Acetyl-NPKY of integrin-ß1 binds KINDLIN2 to control endothelial cell proliferation and junctional integrity.

Integrin-dependent crosstalk between cell-matrix adhesions and cell-cell junctions is critical for controlling endothelial permeability and proliferation in cancer and inflammatory diseases but remains poorly understood. Here, we investigated how acetylation of the distal NPKY-motif of Integrin-ß1 influences endothelial cell physiology and barrier function. Expression of an acetylation-mimetic ß1-K794Q-GFP mutant led to the accumulation of immature cell-matrix adhesions accompanied by a transcriptomic reprograming of endothelial cells, involving genes associated with cell adhesion, proliferation, polarity, and barrier function. ß1-K794Q-GFP induced constitutive MAPK signaling, junctional impairment, proliferation, and reduced contact inhibition at confluence. Structural analysis of Integrin-ß1 interaction with KINDLIN2, biochemical pulldown assay, and binding energy determination by using molecular dynamics simulation showed that acetylation of K794 and the K794Q-mutant increased KINDLIN2 binding affinity to the Integrin-ß1. Thus, enhanced recruitment of KINDLIN2 to Lysine-acetylated Integrin-ß1 and resulting modulation of barrier function, offers new therapeutic possibilities for controlling vascular permeability and disease conditions.

Akt-driven TGF-ß and DKK1 Secretion Impairs F508del Cystic Fibrosis Airway Epithelium Polarity.

Epithelial polarity is fundamental in maintaining barrier integrity and tissue protection. In cystic fibrosis (CF), apicobasal polarity of the airway epithelium is altered, resulting in increased apical fibronectin deposition and enhanced susceptibility to bacterial infections. Here, we evaluated the effect of highly effective modulator treatment (HEMT) on fibronectin apical deposition and investigated the intracellular mechanisms triggering the defect in polarity of the CF airway epithelium. To this end, primary cultures of CF (F508del variant) human airway epithelial cells (HAECs) and a HAEC line, Calu-3, knocked down for CFTR (CF transmembrane conductance regulator) were compared with control counterparts. We show that CFTR mutation in primary HAECs and CFTR knockdown cells promote the overexpression and oversecretion of TGF-ß1 and DKK1 when cultured at an air-liquid interface. These dynamic changes result in hyperactivation of the TGF-ß pathway and inhibition of the Wnt pathway through degradation of ß-catenin leading to imbalanced proliferation and polarization. The abnormal interplay between TGF-ß and Wnt signaling pathways is reinforced by aberrant Akt signaling. Pharmacological manipulation of TGF-ß, Wnt, and Akt pathways restored polarization of the F508del CF epithelium, a correction that was not achieved by HEMT. Our data shed new insights into the signaling pathways that fine-tune apicobasal polarization in primary airway epithelial cells and may provide an explanation to the mitigated efficacy of HEMT on lung infection in people with CF.

Novel 5-aminopyrazoles endowed with anti-angiogenetic properties: Design, synthesis and biological evaluation.

The promising anti-angiogenetic properties of previously synthesized pyrazolyl ureas provided the rationale for the synthesis of novel 5-aminopyrazoles 2-5, differently decorated on the pyrazole nucleus. All the derivatives were tested by MTT assays and proved to be non-cytotoxic against eight different tumor cell lines and normal fibroblasts. An EdU proliferation assay was carried out on human foreskin fibroblasts and VEGF stimulated human umbilical vein endothelial cells which confirmed the absence of cytotoxicity of the compounds on human cells up to 20 µM concentration. To evaluate the influence of the newly synthesized pyrazoles on MAPK and PI3K signaling pathways, the phosphorylation of ERK1/2 and Akt was analyzed by Western blots from HFF and HUVEC cell lysates stimulated with growth factors in the presence or absence of the compounds. Pyrazoles 3b and 3c showed a significant inhibition of Akt phosphorylation in both tested cell lines with lower phosphorylation levels than the reference compound GeGe-3 in HUVEC. Furthermore, derivatives 2 and 3 appeared to strongly affect the migration of HFF cells in a wound healing assay, confirming their potential ability to interfere with the angiogenesis process. The new pyrazole library extends the structure-activity relationships of the previously isolated compounds and highlights the attractiveness of this chemical class for pathological cell migration and angiogenesis.

Influenza A virus exploits transferrin receptor recycling to enter host cells.

Influenza A virus (IAV) enters host cells mostly through clathrin-dependent receptor-mediated endocytosis. A single bona fide entry receptor protein supporting this entry mechanism remains elusive. Here we performed proximity ligation of biotin to host cell surface proteins in the vicinity of attached trimeric hemagglutinin-HRP and characterized biotinylated targets using mass spectrometry. This approach identified transferrin receptor 1 (TfR1) as a candidate entry protein. Genetic gain-of-function and loss-of-function experiments, as well as in vitro and in vivo chemical inhibition, confirmed the functional involvement of TfR1 in IAV entry. Recycling deficient mutants of TfR1 do not support entry, indicating that TfR1 recycling is essential for this function. The binding of virions to TfR1 via sialic acids confirmed its role as a directly acting entry factor, but unexpectedly even headless TfR1 promoted IAV particle uptake in trans. TIRF microscopy localized the entering virus-like particles in the vicinity of TfR1. Our data identify TfR1 recycling as a revolving door mechanism exploited by IAV to enter host cells.

ConFERMing the role of talin in integrin activation and mechanosignaling.

Talin (herein referring to the talin-1 form), is a cytoskeletal adapter protein that binds integrin receptors and F-actin, and is a key factor in the formation and regulation of integrin-dependent cell-matrix adhesions. Talin forms the mechanical link between the cytoplasmic domain of integrins and the actin cytoskeleton. Through this linkage, talin is at the origin of mechanosignaling occurring at the plasma membrane-cytoskeleton interface. Despite its central position, talin is not able to fulfill its tasks alone, but requires help from kindlin and paxillin to detect and transform the mechanical tension along the integrin-talin-F-actin axis into intracellular signaling. The talin head forms a classical FERM domain, which is required to bind and regulate the conformation of the integrin receptor, as well as to induce intracellular force sensing. The FERM domain allows the strategic positioning of protein-protein and protein-lipid interfaces, including the membrane-binding and integrin affinity-regulating F1 loop, as well as the interaction with lipid-anchored Rap1 (Rap1a and Rap1b in mammals) GTPase. Here, we summarize the structural and regulatory features of talin and explain how it regulates cell adhesion and force transmission, as well as intracellular signaling at integrin-containing cell-matrix attachment sites.

New tools to study the interaction between integrins and latent TGFß1.

Transforming growth factor beta (TGFß) 1 regulates cell differentiation and proliferation in different physiological settings, but is also involved in fibrotic progression and protects tumors from the immune system. Integrin αVß6 has been shown to activate latent TGFß1 by applying mechanical forces onto the latency-associated peptide (LAP). While the extracellular binding between αVß6 and LAP1 is well characterized, less is known about the cytoplasmic adaptations that enable αVß6 to apply such forces. Here, we generated new tools to facilitate the analysis of this interaction. We combined the integrin-binding part of LAP1 with a GFP and the Fc chain of human IgG. This chimeric protein, sLAP1, revealed a mechanical rearrangement of immobilized sLAP1 by αVß6 integrin. This unique interaction was not observed between sLAP1 and other integrins. We also analyzed αVß6 integrin binding to LAP2 and LAP3 by creating respective sLAPs. Compared to sLAP1, integrin αVß6 showed less binding to sLAP3 and no rearrangement. These observations indicate differences in the binding of αVß6 to LAP1 and LAP3 that have not been appreciated so far. Finally, αVß6-sLAP1 interaction was maintained even at strongly reduced cellular contractility, highlighting the special mechanical connection between αVß6 integrin and latent TGFß1.

Adaptation of cell spreading to varying fibronectin densities and topographies is facilitated by ß1 integrins.

Cells mechanical behaviour in physiological environments is mediated by interactions with the extracellular matrix (ECM). In particular, cells can adapt their shape according to the availability of ECM proteins, e.g., fibronectin (FN). Several in vitro experiments usually simulate the ECM by functionalizing the surfaces on which cells grow with FN. However, the mechanisms underlying cell spreading on non-uniformly FN-coated two-dimensional substrates are not clarified yet. In this work, we studied cell spreading on variously functionalized substrates: FN was either uniformly distributed or selectively patterned on flat surfaces, to show that A549, BRL, B16 and NIH 3T3 cell lines are able to sense the overall FN binding sites independently of their spatial arrangement. Instead, only the total amount of available FN influences cells spreading area, which positively correlates to the FN density. Immunocytochemical analysis showed that ß1 integrin subunits are mainly responsible for this behaviour, as further confirmed by spreading experiments with ß1-deficient cells. In the latter case, indeed, cells areas do not show a dependency on the amount of available FN on the substrates. Therefore, we envision for ß1 a predominant role in cells for sensing the number of ECM ligands with respect to other focal adhesion proteins.

Paxillin: A Hub for Mechano-Transduction from the ß3 Integrin-Talin-Kindlin Axis.

Focal adhesions are specialized integrin-dependent adhesion complexes, which ensure cell anchoring to the extracellular matrix. Focal adhesions also function as mechano-signaling platforms by perceiving and integrating diverse physical and (bio)chemical cues of their microenvironment, and by transducing them into intracellular signaling for the control of cell behavior. The fundamental biological mechanism of creating intracellular signaling in response to changes in tensional forces appears to be tightly linked to paxillin recruitment and binding to focal adhesions. Interestingly, the tension-dependent nature of the paxillin binding to adhesions, combined with its scaffolding function, suggests a major role of this protein in integrating multiple signals from the microenvironment, and accordingly activating diverse molecular responses. This minireview offers an overview of the molecular bases of the mechano-sensitivity and mechano-signaling capacity of core focal adhesion proteins, and highlights the role of paxillin as a key component of the mechano-transducing machinery based on the interaction of cells to substrates activating the ß3 integrin-talin1-kindlin.

Phosphorylated paxillin and phosphorylated FAK constitute subregions within focal adhesions.

Integrin-mediated adhesions are convergence points for multiple signaling pathways. Their inner structure and diverse functions can be studied with super-resolution microscopy. Here, we examined the spatial organization within focal adhesions by analyzing several adhesion proteins with structured illumination microscopy (SIM). Paxillin (Pax) serves as a scaffold protein and signaling hub in focal adhesions, and focal adhesion kinase (FAK, also known as PTK2) regulates the dynamics of adhesions. We found that their phosphorylated forms, pPax and pFAK, form spot-like, spatially defined clusters within adhesions in several cell lines and confirmed these findings with additional super-resolution techniques. These clusters showed a more regular separation from each other compared with more randomly distributed signals for FAK or paxillin. Mutational analysis indicated that the active (open) FAK conformation is a prerequisite for the pattern formation of pFAK. Live-cell super-resolution imaging revealed that organization in clusters is preserved over time for FAK constructs; however, distance between clusters is dynamic for FAK, while paxillin is more stable. Combined, these data introduce spatial clusters of pPax and pFAK as substructures in adhesions and highlight the relevance of paxillin-FAK binding for establishing a regular substructure in focal adhesions.

Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions.

Focal adhesions (FAs) initiate chemical and mechanical signals involved in cell polarity, migration, proliferation and differentiation. Super-resolution microscopy revealed that FAs are organized at the nanoscale into functional layers from the lower plasma membrane to the upper actin cytoskeleton. Yet, how FAs proteins are guided into specific nano-layers to promote interaction with given targets is unknown. Using single protein tracking, super-resolution microscopy and functional assays, we link the molecular behavior and 3D nanoscale localization of kindlin with its function in integrin activation inside FAs. We show that immobilization of integrins in FAs depends on interaction with kindlin. Unlike talin, kindlin displays free diffusion along the plasma membrane outside and inside FAs. We demonstrate that the kindlin Pleckstrin Homology domain promotes membrane diffusion and localization to the membrane-proximal integrin nano-layer, necessary for kindlin enrichment and function in FAs. Using kindlin-deficient cells, we show that kindlin membrane localization and diffusion are crucial for integrin activation, cell spreading and FAs formation. Thus, kindlin uses a different route than talin to reach and activate integrins, providing a possible molecular basis for their complementarity during integrin activation.

Structural and functional analysis of LIM domain-dependent recruitment of paxillin to αvß3 integrin-positive focal adhesions.

The LIM domain-dependent localization of the adapter protein paxillin to ß3 integrin-positive focal adhesions (FAs) is not mechanistically understood. Here, by combining molecular biology, photoactivation and FA-isolation experiments, we demonstrate specific contributions of each LIM domain of paxillin and reveal multiple paxillin interactions in adhesion-complexes. Mutation of ß3 integrin at a putative paxillin binding site (ß3VE/YA) leads to rapidly inward-sliding FAs, correlating with actin retrograde flow and enhanced paxillin dissociation kinetics. Induced mechanical coupling of paxillin to ß3VE/YA integrin arrests the FA-sliding, thereby disclosing an essential structural function of paxillin for the maturation of ß3 integrin/talin clusters. Moreover, bimolecular fluorescence complementation unveils the spatial orientation of the paxillin LIM-array, juxtaposing the positive LIM4 to the plasma membrane and the ß3 integrin-tail, while in vitro binding assays point to LIM1 and/or LIM2 interaction with talin-head domain. These data provide structural insights into the molecular organization of ß3 integrin-FAs.

Kindlin-2 Mediates Mechanical Activation of Cardiac Myofibroblasts.

We identify the focal adhesion protein kindlin-2 as player in a novel mechanotransduction pathway that controls profibrotic cardiac fibroblast to myofibroblast activation. Kindlin-2 is co-upregulated with the myofibroblast marker α-smooth muscle actin (α-SMA) in fibrotic rat hearts and in human cardiac fibroblasts exposed to fibrosis-stiff culture substrates and pro-fibrotic TGF-ß1. Stressing fibroblasts using ferromagnetic microbeads, stretchable silicone membranes, and cell contraction agonists all result in kindlin-2 translocation to the nucleus. Overexpression of full-length kindlin-2 but not of kindlin-2 missing a putative nuclear localization sequence (∆NLS kindlin-2) results in increased α-SMA promoter activity. Downregulating kindlin-2 with siRNA leads to decreased myofibroblast contraction and reduced α-SMA expression, which is dependent on CC(A/T)-rich GG(CArG) box elements in the α-SMA promoter. Lost myofibroblast features under kindlin-2 knockdown are rescued with wild-type but not ∆NLS kindlin-2, indicating that myofibroblast control by kindlin-2 requires its nuclear translocation. Because kindlin-2 can act as a mechanotransducer regulating the transcription of α-SMA, it is a potential target to interfere with myofibroblast activation in tissue fibrosis.

Induced Pluripotent Stem Cells to Understand Mucopolysaccharidosis. I: Demonstration of a Migration Defect in Neural Precursors.

Background: Mucopolysaccharidosis type I-Hurler (MPS1-H) is a severe genetic lysosomal storage disorder due to loss-of-function mutations in the IDUA gene. The subsequent complete deficiency of alpha l-iduronidase enzyme is directly responsible of a progressive accumulation of glycosaminoglycans (GAG) in lysosomes which affects the functions of many tissues. Consequently, MPS1 is characterized by systemic symptoms (multiorgan dysfunction) including respiratory and cardiac dysfunctions, skeletal abnormalities and early fatal neurodegeneration. Methods: To understand mechanisms underlying MPS1 neuropathology, we generated induced pluripotent stem cells (iPSC) from a MPS1-H patient with loss-of-function mutations in both IDUA alleles. To avoid variability due to different genetic background of iPSC, we established an isogenic control iPSC line by rescuing IDUA expression by a lentivectoral approach. Results: Marked differences between MPS1-H and IDUA-corrected isogenic controls were observed upon neural differentiation. A scratch assay revealed a strong migration defect of MPS1-H cells. Also, there was a massive impact of IDUA deficiency on gene expression (340 genes with an FDR <0.05). Conclusions: Our results demonstrate a hitherto unknown connection between lysosomal degradation, gene expression and neural motility, which might account at least in part for the phenotype of MPS1-H patients.

Crystal structure of the FERM-folded talin head reveals the determinants for integrin binding.

Binding of the intracellular adapter proteins talin and its cofactor, kindlin, to the integrin receptors induces integrin activation and clustering. These processes are essential for cell adhesion, migration, and organ development. Although the talin head, the integrin-binding segment in talin, possesses a typical FERM-domain sequence, a truncated form has been crystallized in an unexpected, elongated form. This form, however, lacks a C-terminal fragment and possesses reduced ß3-integrin binding. Here, we present a crystal structure of a full-length talin head in complex with the ß3-integrin tail. The structure reveals a compact FERM-like conformation and a tightly associated N-P-L-Y motif of ß3-integrin. A critical C-terminal poly-lysine motif mediates FERM interdomain contacts and assures the tight association with the ß3-integrin cytoplasmic segment. Removal of the poly-lysine motif or disrupting the FERM-folded configuration of the talin head significantly impairs integrin activation and clustering. Therefore, structural characterization of the FERM-folded active talin head provides fundamental understanding of the regulatory mechanism of integrin function.

The F1 loop of the talin head domain acts as a gatekeeper in integrin activation and clustering.

Integrin activation and clustering by talin are early steps of cell adhesion. Membrane-bound talin head domain and kindlin bind to the ß integrin cytoplasmic tail, cooperating to activate the heterodimeric integrin, and the talin head domain induces integrin clustering in the presence of Mn2+ Here we show that kindlin-1 can replace Mn2+ to mediate ß3 integrin clustering induced by the talin head, but not that induced by the F2-F3 fragment of talin. Integrin clustering mediated by kindlin-1 and the talin head was lost upon deletion of the flexible loop within the talin head F1 subdomain. Further mutagenesis identified hydrophobic and acidic motifs in the F1 loop responsible for ß3 integrin clustering. Modeling, computational and cysteine crosslinking studies showed direct and catalytic interactions of the acidic F1 loop motif with the juxtamembrane domains of α- and ß3-integrins, in order to activate the ß3 integrin heterodimer, further detailing the mechanism by which the talin-kindlin complex activates and clusters integrins. Moreover, the F1 loop interaction with the ß3 integrin tail required the newly identified compact FERM fold of the talin head, which positions the F1 loop next to the inner membrane clasp of the talin-bound integrin heterodimer.This article has an associated First Person interview with the first author of the paper.

Integrin and autocrine IGF2 pathways control fasting insulin secretion in ß-cells.

Elevated levels of fasting insulin release and insufficient glucose-stimulated insulin secretion (GSIS) are hallmarks of diabetes. Studies have established cross-talk between integrin signaling and insulin activity, but more details of how integrin-dependent signaling impacts the pathophysiology of diabetes are needed. Here, we dissected integrin-dependent signaling pathways involved in the regulation of insulin secretion in ß-cells and studied their link to the still debated autocrine regulation of insulin secretion by insulin/insulin-like growth factor (IGF) 2-AKT signaling. We observed for the first time a cooperation between different AKT isoforms and focal adhesion kinase (FAK)-dependent adhesion signaling, which either controlled GSIS or prevented insulin secretion under fasting conditions. Indeed, ß-cells form integrin-containing adhesions, which provide anchorage to the pancreatic extracellular matrix and are the origin of intracellular signaling via FAK and paxillin. Under low-glucose conditions, ß-cells adopt a starved adhesion phenotype consisting of actin stress fibers and large peripheral focal adhesion. In contrast, glucose stimulation induces cell spreading, actin remodeling, and point-like adhesions that contain phospho-FAK and phosphopaxillin, located in small protrusions. Rat primary ß-cells and mouse insulinomas showed an adhesion remodeling during GSIS resulting from autocrine insulin/IGF2 and AKT1 signaling. However, under starving conditions, the maintenance of stress fibers and the large adhesion phenotype required autocrine IGF2-IGF1 receptor signaling mediated by AKT2 and elevated FAK-kinase activity and ROCK-RhoA levels but low levels of paxillin phosphorylation. This starved adhesion phenotype prevented excessive insulin granule release to maintain low insulin secretion during fasting. Thus, deregulation of the IGF2 and adhesion-mediated signaling may explain dysfunctions observed in diabetes.

Vav3 Mediates Pseudomonas aeruginosa Adhesion to the Cystic Fibrosis Airway Epithelium.

Pseudomonas aeruginosa (Pa) represents the leading cause of airway infection in cystic fibrosis (CF). Early airways colonization can be explained by enhanced adhesion of Pa to the respiratory epithelium. RNA sequencing (RNA-seq) on fully differentiated primary cultures of airway epithelial cells from CF and non-CF donors predict that VAV3, ß1 INTEGRIN, and FIBRONECTIN genes are significantly enriched in CF. Indeed, Vav3 is apically overexpressed in CF, associates with active ß1 integrin luminally exposed, and increases fibronectin deposition. These luminal microdomains, rich in fibronectin and ß1 integrin and regulated by Vav3, mediate the increased Pa adhesion to the CF epithelium. Interestingly, Vav3 inhibition normalizes the CF-dependent fibronectin and ß1-integrin ectopic expression, improves the CF epithelial integrity, and prevents the enhanced Pa trapping to the CF epithelium. Through its capacity to promote a luminal complex with active ß1 integrin and fibronectin that favors bacteria trapping, Vav3 may represent a new target in CF.

The mechano-sensitive response of ß1 integrin promotes SRC-positive late endosome recycling and activation of Yes-associated protein.

Yes-associated protein (YAP) signaling has emerged as a crucial pathway in several normal and pathological processes. Although the main upstream effectors that regulate its activity have been extensively studied, the role of the endosomal system has been far less characterized. Here, we identified the late endosomal/lysosomal adaptor MAPK and mTOR activator (LAMTOR) complex as an important regulator of YAP signaling in a preosteoblast cell line. We found that p18/LAMTOR1-mediated peripheral positioning of late endosomes allows delivery of SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) to the plasma membrane and promotes activation of an SRC-dependent signaling cascade that controls YAP nuclear shuttling. Moreover, ß1 integrin engagement and mechano-sensitive cues, such as external stiffness and related cell contractility, controlled LAMTOR targeting to the cell periphery and thereby late endosome recycling and had a major impact on YAP signaling. Our findings identify the late endosome recycling pathway as a key mechanism that controls YAP activity and explains YAP mechano-sensitivity.

Core Matrisome Protein Signature During Periodontal Ligament Maturation From Pre-occlusal Eruption to Occlusal Function.

The pre-occlusal eruption brings the molars into functional occlusion and initiates tensional strains during mastication. We hypothesized that upon establishment of occlusal contact, the periodontal ligament (PDL) undergoes cell and extracellular matrix maturation to adapt to this mechanical function. The PDL of 12 Wistar male rats were laser microdissected to observe the proteomic changes between stages of pre-occlusal eruption, initial occlusal contact and 1-week after occlusion. The proteome was screened by mass spectrometry and confirmed by immunofluorescence. The PDL underwent maturation upon establishment of occlusion. Downregulation of alpha-fetoprotein stem cell marker and protein synthesis markers indicate cell differentiation. Upregulated proteins were components of the extracellular matrix (ECM) and were characterized with the matrisome project database. In particular, periostin, a major protein of the PDL, was induced following occlusal contact and localized around collagen α-1 (III) bundles. This co-localization coincided with organization of collagen fibers in direction of the occlusal forces. Establishment of occlusion coincides with cellular differentiation and the maturation of the PDL. Co-localization of periostin and collagen with subsequent fiber organization may help counteract tensional forces and reinforce the ECM structure. This may be a key mechanism of the PDL to adapt to occlusal forces and maintain structural integrity.

Induction of ligand promiscuity of αVß3 integrin by mechanical force.

αVß3 integrin can bind to multiple extracellular matrix proteins, including vitronectin (Vn) and fibronectin (Fn), which are often presented to cells in culture as homogenous substrates. However, in tissues, cells experience highly complex and changing environments. To better understand integrin ligand selection in such complex environments, we employed binary-choice substrates of Fn and Vn to dissect αVß3 integrin-mediated binding to different ligands on the subcellular scale. Super-resolution imaging revealed that αVß3 integrin preferred binding to Vn under various conditions. In contrast, binding to Fn required higher mechanical load on αVß3 integrin. Integrin mutations, structural analysis and chemical inhibition experiments indicated that the degree of hybrid domain swing-out is relevant for the selection between Fn and Vn; only a force-mediated, full hybrid domain swing-out facilitated αVß3-Fn binding. Thus, force-dependent conformational changes in αVß3 integrin increased the diversity of available ligands for binding and therefore enhanced the ligand promiscuity of this integrin.This article has an associated First Person interview with the first author of the paper.