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Quantitative kinetic study of the actin-bundling protein L-plastin and of its impact on actin turn-over.

Al Tanoury Z, Schaffner-Reckinger E, Halavatyi A, Hoffmann C, Moes M, Hadzic E, Catillon M, Yatskou M, Friederich E - PLoS ONE (2010)

Bottom Line: Importantly, L-plastin affected actin turn-over by decreasing the actin dissociation rate by four-fold, increasing thereby the amount of F-actin in the focal adhesions, all these effects being promoted by Ser5 phosphorylation.Altogether these findings quantitatively demonstrate for the first time that L-plastin contributes to the fine-tuning of actin turn-over, an activity which is regulated by Ser5 phosphorylation promoting its high affinity binding to the cytoskeleton.In carcinoma cells, PKC-delta signaling pathways appear to link L-plastin phosphorylation to actin polymerization and invasion.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cytoskeleton and Cell Plasticity, Life Sciences Research Unit, University of Luxembourg, Luxembourg City, Luxembourg.

ABSTRACT

Background: Initially detected in leukocytes and cancer cells derived from solid tissues, L-plastin/fimbrin belongs to a large family of actin crosslinkers and is considered as a marker for many cancers. Phosphorylation of L-plastin on residue Ser5 increases its F-actin binding activity and is required for L-plastin-mediated cell invasion.

Methodology/principal findings: To study the kinetics of L-plastin and the impact of L-plastin Ser5 phosphorylation on L-plastin dynamics and actin turn-over in live cells, simian Vero cells were transfected with GFP-coupled WT-L-plastin, Ser5 substitution variants (S5/A, S5/E) or actin and analyzed by fluorescence recovery after photobleaching (FRAP). FRAP data were explored by mathematical modeling to estimate steady-state reaction parameters. We demonstrate that in Vero cell focal adhesions L-plastin undergoes rapid cycles of association/dissociation following a two-binding-state model. Phosphorylation of L-plastin increased its association rates by two-fold, whereas dissociation rates were unaffected. Importantly, L-plastin affected actin turn-over by decreasing the actin dissociation rate by four-fold, increasing thereby the amount of F-actin in the focal adhesions, all these effects being promoted by Ser5 phosphorylation. In MCF-7 breast carcinoma cells, phorbol 12-myristate 13-acetate (PMA) treatment induced L-plastin translocation to de novo actin polymerization sites in ruffling membranes and spike-like structures and highly increased its Ser5 phosphorylation. Both inhibition studies and siRNA knock-down of PKC isozymes pointed to the involvement of the novel PKC-delta isozyme in the PMA-elicited signaling pathway leading to L-plastin Ser5 phosphorylation. Furthermore, the L-plastin contribution to actin dynamics regulation was substantiated by its association with a protein complex comprising cortactin, which is known to be involved in this process.

Conclusions/significance: Altogether these findings quantitatively demonstrate for the first time that L-plastin contributes to the fine-tuning of actin turn-over, an activity which is regulated by Ser5 phosphorylation promoting its high affinity binding to the cytoskeleton. In carcinoma cells, PKC-delta signaling pathways appear to link L-plastin phosphorylation to actin polymerization and invasion.

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L-plastin phosphorylation modulates actin dynamics in focal adhesions.(A). Expression of L-plastin and actin in live Vero cells. Vero cells were cotransfected with monomeric L-plastin-DsRed-N1 fusion variants and GFP-actin. Wild type L-plastin-DsRed is shown here. Scale bar, 20 µm. (B). Normalized FRAP recovery curves obtained for actin in presence of wild type (WT, red), Ser5/Ala (SA, blue) and Ser5/Glu (SE, green) L-plastin-DsRed fusions or DsRed alone (control, orange) are compared to the curves predicted by the one-binding-state model (black curves). Data were obtained from three independent experiments representing 10 FRAP recordings for each condition. (C). Charts representing biochemical parameters obtained from data fitted with a one-binding-state model. Bars represent the mean ± s.d.
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pone-0009210-g002: L-plastin phosphorylation modulates actin dynamics in focal adhesions.(A). Expression of L-plastin and actin in live Vero cells. Vero cells were cotransfected with monomeric L-plastin-DsRed-N1 fusion variants and GFP-actin. Wild type L-plastin-DsRed is shown here. Scale bar, 20 µm. (B). Normalized FRAP recovery curves obtained for actin in presence of wild type (WT, red), Ser5/Ala (SA, blue) and Ser5/Glu (SE, green) L-plastin-DsRed fusions or DsRed alone (control, orange) are compared to the curves predicted by the one-binding-state model (black curves). Data were obtained from three independent experiments representing 10 FRAP recordings for each condition. (C). Charts representing biochemical parameters obtained from data fitted with a one-binding-state model. Bars represent the mean ± s.d.

Mentions: L-plastin appears to affect actin dynamics by binding along actin filaments, as supported by previous biochemical data [9], [20]. To address this issue, FRAP experiments were performed on GFP-actin in Vero cell focal adhesions, co-transfected with GFP-actin and L-plastin variants fused to monomeric DsRed (Fig. 2A). In agreement with previous kinetic studies of GFP-actin in cells [32], the fast recovery phase of GFP-actin involved rapid diffusion of the large free pool of actin monomers into the bleached area. Indeed, diffusion of the free monomers led to recovery in less than one second, which was below the characteristic time of the association/dissociation kinetics and time resolution of our experiments. The measured postbleach fluorescence intensity of GFP-actin was strongly reduced in WT-L-plastin expressing cells as compared to control cells (Fig. 2B), suggesting a decrease of the fraction of free diffusible actin monomers in response to L-plastin expression.


Quantitative kinetic study of the actin-bundling protein L-plastin and of its impact on actin turn-over.

Al Tanoury Z, Schaffner-Reckinger E, Halavatyi A, Hoffmann C, Moes M, Hadzic E, Catillon M, Yatskou M, Friederich E - PLoS ONE (2010)

L-plastin phosphorylation modulates actin dynamics in focal adhesions.(A). Expression of L-plastin and actin in live Vero cells. Vero cells were cotransfected with monomeric L-plastin-DsRed-N1 fusion variants and GFP-actin. Wild type L-plastin-DsRed is shown here. Scale bar, 20 µm. (B). Normalized FRAP recovery curves obtained for actin in presence of wild type (WT, red), Ser5/Ala (SA, blue) and Ser5/Glu (SE, green) L-plastin-DsRed fusions or DsRed alone (control, orange) are compared to the curves predicted by the one-binding-state model (black curves). Data were obtained from three independent experiments representing 10 FRAP recordings for each condition. (C). Charts representing biochemical parameters obtained from data fitted with a one-binding-state model. Bars represent the mean ± s.d.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2821400&req=5

pone-0009210-g002: L-plastin phosphorylation modulates actin dynamics in focal adhesions.(A). Expression of L-plastin and actin in live Vero cells. Vero cells were cotransfected with monomeric L-plastin-DsRed-N1 fusion variants and GFP-actin. Wild type L-plastin-DsRed is shown here. Scale bar, 20 µm. (B). Normalized FRAP recovery curves obtained for actin in presence of wild type (WT, red), Ser5/Ala (SA, blue) and Ser5/Glu (SE, green) L-plastin-DsRed fusions or DsRed alone (control, orange) are compared to the curves predicted by the one-binding-state model (black curves). Data were obtained from three independent experiments representing 10 FRAP recordings for each condition. (C). Charts representing biochemical parameters obtained from data fitted with a one-binding-state model. Bars represent the mean ± s.d.
Mentions: L-plastin appears to affect actin dynamics by binding along actin filaments, as supported by previous biochemical data [9], [20]. To address this issue, FRAP experiments were performed on GFP-actin in Vero cell focal adhesions, co-transfected with GFP-actin and L-plastin variants fused to monomeric DsRed (Fig. 2A). In agreement with previous kinetic studies of GFP-actin in cells [32], the fast recovery phase of GFP-actin involved rapid diffusion of the large free pool of actin monomers into the bleached area. Indeed, diffusion of the free monomers led to recovery in less than one second, which was below the characteristic time of the association/dissociation kinetics and time resolution of our experiments. The measured postbleach fluorescence intensity of GFP-actin was strongly reduced in WT-L-plastin expressing cells as compared to control cells (Fig. 2B), suggesting a decrease of the fraction of free diffusible actin monomers in response to L-plastin expression.

Bottom Line: Importantly, L-plastin affected actin turn-over by decreasing the actin dissociation rate by four-fold, increasing thereby the amount of F-actin in the focal adhesions, all these effects being promoted by Ser5 phosphorylation.Altogether these findings quantitatively demonstrate for the first time that L-plastin contributes to the fine-tuning of actin turn-over, an activity which is regulated by Ser5 phosphorylation promoting its high affinity binding to the cytoskeleton.In carcinoma cells, PKC-delta signaling pathways appear to link L-plastin phosphorylation to actin polymerization and invasion.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cytoskeleton and Cell Plasticity, Life Sciences Research Unit, University of Luxembourg, Luxembourg City, Luxembourg.

ABSTRACT

Background: Initially detected in leukocytes and cancer cells derived from solid tissues, L-plastin/fimbrin belongs to a large family of actin crosslinkers and is considered as a marker for many cancers. Phosphorylation of L-plastin on residue Ser5 increases its F-actin binding activity and is required for L-plastin-mediated cell invasion.

Methodology/principal findings: To study the kinetics of L-plastin and the impact of L-plastin Ser5 phosphorylation on L-plastin dynamics and actin turn-over in live cells, simian Vero cells were transfected with GFP-coupled WT-L-plastin, Ser5 substitution variants (S5/A, S5/E) or actin and analyzed by fluorescence recovery after photobleaching (FRAP). FRAP data were explored by mathematical modeling to estimate steady-state reaction parameters. We demonstrate that in Vero cell focal adhesions L-plastin undergoes rapid cycles of association/dissociation following a two-binding-state model. Phosphorylation of L-plastin increased its association rates by two-fold, whereas dissociation rates were unaffected. Importantly, L-plastin affected actin turn-over by decreasing the actin dissociation rate by four-fold, increasing thereby the amount of F-actin in the focal adhesions, all these effects being promoted by Ser5 phosphorylation. In MCF-7 breast carcinoma cells, phorbol 12-myristate 13-acetate (PMA) treatment induced L-plastin translocation to de novo actin polymerization sites in ruffling membranes and spike-like structures and highly increased its Ser5 phosphorylation. Both inhibition studies and siRNA knock-down of PKC isozymes pointed to the involvement of the novel PKC-delta isozyme in the PMA-elicited signaling pathway leading to L-plastin Ser5 phosphorylation. Furthermore, the L-plastin contribution to actin dynamics regulation was substantiated by its association with a protein complex comprising cortactin, which is known to be involved in this process.

Conclusions/significance: Altogether these findings quantitatively demonstrate for the first time that L-plastin contributes to the fine-tuning of actin turn-over, an activity which is regulated by Ser5 phosphorylation promoting its high affinity binding to the cytoskeleton. In carcinoma cells, PKC-delta signaling pathways appear to link L-plastin phosphorylation to actin polymerization and invasion.

Show MeSH
Related in: MedlinePlus