Purification of Fibronectin

Akiyama, Steven K.

doi:10.1002/0471143030.cb1005s02

Cited by 17 publications

(19 citation statements)

References 28 publications

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“…Fibronectin was purified from outdated human plasma (American Red Cross, Rochester, NY) using gelatin-Sepharose (GE Healthcare) affinity chromatography [38]. Type I collagen was extracted from rat tail tendons, as described [11].…”

Section: Methodsmentioning

confidence: 99%

Cooperative effects of fibronectin matrix assembly and initial cell–substrate adhesion strength in cellular self-assembly

Brennan

Hocking

2016

Acta Biomaterialia

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The cell-dependent polymerization of intercellular fibronectin fibrils can stimulate cells to self-assemble into multicellular structures. The local physical cues that support fibronectin-mediated cellular self-assembly are largely unknown. Here, fibronectin matrix analogs were used as synthetic adhesive substrates to model cell-matrix fibronectin fibrils having different integrin-binding specificity, affinity, and/or density. We utilized this model to quantitatively assess the relationship between adhesive forces derived from cell-substrate interactions and the ability of fibronectin fibril assembly to induce cellular self-assembly. Results indicate that the strength of initial, rather than mature, cell-substrate attachments correlates with the ability of substrates to support fibronectin-mediated cellular self-assembly. The cellular response to soluble fibronectin was bimodal and independent of the integrin-binding specificity of the substrate; increasing soluble fibronectin levels above a critical threshold increased aggregate cohesion on permissive substrates. Once aggregates formed, continuous fibronectin polymerization was necessary to maintain cohesion. During self-assembly, soluble fibronectin decreased cell-substrate adhesion strength and induced aggregate cohesion via a Rho-dependent mechanism, suggesting that the balance of contractile forces derived from fibronectin fibrils within cell-cell versus cell-substrate adhesions controls self-assembly and aggregate cohesion. Thus, initial cell-substrate attachment strength may provide a quantitative basis with which to build predictive models of fibronectin-mediated microtissue fabrication on a variety of substrates.

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Section: Methodsmentioning

confidence: 99%

Cooperative effects of fibronectin matrix assembly and initial cell–substrate adhesion strength in cellular self-assembly

Brennan

Hocking

2016

Acta Biomaterialia

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“…Fibronectin was purified from outdated human plasma (American Red Cross, Rochester, NY) using gelatin-Sepharose (GE Healthcare Life Sciences, Piscataway, NJ) affinity chromatography [27]. Collagen type I was extracted from rat-tail tendons using acetic acid and then precipitated with NaCl, as described previously [21].…”

Section: Methodsmentioning

confidence: 99%

Regional Fibronectin and Collagen Fibril Co-Assembly Directs Cell Proliferation and Microtissue Morphology

2013

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The extracellular matrix protein, fibronectin stimulates cells to self-assemble into three-dimensional multicellular structures by a mechanism that requires the cell-dependent conversion of soluble fibronectin molecules into insoluble fibrils. Fibronectin also binds to collagen type I and mediates the co-assembly of collagen fibrils into the extracellular matrix. Here, the role of collagen-fibronectin binding in fibronectin-induced cellular self-assembly was investigated using fibronectin-null fibroblasts in an in vitro model of tissue formation. High resolution, two-photon immunofluorescence microscopy was combined with second harmonic generation imaging to examine spatial and temporal relationships among fibronectin and collagen fibrils, actin organization, cell proliferation, and microtissue morphology. Time course studies coupled with simultaneous 4-channel multiphoton imaging identified regional differences in fibronectin fibril conformation, collagen fibril remodeling, actin organization, and cell proliferation during three-dimensional cellular self-assembly. Regional differences in cell proliferation and fibronectin structure were dependent on both soluble fibronectin concentration and fibronectin-collagen interactions. Fibronectin-collagen binding was not necessary for either fibronectin matrix formation or intercellular cohesion. However, inhibiting fibronectin binding to collagen reduced collagen fibril remodeling, decreased fibronectin fibril extension, blocked fibronectin-induced cell proliferation, and altered microtissue morphology. Furthermore, continual fibronectin-collagen binding was necessary to maintain both cell proliferation and microtissue morphology. Collectively, these data suggest that the complex changes in extracellular matrix and cytoskeletal remodeling that mediate tissue assembly are driven, in part, by regional variations in cell-mediated fibronectin-collagen co-assembly.

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“…SCA-9 cells were cultured overnight to 70% confluence, washed with 1X PBS, and grown for 18 hours in serum-free DMEM containing vehicle control or inhibitors. To detect fibrillar FN, purified human plasma FN (Akiyama, 1999) that was labeled with Alexa 647 (Larsen et al, 2006) was added to SCA-9 cell cultures at 0.02 μg/μL in serum-free media. Cells were imaged by time-lapse microscopy for 18 hours or immunostained with L8 monoclonal (Chernousov et al, 1987) to label stretched FN and an anti-FN rabbit polyclonal antibody to label total FN.…”

Section: Methodsmentioning

confidence: 99%

Identification of a mechanochemical checkpoint and negative feedback loop regulating branching morphogenesis

Daley

Gulfo

Sequeira

et al. 2009

Developmental Biology

146

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Cleft formation is the initial step in submandibular salivary gland (SMG) branching morphogenesis, and may result from localized actomyosin-mediated cellular contraction. Since ROCK regulates cytoskeletal contraction, we investigated the effects of ROCK inhibition on mouse SMG ex vivo organ cultures. Pharmacological inhibitors of ROCK, isoform-specific ROCK I but not ROCK II siRNAs, as well as inhibitors of myosin II activity stalled clefts at initiation. This finding implies the existence of a mechanochemical checkpoint regulating the transition of initiated clefts into progression-competent clefts. Downstream of the checkpoint, clefts are rendered competent through localized assembly of fibronectin promoted by ROCK I/myosin II. Cleft progression is primarily mediated by ROCK I/myosin II-stimulated cell proliferation with a contribution from cellular contraction. Furthermore, we demonstrate that FN assembly itself promotes epithelial proliferation and cleft progression in a ROCK-dependent manner. ROCK also stimulates a proliferation-independent negative feedback loop to prevent further cleft initiations. These results reveal that cleft initiation and progression are two physically and biochemically distinct processes.

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Purification of Fibronectin

Cited by 17 publications

References 28 publications

Cooperative effects of fibronectin matrix assembly and initial cell–substrate adhesion strength in cellular self-assembly

Cooperative effects of fibronectin matrix assembly and initial cell–substrate adhesion strength in cellular self-assembly

Regional Fibronectin and Collagen Fibril Co-Assembly Directs Cell Proliferation and Microtissue Morphology

Identification of a mechanochemical checkpoint and negative feedback loop regulating branching morphogenesis

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