Structure of Fibronectin and Its Fragments in Electron Microscopy

Price, Todd M.; Rudee, M. L.; Pierschbacher, Michael D.; Ruoslahti, Erkki

doi:10.1111/j.1432-1033.1982.tb07058.x

Cited by 53 publications

(49 citation statements)

References 26 publications

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“…Purified fibronectin molecules visualized after rotary shadowing were about 2 nm thick (Erickson et al, 1981;Engel et al, 1981;Odermatt et al, 1982;Price et al, 1982;Tooney et al, 1983) and were often bent or coiled (Erickson et al, 1981). These properties were similar to those of the filaments observed in our studies at the periphery of microfibrils.…”

Section: Association Between Microfibrils and Fibronectinsupporting

confidence: 73%

Association of fibronectin with the microfibrils of connective tissue

et al. 1989

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The association of fibronectin with the microfibrils of connective tissue was examined in the zonular fibers of the mouse eye by immunohistochemical methods at the light and electron microscopic level. Mouse eyes fixed in formaldehyde were embedded either in paraffin for immunostaining by the peroxidase-antiperoxidase (PAP) method or in Lowicryl for immunolabeling by antirabbit globulin antibodies bound to 5 or 15 nm gold particles. Ultrastructural studies were also carried out after glutaraldehyde perfusion. Both the PAP and immunogold procedures demonstrated the association of fibronectin with microfibrils. After immunolabeling with 5 nm gold particles, examination at high magnification localized fibronectin to fine filaments that appeared to be attached to the surface of microfibrils. The filaments extended outward singly or formed loose aggregates. Their diameter ranged from 1.2 to 3 nm, with a mean of 1.5 nm. Because of their similarity to the fibronectin molecules previously described after rotary shadowing, the filaments were likely to be fibronectin molecules themselves. Since fibronectin is known to have high affinity for the amyloid P component, a model is presented in which fibronectin filaments are bound to the amyloid P component making up the tubular core of microfibrils in mice. Evidence is presented that fibronectin filaments may link microfibrils to one another and thus insure the continuity and strength of zonular fibers. More generally, it is likely that connective-tissue microfibrils, whether or not inserted into elastic fibers, are bonded through fibronectin to surrounding cells, collagen fibrils, or proteoglycans, and thus insure cohesion among connective tissue elements.

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Section: Association Between Microfibrils and Fibronectinsupporting

confidence: 73%

Association of fibronectin with the microfibrils of connective tissue

et al. 1989

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“…Furthermore, these results also suggest that not all domains of Fn are equally influenced by the interaction of the molecule with the substrate. Finally, differences in the conformation of Fn adsorbed onto different surfaces have been independently demonstrated using biophysical techniques, including electron spin resonance (Narasimhan and Lai, 1989), infrared spectroscopy (Pitt et al, 1987), total internal reflection fluorescence (Iwamoto et al, 1985), fluorescence polarization (Williams et al, 1982), and rotary shadowing (Price et al, 1982;Erickson and Carrell, 1983), as well as biological assays, such as antibody binding (Grinnell and Feld, 1982;Underwood et al, 1993;Pettit et al, 1994) and cell adhesion strength (Iuliano et al, 1993;García et al, 1998a). …”

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confidence: 99%

Modulation of Cell Proliferation and Differentiation through Substrate-dependent Changes in Fibronectin Conformation

Garcı́a

Vega

Boettiger

1999

MBoC

612

549

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Integrin-mediated cell adhesion to extracellular matrices provides signals essential for cell cycle progression and differentiation. We demonstrate that substrate-dependent changes in the conformation of adsorbed fibronectin (Fn) modulated integrin binding and controlled switching between proliferation and differentiation. Adsorption of Fn onto bacterial polystyrene (B), tissue culture polystyrene (T), and collagen (C) resulted in differences in Fn conformation as indicated by antibody binding. Using a biochemical method to quantify bound integrins in cultured cells, we found that differences in Fn conformation altered the quantity of bound ␣ 5 and ␤ 1 integrin subunits but not ␣ v or ␤ 3 . C2C12 myoblasts grown on these Fn-coated substrates proliferated to different levels (B Ͼ T Ͼ C). Immunostaining for muscle-specific myosin revealed minimal differentiation on B, significant levels on T, and extensive differentiation on C. Differentiation required binding to the RGD cell binding site in Fn and was blocked by antibodies specific for this site. Switching between proliferation and differentiation was controlled by the levels of ␣ 5 ␤ 1 integrin bound to Fn, and differentiation was inhibited by anti-␣ 5 , but not anti-␣ v , antibodies, suggesting distinct integrin-mediated signaling pathways. Control of cell proliferation and differentiation through conformational changes in extracellular matrix proteins represents a versatile mechanism to elicit specific cellular responses for biological and biotechnological applications. INTRODUCTIONThe adhesion of cells to their substrate through an extracellular matrix provides signals that influence their ability to survive, proliferate, and express specific developmental phenotypes (Menko and Boettiger, 1987;Werb et al., 1989;Adams and Watt, 1990;Streuli et al., 1991;Zhu et al., 1996;Chen et al., 1997). One of the early examples was the development of in vitro culture conditions that permitted the differentiation of avian myogenic cells into contracting myotubes (Hauschka and Konigsberg, 1966;Bischoff and Holtzer, 1968). The critical element for this system was the precoating of tissue culture surfaces with rat tail collagen. This general principle of providing an appropriate substrate to permit the expression of developmental phenotypes has been applied to a wide variety of cells. These include systems that allow the maintenance of neurons and outgrowth of growth cones (Westerfield, 1987) and the recapitulation of the stages of mammary gland development and involution (Li et al., 1987;Barcellos-Hoff et al., 1989). These findings indicate that critical elements of the message directing the expression of a differentiated phenotype are encoded in the extracellular matrix.Cells interact with extracellular matrices primarily through integrins, a widely expressed family of cell surface receptors (Hynes, 1987), and integrin binding to its extracellular ligand is responsible for the downstream effects of the matrix on cell function. For example, in the muscle differentiation syste...

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“…15 In the present study, we extend our previous work and demonstrate that CBD-BMP4 is retained longer at the targeted sites compared to BMP4 alone and induced augmented bone formation even when injected without scaffold. Considering that the 100 kDa of fibronectin fragment that includes CBD (45 kDa) was reported to be about 24 nm, 16 CBD size can be regarded as a nanocarrier. When applied to cranial bone defects, CBD-BMP4 successfully induced new and accelerated bone formation as compared to BMP4 alone.…”

Section: Introductionmentioning

confidence: 99%

“…First-strand complementary deoxyribonucleic acid (cDNA) was constructed from 2 µg of total RNA with oligo (dT) as primers, [12][13][14][15][16][17][18] and the cDNAs were used as a template for polymerase chain reaction (PCR). Quantitative real-time PCR was performed with SYBR PCR master mix (Agilent Technologies, Santa Clara, CA, USA) and specific primers.…”

mentioning

confidence: 99%

Enhanced in vivo osteogenesis by nanocarrier-fused bone morphogenetic protein-4

Sugiyama¹,

Kitajima²,

Mazaki³

et al. 2013

IJN

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Purpose: Bone defects and nonunions are major clinical skeletal problems. Growth factors are commonly used to promote bone regeneration; however, the clinical impact is limited because the factors do not last long at a given site. The introduction of tissue engineering aimed to deter the diffusion of these factors is a promising therapeutic strategy. The purpose of the present study was to evaluate the in vivo osteogenic capability of an engineered bone morphogenetic protein-4 (BMP4) fusion protein.Methods: BMP4 was fused with a nanosized carrier, collagen-binding domain (CBD), derived from fibronectin. The stability of the CBD-BMP4 fusion protein was examined in vitro and in vivo. Osteogenic effects of CBD-BMP4 were evaluated by computer tomography after intramedullary injection without a collagen-sponge scaffold. Recombinant BMP-4, CBD, or vehicle were used as controls. Expressions of bone-related genes and growth factors were compared among the groups. Osteogenesis induced by CBD-BMP4, BMP4, and CBD was also assessed in a bone-defect model. Results: In vitro, CBD-BMP4 was retained in a collagen gel for at least 7 days while BMP4 alone was released within 3 hours. In vivo, CBD-BMP4 remained at the given site for at least 2 weeks, both with or without a collagen-sponge scaffold, while BMP4 disappeared from the site within 3 days after injection. CBD-BMP4 induced better bone formation than BMP4 did alone, CBD alone, and vehicle after the intramedullary injection into the mouse femur. Bone-related genes and growth factors were expressed at higher levels in CBD-BMP4-treated mice than in all other groups, including BMP4-treated mice. Finally, CBD-BMP4 potentiated more bone formation than did controls, including BMP4 alone, when applied to cranial bone defects without a collagen scaffold. Conclusion: Altogether, nanocarrier-CBD enhanced the retention of BMP4 in the bone, thereby promoting augmented osteogenic responses in the absence of a scaffold. These results suggest that CBD-BMP4 may be clinically useful in facilitating bone formation.

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Structure of Fibronectin and Its Fragments in Electron Microscopy

Cited by 53 publications

References 26 publications

Association of fibronectin with the microfibrils of connective tissue

Association of fibronectin with the microfibrils of connective tissue

Modulation of Cell Proliferation and Differentiation through Substrate-dependent Changes in Fibronectin Conformation

Enhanced in vivo osteogenesis by nanocarrier-fused bone morphogenetic protein-4

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