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First published online 20 March 2007
doi: 10.1242/jcs.003954

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Cell adhesion to fibrillin-1: identification of an Arg-Gly-Asp-dependent synergy region and a heparin-binding site that regulates focal adhesion formation

¹ UK Centre for Tissue Engineering, Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK
² Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK
³ Faculty of Medicine, Imperial College London, Exhibition Road, London, SW7 2AZ, UK

View larger version (61K): [in this window] [in a new window]   Fig. 1. (A) Domain organisation of recombinant fibrillin-1 fragments, indicating the levels of cell attachment each fragment supports (+, low attachment; +++++, high attachment) and heparin-binding sites. (B) SDS-PAGE analysis (4-12% Bis-Tris gels) of fibrillin-1 fragments WT PF14, RGAPF14, 19-20PF14, 19-20-21-20PF14, 3-4-5-6PF14, 19-4-5-6PF14 and 3-20-21-6PF14, under non-reducing (i) and reducing (ii) conditions and after PNGaseF digestion (iii). (C) SDS-PAGE analysis (4-12% Bis-Tris gels) of fibrillin-1 fragments WT PF9, R1691APF9, R1692APF9, R1697APF9, R1752APF9, R1691A/R1692APF9 and R1697A/R1752APF9 under non-reducing (i) and reducing (ii) conditions and after PNGaseF digestion (iii).

View larger version (37K): [in this window] [in a new window]   Fig. 2. (A) Dose-dependent HDF attachment to fibrillin-1 fragments PF8 (squares), PF11 (diamonds) and the central cell binding region of fibronectin (50K-FN) (triangles). (B) HDF attachment to 10 µg/ml PF11, PF8 or BSA-blocked plastic in the presence (shaded) or absence (open) of the 1-integrin-activating antibody TS2/16. Data were statistically analysed using unpaired Student's t-tests (GraphPad Prism 2.0). Error bars represent the s.d. of the three experiments. (C) Fluorescence images of actin filaments in phalloidin-stained spread fibroblasts on fibronectin, PF11, PF8 and PF9. There were no spread cells in the BSA control (not shown). (D) Cumulative distance travelled by cells adherent to 50K-FN (solid line), PF8 (long dashed line) or PF11 (short dashed line), as measured by time lapse microscopy. Bar, 50 µm.

View larger version (39K): [in this window] [in a new window]   Fig. 3. (A) HDF attachment to fibrillin-1 fragments PF8, PF9, WT PF14 and PF14 mutants 19-20PF14, 19-20-21-20PF14, 3-4-5-6PF14 and RGA PF14. (B) Phase-contrast photographs of HDFs spreading onto PF8, PF9, WT PF14, 19-20PF14, 19-20-21-20PF14, 3-4-5-6PF14, RGA PF14 and control wells (PBS). (C) Anti-integrin antibody inhibition of HDF attachment to PF8, PF9, WT PF14, 19-20PF14, 19-20-21-20PF14, 3-4-5-6PF14, RGA PF14 and control wells (PBS). (D) HDF attachment to fibrillin-1 fragments PF8, PF11, WT PF14 and the PF14 mutants 19-4-5-6PF14, 3-20-21-6PF14 and 3-4-5-6PF14.

View larger version (87K): [in this window] [in a new window]   Fig. 4. (A) Immunofluorescence images of actin (a,c,e) and vinculin (b,d,f) in REFs adherent to 10 µg/ml fibronectin (a,b) or PF8 (c,d). In e,f, 1 µg/ml PF10 was added in solution for 30 minutes before fixation. (B) The table shows quantification of the number of cells on FN, PF8, or PF8+PF10 fragments that express vinculin-containing and non-vinculin-containing focal plaques (±s.d.).

View larger version (79K): [in this window] [in a new window]   Fig. 5. (A) Immunofluorescence images of actin (a,c,e,g) and vinculin (b,d,f,h) in MEFs adherent to 10 µg/ml PF8 (a-h) for 2 hours. 10 µg/ml of the heparan sulphate binding fragments of fibrillin-1 PF9 (c,d), PF10 (e,f) and PF11 (g,h) were added in solution for 30 minutes before fixing. Quantification revealed that 9% of cells on PF8, 57% of cells on PF8 + soluble PF10, 38% of cells on PF8 + soluble PF9 and 11% of cells on PF8 + soluble mutant PF9 were positive for vinculin and focal adhesions. (B) Immunofluorescence images of actin (a) and vinculin (b) in MEFs adherent to PF8 in the presence of soluble 10 µg/ml heparan sulphate binding mutant R1691A/R1692A PF9.

View larger version (58K): [in this window] [in a new window]   Fig. 6. (A) Immunofluorescence images of actin (a,c,e,g,i,k) and vinculin (b,d,f,h,j,l) in REFs adherent to 10 µg/ml fibronectin (a,c) or to a fragment containing the central cell binding region of fibronectin (110-FN) (c-l) for 2 hours. The heparan sulphate binding fibrillin-1 fragments (10 µg/ml) PF9 (e,f), an RGD-to-RDG mutant PF9 (g,h), PF10 (i,j), or the heparan sulphate binding region of fibronectin (HepII) (k,l) were added in solution for 30 minutes before fixing. (B) The table shows quantification of the number of cells on FN, 110, or 110+fibrillin-1 fragments that express vinculin-containing and non-vinculin-containing focal plaques (±s.d.). (C) Immunofluorescence of actin (a) and vinculin (b) in REFs adherent to 10 µg/ml of 110-fibronectin for 2 hours. Soluble 10 µg/ml heparan sulphate binding mutant R1691A/R1692APF9 was added for 30 minutes before fixing.