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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 has no effect on single-cell migration but does affect cell-contacting behavior. (A) Control U251 cells and those stably expressing OL-pc-HA or OL-pcΔNBS-HA were cultured at a low density, and their phase-contrast time-lapse images were taken for 4 h at 3-min intervals. (left) Tracks of individual cells were collected. Only those not dividing and not in contact with others were sampled. (middle) Statistical analysis of the instantaneous velocities of these cells. Comparison of each experimental group to the control one shows no significant difference (n.s.) at a significance level of α = 0.05. P = 0.13 for OL-pc, and P = 0.27 for OL-pcΔNBS. n = 38, 49, and 39 for control, OL-pc, and OL-pcΔNBS, respectively. (right) Directionality in cell migration defined by the ratio of the direct distance between the starting and ending points for migration to the total track distance. No difference was observed between control (n = 38) and OL-pc–expressing cells (P = 0.28; n = 47) or OL-pcΔNBS–expressing cells (P = 0.22; n = 36). Error bars represent SEM. (B) Time-lapse images of cells making contact with others in low density cultures and their drawings. The numbers denote the time elapsed (in minutes). Arrows indicate the directions of cell movement together with the highest lamellipodial activity. Control cells or OL-pcΔNBS transfectants temporarily move together in the same direction when they meet others, whereas OL-pc transfectants never display such coordination. Also see Video 1 and Video 2 (available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1) for the control and OL-pc transfectant samples, respectively. Bars: (A) 200 μm; (B) 50 μm.
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fig3: OL-pc has no effect on single-cell migration but does affect cell-contacting behavior. (A) Control U251 cells and those stably expressing OL-pc-HA or OL-pcΔNBS-HA were cultured at a low density, and their phase-contrast time-lapse images were taken for 4 h at 3-min intervals. (left) Tracks of individual cells were collected. Only those not dividing and not in contact with others were sampled. (middle) Statistical analysis of the instantaneous velocities of these cells. Comparison of each experimental group to the control one shows no significant difference (n.s.) at a significance level of α = 0.05. P = 0.13 for OL-pc, and P = 0.27 for OL-pcΔNBS. n = 38, 49, and 39 for control, OL-pc, and OL-pcΔNBS, respectively. (right) Directionality in cell migration defined by the ratio of the direct distance between the starting and ending points for migration to the total track distance. No difference was observed between control (n = 38) and OL-pc–expressing cells (P = 0.28; n = 47) or OL-pcΔNBS–expressing cells (P = 0.22; n = 36). Error bars represent SEM. (B) Time-lapse images of cells making contact with others in low density cultures and their drawings. The numbers denote the time elapsed (in minutes). Arrows indicate the directions of cell movement together with the highest lamellipodial activity. Control cells or OL-pcΔNBS transfectants temporarily move together in the same direction when they meet others, whereas OL-pc transfectants never display such coordination. Also see Video 1 and Video 2 (available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1) for the control and OL-pc transfectant samples, respectively. Bars: (A) 200 μm; (B) 50 μm.

Mentions: Nap1 and its associated components are known to regulate cell motility. Therefore, we examined whether OL-pc expression had any effect on cell migration by comparing the parent cells with their transfectants. Cells were plated at low densities to allow their free migration, and their movement was then recorded by time-lapse microscopy. Analysis of the video images did not show any difference in migration speed between the control and OL-pc–transfected cells (Fig. 3 A). We also measured the directionality of cell migration, which was defined previously (Pankov et al., 2005), but could not find any difference in this parameter either between these cells (Fig. 3 A). Careful observation of the videos, however, made us aware that these cells were different in their contacting behavior. When the parent U251 cells made contact with each other, their membrane ruffling was suppressed at the contact sites, and these contact sites as well as the overall polarity of cells, including the lamellipodial directions, were transiently maintained until detachment (Fig. 3 B and Video 1). OL-pc–expressing cells also formed transient contacts; however, these cells in contact randomly moved relative to each other, rapidly changing the lamellipodial positions, suggesting that their peripheral motile activity was not properly controlled by cell–cell contacts (Fig. 3 B and Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1). The association pattern of OL-pcΔNBS–expressing cells was similar to that of the control cells, indicating that the aforementioned action of OL-pc required its binding to Nap1.


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 has no effect on single-cell migration but does affect cell-contacting behavior. (A) Control U251 cells and those stably expressing OL-pc-HA or OL-pcΔNBS-HA were cultured at a low density, and their phase-contrast time-lapse images were taken for 4 h at 3-min intervals. (left) Tracks of individual cells were collected. Only those not dividing and not in contact with others were sampled. (middle) Statistical analysis of the instantaneous velocities of these cells. Comparison of each experimental group to the control one shows no significant difference (n.s.) at a significance level of α = 0.05. P = 0.13 for OL-pc, and P = 0.27 for OL-pcΔNBS. n = 38, 49, and 39 for control, OL-pc, and OL-pcΔNBS, respectively. (right) Directionality in cell migration defined by the ratio of the direct distance between the starting and ending points for migration to the total track distance. No difference was observed between control (n = 38) and OL-pc–expressing cells (P = 0.28; n = 47) or OL-pcΔNBS–expressing cells (P = 0.22; n = 36). Error bars represent SEM. (B) Time-lapse images of cells making contact with others in low density cultures and their drawings. The numbers denote the time elapsed (in minutes). Arrows indicate the directions of cell movement together with the highest lamellipodial activity. Control cells or OL-pcΔNBS transfectants temporarily move together in the same direction when they meet others, whereas OL-pc transfectants never display such coordination. Also see Video 1 and Video 2 (available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1) for the control and OL-pc transfectant samples, respectively. Bars: (A) 200 μm; (B) 50 μm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
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fig3: OL-pc has no effect on single-cell migration but does affect cell-contacting behavior. (A) Control U251 cells and those stably expressing OL-pc-HA or OL-pcΔNBS-HA were cultured at a low density, and their phase-contrast time-lapse images were taken for 4 h at 3-min intervals. (left) Tracks of individual cells were collected. Only those not dividing and not in contact with others were sampled. (middle) Statistical analysis of the instantaneous velocities of these cells. Comparison of each experimental group to the control one shows no significant difference (n.s.) at a significance level of α = 0.05. P = 0.13 for OL-pc, and P = 0.27 for OL-pcΔNBS. n = 38, 49, and 39 for control, OL-pc, and OL-pcΔNBS, respectively. (right) Directionality in cell migration defined by the ratio of the direct distance between the starting and ending points for migration to the total track distance. No difference was observed between control (n = 38) and OL-pc–expressing cells (P = 0.28; n = 47) or OL-pcΔNBS–expressing cells (P = 0.22; n = 36). Error bars represent SEM. (B) Time-lapse images of cells making contact with others in low density cultures and their drawings. The numbers denote the time elapsed (in minutes). Arrows indicate the directions of cell movement together with the highest lamellipodial activity. Control cells or OL-pcΔNBS transfectants temporarily move together in the same direction when they meet others, whereas OL-pc transfectants never display such coordination. Also see Video 1 and Video 2 (available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1) for the control and OL-pc transfectant samples, respectively. Bars: (A) 200 μm; (B) 50 μm.
Mentions: Nap1 and its associated components are known to regulate cell motility. Therefore, we examined whether OL-pc expression had any effect on cell migration by comparing the parent cells with their transfectants. Cells were plated at low densities to allow their free migration, and their movement was then recorded by time-lapse microscopy. Analysis of the video images did not show any difference in migration speed between the control and OL-pc–transfected cells (Fig. 3 A). We also measured the directionality of cell migration, which was defined previously (Pankov et al., 2005), but could not find any difference in this parameter either between these cells (Fig. 3 A). Careful observation of the videos, however, made us aware that these cells were different in their contacting behavior. When the parent U251 cells made contact with each other, their membrane ruffling was suppressed at the contact sites, and these contact sites as well as the overall polarity of cells, including the lamellipodial directions, were transiently maintained until detachment (Fig. 3 B and Video 1). OL-pc–expressing cells also formed transient contacts; however, these cells in contact randomly moved relative to each other, rapidly changing the lamellipodial positions, suggesting that their peripheral motile activity was not properly controlled by cell–cell contacts (Fig. 3 B and Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1). The association pattern of OL-pcΔNBS–expressing cells was similar to that of the control cells, indicating that the aforementioned action of OL-pc required its binding to Nap1.

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