Proteins, Reagents, and Cells

Fibronectin was purified from outdated human plasma (American Red Cross, Rochester, NY) using gelatin-Sepharose (GE Healthcare Life Sciences, Piscataway, NJ) affinity chromatography [27]. Collagen type I was extracted from rat-tail tendons using acetic acid and then precipitated with NaCl, as described previously [21]. The His-tagged peptides, R1R2 and III-11C were expressed in Escherichia coli, purified over Ni-Sepharose, and dialyzed extensively against PBS, as described previously [28]. The 76-mer R1R2 peptide encompasses amino acids Gly¹⁹⁵-Thr²⁵³ from the bacterial adhesin protein, SFS [28,29]. R1R2 binds to fibronectin [29] and inhibits the binding of fibronectin to native and denatured collagens I, II, and III [26]. The concentration of purified peptides was determined using a BCA assay (Pierce). Peptides were filter-sterilized and purity was assessed by SDS-polyacrylamide gel electrophoresis. Peptides were stored in aliquots at -80°C. Antibodies and their sources are as follows: Alexa Fluor⁵⁹⁴-conjugated anti-BrdU mAb, Alexa Fluor⁴⁸⁸-labeled phalloidin, Alexa Fluor⁶⁴⁷-conjugated goat anti-rabbit IgG, (Invitrogen, Carlsbad, CA); polyclonal anti-fibronectin (Sigma). Tissue culture supplies were from Corning/Costar (Cambridge, MA). Unless otherwise indicated, chemical reagents were from Sigma.

Mouse embryonic fibronectin-null fibroblasts (FN-null MEFs) (provided by Dr. Jane Sottile, University of Rochester, Rochester, NY) [16] were cultured on collagen type I-coated dishes at 37°C and 8% CO₂ using a 1:1 mixture of Aim V (Invitrogen) and Cellgro® (Mediatech, Herndon, VA). These media do not contain fibronectin and do not require serum supplementation [16]. Thus, no exogenous source of fibronectin is present during routine culture. FN-null MEFs express α1, αv, α5, β1, and β3 integrin subunits, and do not express α2 and α4 integrin subunits [16]. Both collagens I and III are synthesized by FN-null MEFs [26].

Microscopy

Native collagen gels were prepared as described [21] by mixing 4X concentrated Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies), collagen, and 1X DMEM on ice such that the final mixture contained 1 mg/ml collagen and 1X DMEM. FN-null MEFs were seeded on polymerized collagen gels (5.26 x 10⁴ cells/cm²) in 35 mm tissue culture dishes. Four hours after seeding, fibronectin (25 or 100 nM) was added to wells and cells were incubated for 2, 4, or 6 days. In some experiments, fibronectin was pre-incubated for 60 min with the peptides R1R2 or III-11C (62.5 nM or 2.5 µM), or an equal volume of the vehicle control, PBS.

To visualize actively proliferating cells, bromodeoxyuridine (BrdU; 100 µM; BD Biosciences) was added to wells 4 h prior to fixation. Cells were fixed with 1:1 acetone/methanol (-20°C) or 4% paraformaldehyde (37°C) and permeabilized with 0.05% Triton X-100. Fibronectin was immunolabeled overnight using anti-fibronectin polyclonal antibodies. Cell nuclei, actin, BrdU, and fibronectin were visualized using 4’,6-diamidino-2-phenylindole (DAPI; 60 nM), Alexa⁴⁸⁸-labeled phalloidin (1:200), Alexa⁵⁹⁴-labeled anti-BrdU monoclonal antibody (1:200), and Alexa⁶⁴⁷-labeled goat anti rabbit antibody (1:200), respectively.

Immunofluorescence images were collected using an Olympus Fluoview 1000 AOM-MPM multiphoton microscope equipped with a 25X, 1.05 NA water immersion lens (Olympus). Fluorophores were simultaneously excited at 780 nm using a femtosecond Mai Tai HP Deep See Ti:Sa laser (Spectra-Physics, Mountain View, CA, USA). DAPI, Alexa⁴⁸⁸, and Alexa⁵⁹⁴ emitted fluorescence were separated using a 505 nm long-pass dichroic mirror and filtered using a 460 nm-bandpass filter (#FF01-460-80, Semrock), a 519 nm-bandpass filter (#BA495-546, Olympus), and a 609 nm-bandpass filter (#FF01-609/54-25, Semrock), respectively. Alexa⁶⁴⁷ emitted fluorescence was separated using a 750 nm short-pass dichroic mirror and filtered with a 670 nm-bandpass filter (#FF01-670/30-25, Semrock).

Cross-reactivity was assessed for each secondary antibody utilized. Briefly, Alexa-labeled goat anti-rabbit, goat anti-mouse, and donkey anti-goat secondary antibodies were used to stain non-target primary antibodies, and fluorescence was imaged using two-photon microscopy. Antibody cross-reactivity did not occur with any of the Alexa Fluor-conjugated secondary antibodies tested. Single-labeled controls were also used to assess channel crosstalk [30]. FN-null MEFs, adherent to polymerized collagen gels, were incubated with fibronectin (25 nM) for 4 days. Fibronectin was detected using an anti-fibronectin polyclonal antibody followed by Alexa Fluor⁴⁸⁸-, Alexa Fluor⁵⁹⁴-, or Alexa Fluor⁶⁴⁷-conjugated goat anti-rabbit IgGs, and images were obtained. Crosstalk occurred from the Alexa Fluor⁴⁸⁸ channel into the Alexa Fluor⁵⁹⁴ channel. To correct for 488- to 594-channel crosstalk in 4-channel imaging, Alexa Fluor⁴⁸⁸ images (actin) were subtracted from their respective Alexa Fluor⁵⁹⁴ images (BrdU) using the image calculator function of ImageJ (NIH, Bethesda, Maryland) [31,32].

Collagen was visualized with second-harmonic generation microscopy [33] using the Olympus Fluoview 1000 AOM-MPM multiphoton microscope described above. Collagen second-harmonic fluorescence was separated using a 505 nm long-pass dichroic mirror and filtered using a 390 nm-bandpass filter (#FF01-390/40-25, Semrock). Image brightness and contrast were enhanced offline using Adobe Photoshop®. Images within each figure were brightness and contrast enhanced to the same extent.

Aberrations in the 750 nm short-pass dichroic mirror resulted in Alexa Fluor⁶⁴⁷ images that were unaligned in the x-y direction as compared to images collected using the 505 nm long-pass dichroic mirror (DAPI, Alexa Fluor⁴⁸⁸, and Alexa Fluor⁵⁹⁴). To correct the aberration, Alexa Fluor⁴⁸⁸ emitted fluorescence was collected with both the 505 nm long-pass dichroic mirror (target image) and the 750 nm short-pass dichroic mirror (reference image). Image alignment was performed offline using ImageJ. Target and reference images were merged and the aberration quantified by measuring the lateral and vertical distance between identical landmarks in the merged image. Alexa Fluor⁶⁴⁷ images were then aligned using the translate function in ImageJ with the calculated lateral and vertical aberrations as the respective x- and y-offsets [34].

Image Analysis

The spatial organization of proliferating cells was determined from 4-channel images (DAPI, BrdU, actin, fibronectin) using ImageJ. To determine the relative distance of a BrdU-positive cell from the microtissue centroid (r_a/r_b), actin and BrdU image stacks were merged. r_a/r_b was calculated as the ratio between the straight-line distance from the microtissue centroid to the BrdU-positive cell (r_a) and the straight-line distance from the microtissue centroid to the perimeter of the microtissue (r_b). To determine the microtissue centroid, actin image slices corresponding to the microtissue-collagen substrate interface were thresholded using ImageJ’s Modified IsoData method. The centroid of the microtissue was then calculated from the thresholded actin image using ImageJ’s image moments calculator. The perimeter of a microtissue was defined by actin staining. The ratio, r_a/r_b was calculated for all proliferating cells throughout the vertical height of a microtissue. Nine total multicellular structures per condition (day 2 and day 4 with 25 or 100 nM fibronectin) from 3 independent experiments were used to calculate r_a/r_b.

A theoretical calculation of the expected distribution of r_a/r_b for randomly proliferating cells within a circle was also performed. The circle diameter was defined as 1 unit, and the center of the circle was set at the origin of a Cartesian coordinate system (x =0, y =0). The x-coordinate and y-coordinate for 200 ‘proliferating’ cells within the circle were randomly assigned using a random number generator. The distance from the origin of the circle to each ‘proliferating’ cell (r_a) was calculated using Pythagorean’s theorem. The distance from the origin of the circle to the edge of the circle (r_b) was equal to the radius of the circle (0.5 units). The percent of ‘proliferating’ cells present within a 0.1 unit bin range were recorded on a histogram. The analysis was repeated ten times and the mean percent ‘proliferating’ cells within r_a/r_b ≤ 0.5 and r_a/r_b > 0.5 ± the standard error of the mean (SEM) was calculated using GraphPad Prism software (La Jolla, CA).

To determine the total number of proliferating cells in each microtissue, BrdU image stacks were loaded into ImageJ and BrdU-positive nuclei were counted manually using ImageJ’s cell counter function. Total cell number in microtissues was determined similarly using DAPI image stacks. The percent BrdU-positive cells for each microtissue was calculated as the number of BrdU-positive cells divided by total cell number.

Cell Proliferation

Monodispersed FN-null MEFs were seeded on polymerized collagen gels (5.26 x 10⁴ cells/cm²) in 48-well tissue culture plates. Cells were allowed to adhere to the collagen substrate for 4 h and then treated with fibronectin (25 or 100 nM; 12.5 or 50 μg/ml) in the absence or presence of either R1R2 or a control peptide III-11C (62.5 nM or 2.5 µM for 25 and 100 nM fibronectin, respectively) [26]. For some experiments, fibronectin-treated cells were incubated for 3 days to establish microtissues. Media were then removed and replaced with fresh media containing R1R2- or III-11C-treated fibronectin. For all experiments, fibronectin and peptides were preincubated for 60 min. Cells were incubated for up to 6 days at 37°C and 8% CO₂. Bright-field images were obtained using an Olympus BX60 microscope and photographed using a QImaging MicroPublisher 3.3 RTV digital camera. Cell number was determined using 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (USB, Cleveland, OH), as previously described [21]. Growth assay measurements are expressed as mean ± SEM and represent 1 of at least 3 separate experiments performed in triplicate.

Statistical Analysis - Statistical comparisons were performed using either one-way analysis of variance (ANOVA) followed by Tukey’s post-test or Student’s t-test using GraphPad Prism software. Results were considered statistically significant when p<0.05.

Dynamics of Fibronectin, Actin, and Proliferating Cells within Microtissues

The active assembly of fibronectin fibrils by cells adherent to native collagen substrates stimulates cell proliferation and induces cells to self-assemble into multicellular structures [21,35]. These 3D structures reach a vertical height of ~50 μm five days after a single fibronectin treatment [21]. Microtissue height exhibits a biphasic response to fibronectin concentration, with peak height occurring in response to 25 nM fibronectin [21]. In the present study, 4-channel multi-photon microscopy was used to simultaneously visualize and compare spatial patterns of fibronectin fibrils, actin filaments, and proliferating cells during microtissue assembly. FN-null MEFs, cultured in serum-free media were utilized in this study in order to precisely control the timing and amount of fibronectin added to cells. Additionally, high (100 nM) and low (25 nM) concentrations of fibronectin were applied in order to produce two distinct microtissue morphologies [21]. To clearly illustrate these different morphologies, fibronectin-stained, 3D reconstructed images of day 6 microtissues were projected perpendicular to the imaging plane, and are shown in Figure 1A. FN-null MEFs treated with 25 nM fibronectin produced adherent 3D multicellular aggregates characterized by central dome-shaped cores that extended onto the collagen substrate (Figure 1A). In contrast, cells treated with 100 nM fibronectin formed dense, sheet-like structures with broad surface areas (Figure 1A), having an average vertical height of ~20 μm [21]. In the absence of fibronectin, cells remain dispersed and rounded on the native collagen substrate and do not proliferate [21].

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Figure 1. Spatial and temporal changes in cell proliferation, actin organization, and fibronectin matrix.

FN-null MEFs adherent to native collagen type I gels were treated with 25 or 100 nM fibronectin (FN). (A) After a 6-day incubation, samples were immunostained for fibronectin. Z-slices (1-μm step size) were reconstructed in 3D and projected along the x-y plane. Images represent 1 of 3 experiments performed in triplicate. (B) After either a 2- or 4-day incubation, cells were processed for immunofluorescence microscopy. DAPI, BrdU, actin, and FN images were collected simultaneously along the z-axis at 1-μm intervals using two-photon microscopy. Z-slices corresponding to the microtissue-collagen interface were projected along the z-plane using ImageJ. Scale bar, 50 µm. Images represent 1 of 3 experiments performed in triplicate.

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Time course studies showed that 2 days after addition of 25 nM or 100 nM fibronectin, cells had assembled into discontinuous cell sheets (Fig. 1Ba, b). Proliferating cells were observed throughout the cell sheets (Fig. 1Be, f, i, j). At this time point, actin stress fibers were prominent (Fig. 1Bm, n) and an abundant fibrillar fibronectin matrix was present that extended across the surface of the collagen substrate (Fig. 1Bq, r). By day 4, cell sheets induced by 25 nM fibronectin had reorganized to produce the dome-shaped aggregates (Fig 1Bc, g). The central core of these 3D aggregates contained densely packed, non-proliferating cells (Fig. 1Bg, k), cortical actin (Fig. 1Bo), and pericellular fibronectin (Fig. 1Bs). In contrast to the central core, peripheral regions of the 3D aggregates contained proliferating cells (Fig. 1Bk), actin stress fibers (Fig. 1Bo), and fibrillar fibronectin that radiated out from the central core (Fig 1Bs). In response to 100 nM fibronectin, cell sheets increased in cell density between day 2 and day 4 (Fig. 1Bb, d), but did not transition into the dome-shaped 3D structures. Actin stress fibers and fibrillar fibronectin fibrils were observed throughout the cell sheets (Fig. 1Bp, t). On day 6, structures formed in response to 25 nM or 100 nM fibronectin appeared identical to their respective day 4 structures (not shown).

Merged, 4-channel images are shown in Figure 2. The discontinuous cell sheets formed on day 2 in response to either fibronectin concentration showed similar patterns of co-localized actin stress fibers and fibronectin fibrils, with actively proliferating cells distributed throughout the multicellular structures (Figure 2A; d2, 25 and 100 nM FN). Similar patterns of actin stress fibers, fibronectin fibrils, and proliferating cells were observed on day 4 in response to 100 nM fibronectin (Figure 2A; d4, 100 nM FN). In contrast, 3D aggregates formed on day 4 in response to 25 nM fibronectin showed regional differences in actin and fibronectin staining with cortical actin and pericellular fibronectin co-localized within the compact, central core (Figure 2A; d4, 25 nM FN). Extended, radially aligned fibronectin fibrils and actin stress fibers projected from the edge of the core towards the periphery, where proliferating cells were observed (Figure 2A; d4, 25 nM FN). Higher magnification images obtained from the central and peripheral regions of the 3D aggregate (d4, 25 nM FN) demonstrate distinct organizational patterns of pericellular (Fig. 2Ba) and extended (Fig. 2Bb) fibronectin fibrils, respectively.

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Figure 2. Merged 4-channel images of Day 2 and Day 4 microtissues.

Collagen-adherent FN-null MEFs were treated with 25 or 100 nM fibronectin (FN). After a 2- or 4-day incubation, cells were processed for immunofluorescence microscopy. (A) DAPI (blue), BrdU (red), actin (green), and FN (white) images were collected simultaneously as described in the legend to Figure 1. Z-slices corresponding to the microtissue-collagen interface were projected along the z-plane, merged, and aligned using ImageJ. Scale bar, 50 µm. Images represent 1 of 3 experiments performed in triplicate. (B) Representative FN staining obtained from the core (a) and periphery (b) of day 4 microtissues formed in response to 25 nM FN. Note the different organizational patterns of pericellular (a) and extended (b) fibronectin fibrils. Scale bar, 100 μm.

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To quantitatively assess whether regional differences in cell proliferation occurred as a function of soluble fibronectin concentration, the distance of each proliferating, BrdU-positive cell from the geometric center of the microtissue was measured and the ratio, r_a/r_b, was calculated. A ratio value of 0 or 1.0 corresponds to a proliferating cell located on the centroid or edge of a multicellular structure, respectively. A theoretical calculation of the expected distributions of r_a/r_b values for randomly ‘proliferating’ cells within a circle was performed in parallel. The theoretical mean percent BrdU-positive cells within 0 ≤ r_a/r_b ≤0.5 and r_a/r_b > 0.5 for randomly proliferating cells within a circle was 26% ± 0.7% and 74% ± 1%, respectively.

Proliferating cells were present in all r_a/r_b bins for day 2 (Fig. 3Aa, b) and day 4 cell sheets (Fig. 3Ad). The percentage of BrdU-positive cells located within 0 ≤ r_a/r_b ≤0.5 and r_a/r_b > 0.5 for day 4 cell sheets formed in response to 100 nM fibronectin was 30% ± 1% and 69% ± 1%, respectively, confirming the random appearance of proliferating cells in the immunofluorescence images (Figure 2A). In contrast, proliferating cells were shifted to the outer r_a/r_b bins of 3D aggregates formed in response to 25 nM fibronectin (Fig. 3Ac); the mean percentages of BrdU-positive cells within 0 ≤ r_a/r_b ≤0.5 and r_a/r_b > 0.5 of 3D aggregates on day 4 were 8% ± 3% and 92% ± 3%, respectively. As a final verification of this spatial organization of proliferating cells, images collected on day 4 were reconstructed in 3D and the center z-slices were projected along the x-y plane. Proliferating cells preferentially localized to the peripheral regions of 3D aggregates (Figure 3B; +25 nM FN). In contrast, proliferating cells were randomly distributed throughout the cell sheets (Figure 3B; +100 nM FN). The reduction in cell proliferation found within the central core of the 3D aggregates was not due to an overall decrease in the rate of cell proliferation, as the percent of BrdU-positive cells did not decrease between days 2 and 4 (Figure 3C; 25 nM FN). Moreover, the percentage of proliferating cells was not significantly different between fibronectin concentrations (Figure 3C). Taken together, these data demonstrate the development of regional variations in the location of actively proliferating cells that is dependent on soluble fibronectin concentration.

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Figure 3. Spatial and temporal analysis of fibronectin-induced cell proliferation.

Collagen-adherent FN-null MEFs were treated with 25 nM or 100 nM fibronectin (FN) for either 2 or 4 days, and then processed for immunofluorescence microscopy. Four channel images of fibronectin, actin, BrdU, and DAPI staining were obtained and processed to quantify the spatial distribution of proliferating cells in microtissues. (A) The relative distance of all BrdU-positive cells from the centroid (r _a/r_b) was calculated. Data are grouped to display the frequency of occurrence of proliferating cells from the centroid (r _a/r_b = 0) to the periphery (r_a/r_b = 1). Data are presented as the mean percent of BrdU-positive cells ± SEM of 3 independent experiments. (B) Representative x-y projections showing BrdU (red) and DAPI (blue) staining on day 4. (C) Total numbers of proliferating and non-proliferating cells were determined from BrdU and DAPI staining. Data are presented as mean percent BrdU-positive cells + SEM of 3 experiments performed in triplicate.

https://doi.org/10.1371/journal.pone.0077316.g003

Fibronectin-mediated Collagen Organization, Cell Proliferation, and Cellular Self-assembly

Two photon microscopy and second harmonic generation imaging were utilized to visualize fibronectin and collagen fibril organization within the discontinuous cell sheets that formed 2 days after fibronectin addition, prior to the observed spatial segregation of proliferating cells. In the absence of fibronectin, minimal cell-mediated collagen fibril reorganization was observed (Figure 4a, d). In contrast, addition of fibronectin to collagen-adherent cells triggered the reorganization of collagen fibrils into long, thick bundles (Figure 4g, j). Merged, z-stack images of collagen and fibronectin (Figure 4h, k) staining collected immediately at and above the polymerized collagen gel interface showed extensive co-localization of extended collagen and fibronectin fibrils radiating away from the organized cell networks (Figure 4i, l; arrows). Collagen fibrils were largely absent from the very center of cell sheets, while fibronectin fibril staining was prominent (Figure 4i, l).

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Figure 4. Collagen and fibronectin fibril co-localization within cell networks.

Collagen-adherent FN-null cells were incubated for 2 days in the absence (0 nM FN; d-f) or presence of either 25 nM (g-i) or 100 nM (j-l) fibronectin (FN). Also included are images of polymerized collagen gels incubated for 2 days in the absence of both cells and FN (a-c). Collagen (a,d,g,j) was visualized using second harmonic generation microscopy. FN (b,e,h,k) was visualized using an anti-FN pAb followed by Alexa⁴⁸⁸-conjugated anti-rabbit IgG. FN and collagen were simultaneously visualized along the z-axis at 1-μm intervals using two-photon microscopy and then projected onto the z-plane using ImageJ. Note that images were collected at and above the collagen surface, and do not include images collected below the surface. Merged images of collagen (blue) and FN (green) staining are shown (c,f,i,l). Co-localized signals produce cyan (arrows). Scale bar, 50 μm.

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Side projection images of the day 2 cell sheets confirm the presence of distinct regions where collagen and fibronectin fibrils colocalized (Figure 5). Z-slice images obtained at 1-μm intervals beginning this time below the collagen gel interface and ending above the cell network surface were reconstructed in 3D and projected along the z-plane. Dense nodes of collagen and fibronectin co-localization were prominent at the edges of the individual cell sheets (Figure 5, arrows; cyan). At both fibronectin concentrations, collagen-free fibronectin fibrils were observed on the apical surfaces of the cell sheets (Figure 5; green), while the collagen substrate was clearly visible below the fibronectin layer (Figure 5; blue). These studies demonstrate regional variations in ECM fibronectin and collagen co-assembly within self-organizing cell sheets. These regional differences in ECM organization occur prior to the spatial shift in cell proliferation (Figure 3).

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Figure 5. Collagen-fibronectin co-localization at the microtissue-substrate interface.

Representative x-y projections of 2-day microtissues formed in response to either 25 nM or 100 nM fibronectin (FN). FN (green) and collagen (blue) were visualized as described in the legend to Figure 4. Z-slices (1-μm step size) were collected beginning below the collagen surface and extending above the microtissue. Images were reconstructed in 3D and then projected along the x-y plane. Arrows denote areas of co-localization (cyan). Note the non-overlapping FN and collagen signals on the apical and basal surfaces, respectively.

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To determine whether fibronectin-collagen binding interactions are required for cell proliferation or tissue self-assembly, studies were conducted using the inhibitory peptide, R1R2, a peptide derived from the bacterial adhesin SFS, that has sequence homology to collagen [29]. R1R2 binds to fibronectin [29] and inhibits the binding of fibronectin to native and denatured collagens I, II, and III [26]. Previous work demonstrated that R1R2 inhibits fibronectin-mediated assembly of ECM collagen I fibrils, but does not affect collagen III assembly [26]. We first tested the effects of R1R2 on fibronectin-induced cell proliferation. FN-MEFs seeded on polymerized collagen gels in the absence of fibronectin do not proliferate [21] and remain rounded over the course of 6 days (Fig. 6Ba,f). The increase in cell proliferation that occurs in response to either 25 nM or 100 nM fibronectin was completely inhibited by addition of R1R2 (Figure 6A), and was not affected by addition of the control peptide, III-11C (Figure 6A), demonstrating specificity of the R1R2 fragment. Neither R1R2 nor III-11C reduced cell numbers in the absence of fibronectin (Figure 6A). These data indicate that binding of fibronectin to collagen is required for ECM fibronectin-induced proliferation of cells that are adherent to native collagen substrates.

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Figure 6. Cell proliferation and microtissue morphology require fibronectin-collagen binding.

Collagen-adherent FN-null MEFs were treated with PBS (0 FN) or fibronectin (25 or 100 nM) in the absence or presence of R1R2, the control peptide III-11C, or an equal volume of the vehicle, PBS. (A) Following a 6-day incubation, cell number was determined using MTT. Data are presented as fold increase in cell number versus non-treated (+PBS/PBS) controls + SEM of 3 experiments performed in triplicate. *Significant vs. +PBS/PBS, p<0.001 (ANOVA). ^#Significant vs. respective +FN/PBS and +/III-11C, p<0.001 (ANOVA). (B) Representative phase contrast images collected on days 2 and 6 of culture. Images represent 1 of 3 experiments performed in triplicate. Scale bar, 50 μm.

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The effect of blocking fibronectin-collagen binding on cellular self-assembly was assessed next. Interestingly, addition of R1R2 to fibronectin-treated samples did not block cellular self-assembly but instead, shifted day 2 morphologies from discontinuous cell sheets (Fig. 6Bb, d) to 3D spheroids (Fig. 6Bc, e). Differences in microtissue morphology were also observed on day 6, where addition of R1R2 to 25 nM or 100 nM fibronectin-treated cells also resulted in the formation of spheroids (Fig. 6Bh, j) rather than 3D cell aggregates (Fig. 6Bg) or dense cell sheets (Fig 6Bi). Thus, binding of fibronectin to collagen contributes to the morphology of multicellular structures, but is not required for fibronectin-induced intercellular cohesion.

Fibronectin-Collagen Co-assembly and Microtissue Morphology

To examine further the role of collagen-fibronectin co-assembly in microtissue morphology, we assessed whether fibronectin-collagen binding interactions play a role in determining the structural pattern of ECM fibrils. Collagen-adherent FN-null cells were treated with fibronectin in the presence of either R1R2 or III-11C and cultured for 4 days. Fibronectin and collagen fibrils were visualized simultaneously using two-photon and second harmonic generation imaging, respectively. Similar to the day 2 images shown in Figure 4, elongated fibronectin fibrils (Figure 7a; arrowheads) that co-localized with collagen fibers (Figure 7b, c; arrowheads) were assembled at the periphery of 3D aggregates formed in response to 25 nM fibronectin pretreated with the control peptide, III-11C. In contrast, addition of R1R2 to block binding of fibronectin to collagen resulted in disorganized fibronectin fibrils (Figure 7d; arrows) and reduced cell-mediated collagen remodeling (Figure 7e; arrows). Similar inhibitory effects of R1R2 on fibronectin fibril organization (Figure 7j) and collagen remodeling (Figure 7k) were observed in the presence of 100 nM fibronectin. En face (Figure 7c, f, i, l) and side projection images (Figure 8) demonstrate that addition of R1R2 to fibronectin-treated cultures similarly affected microtissue morphology at either fibronectin concentrations, resulting in the formation of symmetrical 3D spheroids (Figures 7f, l and 8) that contained pericellular fibronectin fibrils (Figure 8).

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Figure 7. Collagen-fibronectin binding is required for ECM fibril organization.

FN-null MEFs adherent to polymerized collagen were treated with fibronectin (25 or 100 nM) in the presence of either R1R2 or the control peptide III-11C. Following a 4-day incubation, cells were processed for immunofluorescence microscopy. Fibronectin (green) and collagen (blue) were visualized using an anti-FN pAb and second harmonic generation imaging, respectively. Images were collected at and above the collagen substrate and then projected onto the z-plane using ImageJ. Dashed red lines denote spheroid location. Arrowheads indicate colocalizing fibronectin and collagen fibrils. Arrows show fibronectin fibrils that do not colocalize with collagen fibrils. Scale bar, 50 μm.

https://doi.org/10.1371/journal.pone.0077316.g007

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Figure 8. Microtissue morphology depends on fibronectin-collagen binding.

Collected images of 4-day microtissues formed in response to either 25 nM or 100 nM fibronectin (FN), pretreated with either R1R2 or III-11C, were reconstructed in 3D and projected onto the x-y plane. Fibronectin (green) and collagen (blue) are shown. Scale bar, 50 μm.

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To determine whether continuous fibronectin-collagen binding interactions are required to maintain microtissue shape or fibronectin-stimulated cell proliferation, microtissues were first allowed to form for 3 days (Fig. 9Aa, b). Media were then removed and replaced with fresh media containing fibronectin and either R1R2 or III-11C peptides. In both cases, addition of R1R2 to either 3D aggregates (Fig. 9Aa) or cell sheets (Fig. 9Ab) triggered a morphological transition to 3D spheroids (Fig. 9Ac, d). Interestingly, addition of R1R2 to preformed 3D aggregates produced numerous small-diameter spheroids (Fig. 9Ac), while addition of R1R2 to preformed cell sheets produced spheroids with larger diameters (Fig. 9Ad). Moreover, the increases in cell number induced by fibronectin over the full course of 6 days were inhibited by addition of R1R2 on day 3 (Figure 9B). Together, these data indicate that binding of fibronectin to collagen is required to maintain microtissue morphologies and fibronectin-mediated cell proliferation. Disruption of collagen-fibronectin interactions after microtissues have formed triggers changes in microtissue shape and disrupts fibronectin-induced cell proliferation.

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Figure 9. Disrupting collagen-fibronectin binding alters microtissue shape and reduces cell proliferation.

Collagen-adherent FN-null MEFs were treated with 25 or 100 nM fibronectin. Following a 3-day incubation, media were removed and replaced with media containing fibronectin (25 or 100 nM) pretreated with R1R2 or III-11C. Cells were then incubated an additional 3 days. (A) Phase contrast images were collected on day 3 prior to treatment with R1R2 (a,b) and again on day 6 (c,d). Images represent 1 of 3 experiments performed in triplicate. Scale bar, 100 μm. (B) Cell number was determined on day 6 using MTT. Data are presented as fold increase in cell number versus non-treated (0 nM FN) controls + SEM of 3 experiments performed in triplicate. *Significant vs. ‘-FN’, p<0.001 (ANOVA). ^#Significant vs. respective ‘+FN’ and ‘+FN/III-11C’, p<0.001 (ANOVA).

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