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NCAM regulates cell motility

Søren Prag^*, Eugene A. Lepekhin, Kateryna Kolkova, Rasmus Hartmann-Petersen, Anna Kawa, Peter S. Walmod, Vadym Belman, Helen C. Gallagher¹, Vladimir Berezin, Elisabeth Bock and Nina Pedersen

Protein Laboratory, Institute of Molecular Pathology, University of Copenhagen, Denmark
¹ Department of Pharmacology, University College Dublin, Belfield, Dublin 4, Ireland
^* Present address: MRC Laboratory for Molecular Cell Biology, University College London, United Kingdom

View larger version (41K): [in a new window]   Fig. 1. Effect of expression of NCAM-140 on single-cell motility of glioma cells. (A) Phase-contrast image of glioma cells plated as single cells on fibronectin and transiently transfected with plasmids encoding NCAM-140 and EGFP overlaid with the fluorescence image of the EGFP-expressing cells. (B) ‘Wind-rose’ plots of tracks with superimposed starting points of glioma cells transiently transfected with a plasmid encoding EGFP and either co-transfected with a plasmid encoding NCAM-140 or a control plasmid. The circles mark the square root of the mean-squared displacement () of the recorded cells. (C) Cell motility measured as rate of diffusion, R; (D) root mean square speed, S, and (E) mean cell speed, S, of glioma cells transiently transfected with vector (control) or a plasmid encoding NCAM-140 (the mean from five experiments was *P<0.05, **P<0.01 using student’s paired t-test). (F,G) The distribution of the S for populations of cells from representative experiments of cells transiently and stably transfected with a control plasmid (F) or a plasmid encoding NCAM-140 (G).

View larger version (35K): [in a new window]   Fig. 2. Effect of NCAM-140 on motility of glioma cells in co-culture with fibroblasts. (A-D) Immunostaining of NCAM (red) of NCAM-140-positive or -negative glioma cell lines permanently transfected to express EGFP (green) in co-culture with NCAM-140-positive and -negative fibroblast cell lines. (E) Rate of diffusion, R, of the EGFP expressing NCAM-140-positive and -negative glioma cells in co-culture (the mean of four experiments was *P<0.05 compared with vector-transfected controls, paired t-test).

View larger version (21K): [in a new window]   Fig. 3. The effect of the intracellular parts of NCAM on glioma-cell motility. (A-E) NCAM-140-positive or -negative glioma cell lines plated as single cells on fibronectin transiently transfected with a plasmid encoding EGFP together with either a plasmid encoding the cytoplasmic part of NCAM-140 (140-cyt) or a control plasmid (vector). (A-D) Immunostaining with antibodies against the cytoplasmic part of NCAM (red) of transiently transfected cells (green). (E) Rate of diffusion, R, of the transiently transfected cells (mean of six experiments, *P<0.05, **P<0.01 compared with vector-transfected control cells, paired t-test). (F) NCAM-140-positive or -negative glioma cell lines plated as single cells on fibronectin transiently transfected with a plasmid encoding EGFP together with either a plasmid encoding the cytoplasmic part of NCAM-180 (180-cyt) or a control plasmid (vector). Rate of diffusion, R, of the transiently transfected cells (mean of 4 experiments, *P<0.05 of 180-cyt-transfected NCAM-140 cells versus vector-transfected NCAM-140 cells and versus 180-cyt-transfected control cells, **P<0.01 for 180-cyt versus vector-transfected control glioma cells, paired t-test).

View larger version (36K): [in a new window]   Fig. 4. Effect of expression of NCAM-140 on cell attachment. IRM images of a single cell from a control cell line (A) and a single cell from an NCAM-140-expressing glioma cell line plated on glass (B). Black and white images were transformed to pseudo-colors: white/yellow reflecting the strongest attachment and blue/purple the weakest. (C) Cumulative distribution of attachment strength determined by quantification of the grey levels after IRM (means of more than 150 cells in each group from a representative experiment). The difference in distribution was highly significant (P<0.001) in three independent experiments. (D) A detachment assay using trypsin on NCAM-140-positive or -negative cell lines plated as single cells on fibronectin (normalized to 100% attached cells for no trypsin added with values from three independent experiments). The difference in attachment was highly significant (P<0.001). (E,F) Immunostaining of focal adhesions of a control (E) and an NCAM-140-expressing cell line (F) plated as single cells on fibronectin using a monoclonal antibody against vinculin. (G,H) Staining of actin stress fibers (F-actin) of a control (G) and an NCAM-140 expressing cell line (H) with Texas-Red conjugated phalloidin.

View larger version (21K): [in a new window]   Fig. 5. Effect of extracellular interactions on cell motility. (A) Rate of diffusion, R, of NCAM-140-positive and -negative glioma cell lines after addition of conditioned medium containing a soluble form of the extracellular part of NCAM (Sol.-NCAM) or control conditioned medium (mean of 6 experiments, *P<0.05, **P<0.001, paired t-test). (B) Rate of diffusion, R, of NCAM-negative glioma cells treated for 20 hours with sodium chlorate (50 mM) or treated for one hour with heparin (50 µg/ml) or recombinant NCAM Ig modules I-II (IgI-II) (100 µg/ml) (mean of 4 experiments, *P<0.05, paired t-test).

View larger version (27K): [in a new window]   Fig. 6. A model of interactions of NCAM-140 that influence cell motility. (A) Optimal attachment for high motility of an NCAM-negative cell. (B) Decreased attachment and low motility owing to NCAM-140 expression.