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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Temporal localization of LIMK1 and SSH1L in SDF-1α–stimulated Jurkat cells. (A) Localization of CFP-LIMK1 (red) and YFP-SSH1L (green) in fixed Jurkat cells unstimulated (0 min) or stimulated for 1, 5, and 20 min with 5 nM SDF-1α. Merged images are shown in the bottom panels. (B) Localization of YFP-actin (red) and CFP-SSH1L (green) in fixed Jurkat cells unstimulated (0 min) or stimulated for 1, 5, and 20 min with 5 nM SDF-1α. Merged images are shown in the bottom panels. Bars, 5 μm.
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fig2: Temporal localization of LIMK1 and SSH1L in SDF-1α–stimulated Jurkat cells. (A) Localization of CFP-LIMK1 (red) and YFP-SSH1L (green) in fixed Jurkat cells unstimulated (0 min) or stimulated for 1, 5, and 20 min with 5 nM SDF-1α. Merged images are shown in the bottom panels. (B) Localization of YFP-actin (red) and CFP-SSH1L (green) in fixed Jurkat cells unstimulated (0 min) or stimulated for 1, 5, and 20 min with 5 nM SDF-1α. Merged images are shown in the bottom panels. Bars, 5 μm.

Mentions: When exposed to SDF-1α, Jurkat cells produced multiple membrane protrusions around the cell at 1 min and became polarized to form a single lamellipodial protrusion in one direction by 5 min (Fig. 2, A and B; and Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200504029/DC1). We examined the temporal changes in the distribution of LIMK1 and SSH1L before and after SDF-1α stimulation by coexpressing CFP-LIMK1 and YFP-SSH1L in Jurkat cells. Fluorescence microscopic analyses after fixing the cells showed that CFP-LIMK1 was diffusely distributed in the cytoplasm both before and after SDF-1α stimulation, whereas YFP-SSH1L was diffusely distributed in the cytoplasm in unstimulated cells but accumulated in the lamellipodial protrusions 1–20 min after SDF-1α stimulation (Fig. 2 A). The three-dimensional projection image more clearly showed the distinct distribution of LIMK1 and SSH1L in Jurkat cells that were stimulated for 5 min (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200504029/DC1). Time-lapse live-cell image analyses of CFP-LIMK1 and YFP-SSH1L also showed that LIMK1 was diffusely distributed and that SSH1L accumulated in membrane protrusions after SDF-1α stimulation (Video 2). We also analyzed the temporal localization of YFP-actin and CFP-SSH1L in fixed (Fig. 2 B) and live Jurkat cells (Video 3). Both analyses revealed that CFP-SSH1L was diffusely distributed in the cytoplasm before SDF-1α stimulation, but it almost colocalized with YFP-actin and accumulated in the F-actin–rich lamellipodia after stimulation. It appears from the merged images that SSH1L is depleted from the tip of the lamellipodium (Fig. 2 B).


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)

Temporal localization of LIMK1 and SSH1L in SDF-1α–stimulated Jurkat cells. (A) Localization of CFP-LIMK1 (red) and YFP-SSH1L (green) in fixed Jurkat cells unstimulated (0 min) or stimulated for 1, 5, and 20 min with 5 nM SDF-1α. Merged images are shown in the bottom panels. (B) Localization of YFP-actin (red) and CFP-SSH1L (green) in fixed Jurkat cells unstimulated (0 min) or stimulated for 1, 5, and 20 min with 5 nM SDF-1α. Merged images are shown in the bottom panels. Bars, 5 μm.
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Related In: Results  -  Collection

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

fig2: Temporal localization of LIMK1 and SSH1L in SDF-1α–stimulated Jurkat cells. (A) Localization of CFP-LIMK1 (red) and YFP-SSH1L (green) in fixed Jurkat cells unstimulated (0 min) or stimulated for 1, 5, and 20 min with 5 nM SDF-1α. Merged images are shown in the bottom panels. (B) Localization of YFP-actin (red) and CFP-SSH1L (green) in fixed Jurkat cells unstimulated (0 min) or stimulated for 1, 5, and 20 min with 5 nM SDF-1α. Merged images are shown in the bottom panels. Bars, 5 μm.
Mentions: When exposed to SDF-1α, Jurkat cells produced multiple membrane protrusions around the cell at 1 min and became polarized to form a single lamellipodial protrusion in one direction by 5 min (Fig. 2, A and B; and Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200504029/DC1). We examined the temporal changes in the distribution of LIMK1 and SSH1L before and after SDF-1α stimulation by coexpressing CFP-LIMK1 and YFP-SSH1L in Jurkat cells. Fluorescence microscopic analyses after fixing the cells showed that CFP-LIMK1 was diffusely distributed in the cytoplasm both before and after SDF-1α stimulation, whereas YFP-SSH1L was diffusely distributed in the cytoplasm in unstimulated cells but accumulated in the lamellipodial protrusions 1–20 min after SDF-1α stimulation (Fig. 2 A). The three-dimensional projection image more clearly showed the distinct distribution of LIMK1 and SSH1L in Jurkat cells that were stimulated for 5 min (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200504029/DC1). Time-lapse live-cell image analyses of CFP-LIMK1 and YFP-SSH1L also showed that LIMK1 was diffusely distributed and that SSH1L accumulated in membrane protrusions after SDF-1α stimulation (Video 2). We also analyzed the temporal localization of YFP-actin and CFP-SSH1L in fixed (Fig. 2 B) and live Jurkat cells (Video 3). Both analyses revealed that CFP-SSH1L was diffusely distributed in the cytoplasm before SDF-1α stimulation, but it almost colocalized with YFP-actin and accumulated in the F-actin–rich lamellipodia after stimulation. It appears from the merged images that SSH1L is depleted from the tip of the lamellipodium (Fig. 2 B).

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