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Ras induces NBT-II epithelial cell scattering through the coordinate activities of Rac and MAPK pathways

Natacha Edme1, Julian Downward2, Jean-Paul Thiery3 and Brigitte Boyer1,*

1 Laboratoire de Régulations Cellulaires et Oncogénése UMR146, Institut Curie Section de Recherche, Centre Universitaire Paris-Sud, 91405 Orsay, France
2 Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK
3 Laboratoire de Compartimentation et Dynamique Cellulaires UMR144, Institut Curie Section de Recherche, 26 rue d'Ulm, 75248 Paris, France



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  		Fig. 1. Active Mek1 promotes NBT-II cell dissociation. (A) Cells were microinjected into the nucleus with 100 ng/µl of constructs coding for MEK1SSDD (a,b), CD2-p110 (c,d) and RalBV23 (e,f), incubated for 18 hours, fixed and double labelled for the expression of the injected construct (a,c,e) and desmoplakin (DP) (b,d,f). Arrows point to microinjected cells, and arrowheads indicate the presence of desmoplakin immunostaining at the cell periphery. (B) For each experiment, several coverslips were analysed and the percentage of desmosome-negative cells was calculated as the ratio between the number of cells without DP at the cell surface and the total number of cells. The results have been averaged from several experiments (n>3) in which at least 100 cells were microinjected. The means and s.e.m. are shown. Bar, 10 µm.

 


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  		Fig. 2. PD98059 and UO126 inhibit EGF-induced desmosome disruption and promotes desmosome reformation in stable RasV12 transfectants. (A) Control NBT-II cells were cultured without (a) or with 30 ng/ml EGF (b) for 15 hours.

Alternatively, 2 days after seeding, cells were incubated with 20 µM of LY80052 (c), 25 µM of FTase2 (d), 100 µM of PD098059 (e) or 100 µM of UO126 (f) for 24 hours before an additional 15 hours incubation with 30 ng/ml of EGF. Cells were fixed and stained for DP expression. Stable RasV12-overexpressing cells (Ras32) (g) were incubated with 20 µM of LY80058 (h), 100 µM of PD098059 (i) or 100 µM of UO126 (j) for 24 hours and 25 µM of FTase2 (k) for 48 hours. (B) Phase contrast micrographs of NBT-II cells (left) and Ras32 cells (right). Note that RasV12 cells exhibit a fibroblastoid phenotype in sharp contrast to the epithelial morphology of the parental NBT-II cells. Bar, 30 µm.

 


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  		Fig. 3. Raf-activating Ras mutants promote desmosome breakdown in NBT-II cells. Cells seeded on coverslips were microinjected into the nucleus with 100 ng/µl of the indicated expression vectors. 18 hours later, they were fixed and immunostained for both Ras and DP expression as described in the Materials and Methods. For each experiment, several coverslips where analysed and the percentages of desmosome-negative cells were calculated as in Fig. 1B. The results have been averaged for several experiments (n>3) in which at least 100 cells were microinjected. The means and s.e.m. are shown.

 


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  		Fig. 4. Rac promotes cell locomotion of MEK1-dissociated cells. (A) Cells were microinjected with 100 ng/µl of the indicated constructs, incubated for 18 hours and then labelled for MEK1 expression. Note that MEK1SSDD-expressing cells retain the round morphology of epithelial NBT-II cells, whereas coexpression of MEK1SSDD and Rac1V12 causes cell spreading and elongation. (B) Cells were microinjected with both 100 ng/µl of the indicated constructs and 25 ng/µl of EGFP encoding construct. After 4 hours of incubation, cells were placed on a Leica motorized microscope connected to a computer using the Metamorph software, and cell motility was recorded by time-lapse videomicroscopy. Velocities were calculated by tracking EGFP-positive cells. Results are expressed as the mean of at least three independent experiments, where at least 40 EGFP-positive cells were recorded±s.e.m. (C) Cells were transfected by the PEI method with the indicated constructs together with the EGPF construct at a 1:4 ratio. 24 hours later, cells were trypsined and re-plated under sparse conditions. Cell migration was recorded 10 hours later as described above. At least three independent experiments were done, and the velocities were averaged for at least 40 cells.

 


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  		Fig. 5. Rac1 activation by downstream pathways of Ras. (A) NBT-II cells were activated with 30 ng/ml of EGF for the indicated times, then lysed with lysis buffer as indicated in the Materials and Methods. Cell extracts were incubated with GST-PAK2 Sepharose beads, washed and immunoblotted with anti-Rac1 antibodies. Levels of endogenous Rac were estimated by immunoblotting total cell extracts with Rac1 antibodies. Alternatively, equivalent amounts of cell extracts were immunoprecipitated with anti-MAPK antibodies and subjected to a kinase assay using MBP as an exogenous substrate. (B-D) Rac pull-down was done on NBT-II cells transiently transfected with the indicated constructs. The upper panel shows the amounts of Rac bound to PAK-GST beads as revealed by Rac1 antibodies. The middle panel shows the levels of Rac as estimated by Rac immunoblotting on total cell extracts. The lower panel shows the levels of EGFP expression measured by EGFP immunoblotting on total cell extracts with anti-GFP antibodies.

 


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  		Fig. 6. Raf-specific mutant forms of Ras promote NBT-II cell migration. Two days after seeding, cells were microinjected with 100 ng/µl of the Ras expression vectors and 25 ng/µl of the EGFP construct. Four hours after microinjection, cells were placed on the motorised stage of a Leica inverted microscope and recorded by videomicroscopy. Velocities were obtained by tracking individual EGFP-positive cells. Each column represents the mean of at least three independent experiments in which at least 40 cells were recorded. (A,B) Clusters of NBT-II cells were transiently transfected with the indicated constructs along with the EGFP reporter construct prior to videocinematography recording. Velocities were measured by tracking at least 40 EGFP-positive cells. For each construct, at least three independent experiments were performed. The mean velocity and s.e.m. are shown. (C) The Rac pull-down assay was done on NBT-II cells transiently transfected with the indicated constructs. The upper panel shows a Rac immunoblot performed on cell extracts purified on PAK-GST Sepharose beads. The lower panel shows a Rac immunoblot of total cell extracts.

 




© The Company of Biologists Ltd 2002