A combinatorial approach for directing the amount of fibronectin fibrils assembled by cells that uses surfaces derivatized with mixtures of fibronectin and cell binding domains

Fibrillar fibronectin (FN) has the crucial role of attracting and attaching cells as well as molecules that mediate tissue repair during wound healing. A previous study demonstrated higher extracellular staining of FN fibrils in cells cultured on surfaces tethered with an equimolar mixture of a FN binding domain and FN's cell binding domain, III1-2 and III9-10 respectively, than on surfaces with III9-10 alone. The effect of varying surface amounts of III1-2 and III9-10 on the quantity of FN fibrils formed by NIH-3T3 fibroblasts was examined. GST tagged III1-2 and III9-10 were conjugated to polyurethane surfaces and ELISAs were used to identify the experimental design space or the range of concentrations of GST-III1-2 and GST-III9-10 that demarcated the limits of protein loading on the surface. When GST-III1-2 was fixed and GST-III9-10 varied within the design space, the amount of FN fibrils measured by immunoblotting detergent insoluble cell lysates was dependent on the ratio of III9-10 to III1-2. When the total protein concentration was fixed and the mixture composition of GST-III1-2 and GST-III9-10 varied such that it optimally covered the design space, a parabolic relationship between FN fibril amount and the ratio of III9-10 to III1-2 was obtained. This relationship had a maximum value when the surface was bonded to equal amounts of III1-2 and III9-10 (P<0.05). Thus the ratio of III9-10 to III1-2 can be utilized to direct the quantity of FN fibrils formed on surfaces.     

Fibronectin regulates assembly of actin filaments and focal contacts in cultured cells via the heparin-binding site in repeat III13

Fibroblasts, when plated on the extracellular matrix protein fibronectin (FN), rapidly spread and form an organized actin cytoskeleton. This process is known to involve both the central alpha5beta1 integrin-binding and the C-terminal heparin-binding regions of FN. We found that within the heparin-binding region, the information necessary for inducing organization of stress fibers and focal contacts was located in a 29-amino acid segment of FN type III module 13 (III13). We did not find a cytoskeleton-organizing role for repeat III14, which had previously been implicated in this process. Within III13, the same five basic amino acids known to be most important for heparin binding were also necessary for actin organization. A substrate of III13 alone was only weakly adhesive but strongly induced formation of filopodia and lamellipodia. Stress fiber formation required a combination of III13 and III7-11 (which contains the integrin alpha5beta1 recognition site), either as a single fusion protein or as separate polypeptides, and the relative amounts of the two binding sites appeared to determine whether stress fibers or filopodia and lamellipodia were the predominant actin structures formed. We propose that a balance of signals from III13 and from integrins regulates the type of actin structures assembled by the cell.     

Identification of a novel heparin-binding site in the alternatively spliced IIICS region of fibronectin: roles of integrins and proteoglycans in cell adhesion to fibronectin splice variants.

The extracellular matrix molecule fibronectin (FN) is a glycoprotein whose major functional property is to support cell adhesion. FN contains at least two distinct cell-binding domains: the central cell-binding domain and the HepII/IIICS region. The HepII region comprises type III repeats 12-14 and contains proteoglycan-binding sites, while the alternatively spliced IIICS segment possesses the major alpha4beta1 integrin-binding sites. Both cell surface proteoglycans and integrins are important for mediating the adhesion of cells to this region of FN. By comparing heparin binding to different recombinant splice variants of the HepII/IIICS region, evidence was obtained for the existence of a novel heparin-binding site in the centre of the IIICS. Site-directed mutagenesis of basic amino acid sequences in this region reduced heparin binding to recombinant HepII/IIICS proteins and, in conjunction with mutations in the HepII region, caused a synergistic loss of activity. Using the H/120 variant of FN, which contains type III repeats 12-15 and the full-length IIICS region, and the H/95 variant of FN, which contains type III repeats 12-15 but lacks the high affinity integrin-binding LDV sequence, the relative roles played by cell-surface proteoglycans and integrins in mediating cell adhesion have been investigated. This was achieved by studying the effects of anti-integrin antibodies and exogenous heparin on A375 melanoma cell attachment to the wild-type and three different mutants of H/120 and H/95 in which the potential proteoglycan-binding sites were partially or completely removed. A375 cell adhesion to H/120 and its mutants was found to involve the co-operative action of both integrin and cell-surface proteoglycan binding, although integrin made a dominant contribution. Anti-integrin antibodies and exogenous heparin were capable of inhibiting melanoma cell adhesion to H/95 and in this case adhesion was due primarily to cell-surface proteoglycan and not integrin binding.     

A novel fibronectin binding site required for fibronectin fibril growth during matrix assembly

Fibronectin (FN) assembly into a fibrillar extracellular matrix is a stepwise process requiring participation from multiple FN domains. Fibril formation is regulated in part by segments within the first seven type III repeats (III1-7). To define the specific function(s) of this region, recombinant FNs (recFNs) containing an overlapping set of deletions were tested for the ability to assemble into fibrils. Surprisingly, recFN lacking type III repeat III1 (FNDeltaIII1), which contains a cryptic FN binding site and has been suggested to be essential for fibril assembly, formed a matrix identical in all respects to a native FN matrix. Similarly, displacement of the cell binding domain in repeats III9-10 to a position close to the NH2-terminal assembly domain, as well as a large deletion spanning repeats III4-7, had no effect on assembly. In contrast, two deletions that included repeat III2, DeltaIII1-2 and DeltaIII2-5, caused significant reductions in fibril elongation, although binding of FN to the cell surface and initiation of assembly still proceeded. Using individual repeats in binding assays, we show that III2 but not III1 contains an FN binding site. Thus, these results pinpoint repeat III2 as an important module for FN-FN interactions during fibril growth.     

Modulation of Cell-adhesive Activity of Fibronectin by the Alternatively Spliced EDA Segment

Fibronectin (FN) has a complex pattern of alternative splicing at the mRNA level. One of the alternatively spliced segments, EDA, is prominently expressed during biological processes involving substantial cell migration and proliferation, such as embryonic development, malignant transformation, and wound healing. To examine the function of the EDA segment, we overexpressed recombinant FN isoforms with or without EDA in CHO cells and compared their cell-adhesive activities using purified proteins. EDA⁺ FN was significantly more potent than EDA⁻ FN in promoting cell spreading and cell migration, irrespective of the presence or absence of a second alternatively spliced segment, EDB. The cell spreading activity of EDA⁺ FN was not affected by antibodies recognizing the EDA segment but was abolished by antibodies against integrin α5 and β1 subunits and by Gly-Arg-Gly-Asp-Ser-Pro peptide, indicating that the EDA segment enhanced the cell-adhesive activity of FN by potentiating the interaction of FN with integrin α5β1. In support of this conclusion, purified integrin α5β1 bound more avidly to EDA⁺ FN than to EDA⁻ FN. Augmentation of integrin binding by the EDA segment was, however, observed only in the context of the intact FN molecule, since the difference in integrin-binding activity between EDA⁺ FN and EDA⁻ FN was abolished after limited proteolysis with thermolysin. Consistent with this observation, binding of integrin α5β1 to a recombinant FN fragment, consisting of the central cell-binding domain and the adjacent heparin-binding domain Hep2, was not affected by insertion of the EDA segment. Since the insertion of an extra type III module such as EDA into an array of repeated type III modules is expected to rotate the polypeptide up to 180° at the position of the insertion, the conformation of the FN molecule may be globally altered upon insertion of the EDA segment, resulting in an increased exposure of the RGD motif in III₁₀ module and/or local unfolding of the module. Our results suggest that alternative splicing at the EDA exon is a novel mechanism for up-regulating integrin-binding affinity of FN operating when enhanced migration and proliferation of cells are required.     

Crystal structure of a heparin- and integrin-binding segment of human fibronectin

The crystal structure of human fibronectin (FN) type III repeats 12-14 reveals the primary heparin-binding site, a clump of positively charged residues in FN13, and a putative minor site approximately 60 A away in FN14. The IDAPS motif implicated in integrin alpha4beta1 binding is at the FN13-14 junction, rendering the critical Asp184 inaccessible to integrin. Asp184 clamps the BC loop of FN14, whose sequence (PRARI) is reminiscent of the synergy sequence (PHSRN) of FN9. Mutagenesis studies prompted by this observation reveal that both arginines of the PRARI sequence are important for alpha4beta1 binding to FN12-14. The PRARI motif may represent a new class of integrin-binding sites. The spatial organization of the binding sites suggests that heparin and integrin may bind in concert.     

Adhesion to fibronectin stimulates inositol lipid synthesis and enhances PDGF-induced inositol lipid breakdown

The aim of these experiments was to investigate whether inositol lipids might mediate some of the effects of extracellular matrix (ECM) on cellular form and functions. The lipid phosphatidylinositol bisphosphate (PIP2) plays a role in cytoskeletal regulation while its hydrolysis products, diacylglycerol and inositol triphosphate, serve as second messengers. We therefore measured the effect of adhesion to fibronectin (FN) on PIP2 and its hydrolysis products, in the presence and absence of the soluble mitogen PDGF. PDGF induced a threefold increase in release of water-soluble inositol phosphates in C3H 10T1/2 fibroblasts when cells were attached to FN, but had little effect in suspended cells. Suppression of inositol phosphate release in unattached cells was not due to dysfunction of the PDGF receptor or failure to activate phospholipase C-gamma; PDGF induced similar tyrosine phosphorylation of PLC-gamma under both conditions. By contrast, the total mass of phosphatidylinositol bisphosphate (PIP2), the substrate for PLC-gamma, was found to decrease by approximately 80% when cells were detached from their ECM attachments and placed in suspension in the absence of PDGF. PIP2 levels were restored when suspended cells were replated on FN, demonstrating that the effect was reversible. Furthermore, a dramatic increase in synthesis of PIP2 could be measured in cells within 2 min after reattachment to FN in the absence of PDGF. These results show that FN acts directly to stimulate PIP2 synthesis, and that it also enhances PIP2 hydrolysis in response to PDGF. The increase in PIP2 induced by adhesion may mediate some of the known effects of FN on cell shape and cytoskeletal organization, while regulation of inositol lipid hydrolysis may provide a means for integrating hormone- and ECM-dependent signaling pathways.     

Fibronectin fragmentation promotes alpha4beta1 integrin-mediated contraction of a fibrin-fibronectin provisional matrix

In injured tissues, the fibrin-fibronectin (FN) provisional matrix provides a framework for cell adhesion, migration, and repair. Effective repair and remodeling require a proper balance between extracellular matrix (ECM) deposition, contraction, and turnover. We utilized a three-dimensional (3D) fibrin-FN provisional matrix model to determine the contributions of the FN-binding integrin receptors alpha5beta1 and alpha4beta1 to matrix contraction. CHOalpha5 cells expressing alpha5beta1, a receptor for FN's RGD cell-binding domain, were highly contractile, and cells were well spread on a 3D fibrin-FN matrix. In contrast, CHOalpha4 cells expressing the alpha4beta1 receptor for FN's alternatively spliced V region attached less efficiently to FN and were deficient in fibrin-FN matrix contraction. Surprisingly, cell adhesion and matrix contraction by CHOalpha4 cells were dramatically enhanced, to levels equivalent to CHOalpha5 cells, when proteolyzed FN was used in place of intact FN in the fibrin-FN matrix. Similar enhancement was observed when ligand binding by alpha4beta1 integrins was activated by treatment with Mn(++), but not by stimulation of actin organization with LPA. Therefore, alpha4beta1-dependent cell responses to the provisional matrix are modulated by cleavage of matrix components.     

Ligation of the fibrin-binding domain by β-strand addition is sufficient for expansion of soluble fibronectin

How fibronectin (FN) converts from a compact plasma protein to a fibrillar component of extracellular matrix is not understood. "Functional upstream domain" (FUD), a polypeptide based on F1 adhesin of Streptococcus pyogenes, binds by anti-parallel β-strand addition to discontinuous sets of N-terminal FN type I modules, (2-5)FNI of the fibrin-binding domain and (8-9)FNI of the gelatin-binding domain. Such binding blocks assembly of FN. To learn whether ligation of (2-5)FNI, (8-9)FNI, or the two sets in combination is important for inhibition, we tested "high affinity downstream domain" (HADD), which binds by β-strand addition to the continuous set of FNI modules, (1-5)FNI, comprising the fibrin-binding domain. HADD and FUD were similarly active in blocking fibronectin assembly. Binding of HADD or FUD to soluble plasma FN exposed the epitope to monoclonal antibody mAbIII-10 in the tenth FN type III module ((10)FNIII) and caused expansion of FN as assessed by dynamic light scattering. Soluble N-terminal constructs truncated after (9)FNI or (3)FNIII competed better than soluble FN for binding of FUD or HADD to adsorbed FN, indicating that interactions involving type III modules more C-terminal than (3)FNIII limit β-strand addition to (1-5)FNI within intact soluble FN. Preincubation of FN with mAbIII-10 or heparin modestly increased binding to HADD or FUD. Thus, ligation of FNIII modules involved in binding of integrins and glycosaminoglycans, (10)FNIII and (12-14)FNIII, increases accessibility of (1-5)FNI. Allosteric loss of constraining interactions among (1-5)FNI, (10)FNIII, and (12-14)FNIII likely enables assembly of FN into extracellular fibrils.     

Coordinated Regulation of Fibronectin Fibril Assembly and Actin Stress Fiber Formation

Assembly of a fibronectin (FN) matrix is a multistep process which influences a number of cellular functions including intracellular cytoskeletal organization and signaling responses. We have previously reported on a recombinant FN (recFN), FNΔIII_1–7, which differs from native FN in its rate of fibril formation. To determine the intracellular consequences of a delay in assembly, we compared the distribution of cytoskeletal proteins during the formation of native and recFN matrices by immunofluorescence at various time points. CHOα5 cell cytoskeleton was reorganized in response to both native and recFN matrix formation. Assembly of native FN induced a rapid reorganization of actin into stress fibers and colocalization of α5131 integrin, focal adhesion kinase (FAK), vinculin, and paxillin to regions of cell-matrix contact. α5β1 integrins and FAK are also clustered upon binding of FNΔIII_1–7 to cells but actin reorganization and focal adhesion formation are delayed and appear to be dependent on the formation of FNΔIII_1–7 fibrils. These results suggest that the structural framework of the matrix plays an important role in the ability of FN to initiate intracellular responses.     

Beta 1 integrin-mediated effects of tenascin-R domains EGFL and FN6-8 on neural stem/progenitor cell proliferation and differentiation in vitro

Neural stem/progenitor cells (NSCs) have the capacity for self-renewal and differentiation into major classes of central nervous system cell types, such as neurons, astrocytes, and oligodendrocytes. The determination of fate of NSCs appears to be regulated by both intrinsic and extrinsic factors. Mounting evidence has shown that extracellular matrix molecules contribute to NSC proliferation and differentiation as extrinsic factors. Here we explore the effects of the epidermal growth factor-like (EGFL) and fibronectin type III homologous domains 6-8 (FN6-8) of the extracellular matrix molecule tenascin-R on NSC proliferation and differentiation. Our results show that domain FN6-8 inhibited NSC proliferation and promoted NSCs differentiation into astrocytes and less into oligodendrocytes or neurons. The EGFL domain did not affect NSC proliferation, but promoted NSC differentiation into neurons and reduced NSC differentiation into astrocytes and oligodendrocytes. Treatment of NSCs with beta 1 integrin function-blocking antibody resulted in attenuation of inhibition of the effect of FN6-8 on NSC proliferation. The influence of EGFL or FN6-8 on NSCs differentiation was inhibited by beta 1 integrin antibody application, implicating beta 1 integrin in proliferation and differentiation induced by EGFL and FN6-8 mediated triggering of NSCs.     

Recombinant expression and characterization of a novel fibronectin isoform expressed in cartilaginous tissues

A novel fibronectin (FN) isoform lacking the segment from IIICS (type III connecting segment) through the I-10 module is expressed predominantly in normal cartilaginous tissues. We expressed and purified recombinant cartilage-type FN using a mammalian expression system and characterized its molecular and biological properties. Although FNs have been shown to be secreted as disulfide-bonded dimers, cartilage-type FN was secreted mainly as a monomer. It was less potent than plasma-type FN in promoting cell adhesion and binding to integrin alpha5beta1, although it was more active than plasma-type FN in binding to chondroitin sulfate E. When added exogenously, cartilage-type FN was poorly assembled into the fibrillar FN matrix, mostly because of its monomeric structure. Given that cartilage is characterized by its non-fibrillar matrix with abundant chondroitin sulfate-containing proteoglycans, it is likely that cartilage-type FN has evolved to adapt itself to the non-fibrillar structure of the cartilage matrix through acquisition of a novel mechanism of alternative pre-mRNA splicing.     

Altered rate of fibronectin matrix assembly by deletion of the first type III repeats

The assembly of fibronectin (FN) into a fibrillar matrix is a complex stepwise process that involves binding to integrin receptors as well as interactions between FN molecules. To follow the progression of matrix formation and determine the stages during which specific domains function, we have developed cell lines that lack an endogenous FN matrix but will form fibrils when provided with exogenous FN. Recombinant FNs (recFN) containing deletions of either the RGD cell- binding sequence (RGD-) or the first type III repeats (FN delta III1-7) including the III1 FN binding site were generated with the baculovirus insect cell expression system. After addition to cells, recFN matrix assembly was monitored by indirect immunofluorescence and by insolubility in the detergent deoxycholate (DOC). In the absence of any native FN, FN delta III1-7 was assembled into fibrils and was converted into DOC-insoluble matrix. This process could be inhibited by the amino- terminal 70 kD fragment of FN, showing that FN delta III1-7 follows an assembly pathway similar to FN. The progression of FN delta III1-7 assembly differed from native FN in that the recFN became DOC-insoluble more quickly. In contrast, RGD- recFNs were not formed into fibrils except when added in combination with native FN. These results show that the RGD sequence is essential for the initiation step but fibrils can form independently of the III1-7 modules. The altered rate of FN delta III1-7 assembly suggests that one function of the missing repeats might be to modulate an early stage of matrix formation.     

The fibronectin receptor is organized by extracellular matrix fibronectin: implications for oncogenic transformation and for cell recognition of fibronectin matrices

Cells interact with extracellular fibronectin (FN) via adhesive fibronectin receptors (FNRs) that are members of the very late antigens (VLAs) subgroup of the integrin family. In stationary fibroblasts, the FNR is highly organized and distributed identically to extracellular FN fibrils. However, in highly migratory neural crest cells and embryonic somatic fibroblasts, this organization is lost and the FNR appears diffuse. Similarly, oncogenic transformation typically leads to disorganization of the FN receptor and loss of matrix FN. Two models can account for these observations. First, the FN matrix may organize the FN receptor at extracellular matrix contacts on the cell surface. Motile cells not depositing FN matrices thus lack organized receptors. Alternatively, as the FNR is required for optimal FN matrix assembly, (McDonald, J. A., B. J. Quade, T. J. Broekelmann, R. LaChance, K. Forseman, K. Hasegawa, and S. Akiyama. 1987. J. Biol. Chem. 272:2957-2967; Roman, J. R. M. LaChance, T. J. Broekelmann, C. J. R. Kennedy, E. A. Wayner, W. G. Carter, J. A. McDonald. 1989. J. Cell Biol. 108:2529-2543) and has putative cytoskeletal links, it could be organized from within the cell helping to position newly forming FN fibrils. To study this question, we developed peptide antibodies specifically recognizing the alpha 5 subunit of the FNR. Using these antibodies, we examined the organization of FN and of the FNR in normal, matrix assembly inhibited, and SV40-transformed human fibroblasts. On FN-coated substrates, the FNR is found in focal contacts rather than diffusely on the basal cell surface, suggesting FNR interaction with intracellular components. However, when FN fibrils are deposited, the FNR is co-distributed with these fibrils. Preventing FN matrix assembly prevents organization of the FNR. Moreover, when fibroblasts with well established FN matrices and co-distributed FNR are incubated briefly with monoclonal antibodies that block FNR binding to FN, the FNR is no longer co-distributed with the FN matrix. Thus, the FN receptor is organized in fibrils on the cell surface in response to extracellular FN. Because exogenous FN restores a FN matrix and receptor organization to SV40-transformed cells, the diffuse FN receptor phenotype appears to be related to loss of the FN matrix rather than to impaired FNR function. These results explain diffusely distributed FNRs in migratory neural crest and embryonic fibroblasts lacking well organized FN matrices and emphasize the existence of separate but related systems controlling FN deposition and recognition by receptor-armed cells.     

Lysophosphatidic acid and microtubule-destabilizing agents stimulate fibronectin matrix assembly through Rho-dependent actin stress fiber formation and cell contraction.

Fibronectin (FN) matrix assembly is a cell-dependent process mediated by cell surface-binding sites for the 70-kDa amino-terminal region of FN. We have shown recently that lysophosphatidic acid (LPA) is a stimulator of FN matrix assembly. Disruption of microtubules has been shown to mimic some of the intracellular effects of LPA including the formation of actin stress fibers and myosin light chain phosphorylation. We compared the effects of microtubule disruption and LPA on FN binding and actin cytoskeleton organization. The disruption of microtubules by nocodazole or vinblastine increased FN binding to adherent cells. The modulation of binding sites was rapid, dynamic, and reversible. Enhanced binding was due to increases in both the number and affinity of binding sites. These effects are similar to the effects of LPA on FN binding. Binding induced by nocodazole was inhibited by the microtubule-stabilizing agent Taxol but not by pretreatment with a concentration of phospholipase B that totally abolished the stimulatory effect of LPA. Fluorescence microscopy revealed a close correlation among actin stress fiber formation, cell contraction, and FN binding. Blockage of the small GTP binding protein Rho or actin-myosin interactions inhibited the effects of both nocodazole and LPA on FN binding. These observations demonstrate that Rho-dependent actin stress fiber formation and cell contraction induce increased FN binding and represent a rapid labile way that cells can modulate FN matrix assembly.     

Tenascin-C suppresses Rho activation

Cell binding to extracellular matrix (ECM) components changes cytoskeletal organization by the activation of Rho family GTPases. Tenascin-C, a developmentally regulated matrix protein, modulates cellular responses to other matrix proteins, such as fibronectin (FN). Here, we report that tenascin-C markedly altered cell phenotype on a three-dimensional fibrin matrix containing FN, resulting in suppression of actin stress fibers and induction of actin-rich filopodia. This distinct morphology was associated with complete suppression of the activation of RhoA, a small GTPase that induces actin stress fiber formation. Enforced activation of RhoA circumvented the effects of tenascin. Effects of active Rho were reversed by a Rho inhibitor C3 transferase. Suppression of GTPase activation allows tenascin-C expression to act as a regulatory switch to reverse the effects of adhesive proteins on Rho function. This represents a novel paradigm for the regulation of cytoskeletal organization by ECM.     

SLLISWD Sequence in the 10FNIII Domain Initiates Fibronectin Fibrillogenesis

Fibronectin (FN) assembly into extracellular matrix is tightly regulated and essential to embryogenesis and wound healing. FN fibrillogenesis is initiated by cytoskeleton-derived tensional forces transmitted across transmembrane integrins onto RGD binding sequences within the tenth FN type III (10FNIII) domains. These forces unfold 10FNIII to expose cryptic FN assembly sites; however, a specific sequence has not been identified in 10FNIII. Our past steered molecular dynamics simulations modeling 10FNIII unfolding by force at its RGD loop predicted a mechanical intermediate with a solvent-exposed N terminus spanning the A and B β-strands. Here, we experimentally confirm that the predicted 23-residue cryptic peptide 1 (CP1) initiates FN multimerization, which is mediated by interactions with 10FNIII that expose hydrophobic surfaces that support 8-anilino-1-napthalenesulfonic acid binding. Localization of multimerization activity to the C terminus led to the discovery of a minimal 7-amino acid “multimerization sequence” (SLLISWD), which induces polymerization of FN and the clotting protein fibrinogen in addition to enhancing FN fibrillogenesis in fibroblasts. A point mutation at Trp-6 that reduces exposure of hydrophobic sites for 8-anilino-1-napthalenesulfonic acid binding and β-structure formation inhibits FN multimerization and prevents physiological cell-based FN assembly in culture. We propose a model for cell-mediated fibrillogenesis whereby cell traction force initiates a cascade of intermolecular exchange starting with the unfolding of 10FNIII to expose the multimerization sequence, which interacts with strand B of another 10FNIII domain via a Trp-mediated β-strand exchange to stabilize a partially unfolded intermediate that propagates FN self-assembly.     

Potentiation of cell migration by adhesion-dependent cooperative signals from the GTPase Rac and Raf kinase

The small GTPase Rac is thought to regulate cell movement by influencing actin cytoskeletal organization and membrane ruffling. However, cell migration also depends on the activation of mitogen-activated protein kinase (MAPK), which can regulate myosin motor function, an event critical for cell contraction. Evidence is provided that, during active cell adhesion to the extracellular matrix, Rac potentiates the MAPK pathway and influences cell migration by selectively synergizing with Raf kinase but not with Ras or MAPK kinase. In fact, the synergy between Rac and Raf kinase increases the chemotactic sensitivity of cells to epidermal growth factor by 1000-fold. Therefore, the role of Rac in cell migration not only depends on its ability to regulate actin cytoskeletal organization but also on its capacity to potentiate chemokine activation of MAPK in a manner that depends on active cell adhesion to the extracellular matrix.     

Hydrogen peroxide induces endothelial cell atypia and cytoskeleton depolymerization

Reactive oxygen intermediates induce cell injury in a variety of pathophysiological conditions. Human umbilical cord vein endothelial cell (HUVEC) cultures were exposed to 1 or 200 microM H2O2 for 15 min, and observed after 15 min, or 1, 4, 24, or 120 h. Factor VIII and the cytoskeletal proteins vimentin and tubulin were visualized immunocytochemically. Release of lactate dehydrogenase (indices of cell membrane injury) did not increase after H2O2 exposure; nor was cellular expression of factor VIII affected. 200 microM H2O2 induced cell contraction after 15 min which disappeared after 1 and 4 h, but was evident again after 24 h. Immediately after exposure, the filamentous structure of vimentin and tubulin disappeared, but normalized after 1 h. After 120 h, the cytoskeleton filaments were coarsened and disorganized, and an abundance of multinucleated giant cells were observed. Catalase (150 U/ml) abolished all effects of H2O2. One microM H2O2 did not induce any changes in HUVEC. Thus, the present concentrations of H2O2 did not induce cell necrosis or altered expression of factor VIII. Early, reversible cell contraction and depolymerization of cytoskeletal proteins were observed, followed by a delayed contraction and cell atypia after 200 microM H2O2.     

Fibronectin aggregation and assembly: the unfolding of the second fibronectin type III domain

The mechanism of fibronectin (FN) assembly and the self-association sites are still unclear and contradictory, although the N-terminal 70-kDa region ((I)1-9) is commonly accepted as one of the assembly sites. We previously found that (I)1-9 binds to superfibronectin, which is an artificial FN aggregate induced by anastellin. In the present study, we found that (I)1-9 bound to the aggregate formed by anastellin and a small FN fragment, (III)1-2. An engineered disulfide bond in (III)2, which stabilizes folding, inhibited aggregation, but a disulfide bond in (III)1 did not. A gelatin precipitation assay showed that (I)1-9 did not interact with anastellin, (III)1, (III)2, (III)1-2, or several (III)1-2 mutants including (III)1-2KADA. (In contrast to previous studies, we found that the (III)1-2KADA mutant was identical in conformation to wild-type (III)1-2.) Because (I)1-9 only bound to the aggregate and the unfolding of (III)2 played a role in aggregation, we generated a (III)2 domain that was destabilized by deletion of the G strand. This mutant bound (I)1-9 as shown by the gelatin precipitation assay and fluorescence resonance energy transfer analysis, and it inhibited FN matrix assembly when added to cell culture. Next, we introduced disulfide mutations into full-length FN. Three disulfide locks in (III)2, (III)3, and (III)11 were required to dramatically reduce anastellin-induced aggregation. When we tested the disulfide mutants in cell culture, only the disulfide bond in (III)2 reduced the FN matrix. These results suggest that the unfolding of (III)2 is one of the key factors for FN aggregation and assembly.