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First published online 15 March 2005
doi: 10.1242/jcs.01732

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The N-terminal Ac-EEED sequence plays a role in -smooth-muscle actin incorporation into stress fibers

Sophie Clément^1,*, Boris Hinz^2,*, Vera Dugina³, Giulio Gabbiani¹ and Christine Chaponnier^1,

¹ Dept of Pathology and Immunology, CMU, Geneva, Switzerland
² Laboratory of Cell Biophysics, Ecole polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland
³ Moscow State University, 119899 Moscow, Russia

View larger version (72K): [in a new window]   Fig. 1. Characterization of transfected cells. REF-52 cells were transfected with -SMA-EGFP and cells stably expressing -SMA-EGFP were obtained after FACS sorting and G418 selection. (A) In the majority of cells, -SMA-EGFP is incorporated into actin stress fibers (see inset), as indicated by indirect double immunofluorescence of fixed cells. (a) -SMA was visualized with anti--sm1 antibody, (b) -SMA-EGFP was visualized with anti-GFP antibody. (c) Overlay of a and b. Scale bar, 50 µm. (B) Transfected REF-52 cells were grown for 5 days in culture medium containing either 2% FCS (a) or 20% FCS (b). Cells were fixed and -SMA-EGFP organization was assessed by fluorescence microscopy. Scale bar, 20 µm. (C) Cell homogenates of non-transfected cells (lanes 1 and 3) and transfected cells (lanes 2 and 4) were analyzed on a 10% SDS-PAGE and immunoblotted with anti-GFP antibody (lanes 1-2) and anti-SMA antibody (lanes 3-4). The anti-GFP antibody detected a single 80 kDa band corresponding to the calculated molecular mass of the fusion protein (42 kDa actin+36 kDa EGFP). This band was also recognized by the anti--SMA antibody. Transfected and non-transfected cells both showed the endogenous -SMA band of 42 kDa.

View larger version (95K): [in a new window]   Fig. 2. Time-lapse observation of -SMA-EGFP in SMA-FP-treated cells and determination of the morphological features of RLSs. (A) Cells were trypsinized and plated into observation chambers for 3 days in DMEM-FCS (2%). Before recording, cells were mounted on an inverted confocal microscope (LSM510, Carl Zeiss) and treated with SMA-FP. EGFP images were collected every 2 minutes for 90 minutes. RLSs appeared mainly in the submembrane area or along stress fibers (arrowhead); fluorescence in the -SMA-EGFP positive stress fibers (arrows) decreases over time. Scale bar, 20 µm. See also videos in supplementary material. (B) Length (a) and diameter (b) of RLSs were determined on digitized confocal images using Openlab 3.0.6 software. Their length varied from 2 to 20 µm and their diameter from 0.1 to 1.2 µm. (C) -SMA-EGFP transfected cells were either non-treated (Cont), or treated for 60 minutes with control peptide SKA-FP or SMA-FP. Reversibility of SMA-FP treatment was assessed by removing the peptide by washing cells with fresh medium for 60 minutes. The percentage of EGFP-positive cells displaying RLSs was estimated by counting a minimum of 200 cells. RLSs were visible in approximately 60% of the transfected cells and the effect of SMA-FP was reversible. Controls in the absence of SMA-FP or in the presence of SKA-FP were close to baseline. Bars represent s.e.m. (**P0.001 compared to control). (D) Non-transfected cells were treated with 5 µg/ml SMA-FP for 30 minutes, fixed and permeabilized with 1% paraformaldehyde for 10 minutes followed by methanol for 3 minutes and stained with anti-actin antibody (see Table 1). By using this detergent-free procedure, RLSs were also found in non-transfected cells. Scale bar, 20 µm.

View larger version (78K): [in a new window]   Fig. 3. Presence of actin isoforms in the RLSs. (A) Overlay images of EGFP fluorescence (green) and (a) staining of ß-cytoplasmic actin (red) or (b) -cytoplasmic actin (red) in non-treated -SMA-EGFP-transfected cells are shown. Scale bar, 10 µm. (B) Cells were treated for 30 minutes with SMA-FP, fixed with 1% paraformaldehyde for 10 minutes followed by methanol incubation for 3 minutes and stained with either (b) anti ß-cytoplasmic actin antibody, (e) -cytoplasmic actin antibody or (h) Alexa 568-phalloidin. (k) The distribution of rhodamine-labeled SMA-FP in -SMA-EGFP-transfected cells was also investigated. -SMA-EGFP patterns are shown in images a, d, g, and j and colocalization was visualized by confocal microscopy. Overlay images are shown in c, f, i and l. Bar, 10 µm.

View larger version (85K): [in a new window]   Fig. 4. Presence of actin-binding proteins in RLS. (A) Gelsolin (a) and cofilin (b) organization in -SMA-EGFP transfected cells in control conditions. Overlay images with -SMA-EGFP show that gelsolin (a, red) completely colocalized with -SMA-EGFP (green) in stress fibers, whereas cofilin (b, red) was additionally found at the periphery of the cell (arrow). (B) Cells were treated for 30 minutes with SMA-FP, fixed with 1% paraformaldehyde for 10 minutes followed by methanol for 3 minutes and stained with a large panel of anti actin-binding protein antibodies (see table 1). EGFP fluorescence (green) and immunofluorescence staining (red) with anti-gelsolin (b) and anti-cofilin (e) showed that they were both present in RLS (yellow RLS in the overlay images c and f), whereas many other actin binding proteins (-actinin, h shown as example) were not found (green RLS in the overlay image i). Bar, 10 µm.

View larger version (61K): [in a new window]   Fig. 5. Visualization of RLSs in spreading REF-52 cells transfected with -SMA-EGFP. Cells were trypsinized, plated in F12 medium, allowed to spread and fixed at different time points with 1% paraformaldehyde for 10 minutes followed by methanol incubation for 3 minutes. (A) After 4 hours of spreading in the absence of SMA-FP, some cells exhibited -SMA-positive stress fibers (a), whereas other cells (b) displayed RLSs that were arranged tangentially to the plasma membrane (see inset). Scale bar, 10 µm. (B) In a minimum of 200 cells, the percentage of cells displaying RLSs (gray bars) or stress fibers (hatched bars) was assessed at different time points after plating (4, 6, 8, and 8 hours). Bars represent s.e.m. (***P0.001, the 4-hour time point was taken as the reference). (C) Cells were seeded in observation chambers in F12 medium supplemented with 2% FCS and allowed to spread for 2 hours. Cell-spreading was recorded using a laser confocal spinning wheel (Nipkow disc) and images were acquired every 10 minutes for 15 hours. Scale bar, 20 µm. During spreading, cells first displayed RLSs that appeared perpendicular to the cell edge (*) and then gradually disappeared while -SMA was organized in stress fibers (arrow).

View larger version (95K): [in a new window]   Fig. 6. Time-lapse FRAP analysis of -SMA-EGFP stress fibers. Photobleached regions appear as dark bars and squares (*). FRAP analysis of cells in control conditions (A) was compared with that of SMA-FP-treated cells (B). In control conditions, bleached -SMA-EGFP stress fibers almost completely recovered their fluorescence within 30 minutes (see square). Bar-shaped bleached zones on individual stress fibers move at different rates during fluorescence recovery, as indicated by the transformation of the straight bleach zone into a zig-zag line (arrows). Lapsed time is indicated at the lower right corner of each image. Scale bars, 10 µm. See also videos in supplementary material. (C) The average rate of movement of bleached zones was quantified by measuring the mean distance of at least ten stress fibers in at least ten cells using Openlab software (2.3±0.2 mm/hour (n=51). In cells pre-treated for 15 minutes with SMA-FP, rates of movement were approximately fivefold slower than in non-treated cells, averaging 0.53±0.09 µm/hour (n=38). Bars represent s.e.m. (**P0.001 compared with control cells).

View larger version (35K): [in a new window]   Fig. 7. FRAP analysis of SMA-EGFP turnover in RLSs. (A) Transfected cells were treated for 30 minutes with SMA-FP. -SMA-EGFP RLSs were photobleached and images were taken at indicated time points. Bar, 5 µm. (B) Fluorescence intensities were measured at different times using Openlab software and related to the intensity measured in the first video frame. Fluorescence before bleaching was normalized to 100%. Each data point represents the mean intensity of fluorescence of 33 RLSs analyzed in eight different cells. Bars represent s.e.m.

View larger version (19K): [in a new window]   Fig. 8. Schematic representation of the proposed mechanism of SMA-FP action and stress-fiber-formation. (a) -SMA incorporation in pre-existing cytoplasmic-actin-containing stress fibers represented as light red dashed lines. -SMA is first recruited in RLSs, a pre-requisite step before its incorporation in stress fibers (see arrows). (b) -SMA incorporation into stress fibers (dark red lines) confers higher cell-contractility (illustrated by the arrows on the top of the cell). When such contractile cells are treated with SMA-FP, -SMA incorporation in stress fibers is inhibited (see zoomed-in part of the cell c), resulting in -SMA accumulation in RLSs, its disappearance from stress fibers, and subsequently `relaxation' of the cell.