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.

Show MeSH

Related in: MedlinePlus

F-actin–mediated activation of SSH1L is critical for polarized lamellipodium formation and chemotaxis. (A) Expression of siRNA-resistant (sr) SSH1L(WT), but not sr-C393S, sr-W458A, or NP mutant, rescues the inhibitory effect of SSH1L siRNA on T cell chemotaxis. Jurkat cells were cotransfected with SSH1L siRNA plasmids together with expression plasmids for sr-SSH1L(WT), sr-C393S, sr-W458A, or NP mutants cultured for 60 h and were subjected to Transwell culture chamber assays as described in Fig. 4. *, P < 0.005; **, P < 0.05, compared with cells transfected with empty vector. Error bars represent SEM. (B) Expression of sr-SSH1L(WT), but not sr-C393S or sr-W458A mutant, recovers the SDF-1α–induced polarized F-actin assembly in SSH1L siRNA cells. Jurkat cells transfected as in A were cultured for 60 h, stimulated with 5 nM SDF-1α for 5 min, and stained with rhodamine-phalloidin for F-actin as described in Fig. 6 B. Bar, 5 μm. (C) Quantitative analysis of cell morphology changes. Jurkat cells transfected with SSH1L siRNA plasmids together with the indicated sr-SSH1L expression plasmids were cultured for 60 h, stimulated with 5 nM SDF-1α for 5 min, and stained as in B. Cells were categorized into three classes, as shown on the right: class 1 (round cells without a lamellipodium), class 2 (cells with a single lamellipodium), and class 3 (cells with multiple lamellipodia around the cells). The percentages of cells in each class are shown as the means of triplicate experiments (200–300 cells were counted in each experiment).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171197&req=5

fig8: F-actin–mediated activation of SSH1L is critical for polarized lamellipodium formation and chemotaxis. (A) Expression of siRNA-resistant (sr) SSH1L(WT), but not sr-C393S, sr-W458A, or NP mutant, rescues the inhibitory effect of SSH1L siRNA on T cell chemotaxis. Jurkat cells were cotransfected with SSH1L siRNA plasmids together with expression plasmids for sr-SSH1L(WT), sr-C393S, sr-W458A, or NP mutants cultured for 60 h and were subjected to Transwell culture chamber assays as described in Fig. 4. *, P < 0.005; **, P < 0.05, compared with cells transfected with empty vector. Error bars represent SEM. (B) Expression of sr-SSH1L(WT), but not sr-C393S or sr-W458A mutant, recovers the SDF-1α–induced polarized F-actin assembly in SSH1L siRNA cells. Jurkat cells transfected as in A were cultured for 60 h, stimulated with 5 nM SDF-1α for 5 min, and stained with rhodamine-phalloidin for F-actin as described in Fig. 6 B. Bar, 5 μm. (C) Quantitative analysis of cell morphology changes. Jurkat cells transfected with SSH1L siRNA plasmids together with the indicated sr-SSH1L expression plasmids were cultured for 60 h, stimulated with 5 nM SDF-1α for 5 min, and stained as in B. Cells were categorized into three classes, as shown on the right: class 1 (round cells without a lamellipodium), class 2 (cells with a single lamellipodium), and class 3 (cells with multiple lamellipodia around the cells). The percentages of cells in each class are shown as the means of triplicate experiments (200–300 cells were counted in each experiment).

Mentions: We next analyzed whether the expression of WT or mutated SSH1L could rescue the inhibitory effect of SSH1L siRNA on chemotaxis. We constructed an siRNA-resistant (sr) SSH1L cDNA bearing two nucleotide mutations in the siRNA target sequence that did not lead to amino acid substitution. Cotransfection of Jurkat cells with sr-SSH1L(WT) along with the SSH1L siRNA plasmid caused chemotactic activity toward SDF-1α in Transwell assays to recover considerably (Fig. 8 A). This indicates that the inhibitory effect of SSH1L siRNA on T cell chemotaxis is attributable to the specific knockdown of endogenous SSH1L expression. Notably, unlike sr-SSH1L(WT), cotransfection with either a phosphatase-dead sr-SSH1L(C393S), an F-actin–insensitive sr-SSH1L(W458A), or the COOH-terminally deleted NP(1–456) mutant (that has no siRNA target sequence and is not activated by F-actin) failed to rescue the chemotactic migration of SSH1L siRNA cells (Fig. 8 A). Phalloidin staining also revealed that SSH1L siRNA cells transfected with sr-SSH1L(WT) induced a relatively normal polarized cell morphology bearing a single lamellipodium, whereas cells that were transfected with sr-SSH1L(C393S), sr-SSH1L(W458A), or NP(1–456) exhibited phenotypes that were similar to those of SSH1L siRNA cells (Fig. 8 B and not depicted). Quantitative analyses showed that control and SSH1L siRNA cells preferentially exhibited cell morphologies that were categorized into class 2 (with a single lamellipodium) and 3 (with multiple lamellipodia around the cell), respectively, after SDF-1α stimulation (Fig. 8 C). The class 3 cell morphology reverted to class 2 morphology in SSH1L siRNA cells upon the expression of sr-SSH1L(WT) but not sr-SSH1L(C393S), sr-SSH1L(W458A), or NP(1–456) (Fig. 8 C). These findings suggest that both phosphatase activity and the F-actin–mediated activation of SSH1L are required for the SDF-1α–induced polarized cell shape formation and chemotactic response of Jurkat cells.


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)

F-actin–mediated activation of SSH1L is critical for polarized lamellipodium formation and chemotaxis. (A) Expression of siRNA-resistant (sr) SSH1L(WT), but not sr-C393S, sr-W458A, or NP mutant, rescues the inhibitory effect of SSH1L siRNA on T cell chemotaxis. Jurkat cells were cotransfected with SSH1L siRNA plasmids together with expression plasmids for sr-SSH1L(WT), sr-C393S, sr-W458A, or NP mutants cultured for 60 h and were subjected to Transwell culture chamber assays as described in Fig. 4. *, P < 0.005; **, P < 0.05, compared with cells transfected with empty vector. Error bars represent SEM. (B) Expression of sr-SSH1L(WT), but not sr-C393S or sr-W458A mutant, recovers the SDF-1α–induced polarized F-actin assembly in SSH1L siRNA cells. Jurkat cells transfected as in A were cultured for 60 h, stimulated with 5 nM SDF-1α for 5 min, and stained with rhodamine-phalloidin for F-actin as described in Fig. 6 B. Bar, 5 μm. (C) Quantitative analysis of cell morphology changes. Jurkat cells transfected with SSH1L siRNA plasmids together with the indicated sr-SSH1L expression plasmids were cultured for 60 h, stimulated with 5 nM SDF-1α for 5 min, and stained as in B. Cells were categorized into three classes, as shown on the right: class 1 (round cells without a lamellipodium), class 2 (cells with a single lamellipodium), and class 3 (cells with multiple lamellipodia around the cells). The percentages of cells in each class are shown as the means of triplicate experiments (200–300 cells were counted in each experiment).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171197&req=5

fig8: F-actin–mediated activation of SSH1L is critical for polarized lamellipodium formation and chemotaxis. (A) Expression of siRNA-resistant (sr) SSH1L(WT), but not sr-C393S, sr-W458A, or NP mutant, rescues the inhibitory effect of SSH1L siRNA on T cell chemotaxis. Jurkat cells were cotransfected with SSH1L siRNA plasmids together with expression plasmids for sr-SSH1L(WT), sr-C393S, sr-W458A, or NP mutants cultured for 60 h and were subjected to Transwell culture chamber assays as described in Fig. 4. *, P < 0.005; **, P < 0.05, compared with cells transfected with empty vector. Error bars represent SEM. (B) Expression of sr-SSH1L(WT), but not sr-C393S or sr-W458A mutant, recovers the SDF-1α–induced polarized F-actin assembly in SSH1L siRNA cells. Jurkat cells transfected as in A were cultured for 60 h, stimulated with 5 nM SDF-1α for 5 min, and stained with rhodamine-phalloidin for F-actin as described in Fig. 6 B. Bar, 5 μm. (C) Quantitative analysis of cell morphology changes. Jurkat cells transfected with SSH1L siRNA plasmids together with the indicated sr-SSH1L expression plasmids were cultured for 60 h, stimulated with 5 nM SDF-1α for 5 min, and stained as in B. Cells were categorized into three classes, as shown on the right: class 1 (round cells without a lamellipodium), class 2 (cells with a single lamellipodium), and class 3 (cells with multiple lamellipodia around the cells). The percentages of cells in each class are shown as the means of triplicate experiments (200–300 cells were counted in each experiment).
Mentions: We next analyzed whether the expression of WT or mutated SSH1L could rescue the inhibitory effect of SSH1L siRNA on chemotaxis. We constructed an siRNA-resistant (sr) SSH1L cDNA bearing two nucleotide mutations in the siRNA target sequence that did not lead to amino acid substitution. Cotransfection of Jurkat cells with sr-SSH1L(WT) along with the SSH1L siRNA plasmid caused chemotactic activity toward SDF-1α in Transwell assays to recover considerably (Fig. 8 A). This indicates that the inhibitory effect of SSH1L siRNA on T cell chemotaxis is attributable to the specific knockdown of endogenous SSH1L expression. Notably, unlike sr-SSH1L(WT), cotransfection with either a phosphatase-dead sr-SSH1L(C393S), an F-actin–insensitive sr-SSH1L(W458A), or the COOH-terminally deleted NP(1–456) mutant (that has no siRNA target sequence and is not activated by F-actin) failed to rescue the chemotactic migration of SSH1L siRNA cells (Fig. 8 A). Phalloidin staining also revealed that SSH1L siRNA cells transfected with sr-SSH1L(WT) induced a relatively normal polarized cell morphology bearing a single lamellipodium, whereas cells that were transfected with sr-SSH1L(C393S), sr-SSH1L(W458A), or NP(1–456) exhibited phenotypes that were similar to those of SSH1L siRNA cells (Fig. 8 B and not depicted). Quantitative analyses showed that control and SSH1L siRNA cells preferentially exhibited cell morphologies that were categorized into class 2 (with a single lamellipodium) and 3 (with multiple lamellipodia around the cell), respectively, after SDF-1α stimulation (Fig. 8 C). The class 3 cell morphology reverted to class 2 morphology in SSH1L siRNA cells upon the expression of sr-SSH1L(WT) but not sr-SSH1L(C393S), sr-SSH1L(W458A), or NP(1–456) (Fig. 8 C). These findings suggest that both phosphatase activity and the F-actin–mediated activation of SSH1L are required for the SDF-1α–induced polarized cell shape formation and chemotactic response of Jurkat cells.

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