azyx-1 is a new gene that overlaps with zyxin and affects its translation in C. elegans, impacting muscular integrity and locomotion.

Overlapping genes are widely prevalent; however, their expression and consequences are poorly understood. Here, we describe and functionally characterize a novel zyx-1 overlapping gene, azyx-1, with distinct regulatory functions in Caenorhabditis elegans. We observed conservation of alternative open reading frames (ORFs) overlapping the 5' region of zyxin family members in several animal species, and find shared sites of azyx-1 and zyxin proteoform expression in C. elegans. In line with a standard ribosome scanning model, our results support cis regulation of zyx-1 long isoform(s) by upstream initiating azyx-1a. Moreover, we report on a rare observation of trans regulation of zyx-1 by azyx-1, with evidence of increased ZYX-1 upon azyx-1 overexpression. Our results suggest a dual role for azyx-1 in influencing zyx-1 proteoform heterogeneity and highlight its impact on C. elegans muscular integrity and locomotion.

Zyxin contributes to coupling between cell junctions and contractile actomyosin networks during apical constriction.

One of the most common cell shape changes driving morphogenesis in diverse animals is the constriction of the apical cell surface. Apical constriction depends on contraction of an actomyosin network in the apical cell cortex, but such actomyosin networks have been shown to undergo continual, conveyor belt-like contractions before the shrinking of an apical surface begins. This finding suggests that apical constriction is not necessarily triggered by the contraction of actomyosin networks, but rather can be triggered by unidentified, temporally-regulated mechanical links between actomyosin and junctions. Here, we used C. elegans gastrulation as a model to seek genes that contribute to such dynamic linkage. We found that α-catenin and ß-catenin initially failed to move centripetally with contracting cortical actomyosin networks, suggesting that linkage is regulated between intact cadherin-catenin complexes and actomyosin. We used proteomic and transcriptomic approaches to identify new players, including the candidate linkers AFD-1/afadin and ZYX-1/zyxin, as contributing to C. elegans gastrulation. We found that ZYX-1/zyxin is among a family of LIM domain proteins that have transcripts that become enriched in multiple cells just before they undergo apical constriction. We developed a semi-automated image analysis tool and used it to find that ZYX-1/zyxin contributes to cell-cell junctions' centripetal movement in concert with contracting actomyosin networks. These results identify several new genes that contribute to C. elegans gastrulation, and they identify zyxin as a key protein important for actomyosin networks to effectively pull cell-cell junctions inward during apical constriction. The transcriptional upregulation of ZYX-1/zyxin in specific cells in C. elegans points to one way that developmental patterning spatiotemporally regulates cell biological mechanisms in vivo. Because zyxin and related proteins contribute to membrane-cytoskeleton linkage in other systems, we anticipate that its roles in regulating apical constriction in this manner may be conserved.

Structured illumination microscopy with noise-controlled image reconstructions.

Super-resolution structured illumination microscopy (SIM) has become a widely used method for biological imaging. Standard reconstruction algorithms, however, are prone to generate noise-specific artifacts that limit their applicability for lower signal-to-noise data. Here we present a physically realistic noise model that explains the structured noise artifact, which we then use to motivate new complementary reconstruction approaches. True-Wiener-filtered SIM optimizes contrast given the available signal-to-noise ratio, and flat-noise SIM fully overcomes the structured noise artifact while maintaining resolving power. Both methods eliminate ad hoc user-adjustable reconstruction parameters in favor of physical parameters, enhancing objectivity. The new reconstructions point to a trade-off between contrast and a natural noise appearance. This trade-off can be partly overcome by further notch filtering but at the expense of a decrease in signal-to-noise ratio. The benefits of the proposed approaches are demonstrated on focal adhesion and tubulin samples in two and three dimensions, and on nanofabricated fluorescent test patterns.

O-GlcNAc transferase promotes the nuclear localization of the focal adhesion-associated protein Zyxin to regulate UV-induced cell death.

Zyxin is a zinc-binding phosphoprotein known to regulate cell migration, adhesion, and cell survival. Zyxin also plays a role in signal transduction between focal adhesions and the nuclear compartment. However, the mechanism of Zyxin shuttling to nucleus is still unclear. Here, we identify that the GlcNAc transferase (O-linked GlcNAc [O-GlcNAc] transferase) can O-GlcNAcylate Zyxin and regulate its nuclear localization. We show that O-GlcNAc transferase O-GlcNAcylates Zyxin at two residues, serine 169 (Ser-169) and Ser-246. In addition, O-GlcNAcylation of Ser-169, but not Ser-246, enhances its interaction with 14-3-3γ, which is a phosphoserine/threonine-binding protein and is reported to bind with phosphorylated Zyxin. Furthermore, we found that 14-3-3γ could promote the nuclear localization of Zyxin after Ser-169 O-GlcNAcylation by affecting the function of the N-terminal nuclear export signal sequence; functionally, UV treatment increases the O-GlcNAcylation of Zyxin, which may enhance the nuclear location of Zyxin. Finally, Zyxin in the nucleus maintains homeodomain-interacting protein kinase 2 stability and promotes UV-induced cell death. In conclusion, we uncover that the nuclear localization of Zyxin can be regulated by its O-GlcNAcylation, and that this protein may regulate UV-induced cell death.

Variants of Focal Adhesion Scaffold Genes Cause Thoracic Aortic Aneurysm.

RATIONALE: Thoracic aortic aneurysm (TAA) leads to substantial mortality worldwide. Familial and syndromic TAAs are highly correlated with genetics. However, the incidence of sporadic isolated TAA (iTAA) is much higher, and the genetic contribution is not yet clear. OBJECTIVE: Here, we examined the genetic characteristics of sporadic iTAA. METHODS AND RESULTS: We performed a genetic screen of 551 sporadic iTAA cases and 1071 controls via whole-exome sequencing. The prevalence of pathogenic mutations in known causal genes was 5.08% in the iTAA cohort. We selected 100 novel candidate genes using a strict strategy, and the suspected functional variants of these genes were significantly enriched in cases compared with controls and carried by 60.43% of patients. We found more severe phenotypes and a lower proportion of hypertension in cases with pathogenic mutations or suspected functional variants. Among the candidate genes, Testin (TES), which encodes a focal adhesion scaffold protein, was identified as a potential TAA causal gene, accounting for 4 patients with 2 missense variants in the LIM1 domain (c.751T>C encoding p.Y251H; c.838T>C encoding p.Y280H) and highly expressed in the aorta. The 2 variants led to a decrease in TES expression. The thoracic aorta was spontaneously dilated in the TesY249H knock-in and Tes-/- mice. Mechanistically, the p.Y249H variant or knockdown of TES led to the repression of vascular smooth muscle cell contraction genes and disturbed the vascular smooth muscle cell contractile phenotype. Interestingly, suspected functional variants of other focal adhesion scaffold genes, including TLN1 (Talin-1) and ZYX (zyxin), were also significantly enriched in patients with iTAA; moreover, their knockdown resulted in decreased contractility of vascular smooth muscle cells. CONCLUSIONS: For the first time, this study revealed the genetic landscape across iTAA and showed that the focal adhesion scaffold genes are critical in the pathogenesis of iTAA.

Zyxin protects from hypertension-induced cardiac dysfunction.

Arterial hypertension causes left ventricular hypertrophy leading to dilated cardiomyopathy. Following compensatory cardiomyocyte hypertrophy, cardiac dysfunction develops due to loss of cardiomyocytes preceded or paralleled by cardiac fibrosis. Zyxin acts as a mechanotransducer in vascular cells that may promote cardiomyocyte survival. Here, we analyzed cardiac function during experimental hypertension in zyxin knockout (KO) mice. In zyxin KO mice, made hypertensive by way of deoxycorticosterone acetate (DOCA)-salt treatment telemetry recording showed an attenuated rise in systolic blood pressure. Echocardiography indicated a systolic dysfunction, and isolated working heart measurements showed a decrease in systolic elastance. Hearts from hypertensive zyxin KO mice revealed increased apoptosis, fibrosis and an upregulation of active focal adhesion kinase as well as of integrins α5 and ß1. Both interstitial and perivascular fibrosis were even more pronounced in zyxin KO mice exposed to angiotensin II instead of DOCA-salt. Stretched microvascular endothelial cells may release collagen 1α2 and TGF-ß, which is characteristic for the transition to an intermediate mesenchymal phenotype, and thus spur the transformation of cardiac fibroblasts to myofibroblasts resulting in excessive scar tissue formation in the heart of hypertensive zyxin KO mice. While zyxin KO mice per se do not reveal a cardiac phenotype, this is unmasked upon induction of hypertension and owing to enhanced cardiomyocyte apoptosis and excessive fibrosis causes cardiac dysfunction. Zyxin may thus be important for the maintenance of cardiac function in spite of hypertension.

The Cytoskeletal Protein Zyxin Inhibits Retinoic Acid Signaling by Destabilizing the Maternal mRNA of the RXRγ Nuclear Receptor.

Zyxin is an LIM-domain-containing protein that regulates the assembly of F-actin filaments in cell contacts. Additionally, as a result of mechanical stress, Zyxin can enter nuclei and regulate gene expression. Previously, we found that Zyxin could affect mRNA stability of the maternally derived stemness factors of Pou5f3 family in Xenopus laevis embryos through binding to Y-box factor1. In the present work, we demonstrate that Zyxin can also affect mRNA stability of the maternally derived retinoid receptor Rxrγ through the same mechanism. Moreover, we confirmed the functional link between Zyxin and Rxrγ-dependent gene expression. As a result, Zyxin appears to play an essential role in the regulation of the retinoic acid signal pathway during early embryonic development. Besides, our research indicates that the mechanism based on the mRNA destabilization by Zyxin may take part in the control of the expression of a fairly wide range of maternal genes.

Mechanosensitive regulation of stanniocalcin-1 by zyxin and actin-myosin in human mesenchymal stromal cells.

Stanniocalcin-1 (STC1) secreted by mesenchymal stromal cells (MSCs) has anti-inflammatory functions, reduces apoptosis, and aids in angiogenesis, both in vitro and in vivo. However, little is known about the molecular mechanisms of its regulation. Here, we show that STC1 secretion is increased only under specific cell-stress conditions. We find that this is due to a change in actin stress fibers and actin-myosin tension. Abolishment of stress fibers by blebbistatin and knockdown of the focal adhesion protein zyxin leads to an increase in STC1 secretion. To also study this connection in 3D, where few focal adhesions and actin stress fibers are present, STC1 expression was analyzed in 3D alginate hydrogels and 3D electrospun scaffolds. Indeed, STC1 secretion was increased in these low cellular tension 3D environments. Together, our data show that STC1 does not directly respond to cell stress, but that it is regulated through mechanotransduction. This research takes a step forward in the fundamental understanding of STC1 regulation and can have implications for cell-based regenerative medicine, where cell survival, anti-inflammatory factors, and angiogenesis are critical.

Actin polymerization state regulates osteogenic differentiation in human adipose-derived stem cells.

BACKGROUND: Actin is an essential cellular protein that assembles into microfilaments and regulates numerous processes such as cell migration, maintenance of cell shape, and material transport. METHODS: In this study, we explored the effect of actin polymerization state on the osteogenic differentiation of human adipose-derived stem cells (hASCs). The hASCs were treated for 7 days with different concentrations (0, 1, 5, 10, 20, and 50 nM) of jasplakinolide (JAS), a reagent that directly polymerizes F-actin. The effects of the actin polymerization state on cell proliferation, apoptosis, migration, and the maturity of focal adhesion-related proteins were assessed. In addition, western blotting and alizarin red staining assays were performed to assess osteogenic differentiation. RESULTS: Cell proliferation and migration in the JAS (0, 1, 5, 10, and 20 nM) groups were higher than in the control group and the JAS (50 nM) group. The FAK, vinculin, paxillin, and talin protein expression levels were highest in the JAS (20 nM) group, while zyxin expression was highest in the JAS (50 nM) group. Western blotting showed that osteogenic differentiation in the JAS (0, 1, 5, 10, 20, and 50 nM) group was enhanced compared with that in the control group, and was strongest in the JAS (50 nM) group. CONCLUSIONS: In summary, our data suggest that the actin polymerization state may promote the osteogenic differentiation of hASCs by regulating the protein expression of focal adhesion-associated proteins in a concentration-dependent manner. Our findings provide valuable information for exploring the mechanism of osteogenic differentiation in hASCs.

Zyxin promotes colon cancer tumorigenesis in a mitotic phosphorylation-dependent manner and through CDK8-mediated YAP activation.

Zyxin is a member of the focal adhesion complex and plays a critical role in actin filament polymerization and cell motility. Several recent studies showed that Zyxin is a positive regulator of Yki/YAP (Yes-associated protein) signaling. However, little is known about the mechanisms by which Zyxin itself is regulated and how Zyxin affects Hippo-YAP activity. We first showed that Zyxin is phosphorylated by CDK1 during mitosis. Depletion of Zyxin resulted in significantly impaired colon cancer cell proliferation, migration, anchorage-independent growth, and tumor formation in xenograft animal models. Mitotic phosphorylation is required for Zyxin activity in promoting growth. Zyxin regulates YAP activity through the colon cancer oncogene CDK8. CDK8 knockout phenocopied Zyxin knockdown in colon cancer cells, while ectopic expression of CDK8 substantially restored the tumorigenic defects of Zyxin-depletion cells. Mechanistically, we showed that CDK8 directly phosphorylated YAP and promoted its activation. Fully activated YAP is required to support the growth in CDK8-knockout colon cancer cells in vitro and in vivo. Together, these observations suggest that Zyxin promotes colon cancer tumorigenesis in a mitotic-phosphorylation-dependent manner and through CDK8-mediated YAP activation.

Bioinformatic Analysis of Structure and Function of LIM Domains of Human Zyxin Family Proteins.

Members of the human Zyxin family are LIM domain-containing proteins that perform critical cellular functions and are indispensable for cellular integrity. Despite their importance, not much is known about their structure, functions, interactions and dynamics. To provide insights into these, we used a set of in-silico tools and databases and analyzed their amino acid sequence, phylogeny, post-translational modifications, structure-dynamics, molecular interactions, and functions. Our analysis revealed that zyxin members are ohnologs. Presence of a conserved nuclear export signal composed of LxxLxL/LxxxLxL consensus sequence, as well as a possible nuclear localization signal, suggesting that Zyxin family members may have nuclear and cytoplasmic roles. The molecular modeling and structural analysis indicated that Zyxin family LIM domains share similarities with transcriptional regulators and have positively charged electrostatic patches, which may indicate that they have previously unanticipated nucleic acid binding properties. Intrinsic dynamics analysis of Lim domains suggest that only Lim1 has similar internal dynamics properties, unlike Lim2/3. Furthermore, we analyzed protein expression and mutational frequency in various malignancies, as well as mapped protein-protein interaction networks they are involved in. Overall, our comprehensive bioinformatic analysis suggests that these proteins may play important roles in mediating protein-protein and protein-nucleic acid interactions.

Zyxin (ZYX) promotes invasion and acts as a biomarker for aggressive phenotypes of human glioblastoma multiforme.

Glioblastoma multiforme (GBM) is characterized by highly invasive growth, which leads to extensive infiltration and makes complete tumor excision difficult. Since cytoskeleton proteins are related to leading processes and cell motility, and through analysis of public GBM databases, we determined that an actin-interacting protein, zyxin (ZYX), may involved in GBM invasion. Our own glioma cohort as well as the cancer genome atlas (TCGA), Rembrandt, and Gravendeel databases consistently showed that increased ZYX expression was related to tumor progression and poor prognosis of glioma patients. In vitro and in vivo experiments further confirmed the oncogenic roles of ZYX and demonstrated the role of ZYX in GBM invasive growth. Moreover, RNA-seq and mass-spectrum data from GBM cells with or without ZYX revealed that stathmin 1 (STMN1) was a potential target of ZYX. Subsequently, we found that both mRNA and protein levels of STMN1 were positively regulated by ZYX. Functionally, STMN1 not only promoted invasion of GBM cells but also rescued the invasion repression caused by ZYX loss. Taken together, our results indicate that high ZYX expression was associated with worse prognosis and highlighted that the ZYX-STMN1 axis might be a potential therapeutic target for GBM.

Phosphorylation of GMFγ by c-Abl Coordinates Lamellipodial and Focal Adhesion Dynamics to Regulate Airway Smooth Muscle Cell Migration.

Airway smooth muscle cells require coordinated protrusion and focal adhesion dynamics to migrate properly. However, the signaling cascades that connect these two processes remain incompletely understood. Glia maturation factor (GMF)-γ has been implicated in inducing actin debranching and inhibiting nucleation. In this study, we discovered that GMFγ phosphorylation at Y104 regulates human airway smooth muscle cell migration. Using high-resolution microscopy coupled with three-dimensional object-based quantitative image analysis software, Imaris 9.2.0, phosphomimetic mutant, Y104D-GMFγ, was enriched at nascent adhesions along the leading edge where it recruited activated neural Wiskott-Aldrich syndrome protein (N-WASP; pY256) to promote actin-branch formation, which enhanced lamellipodial dynamics and limited the growth of focal adhesions. Unexpectedly, we found that nonphosphorylated mutant, Y104F-GMFγ, was enriched in growing adhesions where it promoted a linear branch organization and focal adhesion clustering, and recruited zyxin to increase maturation, thus inhibiting lamellipodial dynamics and cell migration. The localization of GMFγ between the leading edge and focal adhesions was dependent upon myosin activity. Furthermore, c-Abl tyrosine kinase regulated the GMFγ phosphorylation-dependent processes. Together, these results unveil the importance of GMFγ phosphorylation in coordinating lamellipodial and focal adhesion dynamics to regulate cell migration.

Tensile Loads on Tethered Actin Filaments Induce Accumulation of Cell Adhesion-Associated Proteins in Vitro.

Focal adhesions (FAs) and adherens junctions (AJs), which serve as a mechanical interface of cell-matrix and cell-cell interactions, respectively, experience tensile force either originating from the deformation of the surrounding tissues or generated by the actomyosin machinery in the cell. These mechanical inputs cause enlargement of FAs and AJs, while the detailed mechanism for the force-dependent development of FAs and AJs remain unclear. Both FAs and AJs provide sites for tethering of actin filaments and actin polymerization. Here, we develop a cell-free system, in which actin filaments are tethered to glass surfaces, and show that application of tensile force to the tethered filaments in the cell extract induces accumulation of several FA and AJ proteins, associated with further accumulation of actin filaments via de novo actin polymerization. Decline in the tensile force results in a decrease in the amount of the accumulated proteins. These results suggest that the tensile force acting on the tethered actin filaments plays a crucial role in the accumulation of FA and AJ proteins.

New insights into the role of HNF-1ß in kidney (patho)physiology.

Hepatocyte nuclear factor-1ß (HNF-1ß) is an essential transcription factor that regulates the development and function of epithelia in the kidney, liver, pancreas, and genitourinary tract. Humans who carry HNF1B mutations develop heterogeneous renal abnormalities, including multicystic dysplastic kidneys, glomerulocystic kidney disease, renal agenesis, renal hypoplasia, and renal interstitial fibrosis. In the embryonic kidney, HNF-1ß is required for ureteric bud branching, initiation of nephrogenesis, and nephron segmentation. Ablation of mouse Hnf1b in nephron progenitors causes defective tubulogenesis, whereas later inactivation in elongating tubules leads to cyst formation due to downregulation of cystic disease genes, including Umod, Pkhd1, and Pkd2. In the adult kidney, HNF-1ß controls the expression of genes required for intrarenal metabolism and solute transport by tubular epithelial cells. Tubular abnormalities observed in HNF-1ß nephropathy include hyperuricemia with or without gout, hypokalemia, hypomagnesemia, and polyuria. Recent studies have identified novel post-transcriptional and post-translational regulatory mechanisms that control HNF-1ß expression and activity, including the miRNA cluster miR17 ∼ 92 and the interacting proteins PCBD1 and zyxin. Further understanding of the molecular mechanisms upstream and downstream of HNF-1ß may lead to the development of new therapeutic approaches in cystic kidney disease and other HNF1B-related renal diseases.

Genistein Inhibited Estradiol-Induced Vascular Endothelial Cell Injury by Downregulating the FAK/Focal Adhesion Pathway.

BACKGROUND/AIMS: In this study, we aimed to investigate the effects of genistein on the focal adhesion signaling pathway through its regulation of FAK. Genistein ultimately restored and alleviated estradiol-induced vascular endothelial injury. METHODS: Microarray analysis was used to select differentially expressed genes. MTT assay was performed to detect the cell activity, and ROS test and NO test were performed to detect the degree of damage to HUVECs (human umbilical vein endothelial cells). The relative mRNA expression levels and protein expression levels of FAK were tested by western blot and qRT-PCR. GO functional analysis and KEGG pathway analysis were applied to predict the possible relationship between functions and related pathways, and transwell assay was used to detect cell invasion and migration. RESULTS: FAK was highly expressed in the HUVECs treated with estradiol (HU-ESTs). Cell viability and NO level decreased, whereas ROS level increased in the HU-ESTs. Effective knockdown of FAK in HU-ESTs elevated cell viability and NO levels while suppressing ROS levels. In addition, inhibition of FAK greatly decreased cell invasion and migration, while the overexpression of FAK enhanced cell invasion and migration. KEGG further indicated focal adhesion pathways were activated. Genistein elevated HU-EST viability, and NO and ROS level increased in a concentration dependent manner. Transwell and western blot assays revealed that genistein could reduce the FAK expression levels and alleviate the damage to the HU-ESTs. CONCLUSION: FAK overexpression promoted invasion and migration of the HU-ESTs. However, genistein greatly suppressed FAK and estradiol-induced vascular endothelial cell injury.

The cytoskeletal protein Zyxin interacts with the zinc-finger transcription factor Zic1 and plays the role of a scaffold for Gli1 and Zic1 interactions during early development of Xenopus laevis.

We have shown recently that the cytoskeletal protein Zyxin participates in the fine tuning of the neural plate pattering in Xenopus laevis embryos by modulating activity of one of the effectors of Hedgehog (Shh) signaling cascade, the transcription factor Gli1. In the present work, we show that Zyxin can also interact with the potential modulator of the Shh pathway, the transcription factor Zic1. The interaction of proteins occurs primarily by mean of the zinc-finger domain of Zic1 and 2nd LIM domain of Zyxin. Moreover, we have also revealed the ability of the Zyxin, Zic1 and Gli1 to form a ternary complex. The activity of this complex resembles that of the previously described by other authors protein complex formed by Gli1 and Zic1, amplifying effect of the latter. The data obtained provide evidence for the scaffolding role of Zyxin for Gli1 and Zic1 interactions and confirm its role in the regulation of Shh signaling cascade.

A modular toolkit to inhibit proline-rich motif-mediated protein-protein interactions.

Small-molecule competitors of protein-protein interactions are urgently needed for functional analysis of large-scale genomics and proteomics data. Particularly abundant, yet so far undruggable, targets include domains specialized in recognizing proline-rich segments, including Src-homology 3 (SH3), WW, GYF, and Drosophila enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Here, we present a modular strategy to obtain an extendable toolkit of chemical fragments (ProMs) designed to replace pairs of conserved prolines in recognition motifs. As proof-of-principle, we developed a small, selective, peptidomimetic inhibitor of Ena/VASP EVH1 domain interactions. Highly invasive MDA MB 231 breast-cancer cells treated with this ligand showed displacement of VASP from focal adhesions, as well as from the front of lamellipodia, and strongly reduced cell invasion. General applicability of our strategy is illustrated by the design of an ErbB4-derived ligand containing two ProM-1 fragments, targeting the yes-associated protein 1 (YAP1)-WW domain with a fivefold higher affinity.

A Noncanonical Binding Site in the EVH1 Domain of Vasodilator-Stimulated Phosphoprotein Regulates Its Interactions with the Proline Rich Region of Zyxin.

Vasodilator-stimulated phosphoprotein (VASP) is a processive actin polymerase with roles in the control of cell shape and cell migration. Through interaction with the cytoskeletal adaptor protein Zyxin, VASP can localize to damaged stress fibers where it serves to repair and reinforce these structures. VASP localization is mediated by its N-terminal Ena/VASP homology (EVH1) domain, which binds to the (W/F)PxφP motif (most commonly occurring as FPPPP) found in cytoskeletal proteins such as vinculin, lamellipodin, and Zyxin. Sequentially close clusters of four or five of these motifs frequently occur, as in the proline rich region of Zyxin with four such motifs. This suggests that tetrameric VASP might bind very tightly to Zyxin through avidity, with all four EVH1 domains binding to a single Zyxin molecule. Here, quantitative nuclear magnetic resonance titration analysis reveals a dominant bivalent 1:1 (Zyxin:EVH1) interaction between the Zyxin proline rich region and the VASP EVH1 domain that utilizes the EVH1 canonical binding site and a novel secondary binding site on the opposite face of the EVH1 domain. We further show that binding to the secondary binding site is specifically inhibited by mutation of VASP EVH1 domain residue Y39 to E, which mimics Abl-induced phosphorylation of Y39. On the basis of these findings, we propose a model in which phosphorylation of Y39 acts as a stoichiometry switch that governs binding partner selection by the constitutive VASP tetramer. These results have broader implications for other multivalent VASP EVH1 domain binding partners and for furthering our understanding of the role of Y39 phosphorylation in regulating VASP localization and cellular function.

The conserved LIM domain-containing focal adhesion protein ZYX-1 regulates synapse maintenance in Caenorhabditis elegans.

We describe the identification of zyxin as a regulator of synapse maintenance in mechanosensory neurons in C. elegans. zyx-1 mutants lacked PLM mechanosensory synapses as adult animals. However, most PLM synapses initially formed during development but were subsequently lost as the animals developed. Vertebrate zyxin regulates cytoskeletal responses to mechanical stress in culture. Our work provides in vivo evidence in support of such a role for zyxin. In particular, zyx-1 mutant synaptogenesis phenotypes were suppressed by disrupting locomotion of the mutant animals, suggesting that zyx-1 protects mechanosensory synapses from locomotion-induced forces. In cultured cells, zyxin is recruited to focal adhesions and stress fibers via C-terminal LIM domains and modulates cytoskeletal organization via the N-terminal domain. The synapse-stabilizing activity was mediated by a short isoform of ZYX-1 containing only the LIM domains. Consistent with this notion, PLM synaptogenesis was independent of α-actinin and ENA-VASP, both of which bind to the N-terminal domain of zyxin. Our results demonstrate that the LIM domain moiety of zyxin functions autonomously to mediate responses to mechanical stress and provide in vivo evidence for a role of zyxin in neuronal development.