Fig. 3. Overexpression of EGFP/l-caldesmon causes disruption of stress fibres and focal adhesions in smooth muscle cells. (A) A7r5 cells were transfected with a plasmid encoding for EGFP-l-caldesmon for 20 or 48 hours prior to lysis and western blot analysis with a general caldesmon antibody (inset). Densitometry was performed to quantify the level of exogenous EGFP-l-caldesmon expression by normalising the signal to that of endogenous l-Cad. The level of EGFP-l-caldesmon is approximately fivefold higher at 48 hours than at 20 hours. (B-I) A7r5 cells were transfected with a plasmid encoding EGFP/l-caldesmon for 48 hours prior to fixation, and stained using antibodies raised against EGFP (green) and either (B,C,F-I) TRITC-phalloidin (to label F-actin, red) or (D-E) antibodies raised against the focal adhesion protein vinculin (red). (B,D) Expression of high levels of EGFP-l-caldesmon induced the dissolution of stress fibres and focal adhesions (cell 1 in D) in the transfected cells. The untransfected cells contained well-defined stress fibres and large vinculin-stained focal adhesions. (C,E) Moderate to low levels of EGFP-l-caldesmon (cell 2 and 3, respectively, in E) did not cause significant disorganisation of stress fibres but appeared to displace focal adhesions to the cell periphery. (F,G) Confocal micrographs of EGFP-l-caldesmon expressing-cells stimulated with 1 µM PDBu for 30 minutes. Many cells contained large clusters of podosomes, as shown in F, while others had a more scattered distribution of podosomes, as in G. (H,I) Reconstruction of the x-z-profiles of podosomes from cells in F and G showing that EGFP-l-caldesmon co-localised to the actin rich core of the podosome. Bars, 20 µm in B-G; 2 µm in H,I.