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First published online 31 August 2004
doi: 10.1242/jcs.01359

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v-Src accelerates spontaneous motility via phosphoinositide 3-kinase, phospholipase C and phospholipase D, but abrogates chemotaxis in Rat-1 and MDCK cells

¹ CELL Unit, Christian de Duve Institute of Cellular Pathology, Université catholique de Louvain, UCL 75.41, Avenue Hippocrate, 75, 1200 Bruxelles, Belgium
² Laboratory of Toxicology, Université Libre de Bruxelles, Boulevard du Triomphe, 1050 Bruxelles, Belgium

View larger version (33K): [in a new window]   Fig. 5. Generation of, and response to, chemical gradients in Dunn chambers. (A) Alexa 568-dextran (10 kDa) was introduced in the outer well (next to the top of the figure) and the bridge was examined by confocal microscopy after 30 minutes (left; zones 1-3 selected for quantitation of chemotactic response are delimited by horizontal dotted lines). Fluorescence intensity was measured by the Laserpix programme (right). The actual slope of PDGF gradient was calculated according to the method of Zicha et al. (Zicha et al., 1991) and superimposed as a broken line (right). EGF gradient showed a similar slope. (B) Typical trajectories of Rat-1/tsLA29 cells and MDCK/tsLA31 cells during 7 hours of migration in Dunn chambers under chemoattractant gradient (PDGF or EGF, respectively). Increasing growth factor concentration is indicated by the vertical arrow at left. Scale bars: 50 µm.

View larger version (45K): [in a new window]   Fig. 1. Phenotypic comparison of the two cell lines studied. (A) Western blotting. Left: the effect of temperature shift on v-Src content (anti-avian Src antibody EC10) and activation (p-Y418 phosphopeptide-specific antibodies). Right: evidence that pharmacological inhibitors used below do not interfere with v-Src expression and/or activation. (B) Immunolocalization of v-Src and paxillin in comparison with actin filaments. Rat-1/tsLA29 and MDCK/tsLA31 cells were cultured at low density on glass slides and incubated for 20 hours at non-permissive (40°C; upper row) or permissive temperature (34°C; lower row) for v-Src kinase activity, then fixed and permeabilised. v-Src was labelled by anti-avian Src-specific mouse monoclonal IgG2b antibody (clone EC10), and revealed by Alexa 488-labelled goat anti-mouse IgG2 antibodies (green). Paxillin, a focal adhesion marker, was detected by mouse monoclonal IgG1 antibody (clone 165), and revealed by Alexa 647-labelled goat anti-mouse IgG1 antibodies (blue). F-actin was decorated by Alexa 568-phalloidin (red). Cells were analysed by confocal microscopy. Scale bars: 10 µm.

View larger version (28K): [in a new window]   Fig. 2. v-Src increases spontaneous motility in Dunn chambers. To analyse individual movement, cells were plated at low density on glass slides. These were placed in Dunn chambers in the absence of added growth factor and phase-contrast images were recorded at 2-minute intervals. (A) Trajectories during 7 hours of migration; circles indicate initial positions. Scale bar: 200 µm (see also Movies 1-4, supplementary material). (B) Mean speed of migration: data were pooled from 3-4 experiments (n=150 to 180 cell trajectories) in total; box plots represent median (bold vertical line), interquartile range (boxes) and full values range (extremes); ***P<0.001 by Mann-Whitney test.

View larger version (43K): [in a new window]   Fig. 3. v-Src accelerates wound healing. (A) Microscopy. Rat-1/tsLA29 fibroblasts (left) and MDCK/tsLA31 cells (right) were seeded on plastic dishes at high density and incubated overnight at 40°C or 34°C. To prevent growth, cells were treated with mitomycin C for 2 hours before band-stripping, then allowed to migrate into the wounded area for 6 hours in DMEM with 10% FCS, fixed and stained. The photographs present typical examples, with horizontal lines, clearly marked on plastic, indicating the initial edge of the wound (top half of each panel). Scale bar: 100 µm. (B) Quantitation. Spontaneous motility was estimated by counting cells that had colonised the margins of stripped areas after 6 hours. Values are means±s.e.m. of 40 random microscopic fields for each condition, pooled from four experiments (***P<0.001 by Student's t-test).

View larger version (29K): [in a new window]   Fig. 4. Acceleration of motility by v-Src depends on PI 3-kinase, PLC and PLD. Rat-1/tsLA29 fibroblasts (A) and MDCK/tsLA31 cells (B) were pretreated with mitomycin C [except for `mitC(-)' group], and tested for wound healing during 6 hours in DMEM with 10% heat-inactivated FCS supplemented by the indicated concentrations of wortmannin, LY294002 (notice the different concentrations used for the two cell lines), NCDC, 1-butanol or 2-butanol. Values are means±s.e.m. of 20-50 microscopic fields for each condition, pooled from 2-5 experiments and expressed as the percentage of migration at 40°C over the group treated with mitomycin C alone (condition `0').

View larger version (29K): [in a new window]   Fig. 6. Growth factor binding isotherms at 4°C for 2 hours. (A) Rat-1/tsLA29 fibroblasts were incubated in suspension with the indicated final 125I-PDGF concentrations; (B) 125I EGF binding on MDCK/tsLA29 cells was performed in Petri dishes. Values are means±s.d. of specific binding in 6-9 dishes pooled from 3-4 experiments. Curves were drawn after non-linear fitting (r2>0.99), based on the hyperbola y=Bmax*x/(Kd+x), where x is radioligand concentration, y is specific binding, Bmax is a maximum specific binding and Kd is equilibrium dissociation constant. Graphs on the right result from Scatchard transformation.

View larger version (35K): [in a new window]   Fig. 7. Quantification of chemotaxis in Dunn chambers: v-Src abolishes this response. (A) Rat-1/tsLA29 fibroblasts; (B) MDCK/tsLA31 cells. Same procedure as for Fig. 2, except that 1.33 nM PDGF-BB or 1.66 nM EGF were introduced into the outer well of Dunn chambers, respectively. Recording of cell migration started after 30 minutes of chambers assembly, when a linear diffusion gradient of growth factors had been established. For quantification of directionality, orientation of each cell trajectory was determined in the three zones of decreasing growth factor concentrations defined at Fig. 5A (ranges are indicated below bar histograms and numbers of cells analysed in each group are specified in italics). The fraction of cells that had migrated towards the outer well (positive chemotactic response), the inner well, parallel to both wells of the chamber, or non-motile cells, was determined. Values are pooled from 4-8 experiments (***P>0.001, **P<0.01, *P<0.05 by difference-in-probabilities test for the fraction of cells moving towards the growth factor source, as compared with no gradient; or depending on v-Src activation for a comparable level of growth factor/receptor complexes, as indicated by the horizontal bracket).

View larger version (17K): [in a new window]   Fig. 8. (Macro)pinocytic burst in response to growth factors or phorbol ester. (A) Rat-1/tsLA29 fibroblasts; (B) MDCK/tsLA31 cells. Fluid-phase endocytosis was measured by a 5-minutes uptake of HRP in the presence or absence of the indicated concentrations of PDGF-BB, EGF or PMA added concomitantly. Values represent means±s.d. of three dishes from one representative experiment out of three (***P<0.001, **P<0.01 by Student's t-test).

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