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Focal adhesions (FAs) are integrin‐containing protein complexes regulated by a network of hundreds of protein–protein interactions. They are formed in a spatiotemporal manner upon the activation of integrin transmembrane receptors, which is crucial to trigger cell adhesion and many other cellular processes including cell migration, spreading and proliferation. Despite decades of studies, a detailed molecular level understanding on how FAs are organized and function is lacking due to their highly complex and dynamic nature. However, advances have been made on studying key integrin activators, talin and kindlin, and their associated proteins, which are major components of nascent FAs critical for initiating the assembly of mature FAs. This review will discuss the structural and functional findings of talin and kindlin and their immediate interaction network, which will shed light upon the architecture of nascent FAs and how they act as seeds for FA assembly to dynamically regulate diverse adhesion‐dependent physiological and pathological responses. [ABSTRACT FROM AUTHOR]

Aim: Sodium retention is the hallmark of nephrotic syndrome (NS) and mediated by the proteolytic activation of the epithelial sodium channel (ENaC) by aberrantly filtered serine proteases. Plasmin is highly abundant in nephrotic urine and has been proposed to be the principal serine protease responsible for ENaC activation in NS. However, a proof of the essential role of plasmin in experimental NS is lacking. Methods: We used a genetic mouse model of NS based on an inducible podocin knockout (Bl6‐Nphs2tm3.1Antc*Tg(Nphs1‐rtTA*3G)8Jhm*Tg(tetO‐cre)1Jaw or nphs2Δipod). These mice were crossed with plasminogen deficient mice (Bl6‐Plgtm1Jld or plg−/−) to generate double knockout mice (nphs2Δipod*plg−/−). NS was induced after oral doxycycline treatment for 14 days and mice were followed for subsequent 14 days. Results: Uninduced nphs2Δipod*plg−/− mice had normal kidney function and sodium handling. After induction, proteinuria increased similarly in both nphs2Δipod*plg+/+ and nphs2Δipod*plg−/− mice. Western blot revealed the urinary excretion of plasminogen and plasmin in nphs2Δipod*plg+/+ mice which were absent in nphs2Δipod*plg−/− mice. After the onset of proteinuria, amiloride‐sensitive natriuresis was increased compared to the uninduced state in both genotypes. Subsequently, urinary sodium excretion dropped in both genotypes leading to an increase in body weight and development of ascites. Treatment with the serine protease inhibitor aprotinin prevented sodium retention in both genotypes. Conclusions: This study shows that mice lacking urinary plasminogen are not protected from ENaC‐mediated sodium retention in experimental NS. This points to an essential role of other urinary serine proteases in the absence of plasminogen. [ABSTRACT FROM AUTHOR]

Key points A reduction in Kindlin-2 levels in endothelial cells compromises vascular barrier function., Kindlin-2 is a previously unrecognized component of endothelial adherens junctions., By interacting directly and simultaneously with β- or γ-catenin and cortical actin filaments, Kindlin-2 stabilizes adherens junctions., The Kindlin-2 binding sites for β- and γ-catenin reside within its F1 and F3 subdomains. Although Kindlin-2 does not associate directly with tight junctions, its downregulation also destabilizes these junctions. Thus, impairment of both adherens and tight junctions may contribute to enhanced leakiness of vasculature in Kindlin-2+/− mice., Abstract Endothelial cells (EC) establish a physical barrier between the blood and surrounding tissue. Impairment of this barrier can occur during inflammation, ischaemia or sepsis and cause severe organ dysfunction. Kindlin-2, which is primarily recognized as a focal adhesion protein in EC, was not anticipated to have a role in vascular barrier. We tested the role of Kindlin-2 in regulating vascular integrity using several different approaches to decrease Kindlin-2 levels in EC. Reduced levels of Kindlin-2 in Kindlin-2+/- mice aortic endothelial cells (MAECs) from these mice, and human umbilical ECs (HUVEC) treated with Kindlin-2 siRNA showed enhanced basal and platelet-activating factor (PAF) or lipopolysaccharide-stimulated vascular leakage compared to wild-type (WT) counterparts. PAF preferentially disrupted the Kindlin-2+/− MAECs barrier to BSA and dextran and reduced transendothelial resistance compared to WT cells. Kindlin-2 co-localized and co-immunoprecipitated with vascular endothelial cadherin-based complexes, including β- and γ-catenin and actin, components of adherens junctions (AJ). Direct interaction of Kindlin-2 with β- and γ-catenin and actin was demonstrated in co-immunoprecipitation and surface plasmon resonance experiments. In thrombin-stimulated HUVECs, Kindlin-2 and cortical actin dissociated from stable AJs and redistributed to radial actin stress fibres of remodelling focal AJs. The β- and γ-catenin binding site resides within the F1 and F3 subdomains of Kindlin-2 but not the integrin binding site in F3. These results establish a previously unrecognized and vital role of Kindlin-2 with respect to maintaining the vascular barrier by linking Vascuar endothelial cadherin-based complexes to cortical actin and thereby stabilizing AJ. [ABSTRACT FROM AUTHOR]

The FERM domain containing protein Kindlin-3 has been recognized as a major regulator of integrin function in hematopoietic cells, but its role in neoplasia is totally unknown. We have examined the relationship between Kindlin-3 and breast cancer in mouse models and human tissues. Human breast tumors showed a ~7-fold elevation in Kindlin-3 mRNA compared with nonneoplastic tissue by quantitative polymerase chain reaction. Kindlin-3 overexpression in a breast cancer cell line increased primary tumor growth and lung metastasis by 2.5- and 3-fold, respectively, when implanted into mice compared with cells expressing vector alone. Mechanistically, the Kindlin-3-overexpressing cells displayed a 2.2-fold increase in vascular endothelial growth factor (VEGF) secretion and enhanced β1 integrin activation. Increased VEGF secretion resulted from enhanced production of Twist, a transcription factor that promotes tumor angiogenesis. Knockdown of Twist diminished VEGF production, and knockdown of β1 integrins diminished Twist and VEGF production by Kindlin-3-overexpressing cells, while nontargeting small interfering RNA had no effect on expression of these gene products. Thus, Kindlin-3 influences breast cancer progression by influencing the crosstalk between pi integrins and Twist to increase VEGF production. This signaling cascade enhances breast cancer cell invasion and tumor angiogenesis and metastasis. [ABSTRACT FROM AUTHOR]

Thrombospondin-4 (TSP-4) expression increases dramatically in hypertrophic and failing hearts in rodent models and in humans. The aim of this study was to address the function of TSP-4 in the heart. TSP-4-knockout (Thbs4-/-) and wild-type (WT) mice were subjected to transverse aortic constriction (TAC) to increase left ventricle load. After 2 wk, Thbs4-/- mice had a significantly higher heart weight/body weight ratio than WT mice. The additional increase in the heart weight in TAC Thbs4-/- mice was due to increased deposition of extracellular matrix (ECM). The levels of interstitial collagens were higher in the knockout mice, but the size of cardiomyocytes and apoptosis in the myocardium was unaffected by TSP-4 deficiency, suggesting that increased reactive fibrosis was the primary cause of the higher heart weight. The increased ECM deposition in Thbs4-/- mice was accompanied by changes in functional parameters of the heart and decreased vessel density. The expression of inflammatory and fibrotic genes known to be influential in myocardial remodeling changed as a result of TSP-4 deficiency in vivo and as a result of incubation of cells with recombinant TSP-4 in vitro. Thus, TSP-4 is involved in regulating the adaptive responses of the heart to pressure overload, suggesting its important role in myocardial remodeling. Our study showed a direct influence of TSP-4 on heart function and to identify the mechanism of its effects on heart remodeling. [ABSTRACT FROM AUTHOR]

WAVE3, an actin cytoskeleton remodeling protein, is highly expressed in advanced stages of breast cancer and influences tumor cell invasion. Loss of miR-31 has been associated with cancer progression and metastasis. Here, we show that the activity of WAVE3 to promote cancer cell invasion is regulated by miR-31. An inverse correlation was demonstrated between expression levels of WAVE3 and miR-31 in invasive versus noninvasive breast cancer cell lines. miR-31 directly targeted the 3'-UTR of the WAVE3 mRNA and inhibited its expression in the invasive cancer cells, i.e., miR-31-mediated down-regulation of WAVE3 resulted in a significant reduction in the invasive phenotype of cancer cells. This relationship was specific to the loss of WAVE3 expression because re-expression of a miR-31-resistant form of WAVE3 reversed miR-31-mediated inhibition of cancer cell invasion. Furthermore, expression of miR-31 correlates inversely with breast cancer progression in humans, where an increase in expression of miR-31 target genes was observed as the tumors progressed to more aggressive forms. In conclusion, a novel mechanism for the regulation of WAVE3 expression in cancer cells has been identified, which controls the invasive properties of cancer cells. The study also identifies a critical role for WAVE3, downstream of miR-31, in the invasion-metastasis cascade. [ABSTRACT FROM AUTHOR]

Integrin-mediated cell–ECM (extracellular matrix) adhesion is a fundamental process that controls cell behaviour. For correct cell–ECM adhesion, both the ligand-binding affinity and the spatial organization of integrins must be precisely controlled; how integrins are regulated, however, is not completely understood. Kindlins constitute a family of evolutionarily conserved cytoplasmic components of cell–ECM adhesions that bind to β-integrin cytoplasmic tails directly and cooperate with talin in integrin activation. In addition, kindlins interact with many components of cell–ECM adhesions—such as migfilin and integrin-linked kinase—to promote cytoskeletal reorganization. Loss of kindlins causes severe defects in integrin signalling, cell–ECM adhesion and cytoskeletal organization, resulting in early embryonic lethality (kindlin-2), postnatal lethality (kindlin-3) and Kindler syndrome (kindlin-1). It is therefore clear that kindlins, together with several other integrin-proximal proteins, are essential for integrin signalling and cell–ECM adhesion regulation. [ABSTRACT FROM AUTHOR]

The conformation of the carboxy-terminal aspects of the Aα chain of human fibrinogen has been assessed by immunochemically characterizing the Aα 239 - 476 and Aα 518 - 584 regions of the molecule. Two peptides, corresponding to these regions, were isolated from cyanogen bromide digests of the Aα chain by molecular exclusion and high-performance liquid chromatography. Each pep tide reacted with antibodies elicited by immunization with the Aα chain and intact fibrinogen. Aα 239 - 476 appears to be a relatively immunodominant region of the molecule. Competitive inhibition analyses confirmed the accessibility of these regions to antibody in native fibrinogen. Each peptide, however, contained one or more epitopes, which was occult in the native molecule. These occult epitopes were expressed by the intact Aα chain and became accessible when fibrinogen was cleaved with plasmin. With plasmic degradation the epitopes expressed by fibrinogen and contained within these two peptide regions became significantly more reactive with antibody. This change occurred in concert with release of the Aα 518 - 584 region from the core of the molecule but did not require the generation of free Aα 239 - 476. Ultimately the epitopes within both regions were shed from the plasmic-resistant core of fibrinogen. Peptide epitopes were expressed in a similar manner by prolonged plasmic degradation of fibrinogen and fibrin with α chain cross-linking. These results are generally consistent with models depicting the carboxy-terminal aspects of the Aα chain as being surface-oriented but suggest a systematic ordering of structure when these regions are integrated into the native molecule. Plasmic cleavage significantly relaxes the conformational restraints on the organization within this region. [ABSTRACT FROM AUTHOR]

The alkylating agents, iodoacetate and iodoacetamide, were spontaneously incorporated into fibrinogen at pH above 5.5 and 37 'C. These alkylating agents were covalently bound by fibrinogen, and the association was not dependent upon the presence of factor XIII or plasminogen. Approximately 18 mol of either alkylating agent were bound per mol of fibrinogen. By amino acid analysis, no S-carboxymethylcysteine was detected; but the histidine derivatives, 3-carboxymethylhistidine and 1,3-dicarboxymethylhistidine, were identified as the only modified residues, and these were present at an approximate 2 ; 1 ratio. The alkylation reaction was not altered by systematic cleavage of fibrinogen by plasmin, and the alkylated residues in the D - E complex were again restricted to the 3-carboxymethylhistidine and 1,3-dicarboxymethylhistidine derivatives m an approximate 2:1 ratio. Approximately 72% of the incorporated alkylating agent associated with the D region of fibrinogen and 28 % with the E region. Alkylation of fibrinogen prolonged its thrombin time, and alkylated fibrinogen inhibited the coagulation of unmodified fibrinogen. This effect was associated with interference with the polymerization of fibrin monomers. The results indicate that the disulfide bonds of native fibrinogen are stable, and plasmin-mediated catabolism of fibrinogen does not induce disulfide interchange. Specific subsets of surface-oriented histidine residues located within the relatively plasmin- resistant D.E core of fibrinogen are highly susceptible to alkylation, and some of these specific histidine residues may play an important role in the intermolecular alignment and polymerization of fibrin monomers. [ABSTRACT FROM AUTHOR]

Examines the surface expression of intracellular fibrinogen and its involvement in platelet aggregation. Indications that radiolabelled Fab fragments did not interact with non-stimulated platelets but significant binding was observed when the cells were stimulated by various compounds; Mechanism for the surface expression of platelet fibrinogen distinct from that for the binding of plasma fibrinogen.

Fibrinogen participates in platelet aggregation via specific inducible receptors on the cell surface. We have used a photoactivable bifunctional reagent, N‐succinimidyl‐6‐(4′‐azido‐2′‐nitrophenylamino)hexanoate, SANAH, to derivatize 125I‐labeled‐fibrinogen (125I‐Fg) and crosslink it to ADP‐stimulated platelets. Binding experiments established that 125I‐Fg and 125I‐Fg‐SANAH interacted with platelets with the same kinetics and affinity as unlabeled fibrinogen. After photoactivation of the platelet‐bound 125I‐Fg‐SANAH, polyacrylamide gel electrophoresis under reducing conditions revealed formation of a high molecular weight covalent complex with coordinate loss of the Aα chain. 125I‐Fg‐SANAH missing the extreme carboxy‐terminal region of the Aα chain failed to crosslink to the platelets under similar conditions. Crosslinked 125I‐Fg‐SANAH was extracted from the cells in 1% Triton X‐100, and immunoprecipitation with antibodies specific for platelet membrane glycoproteins was used to identify components of the complex. With antibodies to the glycoprotein IIb/III complex (anti‐GP IIb/III), 40 ± 9% of the extracted 125I‐Fg‐SANAH was irnmunoprecipitated. Omission of photoactivation, platelets, or ADP from the reaction or use of unmodified 125I‐Fg resulted in less than 5% immunoprecipitation by the anti‐GP IIb/III. As controls for specificity, anti‐(glycoprotein Ib) or anti‐IgG immunoprecipitated less than 5% of the extracted 125I‐Fg‐SANAH. Under similar conditions, 45% of the GP IIb/III from surface‐labeled platelets was recovered in the anti‐GP IIb/III immunoprecipitate. These results indicate that the Aα chain of fibrinogen comes in close proximity to GP IIb/III when the molecule is bound to its platelet receptor. [ABSTRACT FROM AUTHOR]

Examines how the N700S substitution in thrombospondin-1 (TSP-1) and the A387P substitution in TSP-4 affect calcium binding sites. Alteration of putative calcium binding sites in TSP-1 and TSP-4 variants; Metal ion binding by synthetic peptides corresponding to TSP-1 and TSP-4 variants; Effect of A387P A387P single nucleotide polymorphism in TSP-4 and N700S SNP in TSP-1 on intrinsic tryptophan fluorescence and calcium sensitivity of TSP fragments.

Identifies a mechanism of regulation of cell adhesion molecules (CAM) expression by endothelium that links platelet activation and atherogenesis. Role of thrombospondin-1 (TSP-1) as an inducer of CAM expression and monocyte adhesion to endothelium; Effect of TSP-1 on CAM expression by endothelium; Structure and functional aspects of TSP-1-stimulated up-regulation of CAM; Matrix protein which may trigger the initial stages of inflammation and atherogenesis.