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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 expression accelerates cell movement in wound healing. (A) Confluent cell layers were wounded by cell scraping, and the wounded edges were immediately processed for time-lapse recording. Phase-contrast images were taken for 4 h at 3-min intervals. At the end of the recording, individual cells located along the front of cell sheets were marked with colored circles over their nuclear positions, and the same cells were tracked back to earlier time points. At each time point, the circles were connected with lines in a fixed order. Dashed lines represent wound edges. The numbers denote the time elapsed (in minutes). Control and OL-pcΔNBS transfectants roughly maintain their relative positions during migration, whereas OL-pc transfectants irregularly change their spatial relations with neighbors (e.g., the cell marked with the red circle at the right jumped out to the front position from an initially deep site). Videos 3–5 are available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1. (B) Migration track of individual cells marked with different colors in A, with cell position plotted at 9-min intervals. The arrow indicates the direction of wound healing. Abrupt acceleration of movement is most frequently seen in OL-pc transfectants. (C) Statistical analysis of the instantaneous velocities of cells during wound healing. Comparison of each experimental group with the control cells (n = 38) shows a significant difference for OL-pc (**, P < 0.0005; n = 51) but not for OL-pcΔNBS (P = 0.70; n = 41). (D) Changes in the positions of cells relative to those of their neighbors during migration. At the end of recording (at 240 min after scraping), all cells at the wound edges were marked as in A, and their migration track was drawn as in B. Choosing a pair of cells located next to each other at the 240-min point, the distances between their nuclei at earlier time points were measured at 15-min intervals, and their means as well as the farthest and nearest distances were obtained. The farthest and nearest indices were defined as the ratio of the farthest (Dmax) and nearest (Dmin) distance to the average distance (Davg), respectively, as illustrated in the left panel. The farthest index for OL-pc transfectants is significantly larger (**, P < 0.0005; n = 77) than that for the control cells (n = 58), whereas that for OL-pcΔNBS transfectants (P = 0.39; n = 63) is not. Likewise, the nearest index for OL-pc transfectants is significantly smaller (*, P < 0.005) than that for the controls, whereas that for OL-pcΔNBS (n.s.; P = 0.87) is not. (E) Changes in the mean distance (±SEM [error bars]) of the migrating wound edges from their starting position during a long incubation period up to 11 h. n = 12 for all samples. Bar, 50 μm.
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fig5: OL-pc expression accelerates cell movement in wound healing. (A) Confluent cell layers were wounded by cell scraping, and the wounded edges were immediately processed for time-lapse recording. Phase-contrast images were taken for 4 h at 3-min intervals. At the end of the recording, individual cells located along the front of cell sheets were marked with colored circles over their nuclear positions, and the same cells were tracked back to earlier time points. At each time point, the circles were connected with lines in a fixed order. Dashed lines represent wound edges. The numbers denote the time elapsed (in minutes). Control and OL-pcΔNBS transfectants roughly maintain their relative positions during migration, whereas OL-pc transfectants irregularly change their spatial relations with neighbors (e.g., the cell marked with the red circle at the right jumped out to the front position from an initially deep site). Videos 3–5 are available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1. (B) Migration track of individual cells marked with different colors in A, with cell position plotted at 9-min intervals. The arrow indicates the direction of wound healing. Abrupt acceleration of movement is most frequently seen in OL-pc transfectants. (C) Statistical analysis of the instantaneous velocities of cells during wound healing. Comparison of each experimental group with the control cells (n = 38) shows a significant difference for OL-pc (**, P < 0.0005; n = 51) but not for OL-pcΔNBS (P = 0.70; n = 41). (D) Changes in the positions of cells relative to those of their neighbors during migration. At the end of recording (at 240 min after scraping), all cells at the wound edges were marked as in A, and their migration track was drawn as in B. Choosing a pair of cells located next to each other at the 240-min point, the distances between their nuclei at earlier time points were measured at 15-min intervals, and their means as well as the farthest and nearest distances were obtained. The farthest and nearest indices were defined as the ratio of the farthest (Dmax) and nearest (Dmin) distance to the average distance (Davg), respectively, as illustrated in the left panel. The farthest index for OL-pc transfectants is significantly larger (**, P < 0.0005; n = 77) than that for the control cells (n = 58), whereas that for OL-pcΔNBS transfectants (P = 0.39; n = 63) is not. Likewise, the nearest index for OL-pc transfectants is significantly smaller (*, P < 0.005) than that for the controls, whereas that for OL-pcΔNBS (n.s.; P = 0.87) is not. (E) Changes in the mean distance (±SEM [error bars]) of the migrating wound edges from their starting position during a long incubation period up to 11 h. n = 12 for all samples. Bar, 50 μm.

Mentions: As another way to measure the cell behavior, we conducted a wound-healing assay in which cell migration was stimulated under the conditions in which cells were in contact with each other. When wounds had been produced by cell scraping of confluent cultures, the cells located around the wound edges became polarized so as to migrate toward the open space within 10 min. In control cultures, cells occupying the front line of the wound were arranged in parallel and moved together, maintaining their mutual contacts in which individual cells kept roughly a similar pace, suggesting that their movements were coordinated (Fig. 5 A, control; and Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1). At their lateral contact sites, the membrane ruffling was prohibited, indicating that the contact inhibition of movement was also operating in this system. On the other hand, the movement of OL-pc–expressing cells was rarely coordinated: they dynamically changed their positions relative to those of their neighbors during translocation (Fig. 5 A, OL-pc; and Video 4), as was quantified by defining the farthest and nearest indices (Fig. 5 D). Many of them abruptly moved out of the original positions, leaving their neighbors behind, which resulted in the positioning of themselves at the front-most edge of the wound. This type of uncoordinated movement was also detectable in the control cell cultures, but its frequency was greatly increased in the OL-pc–expressing cells.


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 expression accelerates cell movement in wound healing. (A) Confluent cell layers were wounded by cell scraping, and the wounded edges were immediately processed for time-lapse recording. Phase-contrast images were taken for 4 h at 3-min intervals. At the end of the recording, individual cells located along the front of cell sheets were marked with colored circles over their nuclear positions, and the same cells were tracked back to earlier time points. At each time point, the circles were connected with lines in a fixed order. Dashed lines represent wound edges. The numbers denote the time elapsed (in minutes). Control and OL-pcΔNBS transfectants roughly maintain their relative positions during migration, whereas OL-pc transfectants irregularly change their spatial relations with neighbors (e.g., the cell marked with the red circle at the right jumped out to the front position from an initially deep site). Videos 3–5 are available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1. (B) Migration track of individual cells marked with different colors in A, with cell position plotted at 9-min intervals. The arrow indicates the direction of wound healing. Abrupt acceleration of movement is most frequently seen in OL-pc transfectants. (C) Statistical analysis of the instantaneous velocities of cells during wound healing. Comparison of each experimental group with the control cells (n = 38) shows a significant difference for OL-pc (**, P < 0.0005; n = 51) but not for OL-pcΔNBS (P = 0.70; n = 41). (D) Changes in the positions of cells relative to those of their neighbors during migration. At the end of recording (at 240 min after scraping), all cells at the wound edges were marked as in A, and their migration track was drawn as in B. Choosing a pair of cells located next to each other at the 240-min point, the distances between their nuclei at earlier time points were measured at 15-min intervals, and their means as well as the farthest and nearest distances were obtained. The farthest and nearest indices were defined as the ratio of the farthest (Dmax) and nearest (Dmin) distance to the average distance (Davg), respectively, as illustrated in the left panel. The farthest index for OL-pc transfectants is significantly larger (**, P < 0.0005; n = 77) than that for the control cells (n = 58), whereas that for OL-pcΔNBS transfectants (P = 0.39; n = 63) is not. Likewise, the nearest index for OL-pc transfectants is significantly smaller (*, P < 0.005) than that for the controls, whereas that for OL-pcΔNBS (n.s.; P = 0.87) is not. (E) Changes in the mean distance (±SEM [error bars]) of the migrating wound edges from their starting position during a long incubation period up to 11 h. n = 12 for all samples. Bar, 50 μm.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2483522&req=5

fig5: OL-pc expression accelerates cell movement in wound healing. (A) Confluent cell layers were wounded by cell scraping, and the wounded edges were immediately processed for time-lapse recording. Phase-contrast images were taken for 4 h at 3-min intervals. At the end of the recording, individual cells located along the front of cell sheets were marked with colored circles over their nuclear positions, and the same cells were tracked back to earlier time points. At each time point, the circles were connected with lines in a fixed order. Dashed lines represent wound edges. The numbers denote the time elapsed (in minutes). Control and OL-pcΔNBS transfectants roughly maintain their relative positions during migration, whereas OL-pc transfectants irregularly change their spatial relations with neighbors (e.g., the cell marked with the red circle at the right jumped out to the front position from an initially deep site). Videos 3–5 are available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1. (B) Migration track of individual cells marked with different colors in A, with cell position plotted at 9-min intervals. The arrow indicates the direction of wound healing. Abrupt acceleration of movement is most frequently seen in OL-pc transfectants. (C) Statistical analysis of the instantaneous velocities of cells during wound healing. Comparison of each experimental group with the control cells (n = 38) shows a significant difference for OL-pc (**, P < 0.0005; n = 51) but not for OL-pcΔNBS (P = 0.70; n = 41). (D) Changes in the positions of cells relative to those of their neighbors during migration. At the end of recording (at 240 min after scraping), all cells at the wound edges were marked as in A, and their migration track was drawn as in B. Choosing a pair of cells located next to each other at the 240-min point, the distances between their nuclei at earlier time points were measured at 15-min intervals, and their means as well as the farthest and nearest distances were obtained. The farthest and nearest indices were defined as the ratio of the farthest (Dmax) and nearest (Dmin) distance to the average distance (Davg), respectively, as illustrated in the left panel. The farthest index for OL-pc transfectants is significantly larger (**, P < 0.0005; n = 77) than that for the control cells (n = 58), whereas that for OL-pcΔNBS transfectants (P = 0.39; n = 63) is not. Likewise, the nearest index for OL-pc transfectants is significantly smaller (*, P < 0.005) than that for the controls, whereas that for OL-pcΔNBS (n.s.; P = 0.87) is not. (E) Changes in the mean distance (±SEM [error bars]) of the migrating wound edges from their starting position during a long incubation period up to 11 h. n = 12 for all samples. Bar, 50 μm.
Mentions: As another way to measure the cell behavior, we conducted a wound-healing assay in which cell migration was stimulated under the conditions in which cells were in contact with each other. When wounds had been produced by cell scraping of confluent cultures, the cells located around the wound edges became polarized so as to migrate toward the open space within 10 min. In control cultures, cells occupying the front line of the wound were arranged in parallel and moved together, maintaining their mutual contacts in which individual cells kept roughly a similar pace, suggesting that their movements were coordinated (Fig. 5 A, control; and Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1). At their lateral contact sites, the membrane ruffling was prohibited, indicating that the contact inhibition of movement was also operating in this system. On the other hand, the movement of OL-pc–expressing cells was rarely coordinated: they dynamically changed their positions relative to those of their neighbors during translocation (Fig. 5 A, OL-pc; and Video 4), as was quantified by defining the farthest and nearest indices (Fig. 5 D). Many of them abruptly moved out of the original positions, leaving their neighbors behind, which resulted in the positioning of themselves at the front-most edge of the wound. This type of uncoordinated movement was also detectable in the control cell cultures, but its frequency was greatly increased in the OL-pc–expressing cells.

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