Multivalent anions as universal latent electron donors.

Electrodes with low work functions are required to efficiently inject electrons into semiconductor devices. However, when the work function drops below about 4 electronvolts, the electrode suffers oxidation in air, which prevents its fabrication in ambient conditions. Here we show that multivalent anions such as oxalate, carbonate and sulfite can act as powerful latent electron donors when dispersed as small ion clusters in a matrix, while retaining their ability to be processed in solution in ambient conditions. The anions in these clusters can even n-dope the semiconductor core of π-conjugated polyelectrolytes that have low electron affinities, through a ground-state doping mechanism that is further amplified by a hole-sensitized or photosensitized mechanism in the device. A theoretical analysis of donor levels of these anions reveals that they are favourably upshifted from ionic lattices by a decrease in the Coulomb stabilization of small ion clusters, and by irreversibility effects. We attain an ultralow effective work function of 2.4 electronvolts with the polyfluorene core. We realize high-performance, solution-processed, white-light-emitting diodes and organic solar cells using polymer electron injection layers with these universal anion donors, demonstrating a general approach to chemically designed and ambient-processed Ohmic electron contacts for semiconductor devices.

A microenvironment-inspired synthetic three-dimensional model for pancreatic ductal adenocarcinoma organoids.

Experimental in vitro models that capture pathophysiological characteristics of human tumours are essential for basic and translational cancer biology. Here, we describe a fully synthetic hydrogel extracellular matrix designed to elicit key phenotypic traits of the pancreatic environment in culture. To enable the growth of normal and cancerous pancreatic organoids from genetically engineered murine models and human patients, essential adhesive cues were empirically defined and replicated in the hydrogel scaffold, revealing a functional role of laminin-integrin α3/α6 signalling in establishment and survival of pancreatic organoids. Altered tissue stiffness-a hallmark of pancreatic cancer-was recapitulated in culture by adjusting the hydrogel properties to engage mechano-sensing pathways and alter organoid growth. Pancreatic stromal cells were readily incorporated into the hydrogels and replicated phenotypic traits characteristic of the tumour environment in vivo. This model therefore recapitulates a pathologically remodelled tumour microenvironment for studies of normal and pancreatic cancer cells in vitro.

Talin2 and KANK2 functionally interact to regulate microtubule dynamics, paclitaxel sensitivity and cell migration in the MDA-MB-435S melanoma cell line.

BACKGROUND: Focal adhesions (FAs) are integrin-containing, multi-protein structures that link intracellular actin to the extracellular matrix and trigger multiple signaling pathways that control cell proliferation, differentiation, survival and motility. Microtubules (MTs) are stabilized in the vicinity of FAs through interaction with the components of the cortical microtubule stabilizing complex (CMSC). KANK (KN motif and ankyrin repeat domains) family proteins within the CMSC, KANK1 or KANK2, bind talin within FAs and thus mediate actin-MT crosstalk. We previously identified in MDA-MB-435S cells, which preferentially use integrin αVß5 for adhesion, KANK2 as a key molecule enabling the actin-MT crosstalk. KANK2 knockdown also resulted in increased sensitivity to MT poisons, paclitaxel (PTX) and vincristine and reduced migration. Here, we aimed to analyze whether KANK1 has a similar role and to distinguish which talin isoform binds KANK2. METHODS: The cell model consisted of human melanoma cell line MDA-MB-435S and stably transfected clone with decreased expression of integrin αV (3αV). For transient knockdown of talin1, talin2, KANK1 or KANK2 we used gene-specific siRNAs transfection. Using previously standardized protocol we isolated integrin adhesion complexes. SDS-PAGE and Western blot was used for protein expression analysis. The immunofluorescence analysis and live cell imaging was done using confocal microscopy. Cell migration was analyzed with Transwell Cell Culture Inserts. Statistical analysis using GraphPad Software consisted of either one-way analysis of variance (ANOVA), unpaired Student's t-test or two-way ANOVA analysis. RESULTS: We show that KANK1 is not a part of the CMSC associated with integrin αVß5 FAs and its knockdown did not affect the velocity of MT growth or cell sensitivity to PTX. The talin2 knockdown mimicked KANK2 knockdown i.e. led to the perturbation of actin-MT crosstalk, which is indicated by the increased velocity of MT growth and increased sensitivity to PTX and also reduced migration. CONCLUSION: We conclude that KANK2 functionally interacts with talin2 and that the mechanism of increased sensitivity to PTX involves changes in microtubule dynamics. These data elucidate a cell-type-specific role of talin2 and KANK2 isoforms and we propose that talin2 and KANK2 are therefore potential therapeutic targets for improved cancer therapy.

Talin mechanosensitivity is modulated by a direct interaction with cyclin-dependent kinase-1.

Talin (TLN1) is a mechanosensitive component of adhesion complexes that directly couples integrins to the actin cytoskeleton. In response to force, talin undergoes switch-like behavior of its multiple rod domains that modulate interactions with its binding partners. Cyclin-dependent kinase-1 (CDK1) is a key regulator of the cell cycle, exerting its effects through synchronized phosphorylation of a large number of protein targets. CDK1 activity maintains adhesion during interphase, and its inhibition is a prerequisite for the tightly choreographed changes in cell shape and adhesion that are required for successful mitosis. Using a combination of biochemical, structural, and cell biological approaches, we demonstrate a direct interaction between talin and CDK1 that occurs at sites of integrin-mediated adhesion. Mutagenesis demonstrated that CDK1 contains a functional talin-binding LD motif, and the binding site within talin was pinpointed to helical bundle R8. Talin also contains a consensus CDK1 phosphorylation motif centered on S1589, a site shown to be phosphorylated by CDK1 in vitro. A phosphomimetic mutant of this site within talin lowered the binding affinity of the cytoskeletal adaptor KANK and weakened the response of this region to force as measured by single molecule stretching, potentially altering downstream mechanotransduction pathways. The direct binding of the master cell cycle regulator CDK1 to the primary integrin effector talin represents a coupling of cell proliferation and cell adhesion machineries and thereby indicates a mechanism by which the microenvironment can control cell division in multicellular organisms.

Identification of an Altered Matrix Signature in Kidney Aging and Disease.

BACKGROUND: Accumulation of extracellular matrix in organs and tissues is a feature of both aging and disease. In the kidney, glomerulosclerosis and tubulointerstitial fibrosis accompany the decline in function, which current therapies cannot address, leading to organ failure. Although histologic and ultrastructural patterns of excess matrix form the basis of human disease classifications, a comprehensive molecular resolution of abnormal matrix is lacking. METHODS: Using mass spectrometry-based proteomics, we resolved matrix composition over age in mouse models of kidney disease. We compared the changes in mice with a global characterization of human kidneymatrix during aging and to existing kidney disease datasets to identify common molecular features. RESULTS: Ultrastructural changes in basement membranes are associated with altered cell adhesion and metabolic processes and with distinct matrix proteomes during aging and kidney disease progression in mice. Within the altered matrix, basement membrane components (laminins, type IV collagen, type XVIII collagen) were reduced and interstitial matrix proteins (collagens I, III, VI, and XV; fibrinogens; and nephronectin) were increased, a pattern also seen in human kidney aging. Indeed, this signature of matrix proteins was consistently modulated across all age and disease comparisons, and the increase in interstitial matrix was also observed in human kidney disease datasets. CONCLUSIONS: This study provides deep molecular resolution of matrix accumulation in kidney aging and disease, and identifies a common signature of proteins that provides insight into mechanisms of response to kidney injury and repair.

Conformational equilibria and intrinsic affinities define integrin activation.

We show that the three conformational states of integrin α5ß1 have discrete free energies and define activation by measuring intrinsic affinities for ligand of each state and the equilibria linking them. The 5,000-fold higher affinity of the extended-open state than the bent-closed and extended-closed states demonstrates profound regulation of affinity. Free energy requirements for activation are defined with protein fragments and intact α5ß1 On the surface of K562 cells, α5ß1 is 99.8% bent-closed. Stabilization of the bent conformation by integrin transmembrane and cytoplasmic domains must be overcome by cellular energy input to stabilize extension. Following extension, headpiece opening is energetically favored. N-glycans and leg domains in each subunit that connect the ligand-binding head to the membrane repel or crowd one another and regulate conformational equilibria in favor of headpiece opening. The results suggest new principles for regulating signaling in the large class of receptors built from extracellular domains in tandem with single-span transmembrane domains.

ß1 integrin is a sensor of blood flow direction.

Endothelial cell (EC) sensing of fluid shear stress direction is a critical determinant of vascular health and disease. Unidirectional flow induces EC alignment and vascular homeostasis, whereas bidirectional flow has pathophysiological effects. ECs express several mechanoreceptors that respond to flow, but the mechanism for sensing shear stress direction is poorly understood. We determined, by using in vitro flow systems and magnetic tweezers, that ß1 integrin is a key sensor of force direction because it is activated by unidirectional, but not bidirectional, shearing forces. ß1 integrin activation by unidirectional force was amplified in ECs that were pre-sheared in the same direction, indicating that alignment and ß1 integrin activity has a feedforward interaction, which is a hallmark of system stability. En face staining and EC-specific genetic deletion studies in the murine aorta revealed that ß1 integrin is activated and is essential for EC alignment at sites of unidirectional flow but is not activated at sites of bidirectional flow. In summary, ß1 integrin sensing of unidirectional force is a key mechanism for decoding blood flow mechanics to promote vascular homeostasis.This article has an associated First Person interview with the first author of the paper.

Cyclic mechanical reinforcement of integrin-ligand interactions.

Cells regulate adhesion in response to internally generated and externally applied forces. Integrins connect the extracellular matrix to the cytoskeleton and provide cells with mechanical anchorages and signaling platforms. Here we show that cyclic forces applied to a fibronectin-integrin α5ß1 bond switch the bond from a short-lived state with 1 s lifetime to a long-lived state with 100 s lifetime. We term this phenomenon "cyclic mechanical reinforcement," as the bond strength remembers the history of force application and accumulates over repeated cycles, but does not require force to be sustained. Cyclic mechanical reinforcement strengthens the fibronectin-integrin α5ß1 bond through the RGD binding site of the ligand with the synergy binding site greatly facilitating the process. A flexible integrin hybrid domain is also important for cyclic mechanical reinforcement. Our results reveal a mechanical regulation of receptor-ligand interactions and identify a molecular mechanism for cell adhesion strengthening by cyclic forces.

The Sharpin interactome reveals a role for Sharpin in lamellipodium formation via the Arp2/3 complex.

Sharpin, a multifunctional adaptor protein, regulates several signalling pathways. For example, Sharpin enhances signal-induced NF-κB signalling as part of the linear ubiquitin assembly complex (LUBAC) and inhibits integrins, the T cell receptor, caspase 1 and PTEN. However, despite recent insights into Sharpin and LUBAC function, a systematic approach to identify the signalling pathways regulated by Sharpin has not been reported. Here, we present the first 'Sharpin interactome', which identifies a large number of novel potential Sharpin interactors in addition to several known ones. These data suggest that Sharpin and LUBAC might regulate a larger number of biological processes than previously identified, such as endosomal trafficking, RNA processing, metabolism and cytoskeleton regulation. Importantly, using the Sharpin interactome, we have identified a novel role for Sharpin in lamellipodium formation. We demonstrate that Sharpin interacts with Arp2/3, a protein complex that catalyses actin filament branching. We have identified the Arp2/3-binding site in Sharpin and demonstrate using a specific Arp2/3-binding deficient mutant that the Sharpin-Arp2/3 interaction promotes lamellipodium formation in a LUBAC-independent fashion.This article has an associated First Person interview with the first author of the paper.

Synergistic control of cell adhesion by integrins and syndecans.

The ability of cells to adhere to each other and to their surrounding extracellular matrices is essential for a multicellular existence. Adhesion provides physical support for cells, regulates cell positioning and enables microenvironmental sensing. The integrins and the syndecans are two adhesion receptor families that mediate adhesion, but their relative and functional contributions to cell-extracellular matrix interactions remain obscure. Recent advances have highlighted connections between the signalling networks that are controlled by these families of receptors. Here we survey the evidence that synergistic signalling is involved in controlling adhesive function and the regulation of cell behaviour in response to the external environment.

Relating conformation to function in integrin α5ß1.

Whether ß1 integrin ectodomains visit conformational states similarly to ß2 and ß3 integrins has not been characterized. Furthermore, despite a wealth of activating and inhibitory antibodies to ß1 integrins, the conformational states that these antibodies stabilize, and the relation of these conformations to function, remain incompletely characterized. Using negative-stain electron microscopy, we show that the integrin α5ß1 ectodomain adopts extended-closed and extended-open conformations as well as a bent conformation. Antibodies SNAKA51, 8E3, N29, and 9EG7 bind to different domains in the α5 or ß1 legs, activate, and stabilize extended ectodomain conformations. Antibodies 12G10 and HUTS-4 bind to the ß1 ßI domain and hybrid domains, respectively, activate, and stabilize the open headpiece conformation. Antibody TS2/16 binds a similar epitope as 12G10, activates, and appears to stabilize an open ßI domain conformation without requiring extension or hybrid domain swing-out. mAb13 and SG/19 bind to the ßI domain and ßI-hybrid domain interface, respectively, inhibit, and stabilize the closed conformation of the headpiece. The effects of the antibodies on cell adhesion to fibronectin substrates suggest that the extended-open conformation of α5ß1 is adhesive and that the extended-closed and bent-closed conformations are nonadhesive. The functional effects and binding sites of antibodies and fibronectin were consistent with their ability in binding to α5ß1 on cell surfaces to cross-enhance or inhibit one another by competitive or noncompetitive (allosteric) mechanisms.

The integrin adhesome network at a glance.

The adhesion nexus is the site at which integrin receptors bridge intracellular cytoskeletal and extracellular matrix networks. The connection between integrins and the cytoskeleton is mediated by a dynamic integrin adhesion complex (IAC), the components of which transduce chemical and mechanical signals to control a multitude of cellular functions. In this Cell Science at a Glance article and the accompanying poster, we integrate the consensus adhesome, a set of 60 proteins that have been most commonly identified in isolated IAC proteomes, with the literature-curated adhesome, a theoretical network that has been assembled through scholarly analysis of proteins that localise to IACs. The resulting IAC network, which comprises four broad signalling and actin-bridging axes, provides a platform for future studies of the regulation and function of the adhesion nexus in health and disease.

Ligand-induced Epitope Masking: DISSOCIATION OF INTEGRIN α5ß1-FIBRONECTIN COMPLEXES ONLY BY MONOCLONAL ANTIBODIES WITH AN ALLOSTERIC MODE OF ACTION.

We previously demonstrated that Arg-Gly-Asp (RGD)-containing ligand-mimetic inhibitors of integrins are unable to dissociate pre-formed integrin-fibronectin complexes (IFCs). These observations suggested that amino acid residues involved in integrin-fibronectin binding become obscured in the ligand-occupied state. Because the epitopes of some function-blocking anti-integrin monoclonal antibodies (mAbs) lie near the ligand-binding pocket, it follows that the epitopes of these mAbs may become shielded in the ligand-occupied state. Here, we tested whether function-blocking mAbs directed against α5ß1 can interact with the integrin after it forms a complex with an RGD-containing fragment of fibronectin. We showed that the anti-α5 subunit mAbs JBS5, SNAKA52, 16, and P1D6 failed to disrupt IFCs and hence appeared unable to bind to the ligand-occupied state. In contrast, the allosteric anti-ß1 subunit mAbs 13, 4B4, and AIIB2 could dissociate IFCs and therefore were able to interact with the ligand-bound state. However, another class of function-blocking anti-ß1 mAbs, exemplified by Lia1/2, could not disrupt IFCs. This second class of mAbs was also distinguished from 13, 4B4, and AIIB2 by their ability to induce homotypic cell aggregation. Although the epitope of Lia1/2 was closely overlapping with those of 13, 4B4, and AIIB2, it appeared to lie closer to the ligand-binding pocket. A new model of the α5ß1-fibronectin complex supports our hypothesis that the epitopes of mAbs that fail to bind to the ligand-occupied state lie within, or very close to, the integrin-fibronectin interface. Importantly, our findings imply that the efficacy of some therapeutic anti-integrin mAbs could be limited by epitope masking.

Mechanosensitivity of integrin adhesion complexes: role of the consensus adhesome.

Cell and tissue stiffness have been known to contribute to both developmental and pathological signalling for some time, but the underlying mechanisms remain elusive. Integrins and their associated adhesion signalling complexes (IACs), which form a nexus between the cell cytoskeleton and the extracellular matrix, act as a key force sensing and transducing unit in cells. Accordingly, there has been much interest in obtaining a systems-level understanding of IAC composition. Proteomic approaches have revealed the complexity of IACs and identified a large number of components that are regulated by cytoskeletal force. Here we review the function of the consensus adhesome, an assembly of core IAC proteins that emerged from a meta-analysis of multiple proteomic datasets, in the context of mechanosensing. As IAC components have been linked to a variety of diseases involved with rigidity sensing, the field is now in a position to define the mechanosensing function of individual IAC proteins and elucidate their mechanisms of action.

Highly Emissive Far Red/Near-IR Fluorophores Based on Borylated Fluorene-Benzothiadiazole Donor-Acceptor Materials.

Stille, Suzuki-Miyaura and Negishi cross-coupling reactions of bromine-functionalised borylated precursors enable the facile, high yielding, synthesis of borylated donor-acceptor materials that contain electron-rich aromatic units and/or extended effective conjugation lengths. These materials have large Stokes shifts, low LUMO energies, small band-gaps and significant fluorescence emission >700 nm in solution and when dispersed in a host polymer.

Distinct biophysical mechanisms of focal adhesion kinase mechanoactivation by different extracellular matrix proteins.

Matrix mechanics controls cell fate by modulating the bonds between integrins and extracellular matrix (ECM) proteins. However, it remains unclear how fibronectin (FN), type 1 collagen, and their receptor integrin subtypes distinctly control force transmission to regulate focal adhesion kinase (FAK) activity, a crucial molecular signal governing cell adhesion/migration. Here we showed, using a genetically encoded FAK biosensor based on fluorescence resonance energy transfer, that FN-mediated FAK activation is dependent on the mechanical tension, which may expose its otherwise hidden FN synergy site to integrin α5. In sharp contrast, the ligation between the constitutively exposed binding motif of type 1 collagen and its receptor integrin α2 was surprisingly tension-independent to induce sufficient FAK activation. Although integrin α subunit determines mechanosensitivity, the ligation between α subunit and the ECM proteins converges at the integrin ß1 activation to induce FAK activation. We further discovered that the interaction of the N-terminal protein 4.1/ezrin/redixin/moesin basic patch with phosphatidylinositol 4,5-biphosphate is crucial during cell adhesion to maintain the FAK activation from the inhibitory effect of nearby protein 4.1/ezrin/redixin/moesin acidic sites. Therefore, different ECM proteins either can transmit or can shield from mechanical forces to regulate cellular functions, with the accessibility of ECM binding motifs by their specific integrin α subunits determining the biophysical mechanisms of FAK activation during mechanotransduction.

Genetic Background is a Key Determinant of Glomerular Extracellular Matrix Composition and Organization.

Glomerular disease often features altered histologic patterns of extracellular matrix (ECM). Despite this, the potential complexities of the glomerular ECM in both health and disease are poorly understood. To explore whether genetic background and sex determine glomerular ECM composition, we investigated two mouse strains, FVB and B6, using RNA microarrays of isolated glomeruli combined with proteomic glomerular ECM analyses. These studies, undertaken in healthy young adult animals, revealed unique strain- and sex-dependent glomerular ECM signatures, which correlated with variations in levels of albuminuria and known predisposition to progressive nephropathy. Among the variation, we observed changes in netrin 4, fibroblast growth factor 2, tenascin C, collagen 1, meprin 1-α, and meprin 1-ß. Differences in protein abundance were validated by quantitative immunohistochemistry and Western blot analysis, and the collective differences were not explained by mutations in known ECM or glomerular disease genes. Within the distinct signatures, we discovered a core set of structural ECM proteins that form multiple protein-protein interactions and are conserved from mouse to man. Furthermore, we found striking ultrastructural changes in glomerular basement membranes in FVB mice. Pathway analysis of merged transcriptomic and proteomic datasets identified potential ECM regulatory pathways involving inhibition of matrix metalloproteases, liver X receptor/retinoid X receptor, nuclear factor erythroid 2-related factor 2, notch, and cyclin-dependent kinase 5. These pathways may therefore alter ECM and confer susceptibility to disease.

Rac1 is deactivated at integrin activation sites through an IQGAP1-filamin-A-RacGAP1 pathway.

Cell migration makes a fundamental contribution to both normal physiology and disease pathogenesis. Integrin engagement with extracellular ligands spatially controls, via the cyclical activation and deactivation of the small GTPase Rac1, the dynamic membrane protrusion and cytoskeletal reorganization events that are required for directional migration. Although the pathways that control integrin-mediated Rac1 activation are reasonably well defined, the mechanisms that are responsible for switching off activity are poorly understood. Here, proteomic analysis of activated integrin-associated complexes suggests filamin-A and IQ-motif-containing GTPase-activating protein 1 (IQGAP1) as candidates that link ß1 integrin to Rac1. siRNA-mediated knockdown of either filamin-A or IQGAP1 induced high, dysregulated Rac1 activity during cell spreading on fibronectin. Using immunoprecipitation and immunocytochemistry, filamin-A and IQGAP1 were shown to be part of a complex that is recruited to active ß1 integrin. Mass spectrometric analysis of individual filamin-A, IQGAP1 and Rac1 pull-downs and biochemical analysis, identified RacGAP1 as a novel IQGAP1 binding partner. Further immunoprecipitation and immunocytochemistry analyses demonstrated that RacGAP1 is recruited to IQGAP1 and active ß1 integrin, and that suppression of RacGAP1 expression triggered elevated Rac1 activity during spreading on fibronectin. Consistent with these findings, reduced expression of filamin-A, IQGAP1 or RacGAP1 triggered unconstrained membrane protrusion and disrupted directional cell migration on fibrillar extracellular matrices. These findings suggest a model whereby integrin engagement, followed by filamin-A, IQGAP1 and RacGAP1 recruitment, deactivates Rac1 to constrain its activity spatially and thereby coordinate directional cell migration.

Disruption of integrin-fibronectin complexes by allosteric but not ligand-mimetic inhibitors.

Failure of Arg-Gly-Asp (RGD)-based inhibitors to reverse integrin-ligand binding has been reported, but the prevalence of this phenomenon among integrin heterodimers is currently unknown. In the present study we have investigated the interaction of four different RGD-binding integrins (α5ß1, αVß1, αVß3 and αVß6) with fibronectin (FN) using surface plasmon resonance. The ability of inhibitors to reverse ligand binding was assessed by their capacity to increase the dissociation rate of pre-formed integrin-FN complexes. For all four receptors we showed that RGD-based inhibitors (such as cilengitide) were completely unable to increase the dissociation rate. Formation of the non-reversible state occurred very rapidly and did not rely on the time-dependent formation of a high-affinity state of the integrin, or the integrin leg regions. In contrast with RGD-based inhibitors, Ca2+ (but not Mg2+) was able to greatly increase the dissociation rate of integrin-FN complexes, with a half-maximal response at ~0.4 mM Ca2+ for αVß3-FN. The effect of Ca2+ was overcome by co-addition of Mn2+, but not Mg2+. A stimulatory anti-ß1 monoclonal antibody (mAb) abrogated the effect of Ca2+ on α5ß1-FN complexes; conversely, a function-blocking mAb mimicked the effect of Ca2+. These results imply that Ca2+ acts allosterically, probably through binding to the adjacent metal-ion-dependent adhesion site (ADMIDAS), and that the α1 helix in the ß subunit I domain is the key element affected by allosteric modulators. The data suggest an explanation for the limited clinical efficacy of RGD-based integrin antagonists, and we propose that allosteric antagonists could prove to be of greater therapeutic benefit.