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. 2013 May 24;288(21):14805-14.
doi: 10.1074/jbc.M112.435271. Epub 2013 Apr 15.

Regulation of matrix assembly through rigidity-dependent fibronectin conformational changes

Cara L Carraher  1 , Jean E Schwarzbauer
Affiliations

Regulation of matrix assembly through rigidity-dependent fibronectin conformational changes

Cara L Carraher et al. J Biol Chem. .

Abstract

Cells sense and respond to the mechanical properties of their microenvironment. We investigated whether these properties affect the ability of cells to assemble a fibrillar fibronectin (FN) matrix. Analysis of matrix assembled by cells grown on FN-coated polyacrylamide gels of varying stiffnesses showed that rigid substrates stimulate FN matrix assembly and activation of focal adhesion kinase (FAK) compared with the level of assembly and FAK signaling on softer substrates. Stimulating integrins with Mn(2+) treatment increased FN assembly on softer gels, suggesting that integrin binding is deficient on soft substrates. Guanidine hydrochloride-induced extension of the substrate-bound FN rescued assembly on soft substrates to a degree similar to that of Mn(2+) treatment and increased activation of FAK along with the initiation of assembly at FN matrix assembly sites. In contrast, increasing actin-mediated cell contractility did not rescue FN matrix assembly on soft substrates. Thus, rigidity-dependent FN matrix assembly is determined by extracellular events, namely the engagement of FN by cells and the induction of FN conformational changes. Extensibility of FN in response to substrate stiffness may serve as a mechanosensing mechanism whereby cells use pericellular FN to probe the stiffness of their environment.

Keywords: Extracellular Matrix; Fibroblast; Fibronectin; Integrin; Mechanotransduction; Protein Assembly.

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Figures

FIGURE 1.
FIGURE 1.
FN matrix assembly is up-regulated on the rigid substrate. A, NIH 3T3 cells were plated on substrates ranging between 0.3 and 90 kPa, and at 6 h, NIH 3T3-derived FN matrix was detected by staining with anti-FN antiserum. Predicted stiffness values from Moshayedi et al. (24) are in parentheses; measured kPa values are in bold. Scale bar, 50 μm. B, NIH 3T3 cells were plated on soft, intermediate, or rigid substrates (1.6, 4.3, or 7.4 kPa, respectively) and after 6 or 12 h were fixed and stained with anti-FN antiserum to detect NIH 3T3-derived FN matrix. Scale bar, 25 μm. C, NIH 3T3 cells were allowed to spread on gels for 2 h; then rat plasma FN was added to the culture medium at 10 μg/ml; and after an additional 4 or 10 h, cells were fixed and stained with a rat-specific anti-FN antibody (IC3). Scale bar, 25 μm. D, NIH 3T3 cells were treated as in B and then fixed and stained with both anti-collagen I antibody and anti-FN IC3 antibody. Scale bar, 25 μm.
FIGURE 2.
FIGURE 2.
Analysis of DOC-insoluble FN matrix. A, NIH 3T3 cells were allowed to spread on gels for 2 h, then rat plasma FN was added at 10 μg/ml, and cells were lysed in DOC buffer 6 and 12 h after plating. Rat FN in DOC-soluble (sol) and DOC-insoluble (insol) fractions was detected with IC3 monoclonal antibody. The 6- and 12-h blots are matched ECL exposures except for DOC-insoluble FN for which the 6-h samples were exposed for 60 s and the 12-h samples were exposed for 45 s to prevent overexposure. B, quantification of rat FN in DOC-insoluble matrix at 12 h. The band intensities were measured in the linear range, normalized to GAPDH, and then normalized to the 12-h soft sample (mean ± S.E. (error bars), n = 3). C, NIH 3T3 cell were treated as in A, but cells were lysed 12, 24, and 36 h after plating. DOC-insoluble rat FN was detected with IC3 antibody. In A and C, molecular mass markers of 250 kDa for FN blots and 37 kDa for GAPDH blots are indicated by dashes. interm, intermediate.
FIGURE 3.
FIGURE 3.
LPA and calyculin A do not increase FN matrix assembly on soft substrates. A, NIH 3T3 cells were allowed to spread on gels for 2 h at which time rat FN at 10 μg/ml and 10 μm LPA were added. At 6 h after plating, cells were fixed and stained for rat FN using IC3 antibody. Scale bar, 50 μm. B, cells were plated as in A, but 5 nm calyculin A (Caly A) was added instead of LPA. Scale bar, 50 μm. C, cells treated as in A or B were lysed after 6 h, and DOC-insoluble FN was analyzed by immunoblotting with IC3 antibody. Molecular mass markers of 250 kDa for FN blots and 37 kDa for GAPDH blots are indicated by dashes. interm, intermediate.
FIGURE 4.
FIGURE 4.
Mn2+ treatment increases FN matrix assembly on soft substrates. A, the domain structure of an FN subunit is shown. Cell-binding modules containing synergy (in III9) and RGD (in III10) sites and the N-terminal 70 kDa fragment used to identify matrix assembly sites are labeled and underlined. FN-binding sites are indicated by overbars. B and C, NIH 3T3 cells were allowed to spread on gels for 2 h at which time 10 μg/ml rat FN and 1 mm MnCl2 were added. Cells were fixed or lysed after 6 h. B, cells were fixed and stained for rat FN. Scale bar, 25 μm. C, cells were lysed, and DOC-insoluble FN was analyzed by immunoblotting with IC3 antibody. Molecular mass markers of 250 kDa for FN blots and 37 kDa for GAPDH blots are indicated by dashes. D, band intensities from immunoblots of DOC-insoluble FN were quantified with and without Mn2+ or GdnHCl treatment. The band intensities were normalized to the signal for the untreated rigid sample. Values are the mean ± S.E. (error bars) for at least two experiments. interm, intermediate.
FIGURE 5.
FIGURE 5.
Guanidine HCl treatment of substrate FN rescues FN matrix assembly. A, NIH 3T3 cells were plated on untreated or GdnHCl-treated FN gel substrates and allowed to spread for 2 h before the addition of 10 μg/ml rat FN. Cells were fixed and stained for rat FN after 6 h. Scale bar, 25 μm. B, DOC-insoluble rat FN was detected by immunoblotting of cell lysates treated as in A. Molecular mass markers of 250 kDa for FN blots and 37 kDa for GAPDH blots are indicated by dashes. interm, intermediate.
FIGURE 6.
FIGURE 6.
Enhanced formation of FN matrix assembly sites and FAK activation with GdnHCl-FN. A, NIH 3T3 cells were plated on soft substrates coupled with untreated FN or GdnHCl-treated FN and allowed to spread for 2 h before the addition of 20 μg/ml biotinylated 70-kDa and 10 μg/ml rat FN. Cells were fixed and probed with fluorescently labeled streptavidin 30 min later. Representative images are shown. Scale bars, 10 μm. B, fluorescence signal intensity of FN matrix assembly sites was measured for 17 cells in each condition, and the average intensity per pixel was calculated. Error bars represent S.E. C, NIH 3T3 cells were plated on gels with untreated FN or GdnHCl-FN and lysed in modified radioimmunoprecipitation assay buffer after 2 h. Total FAK and FAK phosphorylated on Tyr-397 were measured by immunoblotting. Blots are representative of two independent experiments.
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