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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SDF-1α–induced changes in P-cofilin levels are regulated by LIMK1 and SSH1L. (A) SDF-1α–induced changes in P-cofilin levels. Jurkat cells were stimulated with 5 nM SDF-1α for the indicated times, and cell lysates were analyzed by immunoblotting with anti–P-cofilin and anticofilin antibodies. The bottom panel shows the relative P-cofilin levels after SDF-1α stimulation as means ± SEM of triplicate experiments. (B) Suppression of endogenous LIMK1, SSH1L, and cofilin expression by siRNA. Jurkat cells were transfected with siRNA plasmids for GFP (control), LIMK1, SSH1L, cofilin, or empty vector (−). After 60 h of culture, cell lysates were analyzed by immunoblotting with antibodies specific for each protein and β-actin. For LIMK1 and SSH1L, the cell lysates were subjected to immunoblotting after immunoprecipitation. (C and D) Effects of LIMK1 or SSH1L siRNA on SDF-1α–induced changes in P-cofilin levels. SSH1L, LIMK1, or GFP (control) siRNA cells were stimulated with 5 nM SDF-1α. Cell lysates, prepared at the indicated times, were analyzed by immunoblotting as in A. The bottom panels indicate the relative P-cofilin levels; the value at time = 0 in control cells is taken as 1.0. Each value represents the mean ± SEM (error bars) of triplicate experiments.
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fig1: SDF-1α–induced changes in P-cofilin levels are regulated by LIMK1 and SSH1L. (A) SDF-1α–induced changes in P-cofilin levels. Jurkat cells were stimulated with 5 nM SDF-1α for the indicated times, and cell lysates were analyzed by immunoblotting with anti–P-cofilin and anticofilin antibodies. The bottom panel shows the relative P-cofilin levels after SDF-1α stimulation as means ± SEM of triplicate experiments. (B) Suppression of endogenous LIMK1, SSH1L, and cofilin expression by siRNA. Jurkat cells were transfected with siRNA plasmids for GFP (control), LIMK1, SSH1L, cofilin, or empty vector (−). After 60 h of culture, cell lysates were analyzed by immunoblotting with antibodies specific for each protein and β-actin. For LIMK1 and SSH1L, the cell lysates were subjected to immunoblotting after immunoprecipitation. (C and D) Effects of LIMK1 or SSH1L siRNA on SDF-1α–induced changes in P-cofilin levels. SSH1L, LIMK1, or GFP (control) siRNA cells were stimulated with 5 nM SDF-1α. Cell lysates, prepared at the indicated times, were analyzed by immunoblotting as in A. The bottom panels indicate the relative P-cofilin levels; the value at time = 0 in control cells is taken as 1.0. Each value represents the mean ± SEM (error bars) of triplicate experiments.

Mentions: Cofilin activity is negatively regulated by phosphorylation at Ser-3. To examine the role cofilin phosphoregulation plays in T cell chemotaxis, we first analyzed time-dependent changes in P-cofilin levels in Jurkat cells after SDF-1α stimulation. The P-cofilin levels increased by 1–5 min as reported previously (Nishita et al., 2002) but reverted to basal levels 20 min after SDF-1α stimulation (Fig. 1 A). Because LIMK1 in Jurkat cells is activated by SDF-1α for up to 20 min (Nishita et al., 2002), the decrease in P-cofilin levels at 20 min probably involves the activation of cofilin phosphatases such as SSH1L. To assess the roles of LIMK1 and SSH1L in the SDF-1α–induced changes in P-cofilin levels, we knocked down LIMK1 and SSH1L expression using siRNA. Endogenous LIMK1 and SSH1L expression was suppressed by the transfection of siRNA plasmids (Fig. 1 B). LIMK1 siRNA reduced the basal P-cofilin levels in unstimulated cells and inhibited the increase in P-cofilin levels that occurred 5 min after SDF-1α stimulation (Fig. 1 C). In contrast, SSH1L siRNA raised the basal P-cofilin levels, and these levels were further augmented at 5 and 20 min after SDF-1α stimulation (Fig. 1 D). These results suggest that LIMK1 plays a critical role in the SDF-1α–induced elevation of P-cofilin levels at 5 min and that SSH1L is involved in decreasing P-cofilin levels in later stages after SDF-1α stimulation.


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)

SDF-1α–induced changes in P-cofilin levels are regulated by LIMK1 and SSH1L. (A) SDF-1α–induced changes in P-cofilin levels. Jurkat cells were stimulated with 5 nM SDF-1α for the indicated times, and cell lysates were analyzed by immunoblotting with anti–P-cofilin and anticofilin antibodies. The bottom panel shows the relative P-cofilin levels after SDF-1α stimulation as means ± SEM of triplicate experiments. (B) Suppression of endogenous LIMK1, SSH1L, and cofilin expression by siRNA. Jurkat cells were transfected with siRNA plasmids for GFP (control), LIMK1, SSH1L, cofilin, or empty vector (−). After 60 h of culture, cell lysates were analyzed by immunoblotting with antibodies specific for each protein and β-actin. For LIMK1 and SSH1L, the cell lysates were subjected to immunoblotting after immunoprecipitation. (C and D) Effects of LIMK1 or SSH1L siRNA on SDF-1α–induced changes in P-cofilin levels. SSH1L, LIMK1, or GFP (control) siRNA cells were stimulated with 5 nM SDF-1α. Cell lysates, prepared at the indicated times, were analyzed by immunoblotting as in A. The bottom panels indicate the relative P-cofilin levels; the value at time = 0 in control cells is taken as 1.0. Each value represents the mean ± SEM (error bars) of triplicate experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171197&req=5

fig1: SDF-1α–induced changes in P-cofilin levels are regulated by LIMK1 and SSH1L. (A) SDF-1α–induced changes in P-cofilin levels. Jurkat cells were stimulated with 5 nM SDF-1α for the indicated times, and cell lysates were analyzed by immunoblotting with anti–P-cofilin and anticofilin antibodies. The bottom panel shows the relative P-cofilin levels after SDF-1α stimulation as means ± SEM of triplicate experiments. (B) Suppression of endogenous LIMK1, SSH1L, and cofilin expression by siRNA. Jurkat cells were transfected with siRNA plasmids for GFP (control), LIMK1, SSH1L, cofilin, or empty vector (−). After 60 h of culture, cell lysates were analyzed by immunoblotting with antibodies specific for each protein and β-actin. For LIMK1 and SSH1L, the cell lysates were subjected to immunoblotting after immunoprecipitation. (C and D) Effects of LIMK1 or SSH1L siRNA on SDF-1α–induced changes in P-cofilin levels. SSH1L, LIMK1, or GFP (control) siRNA cells were stimulated with 5 nM SDF-1α. Cell lysates, prepared at the indicated times, were analyzed by immunoblotting as in A. The bottom panels indicate the relative P-cofilin levels; the value at time = 0 in control cells is taken as 1.0. Each value represents the mean ± SEM (error bars) of triplicate experiments.
Mentions: Cofilin activity is negatively regulated by phosphorylation at Ser-3. To examine the role cofilin phosphoregulation plays in T cell chemotaxis, we first analyzed time-dependent changes in P-cofilin levels in Jurkat cells after SDF-1α stimulation. The P-cofilin levels increased by 1–5 min as reported previously (Nishita et al., 2002) but reverted to basal levels 20 min after SDF-1α stimulation (Fig. 1 A). Because LIMK1 in Jurkat cells is activated by SDF-1α for up to 20 min (Nishita et al., 2002), the decrease in P-cofilin levels at 20 min probably involves the activation of cofilin phosphatases such as SSH1L. To assess the roles of LIMK1 and SSH1L in the SDF-1α–induced changes in P-cofilin levels, we knocked down LIMK1 and SSH1L expression using siRNA. Endogenous LIMK1 and SSH1L expression was suppressed by the transfection of siRNA plasmids (Fig. 1 B). LIMK1 siRNA reduced the basal P-cofilin levels in unstimulated cells and inhibited the increase in P-cofilin levels that occurred 5 min after SDF-1α stimulation (Fig. 1 C). In contrast, SSH1L siRNA raised the basal P-cofilin levels, and these levels were further augmented at 5 and 20 min after SDF-1α stimulation (Fig. 1 D). These results suggest that LIMK1 plays a critical role in the SDF-1α–induced elevation of P-cofilin levels at 5 min and that SSH1L is involved in decreasing P-cofilin levels in later stages after SDF-1α stimulation.

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