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First published online 28 April 2009
doi: 10.1242/jcs.040824

Journal of Cell Science 122, 1647-1653 (2009)
Published by The Company of Biologists 2009
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A novel mode of cell detachment from fibrillar fibronectin matrix under shear

Adam J. Engler1,*, May Chan2, David Boettiger2 and Jean E. Schwarzbauer1,{ddagger}

1 Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
2 Department of Microbiology, University of Pennsylvania, Philadelphia, PA 19104, USA


Figure 1
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  		Fig. 1. Cell detachment under shear. (A) HT1080 cells were allowed to attach to a FN-coated glass coverslip (circles) or to fibrillar FN matrix on a coverslip (squares) for 1 hour before applying shear with the spinning disc device. After spinning, attached cells were counted at various distances from the center, and the fraction of cells attached was normalized to the number at the center of the sample where shear approaches zero. Attached fraction was plotted versus the shear and fit by a sigmoidal curve. The shear needed to detach 50% of the cells was determined from this plot. (B) Low-magnification fluorescence images were captured after spinning. i, ii and iii show cells attached at low, moderate and maximal shear, respectively, for both FN-coated glass and fibrillar FN matrix, as indicated in A.

 

Figure 2
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  		Fig. 2. Effects of adhesion time and matrix stiffness on adhesion strength. (A) Cells were allowed to adhere to substrates for increasing times before spinning. Adhesion strengths ({tau}50) determined from data as in Fig. 1 were plotted as a function of cell adhesion time. Substrates included FN-coated glass (open squares), fibrillar FN matrix (gray squares) and fibrillar FN matrix crosslinked (XL'ed) with 10% formaldehyde (10F, black squares) or with factor XIIIa transglutaminase (fXIIIa, star). (B) A cell attachment assay was used to measure the number of cells bound per image (~3x105 µm2) over time on substrates as indicated in A. Exponential fits in both panels were used to measure adhesion strength or attachment time constants. (C) Atomic force microscopy was used to determine the stiffness of untreated fibrillar FN matrix (none) and those treated with 1% glutaraldehyde (1G), 3.7% or 10% formaldehyde (3.7F or 10F, respectively), or coagulation Factor XIIIa at 5 or 15 µg/ml. *P<0.0001 compared with untreated control. Error bars for all plots represent the mean ± s.d. of triplicate measurements.

 

Figure 3
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  		Fig. 3. Fluorescence imaging of fibrillar matrices after shear. After spinning, FN fibrils were visualized by staining with anti-FN antibodies and rhodamine-labeled secondary antibodies (red). Attached HT1080 cells are labeled with calcein green. (A) Samples from three areas of the coverslip corresponding to 0, 100 and 250 dynes/cm2 are shown. White arrows indicate putative holes in the matrix. (B) Enlarged fluorescence images at known radial positions (i, iii and v) and corresponding thresholded representations (ii, iv and vi) where bright fluorescence from the matrix is shown in white and dark areas are black. Filled arrowheads denote pores in the matrix found throughout all samples while open arrowheads indicate larger features called holes in the matrix. Note the increased number of open arrowheads as a function of shear stress.

 

Figure 4
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  		Fig. 4. Quantitative comparison of hole formation. Features of various sizes identified in thresholded images (as in Fig. 3B) were counted and averaged from triplicate samples, then plotted as histograms. Matrix spun without cells (black line), matrix before spinning with cells (light gray region), matrix after spinning but at an area of minimal shear (gray region) and matrix after high shear spinning (dark gray region) are shown for (A) 0.4 and (B) 4.5 kPa (10% formaldehyde) fibrillar FN matrix. Pores are ~100-300 µm2 and holes are 500-1000 µm2.

 

Figure 5
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  		Fig. 5. Fibril distribution after shear. (A) Summed confocal images of matrix stained with anti-FN antibodies taken in areas of shear corresponding to 0 and 250 dynes/cm2. Note the hole (outlined in red) in the matrix spun at 250 dynes/cm2. (B) Fluorescence intensities under the lines in panel A are plotted versus distance along the line, with red regions indicating areas threefold brighter than the average hole intensity. Assuming that fibril fluorescence intensity is additive, the average peak intensity of single fibrils, 62±15 a.u., was used to establish the scale to the right of the lower plot. Using this scale and the indicated peaks (open arrowheads), we calculated that lines from matrices exposed to 0 and 250 dynes/cm2 shear stress contained 65 and 49 fibrils, respectively. The cross-sectional area under the line was calculated by multiplying 10 µm matrix thickness by the distance along the line (x-axis in panel B, excluding the hole area for the 250 dynes/cm2 line). Fibril density was then calculated by dividing the total number of fibrils by the cross-sectional area, which gave similar fibril densities: 0.08±0.04/µm2 and 0.07±0.03/µm2 for 0 and 250 dynes/cm2, respectively.

 

Figure 6
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  		Fig. 6. Adhesion strength depends on {alpha}5β1 integrin. (A) HT1080 cells were untreated or pretreated with function-blocking antibodies LM609 and BIIG2 against {alpha}vβ3 and {alpha}5β1 integrins, respectively, then allowed to attach to fibrillar matrix for 3 hours. The number of attached cells per image (~3x105 µm2) after washing was measured. Blocking {alpha}5β1 integrin via BIIG2 or a combination of both antibodies resulted in reduced cell binding. Error bars represent the s.d. of triplicate experiments. *P<0.005. (B) Cells were plated on 0.4 or 4.5 kPa matrix in the absence or presence of the indicated concentrations of cyclic RGD or RAD peptides. Adhesion strength, {tau}50, was determined after 3 hours of adhesion. Error bars represent the s.d. of triplicate experiments. *P<0.01. (C) Cells were plated on 0.4 kPa fibrillar matrix in the presence and absence of 10 µM cRGD. Immunofluorescence staining with anti-FN antiserum was performed after spinning. White arrows identify holes in matrix at 250 dynes/cm2.

 

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