Dimeric Fibronectin Research Articles

A disintegrin-like and metalloproteinase domain with thrombospondin type 1 motif 9 (ADAMTS9) regulates fibronectin fibrillogenesis and turnover

The secreted metalloprotease ADAMTS9 has dual roles in extracellular matrix (ECM) turnover and biogenesis of the primary cilium during mouse embryogenesis. Its gene locus is associated with several human traits and disorders, but ADAMTS9 has few known interacting partners or confirmed substrates. Here, using a yeast two-hybrid screen for proteins interacting with its C-terminal Gon1 domain, we identified three putative ADAMTS9-binding regions in the ECM glycoprotein fibronectin. Using solid-phase binding assays and surface plasmon resonance experiments with purified proteins, we demonstrate that ADAMTS9 and fibronectin interact. ADAMTS9 constructs, including those lacking Gon1, co-localized with fibronectin fibrils formed by cultured fibroblasts lacking fibrillin-1, which co-localizes with fibronectin and binds several ADAMTSs. We observed no fibrillar ADAMTS9 staining after blockade of fibroblast fibronectin fibrillogenesis with a peptide based on the functional upstream domain of a Staphylococcus aureus adhesin. These findings indicate that ADAMTS9 binds fibronectin dimers and fibrils directly through multiple sites in both molecules. Proteolytically active ADAMTS9, but not a catalytically inactive variant, disrupted fibronectin fibril networks formed by fibroblasts in vitro, and ADAMTS9-deficient RPE1 cells assembled a robust fibronectin fibril network, unlike WT cells. Targeted LC-MS analysis of fibronectin digested by ADAMTS9-expressing cells identified a semitryptic peptide arising from cleavage at Gly2196-Leu2197 We noted that this scissile bond is in the linker between fibronectin modules III17 and I10, a region targeted also by other proteases. These findings, along with stronger fibronectin staining previously observed in Adamts9 mutant embryos, suggest that ADAMTS9 contributes to fibronectin turnover during ECM remodeling.

Fibronectin assembly during early embryo development: A versatile communication system between cells and tissues.

Fibronectin extracellular matrix is essential for embryogenesis. Its assembly is a cell-mediated process where secreted fibronectin dimers bind to integrin receptors on receiving cells, which actively assemble fibronectin into a fibrillar matrix. During development, paracrine communication between tissues is crucial for coordinating morphogenesis, typically being mediated by growth factors and their receptors. Recent reports of situations where fibronectin is produced by one tissue and assembled by another, with implications on tissue morphogenesis, suggest that fibronectin assembly may also be a paracrine communication event in certain contexts. Here we addressed which tissues express fibronectin (Fn1) while also localizing assembled fibronectin matrix and determining the mRNA expression and/or protein distribution pattern of integrins α5 and αV, α chains of the major fibronectin assembly receptors, during early chick and mouse development. We found evidence supporting a paracrine system in fibronectin matrix assembly in several tissues, including immature mesenchymal tissues, components of central and peripheral nervous system and developing muscle. Thus, similarly to growth factor signaling, fibronectin matrix assembly during early development can be both autocrine and paracrine. We therefore propose that it be considered a cell-cell communication event at the same level and significance as growth factor signaling during embryogenesis.

Occurrence of fibronectin–fibrin complexes in plasma of patients with multimorbidity due to the inflamm-aging phenomenon

BackgroundMultimorbidity is the co-occurrence of chronic diseases associated with low-grade chronic inflammation of connective tissue. Aim of studyFrequency of occurrence and relative amounts of fibronectin (FN) complexes with fibrin (FN–fibrin) and FN monomer were analyzed in 130 plasma samples of 18 to 94-year-old multimorbid patients in relation to concentrations of FN and extra domain A (EDA)-FN, and C-reactive protein (CRP) as well as to age, number of coexisting chronic diseases and presence of specified diseases. ResultsImmunoblotting revealed, besides FN dimer, the presence of FN monomer, and 750-, 1000-, and 1300-kDa FN–fibrin complexes in the multimorbid plasmas. The FN–fibrin complexes appeared more frequently and in higher relative amounts, but FN monomer less frequently and in a lower relative amount in the groups of elderly multimorbid patients, with a higher number of coexisting diseases and with dominance of cardiovascular diseases and osteoarthrosis, and with CRP concentration of 3–5mg/l. In contrast, the normal plasma contained only the FN–fibrin complex of 750kDa in a lower relative amount, but with an increasing amount with normal aging. Moreover, FN concentration increased and EDA-FN decreased with the number of co-existing diseases and aging of patients, although both concentration values were lower than in the age-matched normal groups. FN concentration was the lowest in the exacerbation of a chronic disease and EDA-FN in the stable chronic disease groups. ConclusionThe alterations in plasma FN molecular status were associated with micro-inflammation and micro-coagulation, as well as multimorbidity of subjects and their physiological aging.

A Computational Model of Cell-Generated Traction Forces and Fibronectin Assembly

The extracellular matrix (ECM) is an assembly of proteins that surround cells and serves as the cell substrate in vivo. A primary component of newly synthesized ECM is fibronectin (FN), critical for embryonic development and wound healing. Despite years of research, the mechanism of FN assembly is still not understood. We hypothesize that FN assembly occurs through the revelation of buried FN-FN binding sites within any of the15 Type III FN domains, and that these binding sites are exposed by stretching of FN-III beta strands, allowing attachment of additional FN molecules via a beta strand addition mechanism. To investigate this hypothesis, we developed a biophysical computational model of cell-FN-substrate biomechanical-chemical interactions. In the model, FN-III domains are represented by Hookean springs with distinct stiffnesses; thus, each FN dimer is represented by 30 springs in series. Integrin binding/unbinding is represented by a stochastic first-order reversible chemical reaction with force-dependent off-rate. Model results are validated using in vitro tools: FN fibril growth and traction forces applied over time are measured using microfabricated pillar arrays in a human fibroblast cell line. Beta-strand exposure is probed using Thioflavin T, a fluorescent probe that binds to free beta strands. Results indicate that the computational model recapitulates three unique features that are observed in experimental measurements of FN fibrils: 1) FN fibrils are highly elastic, but have a maximal 4-fold elongation of their resting length; 2) FN fibril length and force are not strongly correlated; and 3) FN fibrils have discrete stable lengths, suggesting local minima of force balance within growing fibrils. These results will be discussed and compared with simulations in which FN fibril assembly is mediated by either a single cryptic binding site or a subset of cryptic binding sites.

A potential role for endogenous proteins as sacrificial sunscreens and antioxidants in human tissues

Excessive ultraviolet radiation (UVR) exposure of the skin is associated with adverse clinical outcomes. Although both exogenous sunscreens and endogenous tissue components (including melanins and tryptophan-derived compounds) reduce UVR penetration, the role of endogenous proteins in absorbing environmental UV wavelengths is poorly defined. Having previously demonstrated that proteins which are rich in UVR-absorbing amino acid residues are readily degraded by broadband UVB-radiation (containing UVA, UVB and UVC wavelengths) here we hypothesised that UV chromophore (Cys, Trp and Tyr) content can predict the susceptibility of structural proteins in skin and the eye to damage by physiologically relevant doses (up to 15.4J/cm2) of solar UVR (95% UVA, 5% UVB). We show that: i) purified suspensions of UV-chromophore-rich fibronectin dimers, fibrillin microfibrils and β- and γ-lens crystallins undergo solar simulated radiation (SSR)-induced aggregation and/or decomposition and ii) exposure to identical doses of SSR has minimal effect on the size or ultrastructure of UV chromophore-poor tropoelastin, collagen I, collagen VI microfibrils and α-crystallin. If UV chromophore content is a factor in determining protein stability in vivo, we would expect that the tissue distribution of Cys, Trp and Tyr-rich proteins would correlate with regional UVR exposure. From bioinformatic analysis of 244 key structural proteins we identified several biochemically distinct, yet UV chromophore-rich, protein families. The majority of these putative UV-absorbing proteins (including the late cornified envelope proteins, keratin associated proteins, elastic fibre-associated components and β- and γ-crystallins) are localised and/or particularly abundant in tissues that are exposed to the highest doses of environmental UVR, specifically the stratum corneum, hair, papillary dermis and lens. We therefore propose that UV chromophore-rich proteins are localised in regions of high UVR exposure as a consequence of an evolutionary pressure to express sacrificial protein sunscreens which reduce UVR penetration and hence mitigate tissue damage.

Bivalent Ligation of the Collagen-binding Modules of Fibronectin by SFS, a Non-anchored Bacterial Protein of Streptococcus equi

SFS is a non-anchored protein of Streptococcus equi subspecies equi that causes upper respiratory infection in horses. SFS has been shown to bind to fibronectin (FN) and block interaction of FN with type I collagen. We have characterized interactions of a recombinant 60-mer polypeptide, R1R2, with FN. R1R2 contains two copies of collagen-like 19-residue repeats. Experiments utilizing various FN fragments and epitope-mapped anti-FN monoclonal antibodies located the binding site to (8-9)FNI modules of the gelatin-binding domain. Fluorescence polarization and competitive enzyme-linked assays demonstrated that R1R2 binds preferentially to compact dimeric FN rather than monomeric constructs containing (8-9)FNI or a large dimeric FN construct that is constitutively in an extended conformation. In contrast to bacterial peptides that bind (2-5)FNI in addition to (8-9)FNI, R1R2 did not cause conformational extension of FN as assessed by a conformationally sensitive antibody. Equilibrium and stopped-flow binding assays and size exclusion chromatography were compatible with a two-step binding reaction in which each of the repeats of R1R2 interacts with one of the subunits of dimeric FN, resulting in a stable complex with a slow koff. In addition to not binding to type I collagen, the R1R2·FN complex incorporated less efficiently into extracellular matrix than free FN. Thus, R1R2 binds to FN utilizing features of compact soluble FN and in doing so interferes with the organization of the extracellular matrix. A similar bivalent binding strategy may underlie the collagen-FN interaction.

The fate of internalized α5 integrin is regulated by matrix-capable fibronectin

BackgroundAssembly of fibronectin matrices is associated with integrin receptor turnover and is an important determinant of tissue remodeling. Although it is well established that fibronectin is the primary ligand for α5β1 receptor, the relationship between fibronectin matrix assembly and the fate of internalized α5 integrin remains poorly characterized. Materials and methodsTo evaluate the effect of fibronectin matrix on the fate of internalized α5 integrin, fibronectin-null Chinese hamster ovary and mouse embryo fibroblast cells were used to track the fate of α5 after exposure to exogenous fibronectin. ResultsIn the absence of matrix-capable fibronectin dimer, levels of internalized α5 decreased rapidly over time. This correlated with a decline in total cellular α5 and was associated with the ubiquitination of α5 integrin. In contrast, internalized and total cellular α5 protein levels were maintained when matrix-capable fibronectin was present in the extracellular space. Further, we show that ubiquitination and degradation of internalized α5 integrin in the absence of fibronectin require the presence of two specific lysine residues in the α5 cytoplasmic tail. ConclusionsOur data demonstrate that α5 integrin turnover is dependent on fibronectin matrix assembly, where the absence of matrix-capable fibronectin in the extracellular space targets the internalized receptor for rapid degradation. These findings have important implications for understanding tissue-remodeling processes found in wound repair and tumor invasion.

ECM protein nanofibers and nanostructures engineered using surface-initiated assembly.

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

Homocysteine Modifies Structural and Functional Properties of Fibronectin and Interferes with the Fibronectin–Fibrillin-1 Interaction

Homocystinuria is a genetic disorder resulting in elevated levels of homocysteine in plasma and tissues. Some of the skeletal and ocular symptoms such as long bone overgrowth, scoliosis, and ectopia lentis overlap with symptoms seen in Marfan syndrome. Marfan syndrome is caused by mutations in the extracellular matrix protein fibrillin-1. We previously showed that fibrillin-1 is a target for homocysteine and that the deposition of homocysteinylated fibrillin-1 in the extracellular matrix is compromised. Since the assembly of fibrillin-1 is critically dependent on fibronectin, we analyzed the consequences of fibronectin homocysteinylation and its interaction with fibrillin-1. Cellular fibronectin and proteolytic fragments were homocysteinylated and tested in various interaction assays with recombinant fibrillin-1 and heparin. Fibronectin homocysteinylation consistently compromised the fibronectin-fibrillin-1 interaction, while the interaction with heparin was not affected. Fibronectin homocysteinylation, but not cysteinylation, reduced the fibronectin dimers to monomers as shown by Western blotting. ELISA analyses of homocysteinylated fibronectin with three monoclonal antibodies demonstrated structural changes in the disulfide-containing FNI domains FNI(2), FNI(4), and FNI(9). Using fluorescently labeled fibronectin, we studied the consequence of fibronectin homocysteinylation on assembly in cell culture. Modified fibronectin showed deficiencies in denovo matrix incorporation and initial assembly. In conclusion, we define here characteristic structural changes of fibronectin upon homocysteinylation that translate into functional deficiencies in the fibronectin-fibrillin-1 interaction and in fibronectin assembly. Since fibronectin is a major organizer of various extracellular protein networks, these structural and functional alterations may contribute to the pathogenesis of homocystinuria and Marfan syndrome.

Fibronectins, Their Fibrillogenesis, and In Vivo Functions

Fibronectin (FN) is a multidomain protein with the ability to bind simultaneously to cell surface receptors, collagen, proteoglycans, and other FN molecules. Many of these domains and interactions are also involved in the assembly of FN dimers into a multimeric fibrillar matrix. When, where, and how FN binds to its various partners must be controlled and coordinated during fibrillogenesis. Steps in the process of FN fibrillogenesis including FN self-association, receptor activities, and intracellular pathways have been under intense investigation for years. In this review, the domain organization of FN including the extra domains and variable region that are controlled by alternative splicing are described. We discuss how FN-FN and cell-FN interactions play essential roles in the initiation and progression of matrix assembly using complementary results from cell culture and embryonic model systems that have enhanced our understanding of this process.

Assembly of Fibronectin Extracellular Matrix

In the process of matrix assembly, multivalent extracellular matrix (ECM) proteins are induced to self-associate and to interact with other ECM proteins to form fibrillar networks. Matrix assembly is usually initiated by ECM glycoproteins binding to cell surface receptors, such as fibronectin (FN) dimers binding to α5ß1 integrin. Receptor binding stimulates FN self-association mediated by the N-terminal assembly domain and organizes the actin cytoskeleton to promote cell contractility. FN conformational changes expose additional binding sites that participate in fibril formation and in conversion of fibrils into a stabilized, insoluble form. Once assembled, the FN matrix impacts tissue organization by contributing to the assembly of other ECM proteins. Here, we describe the major steps, molecular interactions, and cellular mechanisms involved in assembling FN dimers into fibrillar matrix while highlighting important issues and major questions that require further investigation.

Low‐dose ultraviolet radiation selectively degrades chromophore‐rich extracellular matrix components

Photoageing of human skin due to chronic exposure to ultraviolet radiation (UVR) is characterized histologically by extensive remodelling of the dermal elastic fibre system. Whilst enzymatic pathways are thought to play a major role in mediating extracellular matrix (ECM) degeneration in UV-exposed skin, the substrate specificity of UVR-up-regulated and activated matrix metalloproteinases (MMPs) is low. It is unclear, therefore, how such cell-mediated mechanisms alone could be responsible for the reported selective degradation of elastic fibre components such as fibrillin-1 and fibulin-5 during the early stages of photoageing. Here we use atomic force microscopy (AFM) and scanning transmission electron microscopy (STEM) to demonstrate that physiologically attainable doses (20-100 mJ/cm(2)) of direct UV-B radiation can induce profound, dose-dependent, changes in the structure of, and mass distribution within, isolated fibrillin microfibrils. Furthermore, using reducing and native PAGE in combination with AFM, we show that, whilst exposure to low-dose UV-B radiation significantly alters the macromolecular and quaternary structures of both UV chromophore (Cys, His, Phe, Trp and Tyr)-rich fibrillin microfibrils (fibrillin-1, 21.0%) and fibronectin dimers (fibronectin, 12.9%), similar doses have no detectable effect on UV chromophore-poor type I collagen monomers (2.2%). Analysis of the published primary amino acid sequences of 49 dermal ECM components demonstrates that most elastic fibre-associated proteins, but crucially neither elastin nor members of the collagen family, are rich in UV chromophores. We suggest, therefore, that the amino acid composition of elastic fibre-associated proteins [including the fibrillins, fibulins, latent TGFbeta binding proteins (LTBPs) and the lysyl oxidase family of enzymes (LOK/LOXLs)] may predispose them to direct degradation by UVR. As a consequence, this selective acellular photochemical pathway may play an important role in initiating and/or exacerbating cell-mediated ECM remodelling in UVR-exposed skin.

Revisiting the mystery of fibronectin multimers: The fibronectin matrix is composed of fibronectin dimers cross-linked by non-covalent bonds

Fibronectin (FN) matrix fibrils have long been thought to be formed by disulfide-bonded FN multimers, although there is no direct evidence that they are covalently linked with each other. To understand the biochemical properties of these fibrils, we extracted a crude FN matrix from FN-YPet transfected 3T3 cell culture using 0.2% deoxycholate and DNase. The insoluble extracted matrix preserved fibrillar structures and a major portion of the extracted proteins migrated as FN monomers on an SDS gel under reducing conditions. Under non-reducing conditions, some FN molecules appeared to be trapped at the top of the stacking gel. We tested this by mixing fluorescently labeled FN dimers with the extracted matrix just before loading on an SDS gel, and found that most of them were trapped with the extracted proteins at the top of the stacking gel. These results suggested that some components of the extracted matrix plugged the stacking gel and FN dimers were trapped with them. Rotary shadowing electron microscopy showed that the extracted matrix had some fibers that resembled fibrillin microfibrils. Peptide mass fingerprinting confirmed the presence of fibrillin in the extracted matrix. Fibrillin is known to form disulfide-bonded multimers and it is likely to be one of the components that plug the stacking gel and trap FN molecules in this system. The phenomenon by which FN molecules appear to migrate as multimers on SDS gels is thus an artifact rising from the presence of other large components in the extract. We conclude that FN matrix fibrils are made of FN dimers that are further cross-linked by non-covalent protein–protein bonds.

The RGD motif in fibronectin is essential for development but dispensable for fibril assembly

Fibronectin (FN) is secreted as a disulfide-bonded FN dimer. Each subunit contains three types of repeating modules: FN-I, FN-II, and FN-III. The interactions of α5β1 or αv integrins with the RGD motif of FN-III repeat 10 (FN-III10) are considered an essential step in the assembly of FN fibrils. To test this hypothesis in vivo, we replaced the RGD motif with the inactive RGE in mice. FN-RGE homozygous embryos die at embryonic day 10 with shortened posterior trunk, absent tail bud–derived somites, and severe vascular defects resembling the phenotype of α5 integrin–deficient mice. Surprisingly, the absence of a functional RGD motif in FN did not compromise assembly of an FN matrix in mutant embryos or on mutant cells. Matrix assembly assays and solid-phase binding assays reveal that αvβ3 integrin assembles FN-RGE by binding an isoDGR motif in FN-I5, which is generated by the nonenzymatic rearrangement of asparagines (N) into an iso-aspartate (iso-D). Our findings demonstrate that FN contains a novel motif for integrin binding and fibril formation whose activity is controlled by amino acid modification.

The heparin III-binding domain of fibronectin (III4–5 repeats) binds to fibronectin and inhibits fibronectin matrix assembly

Fibronectin matrix assembly involves interactions among various regions of the molecule, which contribute to elongation and stabilization of the fibrils. In this study, we examined the possible role of the heparin III domain of fibronectin (repeats III4–5) in fibronectin fibrillogenesis. We show that a recombinant fragment comprising these repeats (FNIII4–5 fragment) blocked fibronectin fibril formation and the incorporation of 125I-fibronectin into cell layers. Binding assays using a biosensor revealed that FNIII4–5 bound fibronectin and the amino-terminal 70 kDa and 29 kDa fragments. It also bound to itself, indicating a previously unidentified self-association site in repeats III4–5. These interactions were specific since FNIII4–5 did not bind to the FNIII7–10 fragment, representing a central region in fibronectin. The fibronectin-binding property of the III4–5 domain, but not its matrix assembly inhibitory function, was apparently cryptic in larger fragments. By mutating the arginine residues in the WTPPRAQITGYRLTVGLTRR proteoglycan-binding sequence (HBP/III5 site) of FNIII4–5 [Moyano, J.V., Carnemolla, B., Albar, J.P., Leprini, A., Gaggero, B., Zardi, L., Garcia-Pardo, A., 1999. Cooperative role for activated α4β1 integrin and chondroitin sulfate proteoglycans in cell adhesion to the heparin III domain of fibronectin. Identification of a novel heparin and cell binding sequence in repeat III5. J. Biol. Chem. 274, 135–142.], we found that the first two arginine residues in HBP/III5 were involved in the fibronectin-binding property of FNIII4–5, while the last two arginine residues in HBP/III5 were required for inhibition of matrix assembly and the binding of 125I-fibronectin to cell layers. Both properties appear to function independently from each other, depending on the conformation of the fibronectin dimer.

High Fidelity Functional Patterns of an Extracellular Matrix Protein by Electron Beam-Based Inactivation

Controlling the organization of proteins on surfaces provides a powerful biochemical tool for determining how cells interpret the spatial distribution of local signaling molecules. Here, we describe a general high fidelity approach based on electron beam writing to pattern the functional properties of protein-coated surfaces at length scales ranging from tens of nanometers to millimeters. A silicon substrate is first coated with the extracellular matrix protein fibronectin, which is then locally inactivated by exposure to a highly focused electron beam. Biochemical inactivation of the protein is established by the loss of antibody binding to the fibronectin. Functional inactivation is determined by the inability of cells to spread or form focal adhesions on the inactivated substrate, resulting in cell shapes constrained to the pattern, while they do both (and are unconstrained) on the remaining fibronectin. These protein patterns have very high fidelity, and typical patterns agree with the input dimensions of the pattern to within 2%. Further, the feature edges are well defined and approach molecular dimensions in roughness. Inactivation is shown to be dose dependent with observable suppression of the specific binding at 2 microC cm(-2) and complete removal of biochemical activity at approximately 50 microC cm(-2) for 5 keV electrons. The critical dose for inactivation also depends on accelerating voltage, and complete loss of antibody binding was achieved at approximately 4-7 microC cm(-2) for 1 keV electrons, which corresponds to approximately 50-90 electrons per cross-sectional area of a whole fibronectin dimer and ~2-4 electrons per type III fibronectin domain. AFM analysis of the pattern surfaces revealed that electron beam exposure does not remove appreciable amounts of material from the surface, suggesting that the patterning mechanism involves local inactivation rather than the ablation that has been observed in several organic thin film systems.

Characterization of fibronectin assembly by platelets adherent to adsorbed laminin-111.

Various types of laminin (LN) are ubiquitous components of basement membrane and exposed to blood upon localized damage of vascular endothelial cells. Fibronectin is a plasma protein that is insolubilized into fibrils in a regulated fashion by, for example, lysophosphatidic acid (LPA)-stimulated fibroblasts or platelets spread on supportive adhesive ligands. To study assembly of plasma fibronectin by LPA-activated platelets adherent to LN-111 via alpha6beta1 integrin. Platelets adherent to LN-111-bound plasma fibronectin or its N-terminal 70 kD fragment in fibrillar arrays at the periphery of spread platelets under static but not shear conditions. Formation of fibronectin arrays under static conditions was inhibited by co-incubation with the N-terminal 70 kD fragment or with a 49-amino acid peptide that binds to the N-terminal region of fibronectin. Approximately 7000 fibronectin dimers bound per adherent platelet with a K(d) of 50 nm. Bound 70 kD fragment was readily solubilized with deoxycholate (DOC), whereas bound fibronectin became progressively insoluble. Bound 70 kD fragment became resistant to DOC extraction after treatment with a cell-impermeable, reducible crosslinker. Crosslinked 70 kD fragment was found in a high molecular weight complex. As with fibroblasts, signaling molecules modulating actin cytoskeletal organization controlled expression of binding sites for the N-terminal 70 kD region of fibronectin on adherent platelets. These results indicate that platelets adherent to LN-111 via alpha(6)beta(1) support subsequent assembly of fibronectin, but possibly only under conditions of intermittent or stagnant blood flow.

The ins and outs of fibronectin matrix assembly.

Cell phenotype is specified by environmental cues embedded in the architecture and composition of the extracellular matrix (ECM). Much has been learned about matrix organization and assembly through analyses of the ECM protein fibronectin (FN). FN matrix assembly is a cell-mediated process in which soluble dimeric FN is converted into a fibrillar network. Binding of cell surface integrin receptors to FN converts it to an active form, which promotes fibril formation through interactions with other cell-associated FN dimers. As FN fibrils form on the outside of the cell, cytoplasmic domains of integrin receptors organize cytoplasmic proteins into functional complexes inside. Intracellular connections to the actin cytoskeletal network and stimulation of certain key intracellular signaling pathways are essential for FN-integrin interactions and propagation of FN fibril formation. Thus, assembly of native functional ECM depends on exquisite coordination between extracellular events and intracellular pathways.

Alpha5beta1 integrin mediates strong tissue cohesion.

Integrins and cadherins are considered to have distinct and opposing functions. Integrins are traditionally cited for their role in cell-substratum interactions, whereas cadherins are thought to mediate strong intercellular cohesion. Together, these adhesion systems play crucial roles in a wide variety of cellular and developmental processes including cell migration, morphology, differentiation and proliferation. In this manuscript we present evidence that integrins possess the ability to mediate strong intercellular cohesion when cells are grown as 3D aggregates. Much of the data elucidating the role of integrins as mediators of cell-extracellular matrix (ECM) interactions have been generated using conventional cell culture techniques in which cells are plated onto ECM-coated 2D surfaces. In vivo, cells are embedded in a 3D meshwork of ECM proteins. We hypothesized that, within this meshwork, integrin-ECM interactions may impart cohesivity to an aggregate of cells by linking adjacent cells together. To test this hypothesis, we transfected Chinese hamster ovary (CHO-B2) cells to express alpha5beta1 integrin and found that these cells formed compact, spherical aggregates. We measured aggregate cohesivity using tissue surface tensiometry, a novel technique that quantifies cell-cell cohesivity of spheroids under physiological conditions. We determined that alpha5beta1 integrin is capable of conferring strong cohesivity (sigma=8.22+/-0.68 dynes/cm) to aggregates of alpha5-integrin-transfected cells. This cohesion was found to be independent of cadherin expression and was significantly greater than the cohesivity conferred onto CHO-B2 cells transfected with N-cadherin (sigma=3.14+/-0.20 dynes/cm, P</=0.0001), a more traditional cell-cell cohesion system. Fibronectin-null CHO cells that express alpha5beta1 integrin but do not secrete endogenous fibronectin do not form aggregates in fibronectin-depleted medium. Addition of increasing amounts of exogenous dimeric fibronectin to these cells resulted in a dose-dependent compaction. However, compaction failed to occur in the presence of fibronectin monomers. These data indicate that fibronectin is required for alpha5beta1-mediated compaction and that the dimeric structure of fibronectin is essential for this process. Additionally, aggregate formation of the alpha5 integrin transfectants was inhibited by an RGD peptide thus confirming alpha5beta1 integrin specificity. Collectively, these data confirm our hypothesis that alpha5beta1 integrin acts through fibronectin to link adjacent cells together, thus promoting strong intercellular cohesion in 3D cellular aggregates.

Matrix Fibronectin Increases HIV Stability and Infectivity

HIV particles are detected extracellularly in lymphoid tissues, a major reservoir of the virus. We previously reported that a polymerized form of fibronectin (FN), superfibronectin (sFN), as well as a fragment of FN, III1-C, enhanced infection of primary CD4(+) T cells by HIV-1IIIB. We now show that sFN enhances infection of primary CD4(+) T cells by both R5 and X4 strains of HIV-1. Using HIV pseudotyped with different envelope glycoproteins (gp120) and HOS cells transfected with various chemokine receptors alone or in combination with the CD4 molecule, we show that sFN-mediated enhancement requires the CD4 receptor and does not alter the specificity of gp120 for different chemokine receptors. Because the III1-C fragment also resulted in enhancement, we asked whether proteolysis of FN generated fragments capable of enhancing HIV infection. We found that progressive proteolysis of FN by chymotrypsin correlates with an enhancement of HIV infection in both primary CD4(+) T cells and the IG5 reporter cell line. Furthermore, incubation of HIV with sFN significantly prolonged infectivity at 37 degrees C compared with dimeric FN or BSA. In conclusion, these results indicate that polymerized (matrix) or degraded (inflammation-associated), but not dimeric (plasma), FN are capable of enhancing infection by HIV-1, independent of the coreceptor specificity of the strains. Moreover, virions bound to matrix FN maintain infectivity for longer periods of time than do virions in suspension. This study suggests that matrix proteins and their conformational status may play a role in the pathogenesis of HIV.