Limits...
Spatial and temporal regulation of cofilin activity by LIM kinase and Slingshot is critical for directional cell migration.

Nishita M, Tomizawa C, Yamamoto M, Horita Y, Ohashi K, Mizuno K - J. Cell Biol. (2005)

Bottom Line: Cofilin is inactivated by LIM kinase (LIMK)-1-mediated phosphorylation and is reactivated by cofilin phosphatase Slingshot (SSH)-1L.In this study, we show that cofilin activity is temporally and spatially regulated by LIMK1 and SSH1L in chemokine-stimulated Jurkat T cells.We propose that LIMK1- and SSH1L-mediated spatiotemporal regulation of cofilin activity is critical for chemokine-induced polarized lamellipodium formation and directional cell movement.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan.

ABSTRACT
Cofilin mediates lamellipodium extension and polarized cell migration by accelerating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by LIM kinase (LIMK)-1-mediated phosphorylation and is reactivated by cofilin phosphatase Slingshot (SSH)-1L. In this study, we show that cofilin activity is temporally and spatially regulated by LIMK1 and SSH1L in chemokine-stimulated Jurkat T cells. The knockdown of LIMK1 suppressed chemokine-induced lamellipodium formation and cell migration, whereas SSH1L knockdown produced and retained multiple lamellipodial protrusions around the cell after cell stimulation and impaired directional cell migration. Our results indicate that LIMK1 is required for cell migration by stimulating lamellipodium formation in the initial stages of cell response and that SSH1L is crucially involved in directional cell migration by restricting the membrane protrusion to one direction and locally stimulating cofilin activity in the lamellipodium in the front of the migrating cell. We propose that LIMK1- and SSH1L-mediated spatiotemporal regulation of cofilin activity is critical for chemokine-induced polarized lamellipodium formation and directional cell movement.

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Effect of SSH1L, LIMK1, or cofilin siRNA on SDF-1α–induced T cell chemotaxis in Dunn chambers. Jurkat cells were transfected with siRNA plasmids for SSH1L, LIMK1, cofilin, or empty plasmid (control) and were analyzed for their ability to migrate in an SDF-1α gradient in the Dunn chamber during a 50-min period. (A) The migration paths of 30 randomly chosen cells were traced for 50 min. The intersection of the x and y axes was taken to be the starting point of each cell path, whereas the source of SDF-1α was at the top. Magnified views of the paths of control cells and SSH1L siRNA cells are also shown. (B) The net translocation distance (straight distance from the start to the end point) of each cell over the 50-min period is shown as the mean ± SEM (error bars) of the paths of 50 randomly chosen cells. *, P < 0.01 compared with control cells. (C) The migration speed (total length of the migration path per hour) of each cell is shown as the mean ± SEM of the paths of 50 randomly chosen cells. *, P < 0.05; **, P < 0.01 compared with control cells. (D) The directional persistency index (the ratio of the net translocation distance to the cumulative length of migration path) of control and SSH1L siRNA cells. (E) Circular histograms showing the percentage of cells whose final position was located within each of 18 equal sectors (20°). The source of SDF-1α was at the top. Data from control and SSH1L siRNA cells are shown.
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fig5: Effect of SSH1L, LIMK1, or cofilin siRNA on SDF-1α–induced T cell chemotaxis in Dunn chambers. Jurkat cells were transfected with siRNA plasmids for SSH1L, LIMK1, cofilin, or empty plasmid (control) and were analyzed for their ability to migrate in an SDF-1α gradient in the Dunn chamber during a 50-min period. (A) The migration paths of 30 randomly chosen cells were traced for 50 min. The intersection of the x and y axes was taken to be the starting point of each cell path, whereas the source of SDF-1α was at the top. Magnified views of the paths of control cells and SSH1L siRNA cells are also shown. (B) The net translocation distance (straight distance from the start to the end point) of each cell over the 50-min period is shown as the mean ± SEM (error bars) of the paths of 50 randomly chosen cells. *, P < 0.01 compared with control cells. (C) The migration speed (total length of the migration path per hour) of each cell is shown as the mean ± SEM of the paths of 50 randomly chosen cells. *, P < 0.05; **, P < 0.01 compared with control cells. (D) The directional persistency index (the ratio of the net translocation distance to the cumulative length of migration path) of control and SSH1L siRNA cells. (E) Circular histograms showing the percentage of cells whose final position was located within each of 18 equal sectors (20°). The source of SDF-1α was at the top. Data from control and SSH1L siRNA cells are shown.

Mentions: To further investigate this possibility, the chemotactic response of Jurkat cells in an SDF-1α gradient was analyzed using Dunn chambers (Zicha et al., 1991; Allen et al., 1998). Jurkat cells were transfected with siRNA plasmids, and their migration tracks in the bridge of a Dunn chemotaxis chamber were traced by using time-lapse video microscopy. Although control siRNA cells preferentially migrated up the SDF-1α gradient, the chemotactic responses of SSH1L, LIMK1, or cofilin siRNA cells were significantly suppressed (Fig. 5 A). Quantitative analyses revealed that LIMK1 or cofilin siRNA significantly decreased both the net translocation distance (straight distance from the start to the end point) and the migration speed (total length of the migration path per hour; Fig. 5, B and C). In contrast, the net translocation distance and migration speed of SSH1L siRNA cells were 35 and 69% of the values of control cells (Fig. 5, B and C). These data suggest that SSH1L siRNA cells retained their migration potential to appreciable levels but turned more frequently than the control cells. Indeed, we found that SSH1L siRNA cells frequently changed direction compared with control cells (Fig. 5 A, magnified views). As a result, the index of the directional persistency (the ratio of the net translocation distance to the cumulative length of migration path) of SSH1L siRNA cells (0.36) was significantly lower than that of control cells (Fig. 5 D; 0.71). In addition, circular histograms showing the overall directionality of cell migration revealed that although 73% of control cells moved to positions within a 120° arc facing the SDF-1α source, only 33% of the SSH1L siRNA cells moved in this direction (Fig. 5 E), indicating that SSH1L siRNA cells migrated in random directions. Together, these findings suggest that LIMK1 and cofilin play an essential role in cell motility, whereas SSH1L is primarily involved in establishing or maintaining the directionality of cell movement.


Spatial and temporal regulation of cofilin activity by LIM kinase and Slingshot is critical for directional cell migration.

Nishita M, Tomizawa C, Yamamoto M, Horita Y, Ohashi K, Mizuno K - J. Cell Biol. (2005)

Effect of SSH1L, LIMK1, or cofilin siRNA on SDF-1α–induced T cell chemotaxis in Dunn chambers. Jurkat cells were transfected with siRNA plasmids for SSH1L, LIMK1, cofilin, or empty plasmid (control) and were analyzed for their ability to migrate in an SDF-1α gradient in the Dunn chamber during a 50-min period. (A) The migration paths of 30 randomly chosen cells were traced for 50 min. The intersection of the x and y axes was taken to be the starting point of each cell path, whereas the source of SDF-1α was at the top. Magnified views of the paths of control cells and SSH1L siRNA cells are also shown. (B) The net translocation distance (straight distance from the start to the end point) of each cell over the 50-min period is shown as the mean ± SEM (error bars) of the paths of 50 randomly chosen cells. *, P < 0.01 compared with control cells. (C) The migration speed (total length of the migration path per hour) of each cell is shown as the mean ± SEM of the paths of 50 randomly chosen cells. *, P < 0.05; **, P < 0.01 compared with control cells. (D) The directional persistency index (the ratio of the net translocation distance to the cumulative length of migration path) of control and SSH1L siRNA cells. (E) Circular histograms showing the percentage of cells whose final position was located within each of 18 equal sectors (20°). The source of SDF-1α was at the top. Data from control and SSH1L siRNA cells are shown.
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fig5: Effect of SSH1L, LIMK1, or cofilin siRNA on SDF-1α–induced T cell chemotaxis in Dunn chambers. Jurkat cells were transfected with siRNA plasmids for SSH1L, LIMK1, cofilin, or empty plasmid (control) and were analyzed for their ability to migrate in an SDF-1α gradient in the Dunn chamber during a 50-min period. (A) The migration paths of 30 randomly chosen cells were traced for 50 min. The intersection of the x and y axes was taken to be the starting point of each cell path, whereas the source of SDF-1α was at the top. Magnified views of the paths of control cells and SSH1L siRNA cells are also shown. (B) The net translocation distance (straight distance from the start to the end point) of each cell over the 50-min period is shown as the mean ± SEM (error bars) of the paths of 50 randomly chosen cells. *, P < 0.01 compared with control cells. (C) The migration speed (total length of the migration path per hour) of each cell is shown as the mean ± SEM of the paths of 50 randomly chosen cells. *, P < 0.05; **, P < 0.01 compared with control cells. (D) The directional persistency index (the ratio of the net translocation distance to the cumulative length of migration path) of control and SSH1L siRNA cells. (E) Circular histograms showing the percentage of cells whose final position was located within each of 18 equal sectors (20°). The source of SDF-1α was at the top. Data from control and SSH1L siRNA cells are shown.
Mentions: To further investigate this possibility, the chemotactic response of Jurkat cells in an SDF-1α gradient was analyzed using Dunn chambers (Zicha et al., 1991; Allen et al., 1998). Jurkat cells were transfected with siRNA plasmids, and their migration tracks in the bridge of a Dunn chemotaxis chamber were traced by using time-lapse video microscopy. Although control siRNA cells preferentially migrated up the SDF-1α gradient, the chemotactic responses of SSH1L, LIMK1, or cofilin siRNA cells were significantly suppressed (Fig. 5 A). Quantitative analyses revealed that LIMK1 or cofilin siRNA significantly decreased both the net translocation distance (straight distance from the start to the end point) and the migration speed (total length of the migration path per hour; Fig. 5, B and C). In contrast, the net translocation distance and migration speed of SSH1L siRNA cells were 35 and 69% of the values of control cells (Fig. 5, B and C). These data suggest that SSH1L siRNA cells retained their migration potential to appreciable levels but turned more frequently than the control cells. Indeed, we found that SSH1L siRNA cells frequently changed direction compared with control cells (Fig. 5 A, magnified views). As a result, the index of the directional persistency (the ratio of the net translocation distance to the cumulative length of migration path) of SSH1L siRNA cells (0.36) was significantly lower than that of control cells (Fig. 5 D; 0.71). In addition, circular histograms showing the overall directionality of cell migration revealed that although 73% of control cells moved to positions within a 120° arc facing the SDF-1α source, only 33% of the SSH1L siRNA cells moved in this direction (Fig. 5 E), indicating that SSH1L siRNA cells migrated in random directions. Together, these findings suggest that LIMK1 and cofilin play an essential role in cell motility, whereas SSH1L is primarily involved in establishing or maintaining the directionality of cell movement.

Bottom Line: Cofilin is inactivated by LIM kinase (LIMK)-1-mediated phosphorylation and is reactivated by cofilin phosphatase Slingshot (SSH)-1L.In this study, we show that cofilin activity is temporally and spatially regulated by LIMK1 and SSH1L in chemokine-stimulated Jurkat T cells.We propose that LIMK1- and SSH1L-mediated spatiotemporal regulation of cofilin activity is critical for chemokine-induced polarized lamellipodium formation and directional cell movement.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan.

ABSTRACT
Cofilin mediates lamellipodium extension and polarized cell migration by accelerating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by LIM kinase (LIMK)-1-mediated phosphorylation and is reactivated by cofilin phosphatase Slingshot (SSH)-1L. In this study, we show that cofilin activity is temporally and spatially regulated by LIMK1 and SSH1L in chemokine-stimulated Jurkat T cells. The knockdown of LIMK1 suppressed chemokine-induced lamellipodium formation and cell migration, whereas SSH1L knockdown produced and retained multiple lamellipodial protrusions around the cell after cell stimulation and impaired directional cell migration. Our results indicate that LIMK1 is required for cell migration by stimulating lamellipodium formation in the initial stages of cell response and that SSH1L is crucially involved in directional cell migration by restricting the membrane protrusion to one direction and locally stimulating cofilin activity in the lamellipodium in the front of the migrating cell. We propose that LIMK1- and SSH1L-mediated spatiotemporal regulation of cofilin activity is critical for chemokine-induced polarized lamellipodium formation and directional cell movement.

Show MeSH
Related in: MedlinePlus