Mitochondrial integrated stress response controls lung epithelial cell fate.

Alveolar epithelial type 1 (AT1) cells are necessary to transfer oxygen and carbon dioxide between the blood and air. Alveolar epithelial type 2 (AT2) cells serve as a partially committed stem cell population, producing AT1 cells during postnatal alveolar development and repair after influenza A and SARS-CoV-2 pneumonia1-6. Little is known about the metabolic regulation of the fate of lung epithelial cells. Here we report that deleting the mitochondrial electron transport chain complex I subunit Ndufs2 in lung epithelial cells during mouse gestation led to death during postnatal alveolar development. Affected mice displayed hypertrophic cells with AT2 and AT1 cell features, known as transitional cells. Mammalian mitochondrial complex I, comprising 45 subunits, regenerates NAD+ and pumps protons. Conditional expression of yeast NADH dehydrogenase (NDI1) protein that regenerates NAD+ without proton pumping7,8 was sufficient to correct abnormal alveolar development and avert lethality. Single-cell RNA sequencing revealed enrichment of integrated stress response (ISR) genes in transitional cells. Administering an ISR inhibitor9,10 or NAD+ precursor reduced ISR gene signatures in epithelial cells and partially rescued lethality in the absence of mitochondrial complex I function. Notably, lung epithelial-specific loss of mitochondrial electron transport chain complex II subunit Sdhd, which maintains NAD+ regeneration, did not trigger high ISR activation or lethality. These findings highlight an unanticipated requirement for mitochondrial complex I-dependent NAD+ regeneration in directing cell fate during postnatal alveolar development by preventing pathological ISR induction.

A Novel MIP-1-Expressing Macrophage Subtype in BAL Fluid from Healthy Volunteers.

Tissue availability remains an important limitation of single-cell genomic technologies for investigating cellular heterogeneity in human health and disease. BAL represents a minimally invasive approach to assessing an individual's lung cellular environment for diagnosis and research. However, the lack of high-quality, healthy lung reference data is a major obstacle to using single-cell approaches to study a plethora of lung diseases. Here, we performed single-cell RNA sequencing on over 40,000 cells isolated from the BAL of four healthy volunteers. Of the six cell types or lineages we identified, macrophages were consistently the most numerous across individuals. Our analysis confirmed the expression of marker genes defining cell types despite background signals because of the ambient RNA found in many single-cell studies. We assessed the variability of gene expression across macrophages and defined a distinct subpopulation of cells expressing a set of genes associated with Macrophage Inflammatory Protein 1 (MIP-1). RNA in situ hybridization and reanalysis of published lung single-cell data validated the presence of this macrophage subpopulation. Thus, our study characterizes lung macrophage heterogeneity in healthy individuals and provides a valuable resource for future studies to understand the lung environment in health and disease.

Lung Injury Induces Alveolar Type 2 Cell Hypertrophy and Polyploidy with Implications for Repair and Regeneration.

Epithelial polyploidization after injury is a conserved phenomenon recently shown to improve barrier restoration during wound healing. Whether lung injury can induce alveolar epithelial polyploidy is not known. We show that bleomycin injury induces alveolar type 2 cell (AT2) hypertrophy and polyploidy. AT2 polyploidization is also seen in short term ex vivo cultures, where AT2-to-AT1 transdifferentiation is associated with substantial binucleation due to failed cytokinesis. Both hypertrophic and polyploid features of AT2 cells can be attenuated by inhibiting the integrated stress response using the small molecule ISRIB. These data suggest that AT2 hypertrophic growth and polyploidization may be a feature of alveolar epithelial injury. Because AT2 cells serve as facultative progenitors for the distal lung epithelium, a propensity for injury-induced binucleation has implications for AT2 self-renewal and regenerative potential upon reinjury, which may benefit from targeting the integrated stress response.

A spatially restricted fibrotic niche in pulmonary fibrosis is sustained by M-CSF/M-CSFR signalling in monocyte-derived alveolar macrophages.

Ontologically distinct populations of macrophages differentially contribute to organ fibrosis through unknown mechanisms.We applied lineage tracing, single-cell RNA sequencing and single-molecule fluorescence in situ hybridisation to a spatially restricted model of asbestos-induced pulmonary fibrosis.We demonstrate that tissue-resident alveolar macrophages, tissue-resident peribronchial and perivascular interstitial macrophages, and monocyte-derived alveolar macrophages are present in the fibrotic niche. Deletion of monocyte-derived alveolar macrophages but not tissue-resident alveolar macrophages ameliorated asbestos-induced lung fibrosis. Monocyte-derived alveolar macrophages were specifically localised to fibrotic regions in the proximity of fibroblasts where they expressed molecules known to drive fibroblast proliferation, including platelet-derived growth factor subunit A. Using single-cell RNA sequencing and spatial transcriptomics in both humans and mice, we identified macrophage colony-stimulating factor receptor (M-CSFR) signalling as one of the novel druggable targets controlling self-maintenance and persistence of these pathogenic monocyte-derived alveolar macrophages. Pharmacological blockade of M-CSFR signalling led to the disappearance of monocyte-derived alveolar macrophages and ameliorated fibrosis.Our findings suggest that inhibition of M-CSFR signalling during fibrosis disrupts an essential fibrotic niche that includes monocyte-derived alveolar macrophages and fibroblasts during asbestos-induced fibrosis.

Single-Cell Transcriptomic Analysis of Human Lung Provides Insights into the Pathobiology of Pulmonary Fibrosis.

Rationale: The contributions of diverse cell populations in the human lung to pulmonary fibrosis pathogenesis are poorly understood. Single-cell RNA sequencing can reveal changes within individual cell populations during pulmonary fibrosis that are important for disease pathogenesis. Objectives: To determine whether single-cell RNA sequencing can reveal disease-related heterogeneity within alveolar macrophages, epithelial cells, or other cell types in lung tissue from subjects with pulmonary fibrosis compared with control subjects. Methods: We performed single-cell RNA sequencing on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and on one bronchoscopic cryobiospy sample from a patient with idiopathic pulmonary fibrosis. We validated these data using in situ RNA hybridization, immunohistochemistry, and bulk RNA-sequencing on flow-sorted cells from 22 additional subjects. Measurements and Main Results: We identified a distinct, novel population of profibrotic alveolar macrophages exclusively in patients with fibrosis. Within epithelial cells, the expression of genes involved in Wnt secretion and response was restricted to nonoverlapping cells. We identified rare cell populations including airway stem cells and senescent cells emerging during pulmonary fibrosis. We developed a web-based tool to explore these data. Conclusions: We generated a single-cell atlas of pulmonary fibrosis. Using this atlas, we demonstrated heterogeneity within alveolar macrophages and epithelial cells from subjects with pulmonary fibrosis. These results support the feasibility of discovery-based approaches using next-generation sequencing technologies to identify signaling pathways for targeting in the development of personalized therapies for patients with pulmonary fibrosis.

Lrp5/ß-Catenin Signaling Controls Lung Macrophage Differentiation and Inhibits Resolution of Fibrosis.

Previous studies established that attenuating Wnt/ß-catenin signaling limits lung fibrosis in the bleomycin mouse model of this disease, but the contribution of this pathway to distinct lung cell phenotypes relevant to tissue repair and fibrosis remains incompletely understood. Using microarray analysis, we found that bleomycin-injured lungs from mice that lack the Wnt coreceptor low density lipoprotein receptor-related protein 5 (Lrp5) and exhibit reduced fibrosis showed enrichment for pathways related to extracellular matrix processing, immunity, and lymphocyte proliferation, suggesting the contribution of an immune-matrix remodeling axis relevant to fibrosis. Activation of ß-catenin signaling was seen in lung macrophages using the ß-catenin reporter mouse, Axin2+/LacZ. Analysis of lung immune cells by flow cytometry after bleomycin administration revealed that Lrp5-/- lungs contained significantly fewer Siglec Flow alveolar macrophages, a cell type previously implicated as positive effectors of fibrosis. Macrophage-specific deletion of ß-catenin in CD11ccre;ß-cateninflox mice did not prevent development of bleomycin-induced fibrosis but facilitated its resolution by 8 weeks. In a nonresolving model of fibrosis, intratracheal administration of asbestos in Lrp5-/- mice also did not prevent the development of fibrosis but hindered the progression of fibrosis in asbestos-treated Lrp5-/- lungs, phenocopying the findings in bleomycin-treated CD11ccre;ß-cateninflox mice. Activation of ß-catenin signaling using lithium chloride resulted in worsened fibrosis in wild-type mice, further supporting that the effects of loss of Lrp5 are directly mediated by Wnt/ß-catenin signaling. Together, these data suggest that lung myeloid cells are responsive to Lrp5/ß-catenin signaling, leading to differentiation of an alveolar macrophage subtype that antagonizes the resolution of lung fibrosis.

α-Catenin phosphorylation promotes intercellular adhesion through a dual-kinase mechanism.

The cadherin-catenin adhesion complex is a key contributor to epithelial tissue stability and dynamic cell movements during development and tissue renewal. How this complex is regulated to accomplish these functions is not fully understood. We identified several phosphorylation sites in mammalian αE-catenin (also known as catenin α-1) and Drosophila α-Catenin within a flexible linker located between the middle (M)-region and the carboxy-terminal actin-binding domain. We show that this phospho-linker (P-linker) is the main phosphorylated region of α-catenin in cells and is sequentially modified at casein kinase 2 and 1 consensus sites. In Drosophila, the P-linker is required for normal α-catenin function during development and collective cell migration, although no obvious defects were found in cadherin-catenin complex assembly or adherens junction formation. In mammalian cells, non-phosphorylatable forms of α-catenin showed defects in intercellular adhesion using a mechanical dispersion assay. Epithelial sheets expressing phosphomimetic forms of α-catenin showed faster and more coordinated migrations after scratch wounding. These findings suggest that phosphorylation and dephosphorylation of the α-catenin P-linker are required for normal cadherin-catenin complex function in Drosophila and mammalian cells.

α-Catenin is an inhibitor of transcription.

α-Catenin (α-cat) is an actin-binding protein required for cell-cell cohesion. Although this adhesive function for α-cat is well appreciated, cells contain a substantial amount of nonjunctional α-cat that may be used for other functions. We show that α-cat is a nuclear protein that can interact with ß-catenin (ß-cat) and T-cell factor (TCF) and that the nuclear accumulation of α-cat depends on ß-cat. Using overexpression, knockdown, and chromatin immunoprecipitation approaches, we show that α-cat attenuates Wnt/ß-cat-responsive genes in a manner that is downstream of ß-cat/TCF loading on promoters. Both ß-cat- and actin-binding domains of α-cat are required to inhibit Wnt signaling. A nuclear-targeted form of α-cat induces the formation of nuclear filamentous actin, whereas cells lacking α-cat show altered nuclear actin properties. Formation of nuclear actin filaments correlates with reduced RNA synthesis and altered chromatin organization. Conversely, nuclear extracts made from cells lacking α-cat show enhanced general transcription in vitro, an activity that can be partially rescued by restoring the C-terminal actin-binding region of α-cat. These data demonstrate that α-cat may limit gene expression by affecting nuclear actin organization.

Adiponectin inhibits Wnt co-receptor, Lrp6, phosphorylation and ß-catenin signaling.

Adiponectin is a pleiotropic adipokine implicated in obesity, metabolic syndrome and cardiovascular disease. Recent studies have identified adiponectin as a negative regulator of tissue fibrosis. Wnt/ß-catenin signaling has also been implicated in metabolic syndrome and can promote tissue fibrosis, but the extent to which adiponectin cross-regulates Wnt/ß-catenin signaling is unknown. Using primary human dermal fibroblasts and recombinant purified proteins, we show that adiponectin can limit ß-catenin accumulation and downstream gene activation by inhibiting Lrp6 phosphorylation, a key activation step in canonical Wnt signaling. Inhibition of Wnt3a-mediated Lrp6 phospho-activation is relatively rapid (e.g., by 30 min), and is not dependent on established adiponectin G-protein coupled receptors, AdipoR1 and R2, suggesting a more direct relationship to Lrp6 signaling. In contrast, the ability of adiponectin to limit Wnt-induced and baseline collagen production in fibroblasts requires AdipoR1/R2. These results suggest the possibility that the pleiotropic effects of adiponectin may be mediated through distinct cell surface receptor complexes. Accordingly, we propose that the anti-fibrotic activity of adiponectin may be mediated through AdipoR1/R2 receptors, while the ability of adiponectin to inhibit Lrp6 phospho-activation may be relevant to other recently established roles for Lrp6 signaling in glucose metabolism and metabolic syndrome.

Wnt coreceptor Lrp5 is a driver of idiopathic pulmonary fibrosis.

RATIONALE: Wnt/ß-catenin signaling has been implicated in lung fibrosis, but how this occurs and whether expression changes in Wnt pathway components predict disease progression is unknown. OBJECTIVES: To determine whether the Wnt coreceptor Lrp5 drives pulmonary fibrosis in mice and is predictive of disease severity in humans. METHODS: We examined mice with impaired Wnt signaling caused by loss of the Wnt coreceptor Lrp5 in models of lung fibrosis induced by bleomycin or an adenovirus encoding an active form of transforming growth factor (TGF)-ß. We also analyzed gene expression in peripheral blood mononuclear cells (PBMC) from patients with idiopathic pulmonary fibrosis (IPF). MEASUREMENTS AND MAIN RESULTS: In patients with IPF, analysis of peripheral blood mononuclear cells revealed that elevation of positive regulators, Lrp5 and 6, was independently associated with disease progression. LRP5 was also associated with disease severity at presentation in an additional cohort of patients with IPF. Lrp5 null mice were protected against bleomycin-induced pulmonary fibrosis, an effect that was phenocopied by direct inhibition of ß-catenin signaling by the small molecular inhibitor of ß-catenin responsive transcription. Transplantation of Lrp5 null bone marrow cells into wild-type mice did not limit fibrosis. Instead, Lrp5 loss was associated with reduced TGF-ß production by alveolar type 2 cells and leukocytes. Consistent with a role of Lrp5 in the activation of TGF-ß, Lrp5 null mice were not protected against lung fibrosis induced by TGF-ß. CONCLUSIONS: We show that the Wnt coreceptor, Lrp5, is a genetic driver of lung fibrosis in mice and a marker of disease progression and severity in humans with IPF. Evidence that TGF-ß signaling can override a loss in Lrp5 has implications for patient selection and timing of Wnt pathway inhibitors in lung fibrosis.

α-catenin middle- and actin-binding domain unfolding mutants differentially impact epithelial strength and sheet migration.

α-catenin (α-cat) displays force-dependent unfolding and binding to actin filaments through direct and indirect means, but features of adherens junction structure and function most vulnerable to loss of these allosteric mechanisms have not been directly compared. By reconstituting an α-cat F-actin-binding domain unfolding mutant known to exhibit enhanced binding to actin (α-cat-H0-FABD+) into α-cat knockout Madin Darby Canine Kidney (MDCK) cells, we show that partial loss of the α-cat catch bond mechanism (via an altered H0 α-helix) leads to stronger epithelial sheet integrity with greater colocalization between the α-cat-H0-FABD+ mutant and actin. α-cat-H0-FABD+ -expressing cells are less efficient at closing scratch-wounds, suggesting reduced capacity for more dynamic cell-cell coordination. Evidence that α-cat-H0-FABD+ is equally accessible to the conformationally sensitive α18 antibody epitope as WT α-cat and shows similar vinculin recruitment suggests this mutant engages lower tension cortical actin networks, as its M-domain is not persistently open. Conversely, α-cat-M-domain salt-bridge mutants with persistent recruitment of vinculin and phosphorylated myosin light chain show only intermediate monolayer adhesive strengths, but display less directionally coordinated and thereby slower migration speeds during wound-repair. These data show α-cat M- and FABD-unfolding mutants differentially impact cell-cell cohesion and migration properties, and suggest signals favoring α-cat-cortical actin interaction without persistent M-domain opening may improve epithelial monolayer strength through enhanced coupling to lower tension actin networks.

α -catenin phosphorylation is elevated during mitosis to resist apical rounding and epithelial barrier leak.

Epithelial cell cohesion and barrier function critically depend on α -catenin, an actin-binding protein and essential constituent of cadherin-catenin-based adherens junctions. α -catenin undergoes actomyosin force-dependent unfolding of both actin-binding and middle domains to strongly engage actin filaments and its various effectors, where this mechanosensitivity is critical for adherens junction function. We previously showed that α -catenin is highly phosphorylated in an unstructured region that links mechanosensitive middle- and actin-binding domains (known as the P-linker region), but the cellular processes that promote α -catenin phosphorylation have remained elusive. Here, we leverage a previously published phosphor-proteomic data set to show that the α -catenin P-linker region is maximally phosphorylated during mitosis. By reconstituting α -catenin Crispr KO MDCK with wild-type, phospho- mutant and mimic forms of α -catenin, we show that full phosphorylation restrains mitotic cell rounding in the apical direction, strengthening interactions between dividing and non-dividing neighbors to limit epithelial barrier leak. Since major scaffold components of adherens junctions, tight junctions and desmosomes are also differentially phosphorylated during mitosis, we reason that epithelial cell division may be a tractable system to understand how junction complexes are coordinately regulated to sustain barrier function under tension-generating morphogenetic processes.

α-catenin mechanosensitivity as a route to cytokinesis failure through sequestration of LZTS2.

Epithelial cells can become polyploid upon tissue injury, but mechanosensitive cues that trigger this state are poorly understood. Using α-catenin (α-cat) knock-out Madin Darby Canine Kidney (MDCK) cells reconstituted with wild-type and mutant forms of α-cat as a model system, we find that an established α-cat actin-binding domain unfolding mutant designed to reduce force-sensitive binding to F-actin (α-cat-H0-FABD+) can promote cytokinesis failure, particularly along epithelial wound-fronts. Enhanced α-cat coupling to cortical actin is neither sufficient nor mitotic cell-autonomous for cytokinesis failure, but critically requires the mechanosensitive Middle-domain (M1-M2-M3) and neighboring cells. Disease relevant α-cat M-domain missense mutations known to cause a form of retinal pattern dystrophy (α-cat E307K or L436P) are associated with elevated binucleation rates via cytokinesis failure. Similar binucleation rates are seen in cells expressing an α-cat salt-bridge destabilizing mutant (R551A) designed to promote M2-M3 domain unfurling at lower force thresholds. Since binucleation is strongly enhanced by removal of the M1 as opposed to M2-M3 domains, cytokinetic fidelity is most sensitive to α-cat M2-M3 domain opening. To identify α-cat conformation-dependent proximity partners that contribute to cytokinesis, we used a biotin-ligase approach to distinguished proximity partners that show enhanced recruitment upon α-cat M-domain unfurling (R551A). We identified Leucine Zipper Tumor Suppressor 2 (LZTS2), an abscission factor previously implicated in cytokinesis. We confirm that LZTS2 enriches at the midbody, but discover it also localizes to tight and tricellular junctions. LZTS2 knock-down promotes binucleation in both MDCK and Retinal Pigmented Epithelial (RPE) cells. α-cat mutants with persistent M2-M3 domain opening showed elevated junctional enrichment of LZTS2 from the cytosol compared α-cat wild-type cells. These data implicate LZTS2 as a mechanosensitive effector of α-cat that is critical for cytokinetic fidelity. This model rationalizes how persistent mechano-activation of α-cat may drive tension-induced polyploidization of epithelia post-injury and suggests an underlying mechanism for how pathogenic α-cat mutations drive macular dystrophy.

Hypercapnia alters stroma-derived Wnt production to limit ß-catenin signaling and proliferation in AT2 cells.

Persistent symptoms and radiographic abnormalities suggestive of failed lung repair are among the most common symptoms in patients with COVID-19 after hospital discharge. In mechanically ventilated patients with acute respiratory distress syndrome (ARDS) secondary to SARS-CoV-2 pneumonia, low tidal volumes to reduce ventilator-induced lung injury necessarily elevate blood CO2 levels, often leading to hypercapnia. The role of hypercapnia on lung repair after injury is not completely understood. Here - using a mouse model of hypercapnia exposure, cell lineage tracing, spatial transcriptomics, and 3D cultures - we show that hypercapnia limits ß-catenin signaling in alveolar type II (AT2) cells, leading to their reduced proliferative capacity. Hypercapnia alters expression of major Wnts in PDGFRα+ fibroblasts from those maintaining AT2 progenitor activity toward those that antagonize ß-catenin signaling, thereby limiting progenitor function. Constitutive activation of ß-catenin signaling in AT2 cells or treatment of organoid cultures with recombinant WNT3A protein bypasses the inhibitory effects of hypercapnia. Inhibition of AT2 proliferation in patients with hypercapnia may contribute to impaired lung repair after injury, preventing sealing of the epithelial barrier and increasing lung flooding, ventilator dependency, and mortality.

Alpha-T-catenin is expressed in peripheral nerves as a constituent of Schwann cell adherens junctions.

The adherens junction component, alpha-T-catenin (αTcat) is an established contributor to cardiomyocyte junction structure and function, but recent genomic studies link CTNNA3 polymorphisms to diseases with no clear cardiac underpinning, including asthma, autism and multiple sclerosis, suggesting causal contributions from a different cell-type. We show Ctnna3 mRNA is highly expressed in peripheral nerves (e.g. vagus and sciatic), where αTcat protein enriches at paranodes and myelin incisure adherens junctions of Schwann cells. We validate αTcat immunodetection specificity using a new Ctnna3-knock-out fluorescence reporter mouse line yet find no obvious Schwann cell loss-of-function morphology at the light microscopic level. CTNNA3/Ctnna3 mRNA is also abundantly detected in oligodendrocytes of the central nervous system via public databases, supporting a general role for αTcat in these unique cell-cell junctions. These data suggest that the wide range of diseases linked to CTNNA3 may be through its role in maintaining neuroglial functions of central and peripheral nervous systems. This article has a corresponding First Person interview with the co-first authors of the paper.

Nuclear ß-catenin is increased in systemic sclerosis pulmonary fibrosis and promotes lung fibroblast migration and proliferation.

Pulmonary fibrosis is a disease that results in loss of normal lung architecture, but the signaling events that drive tissue destruction are incompletely understood. Wnt/ß-catenin signaling is important in normal lung development, but whether abnormal signaling occurs in lung fibrosis due to systemic sclerosis and the consequences of ß-catenin signaling toward the fibrogenic phenotype remain poorly defined. In this study, we show nuclear ß-catenin accumulation in fibroblastic foci from lungs of patients with systemic sclerosis-associated advanced pulmonary fibrosis. Forced activation of ß-catenin signaling in three independently derived sources of normal human lung fibroblasts promotes proliferation and migratory activities but is not sufficient to activate classic markers of fibroblast activation, such as TGF-ß, type 1 collagen, α-smooth muscle actin, and connective tissue growth factor. These findings indicate that activation of ß-catenin signaling in pulmonary fibroblasts may be a common feature of lung fibrosis, contributing to fibroproliferative and migratory activities associated with the disease.

Beta-catenin/T-cell factor signaling is activated during lung injury and promotes the survival and migration of alveolar epithelial cells.

The Wnt/beta-catenin signaling cascade activates genes that allow cells to adopt particular identities throughout development. In adult self-renewing tissues like intestine and blood, activation of the Wnt pathway maintains a progenitor phenotype, whereas forced inhibition of this pathway promotes differentiation. In the lung alveolus, type 2 epithelial cells (AT2) have been described as progenitors for the type 1 cell (AT1), but whether AT2 progenitors use the same signaling mechanisms to control differentiation as rapidly renewing tissues is not known. We show that adult AT2 cells do not exhibit constitutive beta-catenin signaling in vivo, using the AXIN2(+/LacZ) reporter mouse, or after fresh isolation of an enriched population of AT2 cells. Rather, this pathway is activated in lungs subjected to bleomycin-induced injury, as well as upon placement of AT2 cells in culture. Forced inhibition of beta-catenin/T-cell factor signaling in AT2 cultures leads to increased cell death. Cells that survive show reduced migration after wounding and reduced expression of AT1 cell markers (T1alpha and RAGE). These results suggest that AT2 cells may function as facultative progenitors, where activation of Wnt/beta-catenin signaling during lung injury promotes alveolar epithelial survival, migration, and differentiation toward an AT1-like phenotype.

αT-catenin: A developmentally dispensable, disease-linked member of the α-catenin family.

α-Catenins are actin-filament binding proteins and critical subunits of the cadherin-catenin cell-cell adhesive complex. They are found in nominally-defined epithelial (E), neural (N), and testis (T) forms transcribed from three distinct genes. While most of α-catenin research has focused on the developmentally essential founding member, αE-catenin, this review discusses recent studies on αT-catenin (CTNNA3), a developmentally dispensable isoform that is emerging as relevant to cardiac, allergic and neurological diseases.

Force-dependent allostery of the α-catenin actin-binding domain controls adherens junction dynamics and functions.

α-catenin is a key mechanosensor that forms force-dependent interactions with F-actin, thereby coupling the cadherin-catenin complex to the actin cytoskeleton at adherens junctions (AJs). However, the molecular mechanisms by which α-catenin engages F-actin under tension remained elusive. Here we show that the α1-helix of the α-catenin actin-binding domain (αcat-ABD) is a mechanosensing motif that regulates tension-dependent F-actin binding and bundling. αcat-ABD containing an α1-helix-unfolding mutation (H1) shows enhanced binding to F-actin in vitro. Although full-length α-catenin-H1 can generate epithelial monolayers that resist mechanical disruption, it fails to support normal AJ regulation in vivo. Structural and simulation analyses suggest that α1-helix allosterically controls the actin-binding residue V796 dynamics. Crystal structures of αcat-ABD-H1 homodimer suggest that α-catenin can facilitate actin bundling while it remains bound to E-cadherin. We propose that force-dependent allosteric regulation of αcat-ABD promotes dynamic interactions with F-actin involved in actin bundling, cadherin clustering, and AJ remodeling during tissue morphogenesis.

α-Catenin homodimers are recruited to phosphoinositide-activated membranes to promote adhesion.

A unique feature of α-catenin localized outside the cadherin-catenin complex is its capacity to form homodimers, but the subcellular localization and functions of this form of α-catenin remain incompletely understood. We identified a cadherin-free form of α-catenin that is recruited to the leading edge of migrating cells in a phosphatidylinositol 3-kinase-dependent manner. Surface plasmon resonance analysis shows that α-catenin homodimers, but not monomers, selectively bind phosphatidylinositol-3,4,5-trisphosphate-containing lipid vesicles with high affinity, where three basic residues, K488, K493, and R496, contribute to binding. Chemical-induced dimerization of α-catenin containing a synthetic dimerization domain promotes its accumulation within lamellipodia and elaboration of protrusions with extended filopodia, which are attenuated in the α-cateninKKR<3A mutant. Cells restored with a full-length, natively homodimerizing form of α-cateninKKR<3A display reduced membrane recruitment, altered epithelial sheet migrations, and weaker cell-cell adhesion compared with WT α-catenin. These findings show that α-catenin homodimers are recruited to phosphoinositide-activated membranes to promote adhesion and migration, suggesting that phosphoinositide binding may be a defining feature of α-catenin function outside the cadherin-catenin complex.