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N-WASP activation by a ß1-integrin-dependent mechanism supports PI3K-independent chemotaxis stimulated by urokinase-type plasminogen activator

¹ The Randall Centre for Molecular Mechanisms of Cell Function, New Hunt's House, King's College London, Guy's Campus, London SE1 1UL, UK
² INSERM U461, F-92296, France

View larger version (54K): [in a new window]   Fig. 1. uPA induces cell polarisation, spreading and redistribution of focal adhesion complexes containing ß1-integrins and vinculin. (A,C) Unstimulated cells. (B,D) Cells stimulated with 10 nM mut C uPA for 30 minutes. (A-D) Alexa-568-conjugated phalloidin; (A,B) Anti-ß1-integrins labelled with Alexa-488-conjugated secondary antibody; (C,D) Anti-vinculin labelled with Alexa-488-conjugated secondary antibody. Bar, 10 µm. Images are representative of three experiments.

View larger version (29K): [in a new window]   Fig. 2. Cells assayed for migratory behaviour in Dunn chambers. (A) Growth medium (+ 10% FCS) without chemoattractant gradient. (B-D) starvation medium (+ 0.1% FCS). (B) No chemoattractant gradient. (C) Concentration gradient of EGF (15 ng/ml in outer well). (D) Concentration gradient of mut C uPA (10 nM in outer well). (A-D) Cell trajectories over 5 hours (10 median cell trajectories). The start point for each cell is coordinate (0,0); The end point is marked by a closed circle; the chemoattractant source is at the top of plot; axis graduations, distance (µm). Circular histograms show the proportion of cells with migratory direction lying within each 20° interval (chemoattractant source at top of histogram). Cells that migrated less than 20 µm were excluded from analysis. The red arrow represents the mean direction of migration; the green segment represents the 95% confidence interval determined by the Rayleigh test. Displacement shows the percentage of cells that migrate further than 20 µm. Concentration-dependent response for (E) EGF, (F) wt uPA (open circles) and mut C uPA (closed circles). (G) Effect of ß1-integrin neutralising antibody and mouse IgG antibody on chemotaxis stimulated by 10 nM mut C. More than 40 cells pooled from three to six experiments included in analyses.

View larger version (45K): [in a new window]   Fig. 3. Total Akt and phosphorylated Akt in whole cell lysates determined by SDS PAGE on 10% gels and immunoblotting. Cells were stimulated with 10 ng/ml EGF for 10 minutes or 10 nM mut C uPA. (A) uPA activated Akt phosphorylation. (B) Inhibition of EGF-stimulated Akt phosphorylation by 1-10 µM LY 294002 (pre-treatment 1 hour). Relative band intensities represent the ratio of phospho-Akt to total Akt (band intensity in unstimulated cells, 1.0). The results are representative of three experiments.

View larger version (35K): [in a new window]   Fig. 4. PI3K-independent chemotaxis stimulated by uPA in Dunn chamber chemotaxis assay. (A,B) EGF (15 ng/ml). (C,D) 15 nM mut C uPA. (A,C) Relative cell speeds (basal cell speed=1.0). Positive/negative chemotactic response (+/-) (total number of cells). (B,D) Directionality/displacement of cells stimulated by EGF and uPA±5 µM LY 294002. Mean direction of cell migration (red arrow)/95% confidence interval (green wedge) determined by Rayleigh test. n=total number of cells assayed in three to six experiments. %=proportion of cells that migrate further than 20 µm.

View larger version (39K): [in a new window]   Fig. 5. Effect of LY 294002 on actin cytoskeleton changes induced by uPA and EGF. (A-F) Cells stained with Alexa-568-conjugated phalloidin are shown. Cells were stimulated with 10 nM mut C uPA for (A) 3 minutes, (B) 30 minutes, (D) 3 minutes + LY 294002 (5 µM; 1 hour pre-treatment) or (E) 30 minutes + LY 294002. Cells were stimulated with 10 ng/ml EGF for (C) 30 minutes and (F) 30 minutes + LY 294002. Bar, 10 µm. (G) Quantification of cells with microspikes/lamellipodia. The number of microspikes and lamellipodia were counted after 3 minutes and 30 minutes of stimulation, respectively.

View larger version (29K): [in a new window]   Fig. 6. Cdc42 and Rac1 activation by uPA and EGF. (A,B) Cells were assayed for Cdc42/Rac1 GTPase activation using a PAK-CRIB pull down assay. The levels of total cellular Rac1/Cdc42 and GST-PAK-CRIB that precipitated Cdc42/Rac1 were determined by SDS PAGE on 12% gels and immunoblotting. Temporal activation of (A) Cdc42 and (B) Rac1 by 10 nM uPA and 10 ng/ml EGF are shown. Graphs show ratios of GST-PAK-CRIB bound to total Cdc42/Rac1 (band intensity in unstimulated cells, 1.0). The results represent mean±s.e.m. from three to seven experiments.

View larger version (32K): [in a new window]   Fig. 7. PI3K-independent and ß1-integrin dependent activation of Cdc42 and Rac1 by uPA. Cells were stimulated with 10 nM mut C uPA or 10 ng/ml EGF for 10 min±LY 294002 (1-5 µM, pre-treatment 1 hour). (A,B) shows Rac1 activation, and (C,D) shows Cdc42 activation±LY 294002. (B,D) Ratios of GST-PAK-CRIB bound to total Cdc42/Rac1 (band intensity in unstimulated cells=1.0; mean±s.e.m. from 3-7 separate experiments) are shown. (E) Rac1/Cdc42 activation by 10 nM mut C uPA±3 µg/ml anti-ß1-integrin is shown. (The results are representative of four experiments.)

View larger version (28K): [in a new window]   Fig. 8. uPA and EGF activate N-WASP with divergent requirement for PI3K. (A) Cells were stimulated with 10 ng/ml EGF for 1-10 minutes. EGFR immunoprecipitates were separated on 10% gels and immunoblotted for N-WASP. (B,C) shows N-WASP in Triton X100 soluble cell lysates (cytosolic fraction) and Triton X100 insoluble cell pellets (cytoskeletal fraction). (B) Cells stimulated with 10 ng/ml EGF or 10 nM uPA for 1-30 minutes are shown. (C) Cells stimulated for 10 minutes with 10 nM mut C uPA or 10 ng/ml EGF±LY 294002 are shown (5 µM; pre-treatment 1 hour). The results are representative of three to six experiments.

View larger version (85K): [in a new window]   Fig. 9. uPA and EGF stimulate N-WASP colocalisation to F-actin with divergent requirement for PI3K activity. (A-F) shows cell staining with Alexa-568-conjugated phalloidin and anti-N-WASP labelled with Alexa-488-conjugated secondary antibody. (A,B) shows unstimulated cells in the (A) absence and (B) presence of LY 294002 for 30 minutes (5 µM; pre-treatment 1 hour). (C,D) The result of a 30 minute stimulation with 10 nM mut C uPA in the (C) absence and (D) presence of LY 294002 is shown. (E,F) 30 minutes stimulation with 10 ng/ml EGF in (E) absence and (F) presence of LY 294002 is shown. Images are representative of three experiments. Bar, 10 µm.

View larger version (32K): [in a new window]   Fig. 10. PI3K-independent activation of N-WASP by uPA involves its disassociation from ß1-integrin. (A,B) ß1-integrin immunoprecipitates were separated on 10% gels and immunoblotted for N-WASP. (A) Cells stimulated with 10 nM uPA for 1-30 minutes±LY 294002 (5 µM). (B) Cells stimulated with 10 ng/ml EGF for 1-30 minutes±LY 294002. The results are representative of three experiments.

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