Purification of Fibronectin

Akiyama, Steven K.

doi:10.1002/0471143030.cb1005s60

Cited by 7 publications

(2 citation statements)

References 35 publications

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“…Fibronectin was purified from outdated human plasma (American Red Cross, Rochester, NY) using gelatin-Sepharose (GE Healthcare) affinity chromatography (51). Collagen I was extracted from rat tail tendons with 0.5 M acetic acid and precipitated with sequential NaCl precipitation (52).…”

Section: Reagentsmentioning

confidence: 99%

Assembly of fibronectin fibrils selectively attenuates platelet-derived growth factor–induced intracellular calcium release in fibroblasts

Farrar¹,

Hocking

2018

Journal of Biological Chemistry

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Cellular responses to platelet-derived growth factor (PDGF) are altered in a variety of pathological conditions, including cancers, fibroses, and vascular diseases, making PDGF-induced signaling pathways important therapeutic targets. The limited success of therapies designed to impact PDGF pathways may be overcome with a clearer understanding of how cells integrate signals from PDGF and the extracellular matrix (ECM). Here, we assessed the effects of fibronectin matrix assembly on the responsiveness of mesenchymal cells to PDGF. Our results indicate that fibroblast-mediated assembly of fibronectin fibrils attenuates intracellular calcium release in response to PDGF. The dose-dependent inhibition of PDGF-induced intracellular calcium release was specific to the ECM form of fibronectin. Further, a recombinant protein engineered to mimic ECM fibronectin similarly attenuated intracellular calcium release in response to PDGF. Of note, fibronectin attenuated the PDGFcalcium signaling axis at the level of phosphoinositide 3-kinase (PI3K) activation. Interestingly, ECM fibronectin did not alter other intracellular signals activated by PDGF, including activation of PDGF receptor ␤, AKT Ser/Thr kinase, phospholipase C␥1, and extracellular signal-regulated kinase 1/2 (ERK1/2). Rather, fibronectin inhibited activation of the p55 regulatory subunit of PI3K in response to a variety of stimuli, indicating that ECM fibronectin selectively attenuates the intracellular calcium release cascade while leaving intact other PDGF signaling pathways. Selective regulation of calcium signaling by ECM fibronectin via the p55 regulatory subunit of PI3K represents a mechanism by which cells tune their response to PDGF and may therefore serve as a target to selectively regulate one branch of PDGF signaling.

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Section: Reagentsmentioning

confidence: 99%

Assembly of fibronectin fibrils selectively attenuates platelet-derived growth factor–induced intracellular calcium release in fibroblasts

Farrar¹,

Hocking

2018

Journal of Biological Chemistry

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“…Fibronectin. This can be homemade [12] or purchased from a number of suppliers. Fibronectin stocks are to be preserved at -80ºC in small-sized (~100 µl at ~1mg/ml) aliquots.…”

Section: Methodsmentioning

confidence: 99%

Multiparametric Analysis of Focal Adhesions in Bidimensional Substrates

Talayero

Vicente‐Manzanares

2020

The Integrin Interactome

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Focal adhesions in planar substrates constitute an excellent natural cellular resource to evaluate different parameters related to cell morphology, cytoskeletal organization and adhesive strength. However, their intrinsic heterogeneity in terms of size, molecular composition, orientation, etc., complicates their analysis. Here, we describe a simple and straightforward ImageJ/Fiji-based method to quantify several parameters that describe the morphology and relative composition of focal adhesions. This type of analysis can be implemented in various ways and become useful for drug and shRNA screenings.

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Durotaxis by Human Cancer Cells

DuChez

Doyle

Dimitriadis

et al. 2019

Biophysical Journal

158

153

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Durotaxis is a type of directed cell migration in which cells respond to a gradient of extracellular stiffness. Using automated tracking of positional data for large sample sizes of single migrating cells, we investigated 1) whether cancer cells can undergo durotaxis; 2) whether cell durotactic efficiency varies depending on the regional compliance of stiffness gradients; 3) whether a specific cell migration parameter such as speed or time of migration correlates with durotaxis; and 4) whether Arp2/3, previously implicated in leading edge dynamics and migration, contributes to cancer cell durotaxis. Although durotaxis has been characterized primarily in nonmalignant mesenchymal cells, little is known about its role in cancer cell migration. Diffusible factors are known to affect cancer cell migration and metastasis. However, because many tumor microenvironments gradually stiffen, we hypothesized that durotaxis might also govern migration of cancer cells. We evaluated the durotactic potential of multiple cancer cell lines by employing substrate stiffness gradients mirroring the physiological stiffness encountered by cells in a variety of tissues. Automated cell tracking permitted rapid acquisition of positional data and robust statistical analyses for migrating cells. These durotaxis assays demonstrated that all cancer cell lines tested (two glioblastoma, metastatic breast cancer, and fibrosarcoma) migrated directionally in response to changes in extracellular stiffness. Unexpectedly, all cancer cell lines tested, as well as noninvasive human fibroblasts, displayed the strongest durotactic migratory response when migrating on the softest regions of stiffness gradients (2-7 kPa), with decreased responsiveness on stiff regions of gradients. Focusing on glioblastoma cells, durotactic forward migration index and displacement rates were relatively stable over time. Correlation analyses showed the expected correlation with displacement along the gradient but much less with persistence and none with cell speed. Finally, we found that inhibition of Arp2/3, an actin-nucleating protein necessary for lamellipodial protrusion, impaired durotactic migration.For all live-cell fluorescence experiments, DMEM without phenol red or FluoroBrite DMEM (GIBCO) were used, with each supplemented with a 1:100 ratio of Oxyfluor (Oxyrase, Mansfield, OH) and 10 mM DL-lactate (Sigma, St. Louis, MO) to reduce photobleaching and phototoxicity, Durotaxis by Human Cancer Cells

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Purification of Fibronectin

Cited by 7 publications

References 35 publications

Assembly of fibronectin fibrils selectively attenuates platelet-derived growth factor–induced intracellular calcium release in fibroblasts

Assembly of fibronectin fibrils selectively attenuates platelet-derived growth factor–induced intracellular calcium release in fibroblasts

Multiparametric Analysis of Focal Adhesions in Bidimensional Substrates

Durotaxis by Human Cancer Cells

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