Limits...
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.

Show MeSH

Related in: MedlinePlus

OL-pc alters the distribution of junctional F-actin and N-cadherin. (A) Double immunostaining for N-cadherin and HA tag. N-cadherin signals are linearly arranged along the cell borders in control and OL-pcΔNBS transfectants. N-cadherin is concentrated along the apical-most region of the obliquely formed cell–cell contacts, which was identified by differential focusing. In OL-pc transfectants, however, N-cadherin assumes a streaklike distribution over the OL-pc–positive cell–cell contact areas (arrows). (B) Double immunostaining for N-cadherin and F-actin. In control cells or OL-pcΔNBS transfectants, N-cadherin colocalizes with nonfibrous F-actin signals (arrowheads), whereas in OL-pc transfectants, N-cadherin is associated with the terminals of radial actin fibers (arrows). (C) Ca2+-switch experiment. Cells were preincubated with 1 mM EGTA to inactivate cadherins and were incubated in normal culture medium for 15 min. Double immunostaining for N-cadherin and F-actin shows that their distribution patterns unique to each transfectants are already observable at this time point (arrows). (D) Cells were treated with DMSO (control) or 0.4 μg/ml cytochalasin D for 30 min at 37°C and were stained for N-cadherin and F-actin. The unique distributions of N-cadherin in OL-pc transfectants were abolished by cytochalasin D (arrowheads). (E) Control and OL-pc-HA transfectants were treated with control, Nap1, or WAVE1 siRNA and double stained for N-cadherin (red) and F-actin (green; shown in each of the merged images). Arrows point to OL-pc transfectant-specific distribution of N-cadherin, and arrowheads indicate the restored distribution of N-cadherin. All images were taken with a confocal microscope. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2483522&req=5

fig7: OL-pc alters the distribution of junctional F-actin and N-cadherin. (A) Double immunostaining for N-cadherin and HA tag. N-cadherin signals are linearly arranged along the cell borders in control and OL-pcΔNBS transfectants. N-cadherin is concentrated along the apical-most region of the obliquely formed cell–cell contacts, which was identified by differential focusing. In OL-pc transfectants, however, N-cadherin assumes a streaklike distribution over the OL-pc–positive cell–cell contact areas (arrows). (B) Double immunostaining for N-cadherin and F-actin. In control cells or OL-pcΔNBS transfectants, N-cadherin colocalizes with nonfibrous F-actin signals (arrowheads), whereas in OL-pc transfectants, N-cadherin is associated with the terminals of radial actin fibers (arrows). (C) Ca2+-switch experiment. Cells were preincubated with 1 mM EGTA to inactivate cadherins and were incubated in normal culture medium for 15 min. Double immunostaining for N-cadherin and F-actin shows that their distribution patterns unique to each transfectants are already observable at this time point (arrows). (D) Cells were treated with DMSO (control) or 0.4 μg/ml cytochalasin D for 30 min at 37°C and were stained for N-cadherin and F-actin. The unique distributions of N-cadherin in OL-pc transfectants were abolished by cytochalasin D (arrowheads). (E) Control and OL-pc-HA transfectants were treated with control, Nap1, or WAVE1 siRNA and double stained for N-cadherin (red) and F-actin (green; shown in each of the merged images). Arrows point to OL-pc transfectant-specific distribution of N-cadherin, and arrowheads indicate the restored distribution of N-cadherin. All images were taken with a confocal microscope. Bars, 10 μm.

Mentions: Because Nap1 associating with OL-pc is an actin regulator, we asked whether any changes were induced in actin assembly after OL-pc expression. The overall actin-staining pattern was too complex to compare between these cells. However, we found significant differences in the organization of actin-associated cell junctions, where classical cadherins were localized, between the presence and absence of OL-pc. U251 cells expressed N-cadherin, and this classical cadherin was lineally arranged along cell–cell boundaries, and a similar localization pattern for N-cadherin was observed in OL-pcΔNBS transfectants. Double immunostaining for N-cadherin and OL-pcΔNBS showed that although OL-pcΔNBS was distributed throughout the cell–cell contact regions, N-cadherin tended to be condensed along the apical-most portion of the junctions (Fig. 7 A), as generally seen in epithelial cells. In OL-pc transfectants, on the other hand, N-cadherin was detected as streaklike signals spread over the OL-pc–positive areas, oriented perpendicularly to the cell borders (Fig. 7 A). Cadherin-associated catenins, such as α-catenin, showed a localization pattern similar to that of N-cadherin in each transfectant.


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 alters the distribution of junctional F-actin and N-cadherin. (A) Double immunostaining for N-cadherin and HA tag. N-cadherin signals are linearly arranged along the cell borders in control and OL-pcΔNBS transfectants. N-cadherin is concentrated along the apical-most region of the obliquely formed cell–cell contacts, which was identified by differential focusing. In OL-pc transfectants, however, N-cadherin assumes a streaklike distribution over the OL-pc–positive cell–cell contact areas (arrows). (B) Double immunostaining for N-cadherin and F-actin. In control cells or OL-pcΔNBS transfectants, N-cadherin colocalizes with nonfibrous F-actin signals (arrowheads), whereas in OL-pc transfectants, N-cadherin is associated with the terminals of radial actin fibers (arrows). (C) Ca2+-switch experiment. Cells were preincubated with 1 mM EGTA to inactivate cadherins and were incubated in normal culture medium for 15 min. Double immunostaining for N-cadherin and F-actin shows that their distribution patterns unique to each transfectants are already observable at this time point (arrows). (D) Cells were treated with DMSO (control) or 0.4 μg/ml cytochalasin D for 30 min at 37°C and were stained for N-cadherin and F-actin. The unique distributions of N-cadherin in OL-pc transfectants were abolished by cytochalasin D (arrowheads). (E) Control and OL-pc-HA transfectants were treated with control, Nap1, or WAVE1 siRNA and double stained for N-cadherin (red) and F-actin (green; shown in each of the merged images). Arrows point to OL-pc transfectant-specific distribution of N-cadherin, and arrowheads indicate the restored distribution of N-cadherin. All images were taken with a confocal microscope. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2483522&req=5

fig7: OL-pc alters the distribution of junctional F-actin and N-cadherin. (A) Double immunostaining for N-cadherin and HA tag. N-cadherin signals are linearly arranged along the cell borders in control and OL-pcΔNBS transfectants. N-cadherin is concentrated along the apical-most region of the obliquely formed cell–cell contacts, which was identified by differential focusing. In OL-pc transfectants, however, N-cadherin assumes a streaklike distribution over the OL-pc–positive cell–cell contact areas (arrows). (B) Double immunostaining for N-cadherin and F-actin. In control cells or OL-pcΔNBS transfectants, N-cadherin colocalizes with nonfibrous F-actin signals (arrowheads), whereas in OL-pc transfectants, N-cadherin is associated with the terminals of radial actin fibers (arrows). (C) Ca2+-switch experiment. Cells were preincubated with 1 mM EGTA to inactivate cadherins and were incubated in normal culture medium for 15 min. Double immunostaining for N-cadherin and F-actin shows that their distribution patterns unique to each transfectants are already observable at this time point (arrows). (D) Cells were treated with DMSO (control) or 0.4 μg/ml cytochalasin D for 30 min at 37°C and were stained for N-cadherin and F-actin. The unique distributions of N-cadherin in OL-pc transfectants were abolished by cytochalasin D (arrowheads). (E) Control and OL-pc-HA transfectants were treated with control, Nap1, or WAVE1 siRNA and double stained for N-cadherin (red) and F-actin (green; shown in each of the merged images). Arrows point to OL-pc transfectant-specific distribution of N-cadherin, and arrowheads indicate the restored distribution of N-cadherin. All images were taken with a confocal microscope. Bars, 10 μm.
Mentions: Because Nap1 associating with OL-pc is an actin regulator, we asked whether any changes were induced in actin assembly after OL-pc expression. The overall actin-staining pattern was too complex to compare between these cells. However, we found significant differences in the organization of actin-associated cell junctions, where classical cadherins were localized, between the presence and absence of OL-pc. U251 cells expressed N-cadherin, and this classical cadherin was lineally arranged along cell–cell boundaries, and a similar localization pattern for N-cadherin was observed in OL-pcΔNBS transfectants. Double immunostaining for N-cadherin and OL-pcΔNBS showed that although OL-pcΔNBS was distributed throughout the cell–cell contact regions, N-cadherin tended to be condensed along the apical-most portion of the junctions (Fig. 7 A), as generally seen in epithelial cells. In OL-pc transfectants, on the other hand, N-cadherin was detected as streaklike signals spread over the OL-pc–positive areas, oriented perpendicularly to the cell borders (Fig. 7 A). Cadherin-associated catenins, such as α-catenin, showed a localization pattern similar to that of N-cadherin in each transfectant.

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