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 and chemokinesis. Jurkat cells were transfected with siRNA plasmids for GFP (control), SSH1L, LIMK1, cofilin, or empty vector (−) as indicated. Chemotactic responses toward 5 nM SDF-1α and chemokinetic responses in the presence of 5 nM SDF-1α were determined in the Transwell culture chambers, as described in Materials and methods. The data are expressed as the means ± SEM (error bars) of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.005, compared with cells transfected with the empty vector.
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fig4: Effect of SSH1L, LIMK1, or cofilin siRNA on SDF-1α–induced T cell chemotaxis and chemokinesis. Jurkat cells were transfected with siRNA plasmids for GFP (control), SSH1L, LIMK1, cofilin, or empty vector (−) as indicated. Chemotactic responses toward 5 nM SDF-1α and chemokinetic responses in the presence of 5 nM SDF-1α were determined in the Transwell culture chambers, as described in Materials and methods. The data are expressed as the means ± SEM (error bars) of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.005, compared with cells transfected with the empty vector.

Mentions: To examine the roles that SSH1L, LIMK1, and cofilin play in SDF-1α–induced T cell migration, the expression of each protein in Jurkat cells was suppressed by siRNA (Fig. 1 B), and the directional (chemotactic) and random (chemokinetic) migration of these cells in response to SDF-1α was analyzed by using Transwell culture chambers (Fig. 4). SDF-1α was added only to the lower chamber to analyze the chemotactic response, whereas it was added to both the lower and upper chambers for chemokinesis assays. The knockdown of either SSH1L, LIMK1, or cofilin by siRNA significantly reduced the chemotactic response of Jurkat cells toward SDF-1α (Fig. 4), which indicates that SSH1L, LIMK1, and cofilin all play critical roles in T cell chemotaxis. In contrast, the chemokinetic response of cells was suppressed by LIMK1 or cofilin siRNA but not by SSH1L siRNA (Fig. 4). These results raise the possibility that although LIMK1 and cofilin are required for cell locomotion in general, SSH1L plays a more specific role; namely, in setting the direction 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 and chemokinesis. Jurkat cells were transfected with siRNA plasmids for GFP (control), SSH1L, LIMK1, cofilin, or empty vector (−) as indicated. Chemotactic responses toward 5 nM SDF-1α and chemokinetic responses in the presence of 5 nM SDF-1α were determined in the Transwell culture chambers, as described in Materials and methods. The data are expressed as the means ± SEM (error bars) of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.005, compared with cells transfected with the empty vector.
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Related In: Results  -  Collection

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fig4: Effect of SSH1L, LIMK1, or cofilin siRNA on SDF-1α–induced T cell chemotaxis and chemokinesis. Jurkat cells were transfected with siRNA plasmids for GFP (control), SSH1L, LIMK1, cofilin, or empty vector (−) as indicated. Chemotactic responses toward 5 nM SDF-1α and chemokinetic responses in the presence of 5 nM SDF-1α were determined in the Transwell culture chambers, as described in Materials and methods. The data are expressed as the means ± SEM (error bars) of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.005, compared with cells transfected with the empty vector.
Mentions: To examine the roles that SSH1L, LIMK1, and cofilin play in SDF-1α–induced T cell migration, the expression of each protein in Jurkat cells was suppressed by siRNA (Fig. 1 B), and the directional (chemotactic) and random (chemokinetic) migration of these cells in response to SDF-1α was analyzed by using Transwell culture chambers (Fig. 4). SDF-1α was added only to the lower chamber to analyze the chemotactic response, whereas it was added to both the lower and upper chambers for chemokinesis assays. The knockdown of either SSH1L, LIMK1, or cofilin by siRNA significantly reduced the chemotactic response of Jurkat cells toward SDF-1α (Fig. 4), which indicates that SSH1L, LIMK1, and cofilin all play critical roles in T cell chemotaxis. In contrast, the chemokinetic response of cells was suppressed by LIMK1 or cofilin siRNA but not by SSH1L siRNA (Fig. 4). These results raise the possibility that although LIMK1 and cofilin are required for cell locomotion in general, SSH1L plays a more specific role; namely, in setting the direction 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