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 cofilin, LIMK1, or SSH1L siRNA on F-actin assembly and membrane protrusion formation before and after SDF-1α stimulation. (A) Time-lapse fluorescence analysis. Jurkat cells cotransfected with YFP-actin and siRNA plasmids for mutated SSH1L (control), cofilin, LIMK1, or SSH1L were analyzed by time-lapse fluorescence microscopy, making use of YFP fluorescence. Numbers indicate the times after SDF-1α stimulation. See Videos 4–7 (available at http://www.jcb.org/cgi/content/full/jcb.200504029/DC1). (B) Jurkat cells transfected with siRNA plasmids were left unstimulated (top) or stimulated for 5 min with SDF-1α (bottom) and were fixed and stained with rhodamine-phalloidin to visualize F-actin. Bars, 10 μm.
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fig6: Effect of cofilin, LIMK1, or SSH1L siRNA on F-actin assembly and membrane protrusion formation before and after SDF-1α stimulation. (A) Time-lapse fluorescence analysis. Jurkat cells cotransfected with YFP-actin and siRNA plasmids for mutated SSH1L (control), cofilin, LIMK1, or SSH1L were analyzed by time-lapse fluorescence microscopy, making use of YFP fluorescence. Numbers indicate the times after SDF-1α stimulation. See Videos 4–7 (available at http://www.jcb.org/cgi/content/full/jcb.200504029/DC1). (B) Jurkat cells transfected with siRNA plasmids were left unstimulated (top) or stimulated for 5 min with SDF-1α (bottom) and were fixed and stained with rhodamine-phalloidin to visualize F-actin. Bars, 10 μm.

Mentions: To elucidate the mechanisms by which SSH1L, LIMK1, or cofilin siRNA impaired T cell migration and chemotaxis, alterations in cell morphology and actin cytoskeleton were analyzed by time-lapse fluorescence analysis of live cells expressing YFP-actin (Fig. 6 A and Videos 4–7, available at http://www.jcb.org/cgi/content/full/jcb.200504029/DC1) and rhodamine-phalloidin staining of fixed cells (Fig. 6 B) before and after SDF-1α stimulation. Although control Jurkat cells exhibited a round and symmetrical morphology before SDF-1α stimulation, they generated multiple F-actin–rich membrane protrusions around the cell at 1 min after SDF-1α exposure, and these protrusions were then converted to a single lamellipodial protrusion on one side of the cell by 5 min (Fig. 6, A and B, control; and Video 4). This polarized cell morphology appears to support the directional migration of the cell. The total F-actin content per cell, which was measured by the fluorescence intensity of rhodamine-phalloidin, increased about twofold 10 min after SDF-1α stimulation (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200504029/DC1). In contrast, cofilin siRNA induced aberrant F-actin assembly and multiple large protrusions in the periphery of the cell both before and after SDF-1α stimulation (Fig. 6, A and B, cofilin siRNA; and Video 5). The total F-actin contents in cofilin siRNA cells before and after SDF-1α treatment were significantly higher than those in control cells (Fig. S1). The abnormal accumulation of F-actin suggests that cofilin controls actin filament dynamics by accelerating F-actin disassembly. The inappropriate F-actin assembly in cofilin siRNA cells was not affected by SDF-1α stimulation, suggesting that the cofilin knockdown cells failed to migrate as a result of their inability to rearrange the actin cytoskeleton in response to SDF-1α.


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 cofilin, LIMK1, or SSH1L siRNA on F-actin assembly and membrane protrusion formation before and after SDF-1α stimulation. (A) Time-lapse fluorescence analysis. Jurkat cells cotransfected with YFP-actin and siRNA plasmids for mutated SSH1L (control), cofilin, LIMK1, or SSH1L were analyzed by time-lapse fluorescence microscopy, making use of YFP fluorescence. Numbers indicate the times after SDF-1α stimulation. See Videos 4–7 (available at http://www.jcb.org/cgi/content/full/jcb.200504029/DC1). (B) Jurkat cells transfected with siRNA plasmids were left unstimulated (top) or stimulated for 5 min with SDF-1α (bottom) and were fixed and stained with rhodamine-phalloidin to visualize F-actin. Bars, 10 μm.
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fig6: Effect of cofilin, LIMK1, or SSH1L siRNA on F-actin assembly and membrane protrusion formation before and after SDF-1α stimulation. (A) Time-lapse fluorescence analysis. Jurkat cells cotransfected with YFP-actin and siRNA plasmids for mutated SSH1L (control), cofilin, LIMK1, or SSH1L were analyzed by time-lapse fluorescence microscopy, making use of YFP fluorescence. Numbers indicate the times after SDF-1α stimulation. See Videos 4–7 (available at http://www.jcb.org/cgi/content/full/jcb.200504029/DC1). (B) Jurkat cells transfected with siRNA plasmids were left unstimulated (top) or stimulated for 5 min with SDF-1α (bottom) and were fixed and stained with rhodamine-phalloidin to visualize F-actin. Bars, 10 μm.
Mentions: To elucidate the mechanisms by which SSH1L, LIMK1, or cofilin siRNA impaired T cell migration and chemotaxis, alterations in cell morphology and actin cytoskeleton were analyzed by time-lapse fluorescence analysis of live cells expressing YFP-actin (Fig. 6 A and Videos 4–7, available at http://www.jcb.org/cgi/content/full/jcb.200504029/DC1) and rhodamine-phalloidin staining of fixed cells (Fig. 6 B) before and after SDF-1α stimulation. Although control Jurkat cells exhibited a round and symmetrical morphology before SDF-1α stimulation, they generated multiple F-actin–rich membrane protrusions around the cell at 1 min after SDF-1α exposure, and these protrusions were then converted to a single lamellipodial protrusion on one side of the cell by 5 min (Fig. 6, A and B, control; and Video 4). This polarized cell morphology appears to support the directional migration of the cell. The total F-actin content per cell, which was measured by the fluorescence intensity of rhodamine-phalloidin, increased about twofold 10 min after SDF-1α stimulation (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200504029/DC1). In contrast, cofilin siRNA induced aberrant F-actin assembly and multiple large protrusions in the periphery of the cell both before and after SDF-1α stimulation (Fig. 6, A and B, cofilin siRNA; and Video 5). The total F-actin contents in cofilin siRNA cells before and after SDF-1α treatment were significantly higher than those in control cells (Fig. S1). The abnormal accumulation of F-actin suggests that cofilin controls actin filament dynamics by accelerating F-actin disassembly. The inappropriate F-actin assembly in cofilin siRNA cells was not affected by SDF-1α stimulation, suggesting that the cofilin knockdown cells failed to migrate as a result of their inability to rearrange the actin cytoskeleton in response to SDF-1α.

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