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First published online 4 April 2006
doi: 10.1242/jcs.02881

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Caldesmon is an integral component of podosomes in smooth muscle cells

Department of Biochemistry, and Protein Function Discovery Program, Queen's University, Kingston, ON K7L 3N6, Canada

View larger version (74K): [in a new window]   Fig. 1. Localisation of endogenous l-caldesmon in PDBu-induced podosomes in A7r5 vascular smooth muscle cells. A7r5 cells cultured on glass coverslips and were either unstimulated (A) or stimulated with 1 µM PDBu for 30 minutes (B). Cells were stained with antibodies raised against l-caldesmon and TRITC-phalloidin was used to label F-actin. The two images were merged (overlay) to show the relative localisation of l-cadesmon (green) and F-actin (red). (A) A7r5 cells grown in serum displayed a robust actin cytoskeleton with prominent stress fibres. (B) PDBu-stimulation induced the formation of podosomes containing punctate actin staining. (C) Reconstruction of the x-z profiles of typical podosomes in B; caldesmon co-localised with the actin-rich core of the podosome. Bars, 20 µm in A,B; 2 µm in C. (D) Western blot analysis of rat aortic smooth muscle cell lysate (lane 1) or A7r5 cell lysate (lane 2) probed with a general caldesmon antibody. Whereas aortic smooth muscle expresses the high molecular weight isoform of caldesmon (h-caldesmon), cultured A7r5 cells express the low molecular weight isoform (l-caldesmon). The molecular masses of protein standards (in kDa) are listed on the left.

View larger version (7K): [in a new window]   Fig. 2. Schematic diagram of rat l-caldesmon constructs used in this study. The N-terminal fragment of l-caldesmon (Cad40; amino acids 1-152) harbours the myosin-binding site (hatched) (Velaz et al., 1990; Bogatcheva et al., 1993; Redwood and Marston, 1993; Huber et al., 1995) and a tropomyosin-binding site (black) (Smith et al., 1987; Redwood and Marston, 1993; Redwood et al., 1993). The C-terminal fragment of l-caldesmon (Cad39; amino acids 236-532) contains two tropomyosin-binding sites, two actin-interacting regions (diamonds) (Wang et al., 1997b; Marston et al., 1998), and the two Ca2+/calmodulin binding sites A and B, as indicated (dark-grey boxes) (Wang et al., 1991; Marston et al., 1994; Wang et al., 1996). Both Ca2+/calmodulin-binding sites contain a key tryptophan residue (W454 and W487) that is essential for this interaction (Graether et al., 1997); replacement of these tryptophan residues with alanine (W454A and W487A) generates an l-caldesmon mutant that is unable to interact with Ca2+/calmodulin (CadCamAB).

View larger version (76K): [in a new window]   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.

View larger version (77K): [in a new window]   Fig. 4. Dynamic incorporation of EGFP-caldesmon into podosomes in live cells treated with PDBu. Video images of live A7r5 cells expressing EGFP-l-caldesmon and DS Red-cortactin were recorded either in the absence (A) or presence (B) of 1 µM PDBu. The times in A refer to the time from the beginning of the video; the times in B refer to the time after the onset of PDBu stimulation. Bars, 20 µm.

View larger version (72K): [in a new window]   Fig. 5. Knockdown of endogenous l-caldesmon by siRNA enhances podosome formation. (A,B) A7r5 cells were transfected with the caldesmon-specific siRNA1120 (1120) and siRNA143 (143) or with a negative control siRNA (control) for 48 hours prior to lysis and western blot analysis. Samples were probed with a general l-caldesmon antibody (l-Cad) or ß-actin as a loading control. (A) Blot is representative of three independent experiments. (B) Densitometry was performed to quantify the level of knockdown by each caldesmon siRNA compared with the negative siRNA control, as described in the Materials and Methods. The caldesmon siRNA143 and siRNA1120 reduced the level of caldesmon in the total population of transfected and untransfected cells by 20% and 40%, respectively. This translates to about 40% and 80% knockdown of caldesmon expression, respectively, by siRNA143 and siRNA1120, taking into account of 50% transfection efficiency. (C-E) A7r5 cells co-transfected with siRNA1120 and EGFP-ß-actin were fixed and stained 48 hours after transfection (C) or stimulated with 1 µM PDBu for 30 minutes prior to fixation (D,E). Cells were stained for EGFP (green) to identify siRNA transfected cells and l-caldesmon (red). An overlay of the two channels is shown. (C) Cells transfected with siRNA1120 show a significant decrease in the level of caldesmon but still retain stress fibres. (D,E) Stimulation of cells in which the level of caldesmon has been reduced by siRNA1120 with PDBu show an increase in both the number of cells displaying podosomes and the number of podosomes per cell. Bars, 20 µm in C-E.

View larger version (77K): [in a new window]   Fig. 6. Cad39 but not Cad40 localises to podosomes. A7r5 cells expressing EGFP-Cad40 (A-D) or EGFP-Cad39 (E-H) fragments of caldesmon were fixed and stained 48 hours after transfection (A,B,E,F) or stimulated with 1 µM PDBu for 30 minutes prior to fixation (C,G). Cells were stained for EGFP (green) or F-actin (red) and an overlay of the two channels is shown. (A,B) Cad40 disrupted stress fibres in 50% of transfected cells (A), while cells expressing lower levels of Cad40 retained stress fibres (B), which did not contain Cad40. (C,D) Cad40 did not translocate to podosomes induced by PDBu. (D) A reconstruction of the x-z profiles of podosomes from cells in C. (E,F) Cad39 was less disruptive to stress fibres, affecting only 20% of the transfected cells (E) and the majority (80%) of the cells showed little disorganisation of stress fibres (F). Unlike Cad40, Cad39 localised to stress fibres. (G,H) Cad39 co-localised to the core of the podosome. (H) The x-z profiles of podosomes from cells in G. Bars, 20 µm in A-C,E-G; 2 µm in D,H.

View larger version (82K): [in a new window]   Fig. 7. Interaction with Ca2+/calmodulin is required for the translocation of l-caldesmon to podosomes. A7r5 cells transfected with plasmids encoding EGFP-CadCamAB (green) were either stained with TRITC-phalloidin to identify F-actin (red) (A,B,D,E) or with a monoclonal antibody raised against vinculin (red) to identify focal adhesions (C). The two channels were merged (overlay) to show co-localisation. (A-C) Expression of EGFP-CadCamAB caused severe disruption of actin stress fibres (A) and focal adhesions (C) in some cells; others retain an intact array of stress fibres (B). (D) Confocal micrograph of CadCamAB-transfected cells stimulated with 1 µM PDBu for 30 minutes prior to staining. Insets in D show a 10x10 µm enlargement of the cell to show the exclusion of CadCamAB from the actin core of podosomes. (E) Reconstruction of the x-z profiles of selected podosomes of the cell in D. Bars, 20 µm in A-D; 2 µm in E.

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