Matrix-driven translocation: dependence on interaction of amino-terminal domain of fibronectin with heparin-like surface components of cells or particles.

Newman, Stuart A.; Frenz, Dorothy A.; Hasegawa, Eiichi; Akiyama, Steven K.

doi:10.1073/pnas.84.14.4791

Cited by 55 publications

(34 citation statements)

References 44 publications

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“…Subsequent transfections were done in both T-flask and shaker flask culture at a multiplicity of infection of 5-10. The infected cells were grown for 3 or more days, after which the supernatant was harvested, centrifuged at 5,000 ϫ g for 10 min, and cleared by an additional 10 min of centrifugation at 10,000 ϫ g. Supernatants were analyzed on 12.5% SDS-PAGE gels (35) by Coomassie Blue staining or after electrotransfer to nitrocellulose by immunoblotting with monoclonal antibody 304 (24) and an alkaline phosphataseconjugated goat anti-mouse secondary antibody to confirm and estimate the level of expression of the FnNTDs.…”

Section: Methodsmentioning

confidence: 99%

“…The morphogenetic potential of the interaction between the FnNTD and heparin-like molecules was also demonstrated in experiments that assayed for matrix-driven translocation (MDT), a morphological reorganization that occurs when an artificially constructed extracellular matrix containing heparin-coated polystyrene beads is placed adjacent to similar matrix containing fibronectin (23,24). Under the conditions of the assay the FnNTD was the only domain of fibronectin both necessary and sufficient for MDT to occur (24).…”

mentioning

confidence: 97%

“…Matrix-Driven Translocation Promoting Activity of the FnNTDs-Several of the recombinant FnNTDs were compared with the native fragment for their ability to interact with heparin or heparin-mimetic polystyrene on the surfaces of beads so as to induce MDT (24) (Fig. 6).…”

Section: Fig 2 Production Of Wild Type and Recombinant Fnntds In Thmentioning

confidence: 99%

“…Matrix-driven Translocation-This assay was performed as described previously (23,24). Briefly, "primary gels" were constructed by suspending polystyrene latex beads (6 m, Polysciences, Warrington, PA; final concentration, 5 ϫ 10 6 /ml) in a cooled, soluble collagen solution (1.7-3.5 mg/ml), which was simultaneously adjusted to physiological pH and ionic strength with concentrated Ham's F-12 medium and sodium bicarbonate.…”

Section: Fibronectin-heparin Interactionsmentioning

confidence: 99%

“…Under the conditions of the assay the FnNTD was the only domain of fibronectin both necessary and sufficient for MDT to occur (24). MDT did not occur when dextran sulfate-coated beads were substituted for heparin-coated beads (23) or when tri-or tetrapeptides containing Gly-Arg-Gly were used as competitors of the bead-fibronectin interaction (20) and was greatly reduced when FnNTDs were used in which more than four arginines were blocked by the chemical agent 1,2-cyclohexanedione (20).…”

mentioning

confidence: 99%

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Interaction of the NH2-terminal Domain of Fibronectin with Heparin

Kishore¹,

Samuel²,

Khan³

et al. 1997

Journal of Biological Chemistry

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Determinants of the interaction of the 29-kDa NH 2 -terminal domain of fibronectin with heparin were explored by analysis of normal and mutant recombinant NH 2 -terminal fibronectin fragments produced in an insect cell Baculovirus host vector system. A genomic/ cDNA clone was constructed that specified a secretable human fibronectin NH 2 fragment. With the use of sitedirected mutagenesis a set of 29 kDa fragments was obtained that contained glycine or glutamic acid residues in place of basic residues at various candidate sites for heparin binding in the five type I modules that make up the domain. The recombinant fragment containing the wild type sequence had a nearly normal circular dichroic spectra and a melting profile, as assayed by loss of ellipticity at 228 nm, that was indistinguishable from that of the native fragment obtained by trypsinization of plasma fibronectin. A substantial proportion of the wild type recombinant fragment bound to heparin-Sepharose, where it was eluted at the same NaCl concentration as the native fragment. The wild type fragment was capable of promoting matrix-driven translocation, a morphogenetic effect in artificial extracellular matrices that depends on the interaction of the fibronectin NH 2 terminus with heparin-like molecules on the surfaces of particles. Mutant fragments in which arginines predicted to be most exposed in the folded fragment were converted to glycines retained the same affinity for heparin as the wild type fragment. In contrast, a mutant fragment in which the single basic residue (Arg 99 ) in the minor loop ("⍀-loop") of the second type I module was converted to a glycine had an essentially normal melting profile but exhibited no binding to heparin and failed to promote matrix-driven translocation. A mutant fragment in which the single basic residue (Arg 52 ) of the first type I module was converted to a glycine also completely lacked heparin binding activity, but one in which the single basic residue (Arg 191 ) the fourth type I module was converted to a glycine retained the ability to bind heparin. A mutant fragment in which the single basic residue (Lys 143 ) in the ⍀-loop of the third type I module was converted to a glutamic acid lacked heparin binding activity but had a CD spectrum similar to the heparin-liganded native protein and was capable of promoting matrix-driven translocation. The results indicate that multiple residues in the ⍀-loops of the fibronectin NH 2 -terminal domain participate in its interactions with heparin. In addition, the conformation of one of the nonbinding mutants may mimic the heparin-induced structural alteration in this fibronectin domain required for certain morphogenetic events.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 97%

Section: Fig 2 Production Of Wild Type and Recombinant Fnntds In Thmentioning

confidence: 99%

Section: Fibronectin-heparin Interactionsmentioning

confidence: 99%

mentioning

confidence: 99%

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Interaction of the NH2-terminal Domain of Fibronectin with Heparin

Kishore¹,

Samuel²,

Khan³

et al. 1997

Journal of Biological Chemistry

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Regulation of chondrocyte differentiation and maturation

Hickok

Haas

Tuan

1998

Microsc. Res. Tech.

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Fibronectin mRNA alternative splicing is temporally and spatially regulated during chondrogenesis in vivo and in vitro

et al. 1996

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Fibronectin, a component of the extracellular matrix in a variety of tissues, participates in many critical cellular processes, including differentiation, adhesion, and migration. A positive correlation exists between the presence of fibronectin and the onset of chondrogenesis, the differentiation of mesenchyme into cartilage. Heterogeneity in the structure of fibronectin is largely due to the alternative splicing of at least three exons (IIIB, IIIA, and V) during processing of a single primary transcript. We have previously shown that the fibronectin mRNA splicing patterns change during chondrogenesis (Bennett et al. [1991] J. Biol. Chem. 266:5918–5924). All of the fibronectin mRNAs from prechondrogenic chick limb mesenchyme contain exons IIIB, IIIA, and V (B + A + V +), whereas all of the fibronectin mRNAs from chick cartilage contain exons IIIB and V but do not contain exon IIIA (B + A − V +). In this study, we show that fibronectin mRNAs containing exon IIIA (FN‐A) and/or the mRNAs containing exon IIIB (FN‐B) are expressed in a specific and different spatiotemporal manner in the developing chick limb in vivo, as well as in limb mesenchymal cells undergoing chondrogenesis in vitro. Specifically, in situ hybridization reveals that FN‐B mRNAs are present throughout the various stages (HH 20–30) of limb cartilage development in vivo, whereas FN‐A mRNAs disappear following the condensation phase of chondrogenesis and are absent from the resulting cartilage. Chick limb cartilage fibronectin mRNAs are therefore B + A −, as in other embryonic cartilage tissues. Furthermore, limb mesenchymal cells undergoing chondrogenesis in vitro lose FN‐A mRNAs immediately following condensation, recapitulating the events that occur during chondrogenesis in vivo. These results suggest an important role for fibronectin mRNA alternative splicing during chondrogenic differentiation. © 1996 Wiley‐Liss, Inc.

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Matrix-driven translocation: dependence on interaction of amino-terminal domain of fibronectin with heparin-like surface components of cells or particles.

Cited by 55 publications

References 44 publications

Interaction of the NH2-terminal Domain of Fibronectin with Heparin

Interaction of the NH2-terminal Domain of Fibronectin with Heparin

Regulation of chondrocyte differentiation and maturation

Fibronectin mRNA alternative splicing is temporally and spatially regulated during chondrogenesis in vivo and in vitro

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