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Focal adhesion features during myofibroblastic differentiation are controlled by intracellular and extracellular factors

¹ Moscow State University, 119899 Moscow, Russia
² Department of Pathology, CMU, University of Geneva, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland

View larger version (12K): [in a new window]   Fig. 1. Percentages of FAs showing different area in control and after treatment with TGFß or TGFß plus rED-A.

View larger version (56K): [in a new window]   Fig. 2. Modulation of FAs and stress fiber structure after TGFß treatment. 3D shadow projections, views from the cell basal side. (A) Vinculin (red) and ß-cytoplasmic actin (green) organisation in control fibroblasts. FAs are small and some are connected with cytoplasmic actin stress fibers. Immature (arrow) and mature (arrowhead) FAs. (B) Vinculin (red) and -SMA (green) organisation in fibroblasts treated for 5 days with TGFß. The size of vinculin-containing FAs is dramatically increased in parallel with the appearance of -SMA in stress fibers. (C) Vinculin (red) and ß-cytoplasmic actin (green) organisation in fibroblasts after TGFß-treatment in the presence of rED-A. FAs are smaller and stress fibers are thinner than those in B. -SMA is absent in stress fibers. Bar, 10 µm.

View larger version (57K): [in a new window]   Fig. 3. Codistribution and 3D-structure (shadow projections) of vinculin and 5ß1 integrin in FAs of control and TGFß-treated fibroblasts. (A) Immature (arrow) and mature (arrowhead) FAs of a control fibroblast (view from ventral side): 5ß1 integrin (red) is adjacent to vinculin (green) or colocalised with vinculin (yellow). (B,C) FAs after 5 days of TGFß treatment. (B) Vinculin staining is hidden by 5ß1 integrin staining (basal side of the cell). (C) Vinculin and 5ß1 integrin co-staining (yellow) in FAs appears after removing the first basal optical sections. The terminal portions of FAs appear surrounded by 5ß1 integrin. Bar, 10 µm.

View larger version (116K): [in a new window]   Fig. 4. Organisation of ED-A FN and actin after TGFß treatment. 3D shadow projections. (A) ß-Cytoplasmic actin (red) and ED-A FN (green) in control fibroblasts. View from basal side of the cells showing that some terminal ends of actin stress fibers are connected with FN fibers. (B) -SMA (red) and ED-A FN (green) in fibroblasts treated with TGFß. The appearance of -SMA in actin stress fibers and their thickening are accompanied by an organisation of ED-A FN in parallel bundles and their association with the end of actin stress fibers. The first optical sections from basal surface of extracellular matrix adjacent to fibronexus have been removed. Bar, 10 µm.

View larger version (55K): [in a new window]   Fig. 5. Western blot analysis of cytosolic and cytoskeleton fractions prepared from fibroblasts. Control Dupuytren’s fibroblasts (Lane 1) or TGFß-treated cells (lane 2) and lung fibroblasts (Lane 3) were fractionated in cytosolic pool (left panel) and cytoskeleton pool (right panel) and probed for the presence of cytoskeletal proteins by western blot.

View larger version (91K): [in a new window]   Fig. 6. Incorporation of biotinylated rED-A into the cellular FN network. 3D shadow projections, views from basal side (A,B). Single optical sections (C,D). Bar, 10 µm. (A) Fibroblasts treated with TGFß for 4 days. ED-A FN (green) displays a parallel organisation of thick fibers. (B) TGFß-treated fibroblasts in the presence of rED-A (red). Yellow staining shows zones of colocalisation. The organisation of the FN network is less regular upon rED-A treatment compared to TGFß treated fibroblasts (A). Biotinylated rED-A displays a preferential incorporation into the FN network near the periphery of the cell. Note the thickening of FN fibers mainly at the places of rED-A incorporation. (C,D) Desoxycholate (DOC)-resistant matrix deposited by TGFß-treated cells (C) or by TGFß-treated cells incubated with biotinylated rED-A (D): rED-A (red) and ED-A FN (green). Note a focal incorporation of rED-A into the fibers of DOC-insoluble FN matrix (D). As for B, yellow staining represents zones of colocalisation. (E) DOC-soluble (lanes 1 and 2) and -insoluble (lanes 3 and 4) fractions were prepared from fibroblasts cultured for 4 days in 5 ng/ml of TGFß in the absence (lanes 1 and 3) or in the presence of 100 µg/ml of biotinylated rED-A (lanes 2 and 4). Corresponding amounts of proteins were resolved on 6% gels (FN), 8% gels (vimentin) and 12% gels (rED-A) and probed with a rabbit polyclonal antibody to FN or streptavidin, and with a monoclonal antibody to vimentin. rED-A is incorporated in the DOC-insoluble matrix but does not reduce the level of DOC insoluble FN or of vimentin.

View larger version (124K): [in a new window]   Fig. 7. Incorporation of rED-A into cellular FN fibers. Fibroblasts were cultured for 4 days in TGFß and then for 3 hours in the presence of 50 µg/ml of biotinylated rED-A (A,B). (A) Biotinylated rED-A (red) displays a preferential incorporation (yellow) into thick fibers of the ED-A FN (green) network. (B) Vinculin (red) in FAs is partially overlapping (yellow) with rED-A (green) fibers. (C,D) Overlay binding assays: DOC-insoluble matrix prepared from TGFß-treated fibroblasts was overlaid with biotinylated rED-A (C) or r11A12 (D) at 50 µg/ml, 3 hours. (C) rED-A (red) primarily colocalises with thick fibers of FN (green). (D) The recombinant protein r11A12 does not decorate FN fibers (green). Bars, 10 µm.

View larger version (80K): [in a new window]   Fig. 8. Blocking of the incorporation of rED-A into cellular FN network by inhibitor of actin-myosin contractility. (A) Fibroblasts were incubated for 5 days in TGFß, then 100 µg/ml of biotinylated rED-A was added for 3 hours. A high incorporation (yellow) of rED-A (red) into the ED-A FN network (green) is visible. (B) ML-7 added into the culture medium 1hour before and during incubation with rED-A blocks rED-A incorporation into ED-A FN. Bar, 10 µm.

View larger version (27K): [in a new window]   Fig. 9. Effect of MAPK inhibitor SB230580 on ED-A FN polymerisation. Dupuytren’s (A,C) and lung fibroblasts (B,D) cultured in the presence of various concentration of FN and treated with 20 µM and 50 µM, respectively, of SB230580 for 4 days were either extracted by DOC to recover the polymerised FN fraction or solubilised in loading buffer. Polymerised ED-A FN was probed by western blot on DOC insoluble fraction using IST-9 antibody (A,B, upper line) and -SMA expression tested by western blot of whole cell extracts (A,B, lower line). The production of ED-A FN by the cells was tested by dot blot analysis of culture medium using IST-9 antibody. Western and dot blots were quantified by scanning densitometry to compare the level of soluble and polymerised ED-A FN (C,D).

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