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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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Nap1 and WAVE1 are required for OL-pc function. (A) Control U251 cells and OL-pc-HA transfectants were treated with control, Nap1, or WAVE1 siRNA, and their migration pattern during wound healing was analyzed as described in Fig. 5. See Fig. S4 for the original photographs of these cells. Also see Videos 6 and 7 (available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1) for the videos of Nap1-depleted cells without and with OL-pc, respectively. The arrow indicates the direction of wound healing. (B) Quantification of the instantaneous velocities of those cells. n = 31 and 29 for control and OL-pc transfectants with control siRNA, respectively; n = 40 for both control and OL-pc transfectants with Nap1 siRNA; n = 29 and 34 for control and OL-pc transfectants, respectively, treated with WAVE1 siRNA. **, P < 0.005; ***, P < 0.0005. (C) The farthest and nearest indices obtained as in Fig. 5 D. n = 23 and 22 for control and OL-pc transfectants with control siRNA, respectively; n = 30 for both control and OL-pc transfectants with Nap1 RNAi; n = 23 and 24 for control and OL-pc transfectants with WAVE1 siRNA, respectively. *, P < 0.05; **, P < 0.005. n.s., P = 0.35 and 0.44 for Nap1 siRNA–treated cells and P = 0.83 and 0.06 for WAVE1 siRNA–treated cells for the farthest and nearest indices, respectively. Bar, 50 μm.
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fig6: Nap1 and WAVE1 are required for OL-pc function. (A) Control U251 cells and OL-pc-HA transfectants were treated with control, Nap1, or WAVE1 siRNA, and their migration pattern during wound healing was analyzed as described in Fig. 5. See Fig. S4 for the original photographs of these cells. Also see Videos 6 and 7 (available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1) for the videos of Nap1-depleted cells without and with OL-pc, respectively. The arrow indicates the direction of wound healing. (B) Quantification of the instantaneous velocities of those cells. n = 31 and 29 for control and OL-pc transfectants with control siRNA, respectively; n = 40 for both control and OL-pc transfectants with Nap1 siRNA; n = 29 and 34 for control and OL-pc transfectants, respectively, treated with WAVE1 siRNA. **, P < 0.005; ***, P < 0.0005. (C) The farthest and nearest indices obtained as in Fig. 5 D. n = 23 and 22 for control and OL-pc transfectants with control siRNA, respectively; n = 30 for both control and OL-pc transfectants with Nap1 RNAi; n = 23 and 24 for control and OL-pc transfectants with WAVE1 siRNA, respectively. *, P < 0.05; **, P < 0.005. n.s., P = 0.35 and 0.44 for Nap1 siRNA–treated cells and P = 0.83 and 0.06 for WAVE1 siRNA–treated cells for the farthest and nearest indices, respectively. Bar, 50 μm.

Mentions: Next, we compared the behavior of control and OL-pc transfectants from which Nap1 or WAVE1 was depleted in the wound-healing assay. At the wound edges of Nap1-depleted control U251 cells, these cells appeared more slender than their Nap1-positive counterparts (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1), and, despite their defective lamellipodia formation, they migrated actively (Fig. 6 A and Video 6). When their migration was compared with that of Nap1-depleted OL-pc transfectants, the latter migrated slightly faster than the former (Fig. 6 B and Video 7). However, these Nap1-depleted OL-pc transfectants did not display the uncoordinated migration that was unique to the Nap1-positive OL-pc transfectants but did migrate in a pattern similar to control cells (Fig. 6, A and C). Thus, the migration profile became indistinguishable between the cells with and without OL-pc, except for the migration speed, in the absence of Nap1. WAVE1 depletion produced similar results to that of Nap1, although the overall migration speed of U251 cells was greatly reduced (Fig. 6 and Fig. S4). These results suggest that OL-pc required Nap1 and WAVE1 in inducing the uncoordinated cell movement, although it did not require them for simple enhancement of cell migration.


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

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

Nap1 and WAVE1 are required for OL-pc function. (A) Control U251 cells and OL-pc-HA transfectants were treated with control, Nap1, or WAVE1 siRNA, and their migration pattern during wound healing was analyzed as described in Fig. 5. See Fig. S4 for the original photographs of these cells. Also see Videos 6 and 7 (available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1) for the videos of Nap1-depleted cells without and with OL-pc, respectively. The arrow indicates the direction of wound healing. (B) Quantification of the instantaneous velocities of those cells. n = 31 and 29 for control and OL-pc transfectants with control siRNA, respectively; n = 40 for both control and OL-pc transfectants with Nap1 siRNA; n = 29 and 34 for control and OL-pc transfectants, respectively, treated with WAVE1 siRNA. **, P < 0.005; ***, P < 0.0005. (C) The farthest and nearest indices obtained as in Fig. 5 D. n = 23 and 22 for control and OL-pc transfectants with control siRNA, respectively; n = 30 for both control and OL-pc transfectants with Nap1 RNAi; n = 23 and 24 for control and OL-pc transfectants with WAVE1 siRNA, respectively. *, P < 0.05; **, P < 0.005. n.s., P = 0.35 and 0.44 for Nap1 siRNA–treated cells and P = 0.83 and 0.06 for WAVE1 siRNA–treated cells for the farthest and nearest indices, respectively. Bar, 50 μm.
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fig6: Nap1 and WAVE1 are required for OL-pc function. (A) Control U251 cells and OL-pc-HA transfectants were treated with control, Nap1, or WAVE1 siRNA, and their migration pattern during wound healing was analyzed as described in Fig. 5. See Fig. S4 for the original photographs of these cells. Also see Videos 6 and 7 (available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1) for the videos of Nap1-depleted cells without and with OL-pc, respectively. The arrow indicates the direction of wound healing. (B) Quantification of the instantaneous velocities of those cells. n = 31 and 29 for control and OL-pc transfectants with control siRNA, respectively; n = 40 for both control and OL-pc transfectants with Nap1 siRNA; n = 29 and 34 for control and OL-pc transfectants, respectively, treated with WAVE1 siRNA. **, P < 0.005; ***, P < 0.0005. (C) The farthest and nearest indices obtained as in Fig. 5 D. n = 23 and 22 for control and OL-pc transfectants with control siRNA, respectively; n = 30 for both control and OL-pc transfectants with Nap1 RNAi; n = 23 and 24 for control and OL-pc transfectants with WAVE1 siRNA, respectively. *, P < 0.05; **, P < 0.005. n.s., P = 0.35 and 0.44 for Nap1 siRNA–treated cells and P = 0.83 and 0.06 for WAVE1 siRNA–treated cells for the farthest and nearest indices, respectively. Bar, 50 μm.
Mentions: Next, we compared the behavior of control and OL-pc transfectants from which Nap1 or WAVE1 was depleted in the wound-healing assay. At the wound edges of Nap1-depleted control U251 cells, these cells appeared more slender than their Nap1-positive counterparts (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1), and, despite their defective lamellipodia formation, they migrated actively (Fig. 6 A and Video 6). When their migration was compared with that of Nap1-depleted OL-pc transfectants, the latter migrated slightly faster than the former (Fig. 6 B and Video 7). However, these Nap1-depleted OL-pc transfectants did not display the uncoordinated migration that was unique to the Nap1-positive OL-pc transfectants but did migrate in a pattern similar to control cells (Fig. 6, A and C). Thus, the migration profile became indistinguishable between the cells with and without OL-pc, except for the migration speed, in the absence of Nap1. WAVE1 depletion produced similar results to that of Nap1, although the overall migration speed of U251 cells was greatly reduced (Fig. 6 and Fig. S4). These results suggest that OL-pc required Nap1 and WAVE1 in inducing the uncoordinated cell movement, although it did not require them for simple enhancement of cell migration.

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