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First published online 5 May 2004
doi: 10.1242/jcs.01110

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Control of actin dynamics by p38 MAP kinase – Hsp27 distribution in the lamellipodium of smooth muscle cells

INSERM U 348, IFR Circulation Lariboisière, 41 Boulevard de la Chapelle, 75475 Paris Cedex 10, France

View larger version (14K): [in a new window]   Fig. 1. p38 MAPK is involved in PDGF-induced actin polymerization. Determination of F-actin/G-actin ratio. Suspended SMC were incubated for 30 minutes with 25 µM SB203580 or vehicle and then plated on fibronectin-coated wells for 3 hours in the presence or absence of PDGF (10 ng/ml). Fixed SMC were labeled with TRITC-phalloidin (2 µM) or Alexa 594-DNase I (3 µM). Fluorescence intensities were quantified and relative ratios of F- to G-actin fluorescence were then determined. Data represent the mean±s.e.m. of three independent experiments, carried out in triplicate. **Statistically significant according to Student's t-test: P<0.01.

View larger version (31K): [in a new window]   Fig. 3. p38 MAPK activity is not induced by SMC spreading on fibronectin. Suspended SMC were incubated for 30 minutes with 25 µM SB203580 or vehicle and then plated out in dishes coated with fibronectin for 0, 30 minutes and 3 hours in the presence or absence of PDGF (10 ng/ml). SMC lysates (30 µg) were analyzed by immunoblotting using antibodies directed against p38 MAPK-P and Hsp27-P. Blots were reprobed with an anti--actin antibody to ensure equal loading. These results are representative of four independent experiments.

View larger version (32K): [in a new window]   Fig. 2. p38 MAPK is involved in PDGF-induced chemotaxis and cytoskeletal reorganization. (A) Migration assay. Pre-labeled suspended SMC were incubated for 30 minutes with 25 µM SB203580 (SB, closed circles and closed squares), or vehicle (open circles and open squares) and subjected to the migration assay in the presence (circles) or absence (squares) of PDGF (10 ng/ml) in the lower chamber. The fluorescence of migrating cells was determined at different times. Data represent the mean±s.e.m. of three independent experiments, carried out in triplicate. (B) Suspended SMC were incubated for 30 minutes with vehicle, 25 µM SB203580 or 25 µM SB202474 and then plated on fibronectin-coated coverslips for 3 hours in the presence or absence of PDGF (10 ng/ml). SMC were fixed and F-actin was visualized with Alexa 488-phalloidin. Bar, 10 µm. These results are representative of three independent experiments.

View larger version (53K): [in a new window]   Fig. 4. Hsp27-P is depleted from the leading edge. (A) Suspended SMC were incubated for 30 minutes with 25 µM SB203580 or vehicle and then plated on fibronectin-coated coverslips for 3 hours in the presence or absence of PDGF (10 ng/ml). Fixed SMC were double-labeled for F-actin and Hsp27. Bar, 10 µm. (B) Suspended SMC were plated on fibronectin-coated coverslips for 3 hours in the presence of PDGF. Fixed SMC were double-labeled for F-actin and Hsp27 or Hsp27-P. Bar, 5 µm. These results are representative of three independent experiments.

View larger version (59K): [in a new window]   Fig. 5. p38 MAPK is transiently phosphorylated at the leading edge. Suspended SMC were plated on fibronectin-coated coverslips for 3 hours in the presence or absence of PDGF (10 ng/ml). (A) Fixed SMC were double-labeled for F-actin and p38 MAPK. Bar, 10 µm. (B) Fixed SMC were double-labeled for F-actin and p38 MAPK or p38MAPK-P. Bar, 5 µm. These results are representative of three independent experiments.

View larger version (62K): [in a new window]   Fig. 6. PDGF induces the recruitment of p38 MAPK in the plasma membrane compartment and the association of Hsp27-P with cortical cytoskeleton. Quiescent SMC were pre-incubated with 25 µM SB203580 or vehicle for 30 minutes and incubated in the presence or absence of PDGF (10 ng/ml) for 15 minutes. Particulate membrane fractions were extracted with 0.04% Triton X-100. Equal volumes of Triton-soluble membrane fraction (40 µg) and Triton-insoluble membrane fraction were analyzed by immunoblotting using antibodies directed against Hsp27, Hsp27-P, p38 MAPK, a component of the Arp 2/3 complex, p34 Arc, and paxillin. These results are representative of two independent experiments.

View larger version (40K): [in a new window]   Fig. 7. Hsp27 localization at the leading edge requires free barbed ends. Suspended SMC were plated on fibronectin-coated coverslips for 3 hours in the presence of PDGF (10 ng/ml) and treated with 150 nM cytochalasin D (CD, closed squares), 80 nM latrunculin B (LB, closed triangles) or vehicle (open circles), for the last 30 minutes of spreading. (A) Fixed SMC were double-labeled for F-actin and Hsp27. Bar, 5 µm. These results are representative of four independent experiments. (B) Distributions of F-actin and Hsp27 at the leading edge (0.9 µm from the membrane). Results are expressed as a percentage of the total fluorescence for each fluorophore. Data represent the mean±s.e.m. o f four independent experiments, carried out on at least ten cells in each experimental condition.

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