correlates with their aberrant cytoplasmic localization and their inability to interact with nuclear receptors.

Crosslinking of microtubules and actin filaments by S100A8/S100A9

Microtubules (MT) and actin filaments of phagocytes exhibit highly dynamic properties. The underlying molecular mechanisms are not entirely known. Their exceptional dynamic cytoskeleton is the prerequisite of many phagocyte related functions like exocytosis, phagocytosis, and in particular migration. Cell dynamic/migration is a highly complex process strictly regulated by numerous signaling cascades often initiated by changes in cytosolic calcium concentrations. Therefore, calcium-binding proteins are key elements in signal transduction. The major calcium-binding molecules expressed in granulocytes and monocytes are myeloid-related protein 8 (MRP8 [S100A8]) and MRP14 (S100A9), two members of the S100 protein family. MRP8 and MRP14 form heterodimers in the absence and tetrameric complexes composed of two heterodimers in the presence of calcium. We demonstrate that MRP8/MRP14 tetramers promote MT polymerization as well as MT bundling; moreover, the complex crosslinks MTs to F-Actin in a strictly calcium dependent manner. HEK293 cells transfected with MRP8/MRP14 contain significantly more polymerized tubulin in comparison to mock-transfected cells. These results are in line with reduced level of polymerized tubulin in phagocytes isolated from MRP14 -/- mice. Furthermore, phagocytes of MRP14 knockout mice show altered migration rates compared to wildtype cells. In addition to calcium-induced activation the MRP8/MRP14 complex can be specifically phosphorylated by p38 mitogen-activated protein kinase (MAPK), which abrogates MT/F-Actin crosslinking. Thus MRP8/MRP14 integrates signals of at least two independent signaling pathways. Our results provide evidence, that the MRP8/MRP14 complex fulfils a pivotal role in remodeling cytoskeletal structures necessary for migration of leukocytes.

α-Actinin and Filamin Cooperatively Enhance the Stiffness of Actin Filament Networks

BackgroundThe close subcellular proximity of different actin filament crosslinking proteins suggests that these proteins may cooperate to organize F-actin structures to drive complex cellular functions during cell adhesion, motility and division. Here we hypothesize that α-actinin and filamin, two major F-actin crosslinking proteins that are both present in the lamella of adherent cells, display synergistic mechanical functions.Methodology/Principal FindingsUsing quantitative rheology, we find that combining α-actinin and filamin is much more effective at producing elastic, solid-like actin filament networks than α-actinin and filamin separately. Moreover, F-actin networks assembled in the presence of α-actinin and filamin strain-harden more readily than networks in the presence of either α-actinin or filamin.SignificanceThese results suggest that cells combine auxiliary proteins with similar ability to crosslink filaments to generate stiff cytoskeletal structures, which are required for the production of internal propulsive forces for cell migration, and that these proteins do not have redundant mechanical functions.