The role of the fibronectin IGD motif in stimulating fibroblast migration

The motogenic activity of migration-stimulating factor, a truncated isoform of fibronectin (FN), has been attributed to the IGD motifs present in its FN type 1 modules. The structure-function relationship of various recombinant IGD-containing FN fragments is now investigated. Their structure is assessed by solution state NMR and their motogenic ability tested on fibroblasts. Even conservative mutations in the IGD motif are inactive or have severely reduced potency, while the structure remains essentially the same. A fragment with two IGD motifs is 100 times more active than a fragment with one and up to 10(6) times more than synthetic tetrapeptides. The wide range of potency in different contexts is discussed in terms of cryptic FN sites and cooperativity. These results give new insight into the stimulation of fibroblast migration by IGD motifs in FN.     

Extended binding site on fibronectin for the functional upstream domain of protein F1 of Streptococcus pyogenes

The 49-residue functional upstream domain (FUD) of Streptococcus pyogenes F1 adhesin interacts with fibronectin (FN) in a heretofore unknown manner that prevents assembly of a FN matrix. Biotinylated FUD (b-FUD) bound to adsorbed FN or its recombinant N-terminal 70-kDa fibrin- and gelatin-binding fragment (70K). Binding was blocked by FN or 70K, but not by fibrin- or gelatin-binding subfragments of 70K. Isothermal titration calorimetry showed that FUD binds with K(d) values of 5.2 and 59 nM to soluble 70K and FN, respectively. We tested sets of FUD mutants and epitope-mapped monoclonal antibodies (mAbs) for ability to compete with b-FUD for binding to FN or to block FN assembly by cultured fibroblasts. Deletions or alanine substitutions throughout FUD caused loss of both activities. mAb 4D1 to the (2)FNI module had little effect, whereas mAb 7D5 to the (4)FNI module in the fibrin-binding region, 5C3 to the (9)FNI module in the gelatin-binding region, or L8 to the G-strand of (1)FNIII module adjacent to (9)FNI caused loss of binding of b-FUD to FN and decreased FN assembly. Conversely, FUD blocked binding of 7D5, 5C3, or L8, but not of 4D1, to FN. Circular dichroism indicated that FUD binds to 70K by β-strand addition, a possibility supported by modeling based on crystal structures of peptides bound to (2)FNI-(5)FNI of the fibrin-binding domain and (8)FNI-(9)FNI of the gelatin-binding domain. Thus, the interaction likely involves an extensive anti-parallel β-zipper in which FUD interacts with the E-strands of (2)FNI-(5)FNI and (8)FNI-(9)FNI.     

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.     

On-Off Kinetics of Engagement of FNI Modules of Soluble Fibronectin by β-Strand Addition

Intrinsically disordered sequences within bacterial adhesins bind to E-strands in the β-sheets of multiple FNI modules of fibronectin (FN) by anti-parallel β-strand addition, also called tandem β-zipper formation. The FUD segment of SfbI of Streptococcus pyogenes and Bbk32 segment of BBK32 of Borrelia burgdorferi, despite being imbedded in different adhesins from different bacteria, target the same FNI modules, ^2–5,8–9FNI, in the N-terminal 70-kDa region (FN70K) of FN. To facilitate further comparisons, FUD, Bbk32, two other polypeptides based on SfbI that target ^1–5FNI (HADD) and ^2–5FNI (FRD), and mutant Bbk32 (ΔBbk32) were produced with fluorochromes placed just outside of the binding sequences. Unlabeled FUD competed ~1000-fold better for binding of labeled Bbk32 to FN than unlabeled Bbk32 competed for binding of labeled FUD to FN. Binding kinetics were determined by fluorescence polarization in a stopped-flow apparatus. On-rates for FUD, Bbk32, HADD, and FRD were similar, and all bound more rapidly to FN70K fragment than to full length FN. In stopped-flow displacement and size exclusion chromatographic assays, however, k_off for FUD or HADD to FN70K or FN was considerably lower compared to k_off of FRD or Bbk32. FUD and Bbk32 differ in the spacing between sequences that interact with ³FNI and ⁴FNI or with ⁵FNI and ⁸FNI. ΔBbk32, in which 2 residues were removed from Bbk32 to make the spacing more like FUD, had a k_off intermediate between that of Bbk32 and FUD. These results indicate a “folding-after-binding” process after initial association of certain polypeptide sequences to FN that results in formation of a stable complex and is a function of number of FNI modules engaged by the polypeptide, spacing of engagement sites, and perhaps flexibility within the polypeptide-FN complex. We suggest that contributions of SfbI and BBK32 adhesins to bacterial pathogenicity may be determined in part by stability of adhesin-FN complexes.     

Borrelia burgdorferi Protein BBK32 Binds to Soluble Fibronectin via the N-terminal 70-kDa Region,Causing Fibronectin to Undergo Conformational Extension

BBK32 is a fibronectin (FN)-binding protein expressed on the cell surface of Borrelia burgdorferi, the causative agent of Lyme disease. There is conflicting information about where and how BBK32 interacts with FN. We have characterized interactions of a recombinant 86-mer polypeptide, “Bbk32,” comprising the unstructured FN-binding region of BBK32. Competitive enzyme-linked assays utilizing various FN fragments and epitope-mapped anti-FN monoclonal antibodies showed that Bbk32 binding involves both the fibrin-binding and the gelatin-binding domains of the 70-kDa N-terminal region (FN70K). Crystallographic and NMR analyses of smaller Bbk32 peptides complexed, respectively, with ^2–3FNI and ^8–9FNI, demonstrated that binding occurs by β-strand addition. Isothermal titration calorimetry indicated that Bbk32 binds to isolated FN70K more tightly than to intact FN. In a competitive enzyme-linked binding assay, complex formation with Bbk32 enhanced binding of FN with mAbIII-10 to the ¹⁰FNIII module. Thus, Bbk32 binds to multiple FN type 1 modules of the FN70K region by a tandem β-zipper mechanism, and in doing so increases accessibility of FNIII modules that interact with other ligands. The similarity in the FN-binding mechanism of BBK32 and previously studied streptococcal proteins suggests that the binding and associated conformational change of FN play a role in infection.     

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 ^8-9FNI 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 ^8-9FNI or a large dimeric FN construct that is constitutively in an extended conformation. In contrast to bacterial peptides that bind ^2–5FNI in addition to ^8-9FNI, 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 k_off. 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.     

Assembly of exogenous fibronectin by fibronectin-null cells is dependent on the adhesive substrate

The role of endogenously synthesized fibronectin (FN) in assembly was studied with cells lacking or expressing FN. Cells were cultured as homogeneous or mixed populations on surfaces coated with different matrix proteins. Compared with FN-expressing cells, FN-null cells poorly assembled exogenous plasma FN (pFN) when adhered to vitronectin or the recombinant cell-binding domain (III(7-10)) of FN. Vitronectin had a suppressive effect that was overcome by co-adsorbed pFN or laminin-1 but not by soluble FN. In co-cultures of FN-expressing cells and FN-null cells, endogenous FN was preferentially assembled around FN-expressing cells regardless of the adhesive ligand. If the adhesive ligand was vitronectin, exogenous pFN assembled preferentially around cells expressing cellular FN or recombinant EDa- or EDa+ FN. In co-cultures on vitronectin of FN-null cells and beta(1) integrin subunit-null cells, fibrils of cellular FN and pFN were preferentially deposited by FN-null (beta(1)-expressing) cells immediately adjacent to (FN-secreting) beta(1)-null cells. In co-cultures on vitronectin of FN-null cells and beta(1)-null cells expressing a chimera with the extracellular domain of beta(1) and the cytoplasmic domain of beta(3), preferential assembly was by the chimera-expressing cells. These results indicate that the adhesive ligand is a determinant of FN assembly by cells not secreting endogenous FN (suppressive if vitronectin, non-suppressive but non-supportive if III(7-10), supportive if pFN or laminin-1) and suggest that efficient interaction of freshly secreted cellular FN with a beta(1) integrin, presumably alpha(5)beta(1), substitutes for integrin-mediated adherence to a preformed matrix of laminin-1 or pFN to support assembly of FN.     

Fibronectin binds insulin-like growth factor-binding protein 5 and abolishes Its ligand-dependent action on cell migration

Insulin-like growth factor-binding protein 5 (IGFBP-5) is a secreted protein that binds to insulin-like growth factors (IGFs) and modulates IGF actions on cell proliferation, differentiation, survival, and motility. IGFBP-5 also regulates these cellular events through IGF-independent mechanisms. To elucidate the molecular mechanisms governing these diverse actions of IGFBP-5, we screened a human cDNA library by a yeast two-hybrid system using IGFBP-5 as bait and identified fibronectin (FN) as a potential IGFBP-5-interacting partner. The complex formation of IGFBP-5 and FN was established by glutathione S-transferase pull-down, solution, and solid phase binding assays using glutathione S-transferase-IGFBP-5 and native IGFBP-5 in vitro and by co-immunoprecipitation in vivo. Binding assay using deletion mutants indicated that the IGFBP-5 C domain binds to the 10th and 11th type I repeats of FN. IGFBP-5 potentiated IGF-I-induced cell migration in FN-null, but not in wild-type, mouse embryonic cells. When FN was reintroduced either as an adhesive substrate or in solution to the FN-null cells, the potentiating effect of IGFBP-5 on IGF-I-induced cell migration was abolished. Binding of IGFBP-5 to FN had no effect on the ability of IGFBP-5 to bind IGF-I, but it increased the proteolytic degradation of IGFBP-5. Inhibition of IGFBP-5 proteolysis restored the potentiating effect of IGFBP-5. These results suggest that FN and IGFBP-5 bind to each other, and this binding negatively regulates the ligand-dependent action of IGFBP-5 by triggering IGFBP-5 proteolysis.     

Multi-factorial modulation of IGD motogenic potential in MSF (Migration Stimulating Factor)

Migration Stimulating Factor (MSF) is a genetically truncated isoform of fibronectin (Fn). MSF is a potent stimulator of fibroblast migration, whereas full length Fn is devoid of motogenic activity. MSF and Fn contain four IGD motifs, located in the 3rd, 5th, 7th and 9th type I modules; these modules are referred to as ³FnI, ⁵FnI, ⁷FnI and ⁹FnI, respectively. We have previously reported that mutation of IGD motifs in modules ⁷FnI and ⁹FnI of MSF is sufficient to completely abolish the motogenic response of target adult skin fibroblasts. We now report that the IGD sequences in ³FnI and ⁵FnI are also capable of exhibiting motogenic activity when present within fragments of MSF. When present within ^1-5FnI, these sequences require the presence of serum or vitronectin for their motogenic activity to be manifest, whereas the IGD sequences in ⁷FnI and ⁹FnI are bioactive in the absence of serum factors. All MSF and IGD-containing peptides stimulated the phosphorylation of the integrin binding protein focal adhesion kinase (FAK) but did not necessarily affect migration. These results suggest that steric hindrance determines the motogenic activity of MSF and Fn, and that both molecules contain cryptic bioactive fragments.     

The role of integrin binding sites in fibronectin matrix assembly in vivo

The extracellular matrix (ECM) glycoprotein fibronectin (FN) requires the help of cells to assemble into a functional fibrillar matrix, which then orchestrates the assembly of other ECM proteins and promotes cell adhesion, migration and signalling. Fibrillogenesis is initiated and governed by cell surface integrins that bind to specific sites in the FN molecule. Recent studies identified novel integrin binding sites in FN that can also participate in FN fibril formation and in morphogenetic events during development.     

iso-DGR Sequences Do Not Mediate Binding of Fibronectin N-terminal Modules to Adherent Fibronectin-null Fibroblasts

Fibronectin (FN) without an RGD sequence (FN-RGE), and thus lacking the principal binding site for α5β1 integrin, is deposited into the extracellular matrix of mouse embryos. Spontaneous conversion of ²⁶³NGR and/or ⁵⁰¹NGR to iso-DGR possibly explains this enigma, i.e. ligation of iso-DGR by αvβ3 integrin may allow cells to assemble FN. Partial modification of ²⁶³NGR to DGR or iso-DGR was detected in purified plasma FN by mass spectrometry. To test functions of the conversion, one or both NGR sequences were mutated to QGR in recombinant N-terminal 70-kDa construct of FN (70K), full-length FN, or FN-RGE. The mutations did not affect the binding of soluble 70K to already adherent fibroblasts or the ability of soluble 70K to compete with non-mutant FN or FN-RGE for binding to FN assembly sites. Non-mutant FN and FN-N263Q/N501Q with both NGRs mutated to QGRs were assembled equally well by adherent fibroblasts. FN-RGE and FN-RGE-N263Q/N501Q were also assembled equally well. Although substrate-bound 70K mediated cell adhesion in the presence of 1 mm Mn²⁺ by a mechanism that was inhibited by cyclic RGD peptide, the peptide did not inhibit 70K binding to cell surface. Mutations of the NGR sequences had no effect on Mn²⁺-enhanced cell adhesion to adsorbed 70K but caused a decrease in cell adhesion to reduced and alkylated 70K. These results demonstrate that iso-DGR sequences spontaneously converted from NGR are cryptic and do not mediate the interaction of the 70K region of FN with the cell surface during FN assembly.     

Residues within the conserved helicase motifs of UL9, the origin-binding protein of herpes simplex virus-1, are essential for helicase activity but not for dimerization or origin binding activity

UL9, an essential gene for herpes simplex virus type 1 (HSV-1) DNA replication, exhibits helicase and origin DNA binding activities. It has been hypothesized that UL9 binds and unwinds the HSV-1 origin of replication, creating a replication bubble and promoting the assembly of the viral replication machinery; however, direct confirmation of this hypothesis has not been possible. Based on the presence of conserved helicase motifs, UL9 has been classified as a superfamily II helicase. Mutations in conserved residues of the helicase motifs I-VI of UL9 have been isolated, and most of them fail to complement a UL9 null virus in vivo (Martinez R., Shao L., and Weller S. (1992) J. Virol. 66, 6735-6746). In addition, mutants in motifs I, II, and VI were found to be transdominant (Malik, A. K., and Weller, S. K. (1996) J. Virol. 70, 7859-7866). Here we present the characterization of the biochemical properties of the UL9 helicase motif mutants. We report that mutations in motifs I-IV and VI affect the ATPase activity, and all but the motif III mutation completely abolish the helicase activity. In addition, mutations in these motifs do not interfere with UL9 dimerization or the ability of UL9 to bind the HSV-1 origin of replication. Based on the similarity of the helicase motif sequences between UL9 and UvrB, another superfamily II member with helicase-like activity, we were able to map the UL9 mutations on the structure of the UvrB protein and provide an explanation for the observed phenotypes. Our results indicate that the helicase function of UL9 is indispensable for viral replication, supporting the hypothesis that UL9 is essential for unwinding the HSV-1 origin of replication in vivo. Furthermore, the data presented provide insights into the mechanism of transdominance of the UL9 helicase motif mutants.     

Fibronectin's central cell-binding domain supports focal adhesion formation and Rho signal transduction

Fibroblast adhesion to fibronectin (FN) induces formation of focal adhesions (FAs), structures that have significant effect on cell migration and signaling. FA formation requires actomyosin-based contractility that is regulated by Rho-dependent myosin light chain (MLC) phosphorylation. Previous studies indicated that the FN central cell-binding (and integrin-binding) domain (CBD) is insufficient for FA formation and that the major heparin-binding domain (HepII) facilitates FA formation in a Rho-dependent manner. We describe here conditions under which FN CBD alone is sufficient for FA formation in both human dermal fibroblasts and the FN-null murine fibroblasts. CBD-mediated FA formation is dependent on its surface adsorption and the adhesion activity of the cells. Attachment of FN-null fibroblasts to CBD elicits the same biphasic regulation of Rho activity as seen on intact FN, whereas adhesion to HepII alone does not activate Rho. Activation of Rho requires high levels of integrin occupancy. However, FN or CBD may induce FAs without increased activation of Rho (i.e. the basal level of GTP-Rho induces sufficient phospho-MLC for FA assembly under this condition). In contrast, adhesion to HepII alone does not sustain MLC phosphorylation. Pulse stimulation of cells on CBD or HepII with lysophosphatidic acid elevates Rho GTP loading to the same level, but the lysophosphatidic acid-stimulated MLC phosphorylation is significantly lower in cells on HepII than on CBD. Coating HepII with suboptimal concentrations of CBD induces FAs without increased activation of Rho. Therefore, FN CBD can support FA formation and generate contraction by activating Rho or by facilitating Rho downstream signaling.     

Regulatory role for SRC and phosphatidylinositol 3-kinase in initiation of fibronectin matrix assembly

Fibronectin (FN) matrix assembly is a tightly regulated stepwise process that is initiated by interactions between FN and cell surface integrin receptors. These interactions activate many intracellular signaling pathways that regulate processes such as cell adhesion, migration, and survival. Here we demonstrate that cells lacking Src family kinases showed reduced ability to assemble FN fibrils as detected by immunofluorescence and by analysis of detergent extracts. The amount of FN matrix was further reduced by treatment with the phosphatidylinositol 3 (PI 3-kinase) inhibitor, wortmannin. CHOalpha5 cells, which are dependent on exogenous FN to initiate fibril formation, also showed significant reductions in matrix when treated with inhibitors of Src and PI 3-kinase. Combination of both inhibitors showed an additive inhibitory effect on assembly, which was concomitant with a loss of focal adhesion kinase phosphorylation. Decreased binding of the 70-kDa amino-terminal FN fragment at matrix assembly sites further supports a role for these kinases early during the process. We propose that these two signaling molecules, which lie downstream of integrins and focal adhesion kinase, are essential for efficient initiation of FN matrix assembly.     

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.     

Integrin-dependent and -independent functions of astrocytic fibronectin in retinal angiogenesis

Fibronectin (FN) is a major component of the extracellular matrix and functions in cell adhesion, cell spreading and cell migration. In the retina, FN is transiently expressed and assembled on astrocytes (ACs), which guide sprouting tip cells and deposit a provisional matrix for sprouting angiogenesis. The precise function of FN in retinal angiogenesis is largely unknown. Using genetic tools, we show that astrocytes are the major source of cellular FN during angiogenesis in the mouse retina. Deletion of astrocytic FN reduces radial endothelial migration during vascular plexus formation in a gene dose-dependent manner. This effect correlates with reduced VEGF receptor 2 and PI3K/AKT signalling, and can be mimicked by selectively inhibiting VEGF-A binding to FN through intraocular injection of blocking peptides. By contrast, AC-specific replacement of the integrin-binding RGD sequence with FN-RGE or endothelial deletion of itga5 shows little effect on migration and PI3K/AKT signalling, but impairs filopodial alignment along AC processes, suggesting that FN-integrin α5β1 interaction is involved in filopodial adhesion to the astrocytic matrix. AC FN shares its VEGF-binding function and cell-surface distribution with heparan-sulfate (HS), and genetic deletion of both FN and HS together greatly enhances the migration defect, indicating a synergistic function of FN and HS in VEGF binding. We propose that in vivo the VEGF-binding properties of FN and HS promote directional tip cell migration, whereas FN integrin-binding functions to support filopodia adhesion to the astrocytic migration template.     

The tandem beta-zipper model defines high affinity fibronectin-binding repeats within Staphylococcus aureus FnBPA

Binding of the fibronectin-binding protein FnBPA from Staphylococcus aureus to the human protein fibronectin has previously been implicated in the development of infective endocarditis, specifically in the processes of platelet activation and invasion of the endothelium. We recently proposed a model for binding of fibronectin to FnBPA in which the bacterial protein contains 11 potential binding sites (FnBPA-1 to FnBPA-11), each composed of motifs that bind to consecutive fibronectin type 1 modules in the N-terminal domain of fibronectin. Here we show that six of the 11 sites bind with dissociation constants in the nanomolar range; other sites bind more weakly. The high affinity binding sites include FnBPA-1, the sequence of which had previously been thought to be encompassed by the fibrinogen-binding A domain of FnBPA. Both the number and sequence conservation of the type-1 module binding motifs appears to be important for high affinity binding. The in vivo relevance of the in vitro binding studies is confirmed by the presence of antibodies in patients with S. aureus infections that specifically recognize complexes of these six high affinity repeats with fibronectin.     

Secreted phosphoprotein 1 binds integrins to initiate multiple cell signaling pathways,including FRAP1/mTOR,to support attachment and force-generated migration of trophectoderm cells

Attachment and migration of trophectoderm (Tr) cells, hallmarks of blastocyst implantation in mammals, are unique uterine events. Secreted phosphoprotein 1 (SPP1) in the uterus binds integrins on conceptus Tr and uterine luminal epithelium (LE), affecting cell–cell and cell–matrix interactions. The signal transduction pathways activated by SPP1 and integrins in conceptuses have not been elucidated. Results of this study demonstrate that SPP1 binds αvβ3 and α5β1 integrins to induce focal adhesion assembly, a prerequisite for adhesion and migration of Tr, through activation of: 1) P70S6K via crosstalk between FRAP1/mTOR and MAPK pathways; 2) mTOR, PI3K, MAPK3/MAPK1 (Erk1/2) and MAPK14 (p38) signaling to stimulate Tr cell migration; and 3) focal adhesion assembly and myosin II motor activity to induce migration of Tr cells. These cell signaling pathways, acting in concert, mediate adhesion, migration and cytoskeletal remodeling of Tr cells essential for expansion and elongation of conceptuses and attachment to uterine LE for implantation.     

Probing the Conformation of the Fibronectin III1�C2 Domain by Fluorescence Resonance Energy Transfer

Fibronectin (FN) matrix is crucial for cell and tissue functions during embryonic development, wound healing, and oncogenesis. Assembly of FN matrix fibrils requires FN domains that mediate interactions with integrin receptors and with other FN molecules. In addition, regulation of FN matrix assembly depends on the first two FN type III modules, III₁ and III₂, which harbor FN-binding sites. We propose that interactions between these two modules sequester FN-binding sites in soluble FN and that these sites become exposed by FN conformational changes during assembly. To test the idea that III_1–2 has a compact conformation, we constructed CIIIY, a conformational sensor of III_1–2 based on fluorescent resonance energy transfer between cyan and yellow fluorescent proteins conjugated at its N and C termini. We demonstrate energy transfer in CIIIY and show that fluorescent resonance energy transfer was eliminated by proteolysis and by treatment with mild denaturants that disrupted intramolecular interactions between the two modules. We also show that mutations of key charged residues resulted in conformational changes that exposed binding sites for the N-terminal 70-kDa FN fragment. Collectively, these results support a conformation-dependent mechanism for the regulation of FN matrix assembly by III_1–2.Fibronectin (FN)³ is a 500-kDa modular dimeric protein and a major component of the extracellular matrix. It exists in the blood and other body fluids as a soluble compact molecule and undergoes cell-mediated assembly to form an insoluble three-dimensional fibrillar matrix (reviewed in Ref. 1). The process of FN matrix assembly has been implicated in embryonic development, wound healing, and cancer (2–4). FN is composed of type I–III modules, and sets of these modules comprise binding domains for cells and for other extracellular matrix components (see Fig. 1A). Three of these binding domains are essential for matrix assembly (1). Integrin receptor interactions with the cell-binding domain tether disulfide-bonded FN dimers to the cell surface, where FN-FN interactions involving the N-terminal assembly domain form dimers into fibrils. In addition to these essential domains, other FN-binding sites have been implicated in assembly. In particular, the III_1–2 FN-binding domain plays a regulatory role in matrix assembly. Within this domain reside a cryptic FN-binding site in III₁ and a site available for FN binding in the native form of III₂ (5–8). Recombinant FN lacking III₁ is assembled into a matrix at wild-type levels, but that lacking the III_1–2 domain results in short immature FN fibrils (8). Peptides derived from the III_1–2 domain or antibodies against III_1–2 block matrix assembly by cultured cells (9–11). Furthermore, FN binding to this region is enhanced when FN is mechanically stretched (12). Taken together, these results suggest that conformational changes in the III_1–2 domain may control its interactions during FN assembly.Open in a separate windowFIGURE 1.The FN III_1–2 FRET conformational sensor. A, representation of the domain structure of FN and major interaction sites. FN is composed of repeating modules that form binding domains for other FN molecules, cell receptors, and other extracellular matrix components as indicated. The first two type III modules III₁ and III₂ (black), have FN-binding sites and regulate FN matrix assembly. The N-terminal 70-kDa region contains a matrix assembly domain with FN-binding activity. The cell-binding domain (cell), the heparin-binding domain (heparin), the dimerization site (SS), and the alternatively spliced type IIIA (A), IIIB (B), and variable regions (V) are indicated. 70kD, N-terminal 70-kDa FN fragment. B, schematic of proposed model of III_1–2 domain conformation. Panel i, in solution, the FN-binding sites in III₁ and III₂ (hatched areas) are sequestered through domain orientations that are facilitated by the linker between modules (thin line). Panel ii, binding sites are exposed through conformational changes resulting from cell-mediated extension of FN (arrows). The length of the linker and the height and width of the modules are drawn to scale for a linear peptide and published data on FN type III modules, respectively. C, ribbon diagram representation of CIIIY, a FRET sensor of the model in B (panel i), oriented with N and C termini 50 Å apart. CIIIY consists of the III_1–2 domain with CFP at the N terminus and YFP at the C terminus.To more fully understand the roles of native and cryptic FN-binding sites in matrix assembly, the conformational dynamics of III_1–2 must be characterized. One approach to this problem is to tag III_1–2 with fluorescent probes, which, in conjunction with fluorescent resonance energy transfer (FRET), create a molecular conformational sensor. FRET involves the radiationless transfer of energy from an excited donor fluorophore to an acceptor fluorophore, a process that is very sensitive to the distance between the two fluorophores (13–15). Two fluorescent protein variants, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), are highly related to green fluorescent protein (GFP). Because the emission spectrum of CFP is well matched to the excitation spectrum of YFP, these two fluorophores have been widely used as a donor-acceptor pair in FRET studies (13–15).In this study, we describe a FRET conformational sensor designed to test the idea that intramolecular interactions between III₁ and III₂ sequester key FN-binding and assembly sites. We show that III_1–2 with CFP and YFP fused to the N and C termini, respectively, displays a clear FRET signal, indicating that the attached fluorescent proteins and thus the ends of III_1–2 are in close proximity. FRET data from III_1–2 mutants support the presence of a stabilizing intermodule salt bridge that regulates FN-binding activity.     

Fibronectin Binding Modulates CXCL11 Activity and Facilitates Wound Healing

Engineered biomatrices offer the potential to recapitulate the regenerative microenvironment, with important implications in tissue repair. In this context, investigation of the molecular interactions occurring between growth factors, cytokines and extracellular matrix (ECM) has gained increasing interest. Here, we sought to investigate the possible interactions between the ECM proteins fibronectin (FN) and fibrinogen (Fg) with the CXCR3 ligands CXCL9, CXCL10 and CXCL11, which are expressed during wound healing. New binding interactions were observed and characterized. Heparin-binding domains within Fg (residues 15-66 of the β chain, Fg β15-66) and FN (FNI1-5, but not FNIII12-14) were involved in binding to CXCL10 and CXCL11 but not CXCL9. To investigate a possible influence of FN and Fg interactions with CXCL11 in mediating its role during re-epithelialization, we investigated human keratinocyte migration in vitro and wound healing in vivo in diabetic db/db mice. A synergistic effect on CXCL11-induced keratinocyte migration was observed when cells were treated with CXCL11 in combination with FN in a transmigration assay. Moreover, wound healing was enhanced in full thickness excisional wounds treated with fibrin matrices functionalized with FN and containing CXCL11. These findings highlight the importance of the interactions occurring between cytokines and ECM and point to design concepts to develop functional matrices for regenerative medicine.