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.     

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.     

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.     

Force Dependent Biotinylation of Myosin IIA by α-Catenin Tagged with a Promiscuous Biotin Ligase

Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, β-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only β-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion.     

Essential role of zyxin in platelet biogenesis and glycoprotein Ib-IX surface expression

Platelets are generated from the cytoplasm of megakaryocytes (MKs) via actin cytoskeleton reorganization. Zyxin is a focal adhesion protein and wildly expressed in eukaryotes to regulate actin remodeling. Zyxin is upregulated during megakaryocytic differentiation; however, the role of zyxin in thrombopoiesis is unknown. Here we show that zyxin ablation results in profound macrothrombocytopenia. Platelet lifespan and thrombopoietin level were comparable between wild-type and zyxin-deficient mice, but MK maturation, demarcation membrane system formation, and proplatelet generation were obviously impaired in the absence of zyxin. Differential proteomic analysis of proteins associated with macrothrombocytopenia revealed that glycoprotein (GP) Ib-IX was significantly reduced in zyxin-deficient platelets. Moreover, GPIb-IX surface level was decreased in zyxin-deficient MKs. Knockdown of zyxin in a human megakaryocytic cell line resulted in GPIbα degradation by lysosomes leading to the reduction of GPIb-IX surface level. We further found that zyxin was colocalized with vasodilator-stimulated phosphoprotein (VASP), and loss of zyxin caused diffuse distribution of VASP and actin cytoskeleton disorganization in both platelets and MKs. Reconstitution of zyxin with VASP binding site in zyxin-deficient hematopoietic progenitor cell-derived MKs restored GPIb-IX surface expression and proplatelet generation. Taken together, our findings identify zyxin as a regulator of platelet biogenesis and GPIb-IX surface expression through VASP-mediated cytoskeleton reorganization, suggesting possible pathogenesis of macrothrombocytopenia.Subject terms: Cytoskeleton, Disease genetics     

CRP1, a LIM Domain Protein Implicated in Muscle Differentiation, Interacts with α-Actinin

Members of the cysteine-rich protein (CRP) family are LIM domain proteins that have been implicated in muscle differentiation. One strategy for defining the mechanism by which CRPs potentiate myogenesis is to characterize the repertoire of CRP binding partners. In order to identify proteins that interact with CRP1, a prominent protein in fibroblasts and smooth muscle cells, we subjected an avian smooth muscle extract to affinity chromatography on a CRP1 column. A 100-kD protein bound to the CRP1 column and could be eluted with a high salt buffer; Western immunoblot analysis confirmed that the 100-kD protein is α-actinin. We have shown that the CRP1–α-actinin interaction is direct, specific, and saturable in both solution and solid-phase binding assays. The K_d for the CRP1–α-actinin interaction is 1.8 ± 0.3 μM. The results of the in vitro protein binding studies are supported by double-label indirect immunofluorescence experiments that demonstrate a colocalization of CRP1 and α-actinin along the actin stress fibers of CEF and smooth muscle cells. Moreover, we have shown that α-actinin coimmunoprecipitates with CRP1 from a detergent extract of smooth muscle cells. By in vitro domain mapping studies, we have determined that CRP1 associates with the 27-kD actin–binding domain of α-actinin. In reciprocal mapping studies, we showed that α-actinin interacts with CRP1-LIM1, a deletion fragment that contains the NH₂-terminal 107 amino acids (aa) of CRP1. To determine whether the α-actinin binding domain of CRP1 would localize to the actin cytoskeleton in living cells, expression constructs encoding epitope-tagged full-length CRP1, CRP1-LIM1(aa 1-107), or CRP1-LIM2 (aa 108-192) were microinjected into cells. By indirect immunofluorescence, we have determined that full-length CRP1 and CRP1-LIM1 localize along the actin stress fibers whereas CRP1-LIM2 fails to associate with the cytoskeleton. Collectively these data demonstrate that the NH₂-terminal part of CRP1 that contains the α-actinin–binding site is sufficient to localize CRP1 to the actin cytoskeleton. The association of CRP1 with α-actinin may be critical for its role in muscle differentiation.     

Zyxin is not colocalized with vasodilator-stimulated phosphoprotein (VASP) at lamellipodial tips and exhibits different dynamics to vinculin, paxillin, and VASP in focal adhesions

Actin polymerization is accompanied by the formation of protein complexes that link extracellular signals to sites of actin assembly such as membrane ruffles and focal adhesions. One candidate recently implicated in these processes is the LIM domain protein zyxin, which can bind both Ena/vasodilator-stimulated phosphoprotein (VASP) proteins and the actin filament cross-linking protein alpha-actinin. To characterize the localization and dynamics of zyxin in detail, we generated both monoclonal antibodies and a green fluorescent protein (GFP)-fusion construct. The antibodies colocalized with ectopically expressed GFP-VASP at focal adhesions and along stress fibers, but failed to label lamellipodial and filopodial tips, which also recruit Ena/VASP proteins. Likewise, neither microinjected, fluorescently labeled zyxin antibodies nor ectopically expressed GFP-zyxin were recruited to these latter sites in live cells, whereas both probes incorporated into focal adhesions and stress fibers. Comparing the dynamics of zyxin with that of the focal adhesion protein vinculin revealed that both proteins incorporated simultaneously into newly formed adhesions. However, during spontaneous or induced focal adhesion disassembly, zyxin delocalization preceded that of either vinculin or paxillin. Together, these data identify zyxin as an early target for signals leading to adhesion disassembly, but exclude its role in recruiting Ena/VASP proteins to the tips of lamellipodia and filopodia.     

Mechanical force mobilizes zyxin from focal adhesions to actin filaments and regulates cytoskeletal reinforcement

Organs and tissues adapt to acute or chronic mechanical stress by remodeling their actin cytoskeletons. Cells that are stimulated by cyclic stretch or shear stress in vitro undergo bimodal cytoskeletal responses that include rapid reinforcement and gradual reorientation of actin stress fibers; however, the mechanism by which cells respond to mechanical cues has been obscure. We report that the application of either unidirectional cyclic stretch or shear stress to cells results in robust mobilization of zyxin from focal adhesions to actin filaments, whereas many other focal adhesion proteins and zyxin family members remain at focal adhesions. Mechanical stress also induces the rapid zyxin-dependent mobilization of vasodilator-stimulated phosphoprotein from focal adhesions to actin filaments. Thickening of actin stress fibers reflects a cellular adaptation to mechanical stress; this cytoskeletal reinforcement coincides with zyxin mobilization and is abrogated in zyxin-null cells. Our findings identify zyxin as a mechanosensitive protein and provide mechanistic insight into how cells respond to mechanical cues.     

The LIM domain of zyxin is sufficient for force-induced accumulation of zyxin during cell migration

Cellular responses to mechanical perturbation are vital to cell physiology. In particular, migrating cells have been shown to sense substrate stiffness and alter cell morphology and speed. Zyxin is a focal adhesion protein that responds to external mechanical forces; however, the mechanisms of zyxin recruitment at force-bearing sites are unknown. Using force-sensing microfabricated substrates, we simultaneously measured traction force and zyxin recruitment at force-bearing sites. GFP-tagged zyxin accumulates at force-bearing sites at the leading edge, but not at the trailing edge, of migrating epithelial cells. Zyxin recruitment at force-bearing sites depends on Rho-kinase and myosin II activation, suggesting that zyxin responds not only to the externally applied force, as previously shown, but also to the internally generated actin-myosin force. Zyxin in turn recruits vasodilator-stimulated phosphoprotein, a regulator of actin assembly, to force-bearing sites. To dissect the domains of zyxin that are essential for this unique force-dependent accumulation, we generated two zyxin truncation mutants: one lacking the LIM domain (ΔLIM) and one containing only the LIM domain with all three LIM motifs (LIM). GFP-tagged ΔLIM does not localize to the force-bearing sites, but GFP-tagged zyxin LIM-domain is sufficient for the recruitment to and dynamics at force-bearing focal adhesions. Furthermore, one or two LIM motifs are not sufficient for force-dependent accumulation, suggesting that all three LIM motifs are required. Therefore, the LIM domain of zyxin recruits zyxin to force-bearing sites at the leading edge of migrating cells.     

Distribution of flourescently labeled α-actinin in living and fixed fibroblasts

The distribution of flourescently labeled α-actinin after microinjection into fibroblasts has been determined in both living and fixed cells. We have found that the distribution of the injected tetramethylrhodamine isthiocyanate-labeled protein (TMRITC-α-actinin) in living cells, which is in ruffling membranes, actin microfilament bundles, and polygonal microfilament networks (Feramisco, 1979, Proc. Natl. Acad. Sci. U. S. A. 76:3967-3971), was virtually unaffected by the fixation (3.5 percent formaldehyde) and extraction (absolute acetone) used for the preparation of the cells for immunoflourescence. Also, these patterns were found to coincide with the α-actinin revealed by immunoflourescence. Also, these patterns were found to coincide with the α-actinin revealed by immunoflourescence. These findings offer, for the first time, evidence indicating the validity of the immunoflourescence technique in the localization of α-actinin in cultured cells. With the combination of the injection procedure and the immunoflourescence localization of endogenous structural proteins, it was determined that nearly all of the actin stress fibers were decorated in a periodic manner with the injected α-actinin. Endogenous tropomyosin in the injected cells was found to be distributed with a periodic pattern along the stress fibers that was antiperiodic to the pattern observed for the microinjected α-actinin. The tropomyosin antibody stained the polygonal microfilament networks and was excluded from the foci, whereas the microinjected α-actinin was incorporated into the foci of the networks. Thus, the microinjected fluorescent derivative of α-actinin appears to be incorporated into the functional pools of α-actinin within the living cell and to be utilized by the cell with fidelity.     

Cytoskeleton assembly at endothelial cell-cell contacts is regulated by alphaII-spectrin-VASP complexes

Directed cortical actin assembly is the driving force for intercellular adhesion. Regulated by phosphorylation, vasodilator-stimulated phosphoprotein (VASP) participates in actin fiber formation. We screened for endothelial proteins, which bind to VASP, dependent on its phosphorylation status. Differential proteomics identified αII-spectrin as such a VASP-interacting protein. αII-Spectrin binds to the VASP triple GP₅-motif via its SH3 domain. cAMP-dependent protein kinase–mediated VASP phosphorylation at Ser157 inhibits αII-spectrin–VASP binding. VASP is dephosphorylated upon formation of cell–cell contacts and in confluent, but not in sparse cells, αII-spectrin colocalizes with nonphosphorylated VASP at cell–cell junctions. Ectopic expression of the αII-spectrin SH3 domain at cell–cell contacts translocates VASP, initiates cortical actin cytoskeleton formation, stabilizes cell–cell contacts, and decreases endothelial permeability. Conversely, the permeability of VASP-deficient endothelial cells (ECs) and microvessels of VASP-null mice increases. Reconstitution of VASP-deficient ECs rescues barrier function, whereas αII-spectrin binding-deficient VASP mutants fail to restore elevated permeability. We propose that αII-spectrin–VASP complexes regulate cortical actin cytoskeleton assembly with implications for vascular permeability.     

Molecular dissection of zyxin function reveals its involvement in cell motility

Spatially controlled actin filament assembly is critical for numerous processes, including the vectorial cell migration required for wound healing, cell- mediated immunity, and embryogenesis. One protein implicated in the regulation of actin assembly is zyxin, a protein concentrated at sites where the fast growing ends of actin filaments are enriched. To evaluate the role of zyxin in vivo, we developed a specific peptide inhibitor of zyxin function that blocks its interaction with alpha-actinin and displaces it from its normal subcellular location. Mislocalization of zyxin perturbs cell migration and spreading, and affects the behavior of the cell edge, a structure maintained by assembly of actin at sites proximal to the plasma membrane. These results support a role for zyxin in cell motility, and demonstrate that the correct positioning of zyxin within the cell is critical for its physiological function. Interestingly, the mislocalization of zyxin in the peptide-injected cells is accompanied by disturbances in the distribution of Ena/VASP family members, proteins that have a well-established role in promoting actin assembly. In concert with previous work, our findings suggest that zyxin promotes the spatially restricted assembly of protein complexes necessary for cell motility.     

Distinct forms of the actin cross-linking protein α-actinin support macropinosome internalization and trafficking

The α-actinin family of actin cross-linking proteins have been implicated in driving tumor cell metastasis through regulation of the actin cytoskeleton; however, there has been little investigation into whether these proteins can influence tumor cell growth. We demonstrate that α-actinin 1 and 4 are essential for nutrient uptake through the process of macropinocytosis in pancreatic ductal adenocarcinoma (PDAC) cells, and inhibition of these proteins decreases tumor cell survival in the presence of extracellular protein. The α-actinin proteins play essential roles throughout the macropinocytic process, where α-actinin 4 stabilizes the actin cytoskeleton on the plasma membrane to drive membrane ruffling and macropinosome internalization and α-actinin 1 localizes to actin tails on macropinosomes to facilitate trafficking to the lysosome for degradation. In addition to tumor cell growth, we also observe that the α-actinin proteins can influence uptake of chemotherapeutics and extracellular matrix proteins through macropinocytosis, suggesting that the α-actinin proteins can regulate multiple tumor cell properties through this endocytic process. In summary, these data demonstrate a critical role for the α-actinin isoforms in tumor cell macropinocytosis, thereby affecting the growth and invasive potential of PDAC tumors.     

Zyxin-VASP interactions alter actin regulatory activity in zyxin-VASP complexes

Cell-cell and cell-substrate adhesions are sites of dramatic actin rearrangements and where actin-membrane connections are tightly regulated. Zyxin-VASP complexes localize to sites of cell-cell and cell-substrate adhesion and function to regulate actin dynamics and actin-membrane connections at these sites. To accomplish these functions, zyxin recruits VASP to cellular sites via proline-rich binding sites near zyxin’s amino terminus. While the prevailing thought has been that zyxin simply acts as a scaffold protein for VASP binding, the identification of a LIM domain-VASP interaction could complicate this view. Here we assess how zyxin-VASP binding through both the proline rich motifs and the LIM domains alters specific VASP functions. We find that neither individual interaction alters VASP’s actin regulatory activities. In contrast, however, we find that full-length zyxin dramatically reduces VASPmediated actin bundling and actin assembly. Taken together, these results suggest a model where zyxin-VASP complexes occur in complex organizations with suppressed actin regulatory activity.     

Opposing roles of zyxin/LPP ACTA repeats and the LIM domain region in cell-cell adhesion

Cadherins mediate cell-cell adhesion by linking cell junctions to actin networks. Although several actin regulatory systems have been implicated in cell-cell adhesion, it remains unclear how such systems drive cadherin-actin network formation and how they are regulated to coincide with initiation of adhesion. Previous work implicated VASP in assembly of cell-cell junctions in keratinocytes and the VASP-binding protein zyxin colocalizes with VASP at cell-cell junctions. Here we examine how domains in zyxin and its relative LPP contribute to cell-cell junction assembly. Using a quantitative assay for cell-cell adhesion, we demonstrate that zyxin and LPP function to increase the rate of early cell-cell junction assembly through the VASP-binding ActA repeat region. We also identify the LIM region of zyxin and LPP to be a regulatory domain that blocks function of these proteins. Deletion of the LIM domains drives adhesion and increases VASP level in detergent insoluble cadherin-actin. Dominant-negative zyxin/LPP mutants reduce the rate of adhesion, lower VASP levels in detergent-insoluble cadherin-actin networks, and allow for the accumulation of capping protein at cell-cell contacts. These data implicate the LIM domains of zyxin and LPP in regulating cell-cell junction assembly through VASP.     

Inducible recruitment of Cdc42 or WASP to a cell-surface receptor triggers actin polymerization and filopodium formation

BACKGROUND: Cdc42, a GTP-binding protein of the Rho family, controls actin cytoskeletal organization and helps to generate actin-based protruding structures, such as filopodia. In vitro, Cdc42 regulates actin polymerization by facilitating the creation of free barbed ends - the more rapidly growing ends of actin filaments - and subsequent elongation at these ends. The Wiskott- Aldrich syndrome protein, WASP, which has a pleckstrin-homology domain and a Cdc42/Rac-binding motif, has been implicated in cell signaling and cytoskeleton reorganization. We have investigated the consequences of local recruitment of activated Cdc42 or WASP to the plasma membrane. RESULTS: We used an activated Cdc42 protein that could be recruited to an engineered membrane receptor by adding rapamycin as a bridge, and added antibody-coupled beads to aggregate these receptors. Inducible recruitment of Cdc42 to clusters of receptors stimulated actin polymerization, resulting in the formation of membrane protrusions. Cdc42-induced protrusions were enriched in the vasodilator-stimulated phosphoprotein VASP and the focal-adhesion-associated proteins zyxin and ezrin. The Cdc42 effector WASP could also induce the formation of protrusions, albeit of different morphology. CONCLUSIONS: This is the first demonstration that the local recruitment of activated Cdc42 or its downstream effector, WASP, to a membrane receptor in whole cells is sufficient to trigger actin polymerization that results in the formation of membrane protrusions. Our data suggest that Cdc42-induced actin-based protrusions result from the local and serial recruitment of cytoskeletal proteins including zyxin, VASP, and ezrin.     

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.     

Palladin is a novel binding partner for Ena/VASP family members

Palladin is an actin-associated protein that contains proline-rich motifs within its amino-terminal sequence that are similar to motifs found in zyxin, vinculin, and the Listeria protein ActA. These motifs are known to be potential binding sites for the Vasodilator-Stimulated Phosphoprotein (VASP). Here, we demonstrate that palladin is an additional direct binding partner for VASP, by using co-immunoprecipitation and blot overlay techniques with both endogenous palladin and recombinant myc-tagged palladin. These results show that VASP binds to full-length palladin and also to the amino-terminal half of palladin, where the polyproline motifs are located. Using a synthetic peptide array, two discrete binding sites for VASP were identified within palladin's proline-rich amino-terminal domain. Using double-label immunofluorescence staining of fully-spread and actively-spreading fibroblasts, the extent of co-localization of palladin and VASP was explored. These proteins were found to strongly co-localize along stress fibers, and partially co-localize in focal adhesions, lamellipodia, and focal complexes. These results suggest that the recently described actin-associated protein palladin may play an important role in recruiting VASP to sites of actin filament growth, anchorage, and crosslinking.     

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.