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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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Related in: MedlinePlus

L-plastin phosphorylation modulates its mobility in focal adhesions.(A). Schematic representation of wild-type (WT) L-plastin showing the headpiece domain followed by two independent actin binding domains (ABDs). Residue serine-5 (Ser5) of the headpiece was mutated to alanine (SA) to generate unphosphorylatable L-plastin or to glutamic acid (SE) to generate an L-plastin variant mimicking constitutive phosphorylation. (B). Expression and localization of GFP-coupled L-plastin phosphorylation variants in Vero cells. Vero cells were transfected with cDNA encoding GFP-L-plastin phosphorylation variants. After 48 hours, cells were fixed and processed for immunofluorescence. The localization of L-plastin and F-actin was analyzed with an epifluorescence microscope (Leica DMRX microscope) after staining with Rhodamine-conjugated phalloidin to visualize polymerized actin. Scale bar, 20 µm. (C). A typical FRAP experiment carried out on a Vero cell transfected with WT GFP-L-plastin. The boxed region in the upper panel (scale bar, 10 µm) is shown enlarged in the bottom panels (scale bar, 4 µm). Circular spots, surrounded by a white line, are regions of interest (ROI) that are submitted to photobleaching and that have a diameter of 5 µm. Such spot size was selected to smooth local area effects and visually well-represents the focal adhesion region. Pictures were recorded before bleaching, immediately after bleaching and 90 seconds after bleaching. (D). Normalized FRAP recovery curves of wild type (WT, red), Ser5/Ala (SA, blue) and Ser5/Glu (SE, green) GFP-L-plastin fusions are compared to the curves predicted by the two-binding-state model (black curves). Data were obtained from three independent experiments representing 10 FRAP recordings for each condition. (E). Charts representing biochemical parameters obtained from fitting data with a two-binding-state model. Bars represent the mean ± s.d. P-values were calculated using standard Student's t-test. A p-value<0.05, considered as statistically significant, was obtained for Feq, k*1on and k*2on but not for k1off.
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pone-0009210-g001: L-plastin phosphorylation modulates its mobility in focal adhesions.(A). Schematic representation of wild-type (WT) L-plastin showing the headpiece domain followed by two independent actin binding domains (ABDs). Residue serine-5 (Ser5) of the headpiece was mutated to alanine (SA) to generate unphosphorylatable L-plastin or to glutamic acid (SE) to generate an L-plastin variant mimicking constitutive phosphorylation. (B). Expression and localization of GFP-coupled L-plastin phosphorylation variants in Vero cells. Vero cells were transfected with cDNA encoding GFP-L-plastin phosphorylation variants. After 48 hours, cells were fixed and processed for immunofluorescence. The localization of L-plastin and F-actin was analyzed with an epifluorescence microscope (Leica DMRX microscope) after staining with Rhodamine-conjugated phalloidin to visualize polymerized actin. Scale bar, 20 µm. (C). A typical FRAP experiment carried out on a Vero cell transfected with WT GFP-L-plastin. The boxed region in the upper panel (scale bar, 10 µm) is shown enlarged in the bottom panels (scale bar, 4 µm). Circular spots, surrounded by a white line, are regions of interest (ROI) that are submitted to photobleaching and that have a diameter of 5 µm. Such spot size was selected to smooth local area effects and visually well-represents the focal adhesion region. Pictures were recorded before bleaching, immediately after bleaching and 90 seconds after bleaching. (D). Normalized FRAP recovery curves of wild type (WT, red), Ser5/Ala (SA, blue) and Ser5/Glu (SE, green) GFP-L-plastin fusions are compared to the curves predicted by the two-binding-state model (black curves). Data were obtained from three independent experiments representing 10 FRAP recordings for each condition. (E). Charts representing biochemical parameters obtained from fitting data with a two-binding-state model. Bars represent the mean ± s.d. P-values were calculated using standard Student's t-test. A p-value<0.05, considered as statistically significant, was obtained for Feq, k*1on and k*2on but not for k1off.

Mentions: To investigate the steady-state dynamics of L-plastin and actin in living cells and the role of L-plastin phosphorylation on Ser5 herein (Fig. 1A), we performed confocal microscopy-based fluorescence recovery after photobleaching (FRAP) experiments using previously characterized L-plastin variants in fibroblast-like Vero cells which do not express endogenous L-plastin [9]. FRAP, which is a powerful approach for studying molecular mobility in live cells [26], [27] was combined with mathematical modeling to estimate the steady-state kinetics of L-plastin variants and actin turn-over which reflects actin polymerization and depolymerization reactions [28], [29]. Similar to epitope-tagged wild type L-plastin [9], wild type (WT-) L-plastin fused to GFP colocalized with actin in focal adhesions, membrane protrusions and along stress fibers, as visualised by epifluorescence microscopy (Fig. 1B, upper panels). To study the kinetics of the phosphorylated pool of L-plastin in Vero cells, we took advantage of the fact that transfected WT-L-plastin is phosphorylated on Ser5 and targeted to focal adhesions in these cells [9]. The bleach was therefore performed in a small region of interest (ROI) in focal adhesions (Fig. 1C). For each ROI, the experimental intensity recoveries were normalized and averaged. The obtained curves exhibited a fast and a slow phase of recovery (Fig. 1D). Twenty seconds after photobleaching, 89% of recovery was reached for WT GFP-L-plastin suggesting that the protein is highly mobile, undergoing rapid cycles of association and dissociation, as reported for other crosslinking proteins [26], [30]. Quantitatively, the best fits of the FRAP curves were obtained with a “two-binding-state” model [31]. This model may be applied to a system in which a molecule exhibits two distinct binding states involved in the interaction with the free binding sites of a partner molecule to form a complex. Analysis with the two-binding-state model allowed the separation of FRAP recovery curves into two largely independent phases, a first relatively quick phase from zero to ten seconds (k1off = 0.616 s−1 for WT) and a second much slower phase that represented the plateau (k2off = 0.03 s−1 for WT), such that k1off ≫ k2off. Based on this model, we estimated the equilibrium normalized concentration of free WT-L-plastin molecules Feq (Feq(WT) = 0.513±0.01) and the association rates k*1on (k*1on(WT) = 0.338±0.032 s−1) and k*2on (k*2on(WT) = 0.01±0.001 s−1) by fitting the normalized experimental FRAP curves with equation (5) (Fig. 1E, see Materials and Methods).


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 its mobility in focal adhesions.(A). Schematic representation of wild-type (WT) L-plastin showing the headpiece domain followed by two independent actin binding domains (ABDs). Residue serine-5 (Ser5) of the headpiece was mutated to alanine (SA) to generate unphosphorylatable L-plastin or to glutamic acid (SE) to generate an L-plastin variant mimicking constitutive phosphorylation. (B). Expression and localization of GFP-coupled L-plastin phosphorylation variants in Vero cells. Vero cells were transfected with cDNA encoding GFP-L-plastin phosphorylation variants. After 48 hours, cells were fixed and processed for immunofluorescence. The localization of L-plastin and F-actin was analyzed with an epifluorescence microscope (Leica DMRX microscope) after staining with Rhodamine-conjugated phalloidin to visualize polymerized actin. Scale bar, 20 µm. (C). A typical FRAP experiment carried out on a Vero cell transfected with WT GFP-L-plastin. The boxed region in the upper panel (scale bar, 10 µm) is shown enlarged in the bottom panels (scale bar, 4 µm). Circular spots, surrounded by a white line, are regions of interest (ROI) that are submitted to photobleaching and that have a diameter of 5 µm. Such spot size was selected to smooth local area effects and visually well-represents the focal adhesion region. Pictures were recorded before bleaching, immediately after bleaching and 90 seconds after bleaching. (D). Normalized FRAP recovery curves of wild type (WT, red), Ser5/Ala (SA, blue) and Ser5/Glu (SE, green) GFP-L-plastin fusions are compared to the curves predicted by the two-binding-state model (black curves). Data were obtained from three independent experiments representing 10 FRAP recordings for each condition. (E). Charts representing biochemical parameters obtained from fitting data with a two-binding-state model. Bars represent the mean ± s.d. P-values were calculated using standard Student's t-test. A p-value<0.05, considered as statistically significant, was obtained for Feq, k*1on and k*2on but not for k1off.
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Related In: Results  -  Collection

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

pone-0009210-g001: L-plastin phosphorylation modulates its mobility in focal adhesions.(A). Schematic representation of wild-type (WT) L-plastin showing the headpiece domain followed by two independent actin binding domains (ABDs). Residue serine-5 (Ser5) of the headpiece was mutated to alanine (SA) to generate unphosphorylatable L-plastin or to glutamic acid (SE) to generate an L-plastin variant mimicking constitutive phosphorylation. (B). Expression and localization of GFP-coupled L-plastin phosphorylation variants in Vero cells. Vero cells were transfected with cDNA encoding GFP-L-plastin phosphorylation variants. After 48 hours, cells were fixed and processed for immunofluorescence. The localization of L-plastin and F-actin was analyzed with an epifluorescence microscope (Leica DMRX microscope) after staining with Rhodamine-conjugated phalloidin to visualize polymerized actin. Scale bar, 20 µm. (C). A typical FRAP experiment carried out on a Vero cell transfected with WT GFP-L-plastin. The boxed region in the upper panel (scale bar, 10 µm) is shown enlarged in the bottom panels (scale bar, 4 µm). Circular spots, surrounded by a white line, are regions of interest (ROI) that are submitted to photobleaching and that have a diameter of 5 µm. Such spot size was selected to smooth local area effects and visually well-represents the focal adhesion region. Pictures were recorded before bleaching, immediately after bleaching and 90 seconds after bleaching. (D). Normalized FRAP recovery curves of wild type (WT, red), Ser5/Ala (SA, blue) and Ser5/Glu (SE, green) GFP-L-plastin fusions are compared to the curves predicted by the two-binding-state model (black curves). Data were obtained from three independent experiments representing 10 FRAP recordings for each condition. (E). Charts representing biochemical parameters obtained from fitting data with a two-binding-state model. Bars represent the mean ± s.d. P-values were calculated using standard Student's t-test. A p-value<0.05, considered as statistically significant, was obtained for Feq, k*1on and k*2on but not for k1off.
Mentions: To investigate the steady-state dynamics of L-plastin and actin in living cells and the role of L-plastin phosphorylation on Ser5 herein (Fig. 1A), we performed confocal microscopy-based fluorescence recovery after photobleaching (FRAP) experiments using previously characterized L-plastin variants in fibroblast-like Vero cells which do not express endogenous L-plastin [9]. FRAP, which is a powerful approach for studying molecular mobility in live cells [26], [27] was combined with mathematical modeling to estimate the steady-state kinetics of L-plastin variants and actin turn-over which reflects actin polymerization and depolymerization reactions [28], [29]. Similar to epitope-tagged wild type L-plastin [9], wild type (WT-) L-plastin fused to GFP colocalized with actin in focal adhesions, membrane protrusions and along stress fibers, as visualised by epifluorescence microscopy (Fig. 1B, upper panels). To study the kinetics of the phosphorylated pool of L-plastin in Vero cells, we took advantage of the fact that transfected WT-L-plastin is phosphorylated on Ser5 and targeted to focal adhesions in these cells [9]. The bleach was therefore performed in a small region of interest (ROI) in focal adhesions (Fig. 1C). For each ROI, the experimental intensity recoveries were normalized and averaged. The obtained curves exhibited a fast and a slow phase of recovery (Fig. 1D). Twenty seconds after photobleaching, 89% of recovery was reached for WT GFP-L-plastin suggesting that the protein is highly mobile, undergoing rapid cycles of association and dissociation, as reported for other crosslinking proteins [26], [30]. Quantitatively, the best fits of the FRAP curves were obtained with a “two-binding-state” model [31]. This model may be applied to a system in which a molecule exhibits two distinct binding states involved in the interaction with the free binding sites of a partner molecule to form a complex. Analysis with the two-binding-state model allowed the separation of FRAP recovery curves into two largely independent phases, a first relatively quick phase from zero to ten seconds (k1off = 0.616 s−1 for WT) and a second much slower phase that represented the plateau (k2off = 0.03 s−1 for WT), such that k1off ≫ k2off. Based on this model, we estimated the equilibrium normalized concentration of free WT-L-plastin molecules Feq (Feq(WT) = 0.513±0.01) and the association rates k*1on (k*1on(WT) = 0.338±0.032 s−1) and k*2on (k*2on(WT) = 0.01±0.001 s−1) by fitting the normalized experimental FRAP curves with equation (5) (Fig. 1E, see Materials and Methods).

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