Journal: PLoS Biology

Article Title: Force-Induced Unfolding of Fibronectin in the Extracellular Matrix of Living Cells

doi: 10.1371/journal.pbio.0050268

Figure Lengend Snippet: Spatial ratiometric images and histograms of all pixels within each field of view are shown for dimeric amine/cys Fn-DA in 0 and 1 M GdnHCl and monomeric amine/cys Fn-DA in 1 and 4 M GdnHCl (A). Amine/cys Fn-DA was added to the culture medium of fibroblasts for 24 h, and excess Fn-u was added to suppress intermolecular energy transfer. Confocal microscopic images of acceptor and donor peak intensities taken 1 μm above the glass–cell interface were background subtracted, averaged, and thresholded, and the I A / I D ratiometric image of acceptor to donor was color-coded within the range of 0.05 to 1.0. A histogram (B) for all pixels of amine/cys Fn-DA–containing ECM (C) and an overlay of I A / I D on the DIC image (D) are shown in a region in which the matrix showed a transition from low to intermediate I A / I D within a single Fn fiber. Histograms are overlaid in (B) for regions of extended (E; purple) and unfolded Fn (F; pink). Histograms were generated with 0.01-ratio-unit bin widths. Scale bars = 25 μm.

Article Snippet: Primary human dermal fibroblast cells derived from foreskins (PromoCell, http://www.promocell.com/ ) were maintained for less than eight passages in Fibroblast Growth Medium plus Supplement (PromoCell).

Techniques: Generated

Journal: PLoS Biology

Article Title: Force-Induced Unfolding of Fibronectin in the Extracellular Matrix of Living Cells

doi: 10.1371/journal.pbio.0050268

Figure Lengend Snippet: Amine/cys Fn-DA and excess Fn-u were added to the culture medium of fibroblasts for 24 h. Color-coded I A / I D ratiometric images are shown for control cells (A), extracted cell-free matrix (B), and fibroblast cells after 60 min exposure to the ROCK inhibitor Y-27632 (C). Histograms with 0.01-ratio-unit bin widths for all pixels of control (black), cell-free (purple), and ROCK-inhibited matrix (pink) were derived from three random fields of view each from three separate experiments in each group (D). Solution denaturation values for dimeric Fn-DA in 0 M GdnHCl and monomeric Fn-DA in 1 and 4 M GdnHCl are shown as red, green, and blue lines, respectively. Scale bars = 50 μm.

Article Snippet: Primary human dermal fibroblast cells derived from foreskins (PromoCell, http://www.promocell.com/ ) were maintained for less than eight passages in Fibroblast Growth Medium plus Supplement (PromoCell).

Techniques: Control, Derivative Assay

Journal: PLoS Biology

Article Title: Force-Induced Unfolding of Fibronectin in the Extracellular Matrix of Living Cells

doi: 10.1371/journal.pbio.0050268

Figure Lengend Snippet: A schematic of the strain device is shown in the relaxed configuration with length L before (A) and length L + DL after (B) application of strain. PDMS sheets were covalently modified with Fn-u as described in Materials and Methods, and fibroblast cells were cultured for 24 h in the presence of amine/cys Fn-DA and excess Fn-u. Cells were extracted in mild detergent. Color-coded I A / I D ratiometric images are shown for a field of view without application of stretch (C) and after application of 70% elongation strain with 28% transverse compression (D). Region of interest analysis on individual fibrils was used to determine the impact of elongation on I A / I D on a per fibril basis (circles, mean ± standard deviation), and binned averages were calculated for fibrils between −37% and −20%, −20% and −10%, −10% and 10%, 10% and 40%, and 40% and 73% strain (red squares, mean ± standard deviations) (E). Abscissa is also plotted as relative length change. Solution values for dimeric Fn-DA in 0 M GdnHCl and monomeric Fn-DA in 1 and 4 M GdnHCl are shown as horizontal red, green, and blue lines, respectively. Scale bars = 50 μm.

Article Snippet: Primary human dermal fibroblast cells derived from foreskins (PromoCell, http://www.promocell.com/ ) were maintained for less than eight passages in Fibroblast Growth Medium plus Supplement (PromoCell).

Techniques: Modification, Cell Culture, Standard Deviation

Journal: PLoS Biology

Article Title: Force-Induced Unfolding of Fibronectin in the Extracellular Matrix of Living Cells

doi: 10.1371/journal.pbio.0050268

Figure Lengend Snippet: Cys/cys Fn-DA (A–C) or amine/cys Fn-DA (D–G) was incorporated into fibroblast matrix on Fn-u that was adsorbed to plasma cleaned PDMS, and after cell extraction the substrate was relaxed to 4/5 (A and B; 3.7% transverse stretch) or 3/5 the starting length (D–F; 10% transverse stretch). I A / I D ratiometric images of cys/cys Fn-DA–containing matrix are shown at the PDMS–ECM interface (A), where a portion of the cell-free fibers are still attached to the substrate, and from the same field of view but acquired 3 μm above the PDMS surface (B), where the strain-free Fn mat randomly diffused around its points of attachment to the underlying ECM. Histograms are shown for all pixels within the field of view at the substrate (C; black) and from the upper, strain-free confocal slice (C; pink). An I A / I D ratiometric image of amine/cys Fn-DA is shown with both detached (E) and still-attached (F) regions of matrix within the same confocal slice. Region of interest analysis was used to generate histograms (G0 for all pixels within the detached (E and G; purple) and attached (F and G; pink) regions of matrix, which were overlaid on a histogram of all pixels in the field of view (black). Scale bars = 50 μm.

Article Snippet: Primary human dermal fibroblast cells derived from foreskins (PromoCell, http://www.promocell.com/ ) were maintained for less than eight passages in Fibroblast Growth Medium plus Supplement (PromoCell).

Techniques: Clinical Proteomics, Extraction