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Contact-dependent promotion of cell migration by the OL-protocadherin-Nap1 interaction.

Nakao S, Platek A, Hirano S, Takeichi M - J. Cell Biol. (2008)

Bottom Line: Although OL-pc expression had no effect on the motility of solitary U251 cells, it accelerated their movement when they were in contact with one another, causing concomitant reorganization of F-actin and N-cadherin at cell junctions.OL-pc mutants lacking the Nap1-binding site exhibited no such effect.These results suggest that OL-pc remodels the motility and adhesion machinery at cell junctions by recruiting the Nap1-WAVE1 complex to these sites and, in turn, promotes the migration of cells.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

ABSTRACT
OL-protocadherin (OL-pc) is a transmembrane protein belonging to the cadherin superfamily, which has been shown to accumulate at cell-cell contacts via its homophilic interaction, but its molecular roles remain elusive. In this study, we show that OL-pc bound Nck-associated protein 1 (Nap1), a protein that regulates WAVE-mediated actin assembly. In astrocytoma U251 cells not expressing OL-pc, Nap1 was localized only along the lamellipodia. However, exogenous expression of OL-pc in these cells recruited Nap1 as well as WAVE1 to cell-cell contact sites. Although OL-pc expression had no effect on the motility of solitary U251 cells, it accelerated their movement when they were in contact with one another, causing concomitant reorganization of F-actin and N-cadherin at cell junctions. OL-pc mutants lacking the Nap1-binding site exhibited no such effect. N-cadherin knockdown mimicked OL-pc expression in enhancing cell movement. These results suggest that OL-pc remodels the motility and adhesion machinery at cell junctions by recruiting the Nap1-WAVE1 complex to these sites and, in turn, promotes the migration of cells.

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OL-pc recruits Nap1 and WAVE1 to cell–cell contacts. (A, left) Control (vain vector transfected) U251 cells and those stably expressing OL-pc-HA or OL-pcΔNBS-HA were cultured in a low density and double immunostained for Nap1 and HA tag. Arrows point to Nap1-positive lamellipodia, and arrowheads here as well as in B and C indicate cell–cell contact sites. Note that Nap1 is localized at cell–cell contacts in the transfectants of OL-pc but not in those of OL-pcΔNBS. (right) Double staining for F-actin and HA. Both OL-pc and OL-pcΔNBS colocalize with F-actin in the lamellipodia. (B) Cells in confluent cultures were double immunostained for Nap1 and HA after extraction with 0.5% Triton X-100. (C) WAVE1-GFP was transiently introduced into cells followed by double immunostaining for OL-pc and GFP. Note the accumulation of WAVE1 at OL-pc–positive but not OL-pcΔNBS–positive cell–cell contact sites. Images were taken with a CCD camera for A and B and with a confocal microscope for C. Bars, 20 μm.
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fig2: OL-pc recruits Nap1 and WAVE1 to cell–cell contacts. (A, left) Control (vain vector transfected) U251 cells and those stably expressing OL-pc-HA or OL-pcΔNBS-HA were cultured in a low density and double immunostained for Nap1 and HA tag. Arrows point to Nap1-positive lamellipodia, and arrowheads here as well as in B and C indicate cell–cell contact sites. Note that Nap1 is localized at cell–cell contacts in the transfectants of OL-pc but not in those of OL-pcΔNBS. (right) Double staining for F-actin and HA. Both OL-pc and OL-pcΔNBS colocalize with F-actin in the lamellipodia. (B) Cells in confluent cultures were double immunostained for Nap1 and HA after extraction with 0.5% Triton X-100. (C) WAVE1-GFP was transiently introduced into cells followed by double immunostaining for OL-pc and GFP. Note the accumulation of WAVE1 at OL-pc–positive but not OL-pcΔNBS–positive cell–cell contact sites. Images were taken with a CCD camera for A and B and with a confocal microscope for C. Bars, 20 μm.

Mentions: To study the biological role of the OL-pc–Nap1 complex, we transfected U251 cells with cDNAs for OL-pc and OL-pcΔNBS and isolated their stable transfectant lines. Each line was a mixture of uncloned transfectants, which allowed us to avoid observing clone-specific phenotypes. Despite the description in the GEO DataSets (National Center for Biotechnology Information) that OL-pc mRNA is expressed in the U251 line, we did not detect any endogenous OL-pc protein in it. In low cell density cultures, U251 cells were highly motile, exhibiting a polarized fanlike shape with lamellipodia at the leading edge (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1). When these cells had collided, they formed transient contacts, although they soon became separated from one another as a result of the high locomotive activities. In higher densities, they more stably contacted each other. Irrespective of the cell densities, the exogenous OL-pc and OL-pcΔNBS were always concentrated at cell–cell contact sites (Fig. 2 A). These molecules were also detectable in the lamellipodial regions, where they were well colocalized with actin fibers (Fig. 2 A, right).


Contact-dependent promotion of cell migration by the OL-protocadherin-Nap1 interaction.

Nakao S, Platek A, Hirano S, Takeichi M - J. Cell Biol. (2008)

OL-pc recruits Nap1 and WAVE1 to cell–cell contacts. (A, left) Control (vain vector transfected) U251 cells and those stably expressing OL-pc-HA or OL-pcΔNBS-HA were cultured in a low density and double immunostained for Nap1 and HA tag. Arrows point to Nap1-positive lamellipodia, and arrowheads here as well as in B and C indicate cell–cell contact sites. Note that Nap1 is localized at cell–cell contacts in the transfectants of OL-pc but not in those of OL-pcΔNBS. (right) Double staining for F-actin and HA. Both OL-pc and OL-pcΔNBS colocalize with F-actin in the lamellipodia. (B) Cells in confluent cultures were double immunostained for Nap1 and HA after extraction with 0.5% Triton X-100. (C) WAVE1-GFP was transiently introduced into cells followed by double immunostaining for OL-pc and GFP. Note the accumulation of WAVE1 at OL-pc–positive but not OL-pcΔNBS–positive cell–cell contact sites. Images were taken with a CCD camera for A and B and with a confocal microscope for C. Bars, 20 μm.
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fig2: OL-pc recruits Nap1 and WAVE1 to cell–cell contacts. (A, left) Control (vain vector transfected) U251 cells and those stably expressing OL-pc-HA or OL-pcΔNBS-HA were cultured in a low density and double immunostained for Nap1 and HA tag. Arrows point to Nap1-positive lamellipodia, and arrowheads here as well as in B and C indicate cell–cell contact sites. Note that Nap1 is localized at cell–cell contacts in the transfectants of OL-pc but not in those of OL-pcΔNBS. (right) Double staining for F-actin and HA. Both OL-pc and OL-pcΔNBS colocalize with F-actin in the lamellipodia. (B) Cells in confluent cultures were double immunostained for Nap1 and HA after extraction with 0.5% Triton X-100. (C) WAVE1-GFP was transiently introduced into cells followed by double immunostaining for OL-pc and GFP. Note the accumulation of WAVE1 at OL-pc–positive but not OL-pcΔNBS–positive cell–cell contact sites. Images were taken with a CCD camera for A and B and with a confocal microscope for C. Bars, 20 μm.
Mentions: To study the biological role of the OL-pc–Nap1 complex, we transfected U251 cells with cDNAs for OL-pc and OL-pcΔNBS and isolated their stable transfectant lines. Each line was a mixture of uncloned transfectants, which allowed us to avoid observing clone-specific phenotypes. Despite the description in the GEO DataSets (National Center for Biotechnology Information) that OL-pc mRNA is expressed in the U251 line, we did not detect any endogenous OL-pc protein in it. In low cell density cultures, U251 cells were highly motile, exhibiting a polarized fanlike shape with lamellipodia at the leading edge (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1). When these cells had collided, they formed transient contacts, although they soon became separated from one another as a result of the high locomotive activities. In higher densities, they more stably contacted each other. Irrespective of the cell densities, the exogenous OL-pc and OL-pcΔNBS were always concentrated at cell–cell contact sites (Fig. 2 A). These molecules were also detectable in the lamellipodial regions, where they were well colocalized with actin fibers (Fig. 2 A, right).

Bottom Line: Although OL-pc expression had no effect on the motility of solitary U251 cells, it accelerated their movement when they were in contact with one another, causing concomitant reorganization of F-actin and N-cadherin at cell junctions.OL-pc mutants lacking the Nap1-binding site exhibited no such effect.These results suggest that OL-pc remodels the motility and adhesion machinery at cell junctions by recruiting the Nap1-WAVE1 complex to these sites and, in turn, promotes the migration of cells.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

ABSTRACT
OL-protocadherin (OL-pc) is a transmembrane protein belonging to the cadherin superfamily, which has been shown to accumulate at cell-cell contacts via its homophilic interaction, but its molecular roles remain elusive. In this study, we show that OL-pc bound Nck-associated protein 1 (Nap1), a protein that regulates WAVE-mediated actin assembly. In astrocytoma U251 cells not expressing OL-pc, Nap1 was localized only along the lamellipodia. However, exogenous expression of OL-pc in these cells recruited Nap1 as well as WAVE1 to cell-cell contact sites. Although OL-pc expression had no effect on the motility of solitary U251 cells, it accelerated their movement when they were in contact with one another, causing concomitant reorganization of F-actin and N-cadherin at cell junctions. OL-pc mutants lacking the Nap1-binding site exhibited no such effect. N-cadherin knockdown mimicked OL-pc expression in enhancing cell movement. These results suggest that OL-pc remodels the motility and adhesion machinery at cell junctions by recruiting the Nap1-WAVE1 complex to these sites and, in turn, promotes the migration of cells.

Show MeSH
Related in: MedlinePlus