Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Feb 15;319(4):546-55.
doi: 10.1016/j.yexcr.2012.10.005. Epub 2012 Oct 29.

Fibroblast cluster formation on 3D collagen matrices requires cell contraction dependent fibronectin matrix organization

Bruno da Rocha-Azevedo  1 , Chin-Han Ho, Frederick Grinnell
Affiliations

Fibroblast cluster formation on 3D collagen matrices requires cell contraction dependent fibronectin matrix organization

Bruno da Rocha-Azevedo et al. Exp Cell Res. .

Abstract

Fibroblasts incubated on 3D collagen matrices in serum or lysophosphatidic acid (LPA)-containing medium self-organize into clusters through a mechanism that requires cell contraction. However, in platelet-derived growth factor (PDGF)-containing medium, cells migrate as individuals and do not form clusters even though they constantly encounter each other. Here, we present evidence that a required function of cell contraction in clustering is formation of fibronectin (FN) fibrillar matrix. We found that in serum or LPA but not in PDGF or basal medium, cells organized FN (both serum and cellular) into a fibrillar, detergent-insoluble matrix. Cell clusters developed concomitant with FN matrix formation. FN fibrils accumulated beneath cells and along the borders of cell clusters in regions of cell-matrix tension. Blocking Rho kinase or myosin II activity prevented FN matrix assembly and cell clustering. Using siRNA silencing and function-blocking antibodies and peptides, we found that cell clustering and FN matrix assembly required α5β1 integrins and fibronectin. Cells were still able to exert contractile force and compact the collagen matrix under the latter conditions, which showed that contraction was not sufficient for cell clustering to occur. Our findings provide new insights into how procontractile (serum/LPA) and promigratory (PDGF) growth factor environments can differentially regulate FN matrix assembly by fibroblasts interacting with collagen matrices and thereby influence mesenchymal cell morphogenetic behavior under physiologic circumstances such as wound repair, morphogenesis and malignancy.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Fibroblast clustering in DMEM medium containing FBS
(A) Time-lapse phase contrast images (from Supplemental Movie 1) of cells cultured for 18h on collagen matrices in DMEM/FBS. Images shown are from the same microscopic field at the times indicated. Bar = 100 μm. (B) Scanning electron microscopic image of a collagen matrix incubated with fibroblasts in DMEM/FBS for 18h. At the end of the incubation, samples were fixed and processed for SEM. Bar = 10 μm.
Figure 2
Figure 2. Structural organization and molecular composition of cell clusters
(A) Fluorescence microscopy image of a fibroblast cluster formed during 18h in DMEM/FBS fixed and stained for actin and nuclei (PI). (B) Same as “A” except stained for α5 integrin (green), FN (red), and cell nuclei (Hoechst, blue). (C) Same as “A” except washed once with cold PBS, and treated for 10 min at 37°C in PBS without or with 1% deoxycholate (DOC) as indicated after which the samples were washed three times and fixed and stained for fibronectin. Bar = 100 μm.
Figure 3
Figure 3. Rhodamine-fibronectin organization during cell cluster formation
Time-lapse phase contrast/fluorescence images (from Supplemental Movie 2) of cells cultured 18h on collagen matrices in DMEM/FBS containing 5 μg/ml rhodamine-FN. Images shown are from the same microscopic field at the times indicated. (A) 1 h; (B) 4h; (D) 8h; (D) 18 h. Bar = 100 μm.
Figure 4
Figure 4. FN organization and cell cluster formation in LPA-containing medium
(A) Fibroblasts were cultured for 18h on collagen matrices in DMEM/FBS and DMEM/LPA as indicated. At the end of the incubations, samples were fixed and stained for FN and Hoechst 33342. Bar = 100 μm. (B) Z-plane distribution of FN in a fibroblast cluster formed during 18h in DMEM/LPA fixed and stained for FN (red), α5 integrin (green), and nuclei (blue). (i) top area of the cluster; (ii) upper part of the cluster; (iii) lower part of the cluster; (iv) bottom of the cluster. Supplemental Movie 3 shows the full set of confocal microscopy-generated 0.5 μm Z stacks. Bar = 50 μm.
Figure 5
Figure 5. Effect of varying the growth factor environment on FN distribution and cell cluster formation
Cells were cultured for 18h on collagen matrices in DMEM containing FBS, PDGF and no growth factor (basal) as indicated. The PDGF and basal incubations contained 20 μg/ml plasma FN. At the end of the incubations, samples were fixed and stained for actin, FN and nuclei (Hoechst) as indicated. Bar = 100 μm.
Figure 6
Figure 6. Blocking Rho kinase and myosin II contraction inhibits FN matrix assembly and cell cluster formation
Fluorescence microscopy images of fibroblasts on collagen matrices during 18h in DMEM/FBS containing 5 μM Y27632 and 20 μM blebbistatin as indicated. At the end of the incubations, samples were fixed and stained for actin, FN and nuclei (Hoechst). Bar = 100 μm.
Figure 7
Figure 7. Effect of function blocking antibodies against α5 and β1 integrin subunits on fibroblast cluster formation
(A) Cells were cultured for 18h on collagen matrices in DMEM/FBS containing 100 μg/ml monoclonal antibodies against β1 (MAb13) or α5 (MAb16) as indicated. At the end of the incubations, samples were fixed and stained for actin and nuclei (PI). (B) Samples were incubated on FN-coated coverslips for 1h and then fixed and stained for actin. Bar =100 μm.
Figure 8
Figure 8. Silencing α5 integrin inhibits FN matrix organization and cell cluster formation
(A) Western blotting results showing α5 integrin and actin detection for cells after transfection with specific α5 or mock siRNA. (B) Extent of matrix compaction by mock and α5-silenced cells cultured 18h on collagen matrices in DMEM/FBS. Compaction was calculated as final matrix height/starting matrix height. (C) Mock and α5 integrin silenced cells were cultured for 18h on collagen matrices in DMEM/FBS. At the end of the incubations, samples were fixed and stained for actin and PI. (D) same as “C” except the cells were stained for α5 integrin and FN. Bar = 100 μm.
Figure 9
Figure 9. Blocking FN inhibits cell cluster formation
(A) Fibroblasts were cultured for 18h on collagen matrices in DMEM/FBS containing 50 μg/ml of monoclonal antibody against FN (HFN 7.1) as indicated. At the end of the incubations, samples were fixed and stained for actin. (B) Fibroblasts were cultured for 18h on collagen matrices in DMEM/FBS containing 200 μg/ml of cyclo-peptides as indicated. At the end of the incubations, samples were fixed and stained for actin and PI. Bar = 100 μm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Griffith LG, Swartz MA. Capturing complex 3D tissue physiology in vitro. Nat Rev Mol Cell Biol. 2006;7:211–24. - PubMed
    1. Wozniak MA, Chen CS. Mechanotransduction in development: a growing role for contractility. Nat Rev Mol Cell Biol. 2009;10:34–43. - PMC - PubMed
    1. Grinnell F, Petroll WM. Cell motility and mechanics in three-dimensional collagen matrices. Annu Rev Cell Dev Biol. 2010;26:335–61. - PubMed
    1. Geiger B, Yamada KM. Molecular architecture and function of matrix adhesions. Cold Spring Harb Perspect Biol. 2011;3:a005033. - PMC - PubMed
    1. DuFort CC, Paszek MJ, Weaver VM. Balancing forces: architectural control of mechanotransduction. Nat Rev Mol Cell Biol. 2011;12:308–19. - PMC - PubMed

Publication types

MeSH terms