Tandem zyxin LIM sequences do not enhance force sensitive accumulation

The ability to sense mechanical forces is vital to cell physiology. Yet, the molecular basis of mechano-signaling remains unclear. Previous studies have shown that zyxin, a focal adhesion protein, is recruited at force-bearing sites on the actin cytoskeleton and, therefore, identifying zyxin as a mechano-sensing protein candidate. Furthermore, zyxin accumulation at force-bearing sites requires the LIM domain located at the C-terminus of zyxin. The zyxin LIM domain consists of three LIM motifs, each containing two zinc-binding sites. Since individual LIM motifs do not accumulate at focal adhesions or force-bearing sites, we hypothesize that multiple zyxin LIM domains increase force sensitivity. Using a miniature force sensor and GFP-tagged LIM variants, we quantified the relationship between single, tandem dimer and trimer LIM protein localization and traction forces. While the presence of extra LIM domains affected VASP recruitment to focal adhesions, force sensitivity was not enhanced over the single LIM domain. Therefore, zyxin force sensitivity is optimal with a single LIM domain, while additional LIM domains fail to enhance force sensitivity.     

Targeting of zyxin to sites of actin membrane interaction and to the nucleus

The localization of proteins to particular intracellular compartments often regulates their functions. Zyxin is a LIM protein found prominently at sites of cell adhesion, faintly in leading lamellipodia, and transiently in cell nuclei. Here we have performed a domain analysis to identify regions in zyxin that are responsible for targeting it to different subcellular locations. The N-terminal proline-rich region of zyxin, which harbors binding sites for alpha-actinin and members of the Ena/VASP family, concentrates in lamellipodial extensions and weakly in focal adhesions. The LIM region of zyxin displays robust targeting to focal adhesions. When overexpressed in cells, the LIM region of zyxin causes displacement of endogenous zyxin from focal adhesions. Upon mislocalization of full-length zyxin, at least one member of the Ena/VASP family is also displaced, and the organization of the actin cytoskeleton is perturbed. Zyxin also has the capacity to shuttle between the nucleus and focal adhesion sites. When nuclear export is inhibited, zyxin accumulates in cell nuclei. The nuclear accumulation of zyxin occurs asynchronously with approximately half of the cells exhibiting nuclear localization of zyxin within 2.3 h of initiating leptomycin B treatment. Our results provide insight into the functions of different zyxin domains.     

A zyxin head-tail interaction regulates zyxin-VASP complex formation

Zyxin is an adhesion protein that regulates actin assembly by binding to VASP family members through N-terminal proline-rich motifs. Evidence suggests that zyxin’s C-terminal LIM domains function as a negative regulator of zyxin-VASP complexes. Zyxin LIM domains access to binding partners is negatively regulated by an unknown mechanism. One possibility is that zyxin LIM domains mediate a head-tail interaction, blocking interactions with other proteins. Such a mechanism might prevent both zyxin-VASP complexes activity and LIM domain access. In this report, the effect of LIM domains on zyxin-VASP complex assembly is defined. We find that zyxin LIM domains associate with zyxin’s VASP binding sites, preventing zyxin from binding to PKA-phosphorylated VASP. Unphosphorylated VASP overcomes the head-tail interaction, a result of a direct interaction with the LIM domain region. Zyxin, like a growing number of actin regulators, is controlled by intramolecular interactions.     

Spatial proximity of proteins surrounding zyxin under force-bearing conditions

Sensing physical forces is a critical first step in mechano-transduction of cells. Zyxin, a LIM domain-containing protein, is recruited to force-bearing actin filaments and is thought to repair and strengthen them. Yet, the precise force-induced protein interactions surrounding zyxin remain unclear. Using BioID analysis, we identified proximal proteins surrounding zyxin under normal and force-bearing conditions by label-free mass spectrometry analysis. Under force-bearing conditions, increased biotinylation of α-actinin 1, α-actinin 4, and AFAP1 were detected, and these proteins accumulated along force-bearing actin fibers independently from zyxin, albeit at a lower intensity than zyxin. VASP also accumulated along force-bearing actin fibers in a zyxin-dependent manner, but the biotinylation of VASP remained constant regardless of force, supporting the model of a free zyxin–VASP complex in the cytoplasm being corecruited to tensed actin fibers. In addition, ARHGAP42, a RhoA GAP, was also identified as a proximal protein of zyxin and colocalized with zyxin along contractile actin bundles. The overexpression of ARHGAP42 reduced the rate of small wound closure, a zyxin-dependent process. These results demonstrate that the application of proximal biotinylation can resolve the proximity and composition of protein complexes as a function of force, which had not been possible with traditional biochemical analysis.     

Zyxin-mediated Actin Assembly Is Required for Efficient Wound Closure

Cytoskeletal regulation of cell adhesion is vital to the organization of multicellular structures. The focal adhesion protein zyxin emerged as a key regulator of actin assembly because zyxin recruits Enabled/vasodilator-stimulated phospho-proteins (Ena/VASP) to promote actin assembly. Zyxin also localizes to the sites of cell-cell adhesion and is thought to promote actin assembly with Ena/VASP. Using shRNA targeted to zyxin, we analyzed the roles of zyxin at adhesive contacts. In zyxin-deficient cells, the actin assembly at both focal adhesion and cell-cell adhesion was limited, but their migration rate was unchanged. Cell spreading on E-cadherin-coated surfaces and the formation of cell clusters were slower for zyxin-deficient cells than wild type cells. By ablating a single cell within a cell monolayer, we quantified the rate of wound closure driven by a contractile circumferential actin ring. Zyxin-deficient cells failed to recruit VASP to cell-cell junctions at the wound edge and had a slower wound closure rate than wild type cells. Our results suggest that, by recruiting VASP, zyxin regulates actin assembly at the sites of force-bearing cell-cell adhesion.     

Zyxin phosphorylation at serine 142 modulates the zyxin head-tail interaction to alter cell-cell adhesion

Zyxin is an actin regulatory protein that is concentrated at sites of actin–membrane association, particularly cell junctions. Zyxin participates in actin dynamics by binding VASP, an interaction that occurs via proline-rich N-terminal ActA repeats. An intramolecular association of the N-terminal LIM domains at or near the ActA repeats can prevent VASP and other binding partners from binding full-length zyxin. Such a head–tail interaction likely accounts for how zyxin function in actin dynamics, cell adhesion, and cell migration can be regulated by the cell. Since zyxin binding to several partners, via the LIM domains, requires phosphorylation, it seems likely that zyxin phosphorylation might alter the head–tail interaction and, thus, zyxin activity. Here we show that zyxin point mutants at a known phosphorylation site, serine 142, alter the ability of a zyxin fragment to directly bind a separate zyxin LIM domains fragment protein. Further, expression of the zyxin phosphomimetic mutant results in increased localization to cell–cell contacts of MDCK cells and generates a cellular phenotype, namely inability to disassemble cell–cell contacts, precisely like that produced by expression of zyxin mutants that lack the entire regulatory LIM domain region. These data suggest that zyxin phosphorylation at serine 142 results in release of the head–tail interaction, changing zyxin activity at cell–cell contacts.     

Delineating the Tes Interaction Site in Zyxin and Studying Cellular Effects of Its Disruption

Focal adhesions are integrin-based structures that link the actin cytoskeleton and the extracellular matrix. They play an important role in various cellular functions such as cell signaling, cell motility and cell shape. To ensure and fine tune these different cellular functions, adhesions are regulated by a large number of proteins. The LIM domain protein zyxin localizes to focal adhesions where it participates in the regulation of the actin cytoskeleton. Because of its interactions with a variety of binding partners, zyxin has been proposed to act as a molecular scaffold. Here, we studied the interaction of zyxin with such a partner: Tes. Similar to zyxin, Tes harbors three highly conserved LIM domains of which the LIM1 domain directly interacts with zyxin. Using different zyxin variants in pull-down assays and ectopic recruitment experiments, we identified the Tes binding site in zyxin and showed that four highly conserved amino acids are crucial for its interaction with Tes. Based upon these findings, we used a zyxin mutant defective in Tes-binding to assess the functional consequences of abrogating the zyxin-Tes interaction in focal adhesions. Performing fluorescence recovery after photobleaching, we showed that zyxin recruits Tes to focal adhesions and modulates its turnover in these structures. However, we also provide evidence for zyxin-independent localization of Tes to focal adhesions. Zyxin increases focal adhesion numbers and reduces focal adhesion lifetimes, but does so independent of Tes. Quantitative analysis showed that the loss of interaction between zyxin and Tes affects the process of cell spreading. We conclude that zyxin influences focal adhesion dynamics, that it recruits Tes and that this interaction is functional in regulating cell spreading.     

Molecular and phylogenetic characterization of Zyx102, a Drosophila orthologue of the zyxin family that interacts with Drosophila Enabled

Adherens junctions, which are cadherin-mediated junctions between cells, and focal adhesions, which are integrin-mediated junctions between cells and the extracellular matrix, are protein complexes that link the actin cytoskeleton to the plasma membrane and, in turn, to the extracellular environment. Zyxin is a LIM domain protein that is found in vertebrate adherens junctions and focal adhesions. Zyxin's molecular architecture and binding partner repertoire suggest roles in actin assembly and dynamics, cell motility, and nuclear-cytoplasmic communication. In order to study the function of zyxin in development, we have identified a zyxin orthologue in Drosophila melanogaster that we have termed Zyx102. Like its vertebrate counterparts, Zyx102 displays three carboxy-terminal LIM domains, a potential nuclear export signal, and three proline-rich motifs, one of which matches the consensus for mediating an interaction with Ena/VASP (Drosophila Enabled/Vasodilator-stimulated phosphoprotein) proteins. Here we show that Zyx102 and Enabled (Ena), the Drosophila member of the Ena/VASP family, can interact specifically in vitro and that this interaction does not occur when a particular mutant form of Ena, encoded by the lethal ena210 allele, is used. Lastly, we show that the zyx102 gene and Drosophila Ena are co-expressed during oogenesis and early embryogenesis, indicating that the two proteins may be able to interact during the development of the Drosophila egg chamber and early embryo.     

Stretch-induced actin remodeling requires targeting of zyxin to stress fibers and recruitment of actin regulators

Reinforcement of actin stress fibers in response to mechanical stimulation depends on a posttranslational mechanism that requires the LIM protein zyxin. The C-terminal LIM region of zyxin directs the force-sensitive accumulation of zyxin on actin stress fibers. The N-terminal region of zyxin promotes actin reinforcement even when Rho kinase is inhibited. The mechanosensitive integrin effector p130Cas binds zyxin but is not required for mitogen-activated protein kinase-dependent zyxin phosphorylation or stress fiber remodeling in cells exposed to uniaxial cyclic stretch. α-Actinin and Ena/VASP proteins bind to the stress fiber reinforcement domain of zyxin. Mutation of their docking sites reveals that zyxin is required for recruitment of both groups of proteins to regions of stress fiber remodeling. Zyxin-null cells reconstituted with zyxin variants that lack either α-actinin or Ena/VASP-binding capacity display compromised response to mechanical stimulation. Our findings define a bipartite mechanism for stretch-induced actin remodeling that involves mechanosensitive targeting of zyxin to actin stress fibers and localized recruitment of actin regulatory machinery.     

LIM Domains Target Actin Regulators Paxillin and Zyxin to Sites of Stress Fiber Strain

Contractile actomyosin stress fibers are critical for maintaining the force balance between the interior of the cell and its environment. Consequently, the actin cytoskeleton undergoes dynamic mechanical loading. This results in spontaneous, stochastic, highly localized strain events, characterized by thinning and elongation within a discrete region of stress fiber. Previous work showed the LIM-domain adaptor protein, zyxin, is essential for repair and stabilization of these sites. Using live imaging, we show paxillin, another LIM-domain adaptor protein, is also recruited to stress fiber strain sites. Paxillin recruitment to stress fiber strain sites precedes zyxin recruitment. Zyxin and paxillin are each recruited independently of the other. In cells lacking paxillin, actin recovery is abrogated, resulting in slowed actin recovery and increased incidence of catastrophic stress fiber breaks. For both paxillin and zyxin, the LIM domains are necessary and sufficient for recruitment. This work provides further evidence of the critical role of LIM-domain proteins in responding to mechanical stress in the actin cytoskeleton.     

Akt phosphorylation of zyxin mediates its interaction with acinus-S and prevents acinus-triggered chromatin condensation

Zyxin, a focal adhesion molecule, contains LIM domains and shuttles between the cytoplasm and the nucleus. Nuclear zyxin promotes cardiomyocyte survival, which is mediated by nuclear-activated Akt. However, the molecular mechanism of how zyxin antagonizes apoptosis remains elusive. Here, we report that zyxin binds to acinus-S, a nuclear speckle protein inducing apoptotic chromatin condensation after cleavage by caspases, and prevents its apoptotic action, which is regulated by Akt. Akt binds and phosphorylates zyxin on serine 142, leading to its association with acinus. Interestingly, 14-3-3gamma, but not zeta isoform selectively, triggers zyxin nuclear translocation, which is Akt phosphorylation dependent. Zyxin is also a substrate of caspases, but Akt phosphorylation is unable to prevent its apoptotic cleavage. Expression of zyxin S142D, a phosphorylation mimetic mutant, diminishes acinus proteolytic cleavage and chromatin condensation; by contrast, wild-type zyxin or unphosphorylated S142A mutant fails. Thus, Akt regulates zyxin/acinus complex formation in the nucleus, contributing to suppression of apoptosis.     

Zyxin interacts with the SH3 domains of the cytoskeletal proteins LIM-nebulette and Lasp-1

Zyxin is a versatile component of focal adhesions in eukaryotic cells. Here we describe a novel binding partner of zyxin, which we have named LIM-nebulette. LIM-nebulette is an alternative splice variant of the sarcomeric protein nebulette, which, in contrast to nebulette, is expressed in non-muscle cells. It displays a modular structure with an N-terminal LIM domain, three nebulin-like repeats, and a C-terminal SH3 domain and shows high similarity to another cytoskeletal protein, Lasp-1 (LIM and SH3 protein-1). Co-precipitation studies and results obtained with the two-hybrid system demonstrate that LIM-nebulette and Lasp-1 interact specifically with zyxin. Moreover, the SH3 domain from LIM-nebulette is both necessary and sufficient for zyxin binding. The SH3 domains from Lasp-1 and nebulin can also interact with zyxin, but the SH3 domains from more distantly related proteins such as vinexin and sorting nexin 9 do not. On the other hand, the binding site in zyxin is situated at the extreme N terminus as shown by site-directed mutagenesis. LIM-nebulette and Lasp-1 use the same linear binding motif. This motif shows some similarity to a class II binding site but does not contain the classical PXXP sequence. LIM-nebulette reveals a subcellular distribution at focal adhesions similar to Lasp-1. Thus, LIM-nebulette, Lasp-1, and zyxin may play an important role in the organization of focal adhesions.     

An alpha-actinin binding site of zyxin is essential for subcellular zyxin localization and alpha-actinin recruitment

The LIM domain protein zyxin is a component of adherens type junctions, stress fibers, and highly dynamic membrane areas and appears to be involved in microfilament organization. Chicken zyxin and its human counterpart display less than 60% sequence identity, raising concern about their functional identity. Here, we demonstrate that human zyxin, like the avian protein, specifically interacts with alpha-actinin. Furthermore, we map the interaction site to a motif of approximately 22 amino acids, present in the N-terminal domain of human zyxin. This motif is both necessary and sufficient for alpha-actinin binding, whereas a downstream region, which is related in sequence, appears to be dispensable. A synthetic peptide comprising human zyxin residues 21-42 specifically binds to alpha-actinin in solid phase binding assays. In contrast to full-length zyxin, constructs lacking this motif do not interact with alpha-actinin in blot overlays and fail to recruit alpha-actinin in living cells. When zyxin lacking the alpha-actinin binding site is expressed as a fusion protein with green fluorescent protein, association of the recombinant protein with stress fibers is abolished, and targeting to focal adhesions is grossly impaired. Our results suggest a crucial role for the alpha-actinin-zyxin interaction in subcellular zyxin localization and microfilament organization.     

Synemin interacts with the LIM domain protein zyxin and is essential for cell adhesion and migration

Synemin is a unique cytoplasmic intermediate filament protein for which there is limited understanding of its exact cellular functions. The single human synemin gene encodes at least two splice variants named α-synemin and β-synemin, with the larger α-synemin containing an additional 312 amino acid insert within the C-terminal tail domain. We report herein that, by using the entire tail domain of the smaller β-synemin as the bait in a yeast two-hybrid screen of a human skeletal muscle cDNA library, the LIM domain protein zyxin was identified as an interaction partner for human synemin. The synemin binding site in human zyxin was subsequently mapped to the C-terminal three tandem LIM-domain repeats, whereas the binding site for zyxin within β-synemin is within the C-terminal 332 amino acid region (SNβTII) at the end of the long tail domain. Transient expression of SNβTII within mammalian cells markedly reduced zyxin protein level, blocked localization of zyxin at focal adhesion sites and resulted in decreased cell adhesion and increased motility. Knockdown of synemin expression with siRNAs within mammalian cells resulted in significantly compromised cell adhesion and cell motility. Our results suggest that synemin participates in focal adhesion dynamics and is essential for cell adhesion and migration.     

Mechanical forces alter zyxin unbinding kinetics within focal adhesions of living cells

The formation of focal adhesions that mediate alterations of cell shape and movement is controlled by a mechanochemical mechanism in which cytoskeletal tensional forces drive changes in molecular assembly; however, little is known about the molecular biophysical basis of this response. Here, we describe a method to measure the unbinding rate constant k(OFF) of individual GFP-labeled focal adhesion molecules in living cells by modifying the fluorescence recovery after photobleaching (FRAP) technique and combining it with mathematical modeling. Using this method, we show that decreasing cellular traction forces on focal adhesions by three different techniques--chemical inhibition of cytoskeletal tension generation, laser incision of an associated actin stress fiber, or use of compliant extracellular matrices--increases the k(OFF) of the focal adhesion protein zyxin. In contrast, the k(OFF) of another adhesion protein, vinculin, remains unchanged after tension dissipation. Mathematical models also demonstrate that these force-dependent increases in zyxin's k(OFF) that occur over seconds are sufficient to quantitatively predict large-scale focal adhesion disassembly that occurs physiologically over many minutes. These findings demonstrate that the molecular binding kinetics of some, but not all, focal adhesion proteins are sensitive to mechanical force, and suggest that force-dependent changes in this biophysical parameter may govern the supramolecular events that underlie focal adhesion remodeling in living cells.     

Characterization of the interaction between zyxin and members of the Ena/vasodilator-stimulated phosphoprotein family of proteins

Zyxin contains a proline-rich N-terminal domain that is similar to the C-terminal domain in the ActA protein of the bacteria, Listeria monocytogenes. We screened the entire amino acid sequence of human zyxin for Mena-interacting peptides and found that, as with ActA, proline-rich sequences were the sole zyxin sequences capable of binding to Ena/vasodilator-stimulated phosphoprotein (VASP) family members in vitro. From this information, we tested zyxin mutants in which the proline-rich sequences were altered. The reduction in Mena/VASP binding was confirmed by peptide tests, immunoprecipitation, and ectopic expression of zyxin variants at the surface of mitochondria. By transfection assays we showed that zyxin interaction with Mena/VASP in vivo enhances the production of actin-rich structures at the apical surface of cells. Microinjection into cells of peptides corresponding to the first proline-rich sequence of zyxin caused the loss of Mena/VASP from focal contacts. Furthermore, these peptides reduced the degree of spreading of cells replated after trypsinization. We conclude that zyxin and proteins that harbor similar proline-rich repeats contribute to the positioning of Mena/VASP proteins. The positioning of Ena/VASP family members appears to be important when the actin cytoskeleton is reorganized, such as during spreading.     

Members of the Zyxin family of LIM proteins interact with members of the p130Cas family of signal transducers.

Integrin binding to extracellular matrix proteins induces formation of signaling complexes at focal adhesions. Zyxin co-localizes with integrins at sites of cell-substratum adhesion and is postulated to serve as a docking site for the assembly of multimeric protein complexes involved in regulating cell motility. Recently, we identified a new member of the zyxin family called TRIP6. TRIP6 is localized at focal adhesions and overexpression of TRIP6 slows cell migration. In an effort to define the molecular mechanism by which TRIP6 affects cell migration, the yeast two-hybrid assay was employed to identify proteins that directly bind to TRIP6. This assay revealed that both TRIP6 and zyxin interact with CasL/HEF1, a member of the Cas family. This association is mediated by the LIM region of the zyxin family members and the SH2 domain-binding region of CasL/HEF1. Furthermore, the association between p130(Cas) and the two zyxin family members was demonstrated to occur in vivo by co-immunoprecipitation. Zyxin and Cas family members may cooperate to regulate cell motility.     

An interaction between zyxin and alpha-actinin

Zyxin is an 82-kD protein first identified as a component of adhesion plaques and the termini of stress fibers near where they associate with the cytoplasmic face of the adhesive membrane. We report here that zyxin interacts with the actin cross-linking protein alpha-actinin. Zyxin cosediments with filamentous actin in an alpha-actinin-dependent manner and an association between zyxin and alpha-actinin is observed in solution by analytical gel filtration. The specificity of the interaction between zyxin and alpha-actinin was demonstrated by blot overlay experiments in which 125I-zyxin recognizes most prominently alpha-actinin among a complex mixture of proteins extracted from avian smooth muscle. By these blot overlay binding studies, we determined that zyxin interacts with the NH2-terminal 27-kD domain of alpha-actinin, a region that also contains the actin binding site. Solid phase binding assays were performed to evaluate further the specificity of the binding and to determine the affinity of the zyxin-alpha-actinin interaction. By these approaches we have demonstrated a specific, saturable, moderate-affinity interaction between zyxin and alpha-actinin. Furthermore, double-label immunofluorescence reveals that zyxin and alpha-actinin exhibit extensive overlap in their subcellular distributions in both chicken embryo fibroblasts and pigmented retinal epithelial cells. The significant colocalization of the two proteins is consistent with the possibility that the interaction between zyxin and alpha-actinin has a biologically relevant role in coordinating membrane-cytoskeletal interactions.