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First published online 24 July 2008
doi: 10.1242/jcs.029819

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Fibrillin-1 microfibril deposition is dependent on fibronectin assembly

¹ Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Science, Michael Smith Building, Oxford Road, University of Manchester, Manchester M13 9PT, UK
² Max Planck Institute of Biochemistry, Department of Molecular Medicine, Am Klopferspitz 18, 82152 Martinsried, Germany

View larger version (99K): [in this window] [in a new window]   Fig. 1. Fibrillin-1 microfibril assembly by human dermal fibroblasts. (A) Representative confocal microscopy images of the assembly of microfibrils (using rabbit anti-proline-rich region pAb; red) and fibronectin (FN) (using FN-3E2 mAb to cellular fibronectin; green) by HDFs at 24 hours and 5 days. Cell nuclei were stained with DAPI (blue). Scale bars: 50 µm. (B) Enlarged images from A. These experiments were repeated several times with similar results.

View larger version (63K): [in this window] [in a new window]   Fig. 2. Fibronectin RNAi disrupts fibrillin-1 microfibril assembly by human dermal fibroblasts. (A) RT-PCR analysis of RNAi knockdown of fibronectin in HDFs. FN1 and FN2 are knockdowns using different oligonucleotides (see Materials and Methods). FN2 scr, scrambled oligonucleotide control; Col I, collagen I; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (B) Immunoblotting of fibrillin-1 [using rabbit anti-proline-rich region (PRO) pAb] and fibronectin (using FN-3E2 mAb to cellular fibronectin) on lysates and medium from knockdown and control cultures, as outlined in A, with β-actin loading controls (using mAb AC-74) for the cell lysates. (C) Representative confocal microscopy images of fibronectin assembly (using FN-3E2 mAb to cellular fibronectin) in the RNAi and control cultures, from 1-7 days. (D) Representative confocal microscopy images of microfibril assembly (using rabbit anti-proline-rich region (PRO) pAb; red) and fibronectin (using FN-3E2 mAb to cellular fibronectin; green) in control cultures, fibronectin RNAi cultures, and fibronectin RNAi cultures supplemented with cellular fibronectin (10 µg/ml), all at 5 days. Microfibril assembly was grossly disrupted in the fibronectin siRNA cells, and partially rescued by cellular fibronectin. Scale bars: 50 µm. (E) Enlarged images from D. All experiments were repeated at least three times.

View larger version (62K): [in this window] [in a new window]   Fig. 3. Blocking 5β1 integrins disrupts fibrillin-1 microfibril assembly by human dermal fibroblasts. (A) FACS analysis of HDFs, confirming expression of 5β1 and vβ3 integrins. The antibodies used were mAb13 (β1 integrin), mAb16 (5 integrin), 17E6 (v integrin) and 23C6 (vβ3 integrin). (B) HDFs were incubated for 24 hours or 5 days in the presence of integrin-blocking antibodies mAb13 (β1) and mAb16 (5) or a rabbit IgG control (all at 10 µg/ml). Medium and cell lysates were immunoblotted for fibrillin-1 [using the rabbit anti-proline-rich region (PRO) pAb] or fibronectin (using FN-3E2 mAb), with β-actin (using mAb AC-74) loading controls for the cell lysates. Molecular masses are indicated (in kDa). Quantitative analysis on the media was done by densitometry as a measure of pixel density. Cell lysates were quantified using densitometry with data normalised against β-actin. All data are represented as the mean ± s.d. of two repeated experiments. There was no statistical difference between treated samples and the control when using the Student's t-test. (C) Representative confocal microscopy images of the assembly of microfibrils (using PRO pAb; red) and fibronectin (using FN-3E2 mAb to cellular fibronectin; green), in the presence or absence of mAb13 (blocks β1 integrins) and mAb16 (blocks 5 integrin), at 5 days. Cell nuclei were stained with DAPI (blue). Microfibril assembly was grossly disrupted in the antibody-treated cells. Scale bars: 50 µm. Enlarged images are also shown. All experiments were repeated at least three times.

View larger version (51K): [in this window] [in a new window]   Fig. 4. Fibrillin-1 microfibril assembly is disrupted in β1-null murine embryonic fibroblasts. (A) FACS analysis detected 5 (5H10 mAb) or β1 (KM16 mAb) integrin subunits in wild-type but not β1-integrin-null MEFs. (B) Representative confocal microscopy images of microfibril assembly (using PRO pAb; red) and fibronectin (using FN-3E2 mAb to cellular fibronectin; green) in wild-type and β1-integrin-null MEFs, at 3 days. Cell nuclei were stained with DAPI (blue). fibronectin and microfibril assembly was markedly reduced in the β1-integrin-null cells. Scale bars: 50 µm. Enlarged images are also shown. These experiments were repeated three times.

View larger version (116K): [in this window] [in a new window]   Fig. 5. Rho kinase inhibitors disrupt fibrillin-1 microfibril assembly by human dermal fibroblasts. (A) Representative confocal microscopy images of microfibril assembly (using PRO pAb; red) and fibronectin (using FN-3E2 mAb to cellular fibronectin; green) in the presence or absence of either of two ROCK inhibitors (H1152 or Y27632) or DMSO (dimethylsulphoxide) control, at 5 days. Cell nuclei were stained with DAPI (blue). Microfibril assembly was grossly disrupted in the ROCK-inhibitor-treated cells. Scale bars: 50 µm. (B) Representative confocal microscopy images of F-actin stress fibres, stained with phalloidin, in the presence or absence of either H1152 or Y27632 or DMSO control, at 5 days. Stress fibres were markedly reduced in the inhibitor-treated cultures. These experiments were repeated three times.

View larger version (119K): [in this window] [in a new window]   Fig. 6. Fibrillin-1 microfibril assembly is disrupted in murine embryonic fibroblasts expressing mutant fibronectinRGE/RGE. Representative confocal microscopy images of microfibril assembly [using Santa Cruz N-19 anti-fibrillin-1 pAb (SC-7541); green, FITC] and fibronectin assembly (using FN-3E2 mAb to cellular fibronectin; Texas Red) by nonpermeabilised wild-type or fibronectinRGE/RGE mutant MEFs, at 14 days. Cell nuclei were stained with DAPI (blue). Mutant cells deposited reduced fibronectin and very little extracellular fibrillin-1. Scale bars: 50 µm. Enlarged images are also shown. These experiments were repeated three times.

View larger version (32K): [in this window] [in a new window]   Fig. 7. Model of pericellular fibronectin and fibrillin-1 assembly. Diagram illustrating how fibronectin and 5β1 integrin may contribute to fibrillin-1 microfibril assembly. The model predicts that activated 5β1 integrin and associated cytoskeletal changes drive fibronectin fibrillogenesis, which in turn facilitates the deposition of extensive microfibril arrays. Although fibrillin-1 may associate with pericellular fibronectin even when fibronectin cannot assemble through 5β1 (Takahashi et al., 2007), in these conditions there is little microfibril assembly (see Figs 4, 6).

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