Fibronectin's major binding sites and an example of module unfolding under tensile stress. (A) Fibronectins are dimeric molecules composed of over 50 repeats of three different β-sheet modules (FnI, FnII, and FnIII). One monomer of the type of fibronectin found in human blood plasma is shown in (A); fibronectin produced by cells may contain additional alternatively spliced modules, as indicated. Fibronectins contain a large number of molecular recognition and cryptic sites, including the cell binding site RGD, which is recognized by multiple integrins;77 the synergy site PHSRN, which is recognized by α5β1 and αIIbβ3 integrins;78,79 the sequence IDAPS at the FnIII₁₃₋₁₄ junction in the Heparin II region of fibronectin, which supports α4β1-dependent cell adhesion;80,81 and the NGR motif in FnI₅, which is non-enzymatically converted to isoDGR and can then bind the αvβ3 integrin.82,83 A similar, highly conserved, NGR motif occurs in FnI₇, but has not been extensively studied.83 The cryptic sites include various Fn self-assembly sites whose exposure is needed to induce fibronectin fibrillo-genesis,51 a cryptic fragment from FnIII₁ that localizes to lipid rafts and stimulates cell growth and contractility,84 and a binding site for tenascin.85 One cryptic site with enzymatic activity is the FnCol-ase, which is a metalloprotease in the collagen binding domain of plasma fibronectin capable of digesting gelatin, helical type II and type IV collagen, α- and β-casein, and insulin β-chain.86 Other enzymatically active cryptic sites include Fn-ase, a proteinase specific to Fn, actin, and myosin;87 and a disulfide isomerase.88 Finally, there are two cryptic, non-disulfide-bonded cysteines on each monomer, in modules FnIII₇ and FnIII₁₅ which are utilized in this study to site-specifically attach the acceptor fluorophores. (B) Tensile stress applied to Fn fibers causes changes in the quaternary, tertiary, and secondary structure of Fn molecules.33 Figure B shows three FnIII modules with intact secondary structure (top) and with the partial unfolding of one module due to increased tensile stress (bottom). (Unfolding of the center module was simulated with the molecular dynamics program NAMD. The ribbon diagrams were assembled using Maya (Autodesk) software.).

Fn matrix assembly and unfolding on rigid and flexible polyacrylamide surfaces. (A–D) Fluorescence and phase contrast images of Fn matrices and fibroblasts on rigid and flexible polyacrylamide surfaces (4 h culture). Fn matrix assembly occurs on both rigid and flexible polyacrylamide surfaces with differences in matrix organization and cell shape. (A) On rigid surfaces, matrix is abundant, and fibrils appear linear and are formed both around the cell periphery and underneath cells. (B) Cells on rigid surfaces are well spread, with many protrusions extending from the main cell body. (C) On flexible surfaces, the matrix is more sparse and concentrated mostly underneath the cell body. (D) Cells on flexible surfaces are not well spread and cover a smaller surface area than cells on rigid surfaces. Scale bar 15 mm. (E) FRET distributions from matrix fibrils on rigid and flexible surfaces after 4 h of culture (approximately 200−400 fibrils). The boxes represent the 25th to the 75th percentile and the ‘whiskers’ show the 2nd and 98th percentiles. FRET from fibrils on the rigid surface falls below the 1 M calibration point, indicating that Fn is partially unfolded on rigid surfaces. FRET values on the flexible surface shows that Fn is primarily extended. Differences are statistically significant (ANOVA test, p ⪡ 0.001).

Staining for β1 integrin on rigid and flexible polyacrylamide surfaces and in 3D matrices. Top: Cells on polyacrylamide gels. (A) Vigorous recruitment of β1 into fibrillar adhesions on rigid surfaces. (B) Reduced integrin β1 staining is visible on flexible surfaces in structures reminiscent of fibrillar adhesions. Scale bar is 15 μm. (C) Box and whisker plot of adhesion lengths on rigid (n = 242) and flexible surfaces (n = 309). The top and bottom vertical lines denote the 98th and 2nd percentile values. Distributions are significantly different, the * denotes p < 0.001. Bottom: Cells on glass. (D–F) Typical adhesion structures containing β1 integrins shown over time. (D) At 24 h very few adhesion-like structures were present and those tended to be very short. (E) Over time the β1-containing adhesions became increasingly elon-gated, seeming to reach a kind of steady state by 72 h. Scale bar is 10 mm. (F) Box and whisker plots of integrin lengths at different timepoints. The * denotes a significance of p < 0.001; N = 195, 200, 160, 160, and 143 for each time point, respectively.

FRET distributions at different time points in Fn matrix assembly. (A) FRET distributions as a function of time shown as percentiles. The vertical lines show the 2nd and the 98th percentile values; the boxes show the 25th and 75th percentile values. (B) FRET from 24 h and 96 h matrices before and after cells were extracted. In 24 h matrices (left) extraction of cells produced a modest increase in FRET mostly due to the loss of the lower FRET values (N = 345). In 96 h matrices (right) a large increase in FRET occurred after removal of cells, although not to the same level as observed in denuded 24 h matrices (N = 455).

Location and angular arrangement of newly made fibrils compared to the oldest (0−24 h) and intermediate (24−48 h) fibrils in a 72 h matrix. (A) Comparison of matrix fibrils made from 0−24 h and with matrix undergoing active assembly at the 72 h time point based on staining with the 70 kDa Fn fragment (see Materials and Methods). Since the angle of bisection can be measured from either side, in order to increase signal to noise the acute angle was chosen. The angular distribution clustered around 0 and 90 degrees. N = 124. (B) Same comparison as described above, but newly made fibrils are compared to intermediate fibrils (24−72 h). The distribution is sharply centered around 0 degrees, indicating that most fibrils are made tangentially to their immediate predecessors. N = 115. Representative images are also shown.

Matrix assembled by cells during the last 1 h of assembly. (A) Labeled Fn (black bars) was added to 24, 48 or 72 h matrices during the last hour of assembly. (B) The morphology of newly added fibrils appears similar at each time point. (C) FRET measurements show major differences in unfolding in Fn in newly assembled matrix. At 72 h, Fn is unfolded to a much greater extent than in either 48 or 24 h cultures.

Images of cells and extracellular matrix at 24 and 96 h. (A–B) At 24 h, large numbers of non-polarized cells populate isotropically oriented matrix fibrils. (C–D) At 4 days, the matrix is very dense and aligned with the long axis of cells; cells have acquired a highly elongated in vivo-like spindle shapes. Scale bar is 20 μm.

Pulse-chase experiment in a 72 h matrix. (A) Time periods in which samples were given doubly-labeled Fn (black) or unlabled Fn (gray). (B) Representative images of matrix morphology for fibrils made from 0−24 h (1), 24−48 h (2), or 48−72 h (3). (C) FRET (I_A/I_D) distributions of fibers produced during the three, 24 h time periods. Not all matrix Fn is stretched to the same extent, but rather there is progressive unfolding that results in the older matrix being more unfolded than the recently assembled.