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First published online 13 September 2005
doi: 10.1242/jcs.02566

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Stimulatory effects of a three-dimensional microenvironment on cell-mediated fibronectin fibrillogenesis

Department of Molecular Biology, Schultz Laboratory, Princeton University, Princeton, NJ 08544, USA

View larger version (40K): [in a new window]   Fig. 1. Three-dimensionality of cell-free 3D fibronectin matrix. 3D matrix prepared from WI-38 (VA13) cells (A) and a monolayer of WI-38(VA13) cells (B) were immunostained with anti-human fibronectin antibody HFN7.1 and analyzed by confocal microscopy. 0° is the projected image and 90° the 3D image rotated through 90 degrees to show thickness. The basal cell surfaces are to the left. (C) Equivalent proportions of NIH3T3 3D matrix (lane 1) and confluent cell culture (lane 2) solubilized with SDS sample buffer were compared by SDS-PAGE using 6% polyacrylamide and silver staining. The major bands in lanes 1 and 2 were confirmed as fibronectin by immunoblotting (not shown). The location of fibronectin at 250 kDa and molecular mass markers (180 kDa and 116 kDa) are indicated.

View larger version (63K): [in a new window]   Fig. 2. Increased fibronectin matrix assembly by WI-38 (VA13) cells on a 3D fibronectin matrix. WI-38(VA13) cells were seeded on 3D matrix prepared from NIH3T3 cells (3D) or on surfaces coated with 10 µg/ml rat pFN (2D). (A) DOC lysates were prepared at the indicated times. DOC-insoluble samples were analyzed on 5% polyacrylamide-SDS gels and immunoblotted with HFN7.1. (B) Cells were seeded in 24-well dishes at the indicated cell densities. After incubation for 6 hours, DOC-insoluble samples were prepared and analyzed as described in A. (C) WI-38 (VA13) cells seeded for 8 hours on 3D matrix (3D, left) or fibronectin-coated coverslips (2D, right) were fixed and immunostained with HFN7.1 followed by fluorescein goat anti-mouse IgG. (D) DOC-insoluble fibronectin matrix formed by WI-38 human lung fibroblasts (left) and human dermal fibroblasts (right) was analyzed at 8 hours as in A. Bar, 20 µm.

View larger version (14K): [in a new window]   Fig. 3. Quantification of matrix levels. WI-38 (VA13) or NIH3T3 cells were seeded on 3D or 2D substrates in [35S]methionine labeling medium for 6 hours. Fibronectin was isolated from DOC fractions and culture media, resolved by SDS-PAGE, and quantified by analysis with a phosphorimager. Phosphorimager counts for 3D samples were normalized to 2D samples, which were set to 1 for each sample pair. (A) Relative amounts of DOC-insoluble fibronectin compared to total fibronectin synthesized. (B) Relative amounts of total fibronectin (sum of DOC-soluble, DOC-insoluble and secreted).

View larger version (71K): [in a new window]   Fig. 4. Matrix incorporation of exogenous fibronectin on 2D and 3D fibronectin substrates. (A) CHO(B2)5 cells were plated on 2D and 3D substrates in the presence of 10 µg/ml rat pFN. Fibronectin levels in DOC-insoluble matrix were detected with IC3 anti-rat fibronectin antibody at the indicated times. Exogenous fibronectin was also incubated with 3D matrix in the absence of cells and background binding was analyzed at 23 hours (far right-hand lane). (B) 3x105 CHO(B2)5 cells were plated on substrates with 10 µg/ml rat pFN with or without 125 µg/ml 70 kDa fibronectin fragment. DOC-insoluble samples were collected and analyzed by immunoblotting after 7 hours incubation. The right lane shows no background fibronectin binding in the absence of cells. (C) CHO(B2)5 cells plated with 4 µg/ml exogenous rat pFN on 3D (left) and 2D (right) substrates for 15 hours were fixed and stained with IC3 antibody and fluorescein-goat anti-mouse IgG plus rhodamine-conjugated phalloidin. Bars, 20 µm.

View larger version (80K): [in a new window]   Fig. 5. Distribution of newly assembled and pre-existing fibrils formed on a 3D matrix. CHO(B2)5 cells were incubated in medium with 4 µg/ml rat pFN on coverslips with 3D human fibronectin matrix. After incubation for 15 hours, samples were fixed and stained sequentially with IC3 and fluorescein-goat anti-mouse IgG to detect rat fibronectin fibrils followed by biotinylated HFN7.1 and rhodamine-streptavidin to detect human fibronectin 3D matrix. (A) Projected confocal images of newly assembled rat pFN fibrils (left), 3D human fibronectin matrix (middle) and the merged image (right). (B) Fluorescence intensity profiles of four areas (lines 1-4 in merged image of A) were analyzed using laser-scanning microscopy LSM 510 version 3.2 software (Zeiss).

View larger version (43K): [in a new window]   Fig. 6. 3D architecture plays a key role in stimulating fibronectin matrix assembly. (A) CHO(B2)5 cells in medium supplemented with rat fibronectin (FN) were plated on 3D matrix or on 2D fibronectin-coated substrates with no other additions (3D, 2D) or with conditioned medium (+CM) or resuspended matrix added (+resus matrix). 3D matrix was incubated with rat fibronectin but without cells (3D, right lane). DOC-insoluble material was prepared after 16 hours and fibronectin was detected with IC3 antibody. (B) Confluent WI-38(VA13) cells were lysed and incubated with a 2D fibronectin substrate. CHO(B2)5 cells were plated on 3D, 2D and 2D+lysate substrates. DOC-insoluble fractions were prepared and analyzed as in A. (C) CHO(B2)5 cells were plated on 3D fibronectin matrix (3D), on compressed matrix (Comp) or on 2D substrate in the presence of 5 µg/ml rat pFN. After incubation for 7 hours and 18 hours, DOC-insoluble fractions were prepared and analyzed. The immunoblot was developed with ECL plus western blotting detection system and band intensities were quantified. Numbers below each lane represent relative amounts of fibronectin normalized to 3D samples, which were set to 1 for each time point.

View larger version (40K): [in a new window]   Fig. 7. Controls for the effects of 3D matrix pliability. (A) CHO(B2)5 cells were plated in medium supplemented with rat fibronectin on Matrigel (MG), Matrigel containing 60 µg/ml fibronectin (MG+FN), 3D fibronectin matrix (3D) or 2D fibronectin substrate (2D). (B) Cells were plated in medium supplemented with rat fibronectin (FN) on 3D fibronectin matrix (3D), 2D fibronectin substrate (2D), or 3D fibronectin matrix that had been fixed in 4% paraformaldehyde (fixed 3D). 3D and fixed 3D (right lanes) were incubated with fibronectin but without cells. (C) Cells were seeded on 3D substrates in the absence (-) and presence (+) of 100 µg/ml 9D2 antibody for 7 hours. DOC-insoluble fractions were analyzed by immunoblotting with IC3.

View larger version (44K): [in a new window]   Fig. 8. Stimulatory effects of 3D matrix on integrin activities. (A) HT1080 cells were plated on substrates in the presence of 20 µg/ml rat pFN. After 15 hours, 1 mM MnCl2 was added to one set of cells (+). After an additional 4-hour incubation, DOC-insoluble samples were prepared and analyzed by immunoblotting with IC3. (B) CHO(B2)vß3 or CHO(B2) cells were allowed to attach for 2 hours on substrates prepared from WI-38 (VA13) cells (3D) or coated with human pFN (2D). Rat pFN was added at the indicated concentrations followed by incubation for another 15 hours. The DOC-insoluble fractions were analyzed by immunoblotting with IC3 antibody against rat fibronectin. (C) CHO(B2)vß3 cells were plated as described in B but with 40 µg/ml rat pFN for 27 hours followed by an additional 4 hours with 1 mM MnCl2. The DOC-insoluble samples were prepared as in B. (D) CHO(B2)5 and CHO(B2)vß3 cells were allowed to attach to 3D fibronectin matrices for 1 hour. 1 µg/ml SAM-1 anti-5 function-blocking antibody or 10 µg/ml LM609 anti-vß3 function-blocking antibody were then added to the cells with 10 µg/ml rat fibronectin (+). After incubation for 16 hours, the DOC-insoluble samples were collected and analyzed by immunoblotting with IC3 antibody.