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p21-Activated kinase (PAK) regulates cytoskeletal reorganization and directional migration in human neutrophils.

Itakura A, Aslan JE, Kusanto BT, Phillips KG, Porter JE, Newton PK, Nan X, Insall RH, Chernoff J, McCarty OJ - PLoS ONE (2013)

Bottom Line: In this study, we characterized the role of p21-activated kinase (PAK) downstream of Rho GTPases in cytoskeletal remodeling and chemotactic processes of human neutrophils.We found that PAK activation occurred upon stimulation of neutrophils with f-Met-Leu-Phe (fMLP), and PAK accumulated at the actin-rich leading edge of stimulated neutrophils, suggesting a role for PAK in Rac-dependent actin remodeling.Treatment with the pharmacological PAK inhibitor, PF3758309, abrogated the integrity of RhoA-mediated actomyosin contractility and surface adhesion.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Oregon Health and Science University, Portland, Oregon, United States of America.

ABSTRACT
Neutrophils serve as a first line of defense in innate immunity owing in part to their ability to rapidly migrate towards chemotactic factors derived from invading pathogens. As a migratory function, neutrophil chemotaxis is regulated by the Rho family of small GTPases. However, the mechanisms by which Rho GTPases orchestrate cytoskeletal dynamics in migrating neutrophils remain ill-defined. In this study, we characterized the role of p21-activated kinase (PAK) downstream of Rho GTPases in cytoskeletal remodeling and chemotactic processes of human neutrophils. We found that PAK activation occurred upon stimulation of neutrophils with f-Met-Leu-Phe (fMLP), and PAK accumulated at the actin-rich leading edge of stimulated neutrophils, suggesting a role for PAK in Rac-dependent actin remodeling. Treatment with the pharmacological PAK inhibitor, PF3758309, abrogated the integrity of RhoA-mediated actomyosin contractility and surface adhesion. Moreover, inhibition of PAK activity impaired neutrophil morphological polarization and directional migration under a gradient of fMLP, and was associated with dysregulated Ca(2+) signaling. These results suggest that PAK serves as an important effector of Rho-family GTPases in neutrophil cytoskeletal reorganization, and plays a key role in driving efficient directional migration of human neutrophils.

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PAK inhibition leads to an accumulation of active RhoA and phosphorylated myosin light chain.(A) Human neutrophils adherent on fibronectin surfaces were pretreated with vehicle (0.1% DMSO) or PF3758309 (PF; PAK inhibitor, 10 µM), and stimulated with fMLP (10 nM) for 3 min and stained for phospho-PAK1/2 Thr423/402 (green) and F-actin (red). (B) The mean percentage of cells displaying phospho-PAK1/2 immunofluorescence at basal level (white bars) or after stimulation (black bars) was quantified from at least 50 cells per treatment. (C) Cell circularity was analyzed using MATLAB and presented as aspect ratio of individual neutrophils at basal level (white bars) or after fMLP stimulation (black bars). (D) Neutrophils were pretreated as indicated in (A) and stimulated with fMLP (10 nM). Cells were stained for active RhoA-GTP (green) and F-actin (red). (E) The mean relative intensity of RhoA-GTP immunofluorescence or (F) the mean percentage of RhoA-GTP/actin colocalization of individual cell area at basal level (white bars) or after fMLP stimulation (black bars). (G) Neutrophils were pretreated with the inhibitors indicated in (A), or with Y27632 (ROCK inhibitor, 10 µM), or a combination of 10 µM PF3758309 and 10 µM Y27632 (PF+Y27632) and stained for phospho-myosin light chain (p-MLC; green) and F-actin (red). (H) Relative p-MLC fluorescence intensity or (I) the distance between DIC cell centroid and p-MLC fluorescence centroid at basal level (white bars) or after fMLP stimulation (black bars). Representative images obtained from at least 3 independent experiments are shown. * P<0.05 compared to the basal level; # P<0.05, compared to DMSO-treated cells; ** P<0.05, compared to cells treated with Y27632 alone. Scale ba = (A) and (G) 5 µm; (D) 10 µm.
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pone-0073063-g002: PAK inhibition leads to an accumulation of active RhoA and phosphorylated myosin light chain.(A) Human neutrophils adherent on fibronectin surfaces were pretreated with vehicle (0.1% DMSO) or PF3758309 (PF; PAK inhibitor, 10 µM), and stimulated with fMLP (10 nM) for 3 min and stained for phospho-PAK1/2 Thr423/402 (green) and F-actin (red). (B) The mean percentage of cells displaying phospho-PAK1/2 immunofluorescence at basal level (white bars) or after stimulation (black bars) was quantified from at least 50 cells per treatment. (C) Cell circularity was analyzed using MATLAB and presented as aspect ratio of individual neutrophils at basal level (white bars) or after fMLP stimulation (black bars). (D) Neutrophils were pretreated as indicated in (A) and stimulated with fMLP (10 nM). Cells were stained for active RhoA-GTP (green) and F-actin (red). (E) The mean relative intensity of RhoA-GTP immunofluorescence or (F) the mean percentage of RhoA-GTP/actin colocalization of individual cell area at basal level (white bars) or after fMLP stimulation (black bars). (G) Neutrophils were pretreated with the inhibitors indicated in (A), or with Y27632 (ROCK inhibitor, 10 µM), or a combination of 10 µM PF3758309 and 10 µM Y27632 (PF+Y27632) and stained for phospho-myosin light chain (p-MLC; green) and F-actin (red). (H) Relative p-MLC fluorescence intensity or (I) the distance between DIC cell centroid and p-MLC fluorescence centroid at basal level (white bars) or after fMLP stimulation (black bars). Representative images obtained from at least 3 independent experiments are shown. * P<0.05 compared to the basal level; # P<0.05, compared to DMSO-treated cells; ** P<0.05, compared to cells treated with Y27632 alone. Scale ba = (A) and (G) 5 µm; (D) 10 µm.

Mentions: Neutrophil morphological polarity is maintained through a balance of ‘frontness’ and ‘backness’ signals which have been shown to be mediated by the Rho GTPases Rac/Cdc42 GTPases and RhoA, respectively [2]. The putative roles of the Rho GTPases and PAK in leading edge dynamics led us to hypothesize that PAKs may maintain neutrophil ‘frontness’ by modulating ‘backness’ during neutrophil polarization. To test this hypothesis, we assessed the effects of PAK inhibition on RhoA GTPase activation and localization using the pharmacological PAK inhibitor, PF3758309 [12], [15], [16], which inhibited the phosphorylation of PAK1/2 Thr423/402 (Fig. 2A and 2B) and PAK2 Ser20 (data not shown) in fMLP-stimulated neutrophils. While neutrophils developed a morphological polarity following fMLP stimulation, characterized by a significant increase in aspect ratio (a function of the largest cell diameter and the smallest diameter), PF3758309-treated cells underwent polarization in the absence of any stimulation (Fig. 2C). In vehicle-treated cells, fMLP-induced polarization was associated with an increase in the immunofluorescence of active RhoA-GTP (Fig. 2D and 2E) and a decrease in the colocalization of RhoA-GTP with actin (Fig. 2D and 2F), suggesting that fMLP induced active RhoA accumulation at the uropod. In contrast, PF3758309-treated cells displayed similar signatures of cell polarization in the absence of fMLP with a significantly higher level of active RhoA as compared to control cells (Fig. 2D–F). Stimulation of PAK-inhibited cells with fMLP resulted in a comparable level of active RhoA to fMLP-stimulated control cells (Fig. 2D–F). These results suggest that PAK contributes to neutrophil cytoskeletal dynamics under basal conditions by suppressing RhoA-GTP accumulation and relocalization.


p21-Activated kinase (PAK) regulates cytoskeletal reorganization and directional migration in human neutrophils.

Itakura A, Aslan JE, Kusanto BT, Phillips KG, Porter JE, Newton PK, Nan X, Insall RH, Chernoff J, McCarty OJ - PLoS ONE (2013)

PAK inhibition leads to an accumulation of active RhoA and phosphorylated myosin light chain.(A) Human neutrophils adherent on fibronectin surfaces were pretreated with vehicle (0.1% DMSO) or PF3758309 (PF; PAK inhibitor, 10 µM), and stimulated with fMLP (10 nM) for 3 min and stained for phospho-PAK1/2 Thr423/402 (green) and F-actin (red). (B) The mean percentage of cells displaying phospho-PAK1/2 immunofluorescence at basal level (white bars) or after stimulation (black bars) was quantified from at least 50 cells per treatment. (C) Cell circularity was analyzed using MATLAB and presented as aspect ratio of individual neutrophils at basal level (white bars) or after fMLP stimulation (black bars). (D) Neutrophils were pretreated as indicated in (A) and stimulated with fMLP (10 nM). Cells were stained for active RhoA-GTP (green) and F-actin (red). (E) The mean relative intensity of RhoA-GTP immunofluorescence or (F) the mean percentage of RhoA-GTP/actin colocalization of individual cell area at basal level (white bars) or after fMLP stimulation (black bars). (G) Neutrophils were pretreated with the inhibitors indicated in (A), or with Y27632 (ROCK inhibitor, 10 µM), or a combination of 10 µM PF3758309 and 10 µM Y27632 (PF+Y27632) and stained for phospho-myosin light chain (p-MLC; green) and F-actin (red). (H) Relative p-MLC fluorescence intensity or (I) the distance between DIC cell centroid and p-MLC fluorescence centroid at basal level (white bars) or after fMLP stimulation (black bars). Representative images obtained from at least 3 independent experiments are shown. * P<0.05 compared to the basal level; # P<0.05, compared to DMSO-treated cells; ** P<0.05, compared to cells treated with Y27632 alone. Scale ba = (A) and (G) 5 µm; (D) 10 µm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3760889&req=5

pone-0073063-g002: PAK inhibition leads to an accumulation of active RhoA and phosphorylated myosin light chain.(A) Human neutrophils adherent on fibronectin surfaces were pretreated with vehicle (0.1% DMSO) or PF3758309 (PF; PAK inhibitor, 10 µM), and stimulated with fMLP (10 nM) for 3 min and stained for phospho-PAK1/2 Thr423/402 (green) and F-actin (red). (B) The mean percentage of cells displaying phospho-PAK1/2 immunofluorescence at basal level (white bars) or after stimulation (black bars) was quantified from at least 50 cells per treatment. (C) Cell circularity was analyzed using MATLAB and presented as aspect ratio of individual neutrophils at basal level (white bars) or after fMLP stimulation (black bars). (D) Neutrophils were pretreated as indicated in (A) and stimulated with fMLP (10 nM). Cells were stained for active RhoA-GTP (green) and F-actin (red). (E) The mean relative intensity of RhoA-GTP immunofluorescence or (F) the mean percentage of RhoA-GTP/actin colocalization of individual cell area at basal level (white bars) or after fMLP stimulation (black bars). (G) Neutrophils were pretreated with the inhibitors indicated in (A), or with Y27632 (ROCK inhibitor, 10 µM), or a combination of 10 µM PF3758309 and 10 µM Y27632 (PF+Y27632) and stained for phospho-myosin light chain (p-MLC; green) and F-actin (red). (H) Relative p-MLC fluorescence intensity or (I) the distance between DIC cell centroid and p-MLC fluorescence centroid at basal level (white bars) or after fMLP stimulation (black bars). Representative images obtained from at least 3 independent experiments are shown. * P<0.05 compared to the basal level; # P<0.05, compared to DMSO-treated cells; ** P<0.05, compared to cells treated with Y27632 alone. Scale ba = (A) and (G) 5 µm; (D) 10 µm.
Mentions: Neutrophil morphological polarity is maintained through a balance of ‘frontness’ and ‘backness’ signals which have been shown to be mediated by the Rho GTPases Rac/Cdc42 GTPases and RhoA, respectively [2]. The putative roles of the Rho GTPases and PAK in leading edge dynamics led us to hypothesize that PAKs may maintain neutrophil ‘frontness’ by modulating ‘backness’ during neutrophil polarization. To test this hypothesis, we assessed the effects of PAK inhibition on RhoA GTPase activation and localization using the pharmacological PAK inhibitor, PF3758309 [12], [15], [16], which inhibited the phosphorylation of PAK1/2 Thr423/402 (Fig. 2A and 2B) and PAK2 Ser20 (data not shown) in fMLP-stimulated neutrophils. While neutrophils developed a morphological polarity following fMLP stimulation, characterized by a significant increase in aspect ratio (a function of the largest cell diameter and the smallest diameter), PF3758309-treated cells underwent polarization in the absence of any stimulation (Fig. 2C). In vehicle-treated cells, fMLP-induced polarization was associated with an increase in the immunofluorescence of active RhoA-GTP (Fig. 2D and 2E) and a decrease in the colocalization of RhoA-GTP with actin (Fig. 2D and 2F), suggesting that fMLP induced active RhoA accumulation at the uropod. In contrast, PF3758309-treated cells displayed similar signatures of cell polarization in the absence of fMLP with a significantly higher level of active RhoA as compared to control cells (Fig. 2D–F). Stimulation of PAK-inhibited cells with fMLP resulted in a comparable level of active RhoA to fMLP-stimulated control cells (Fig. 2D–F). These results suggest that PAK contributes to neutrophil cytoskeletal dynamics under basal conditions by suppressing RhoA-GTP accumulation and relocalization.

Bottom Line: In this study, we characterized the role of p21-activated kinase (PAK) downstream of Rho GTPases in cytoskeletal remodeling and chemotactic processes of human neutrophils.We found that PAK activation occurred upon stimulation of neutrophils with f-Met-Leu-Phe (fMLP), and PAK accumulated at the actin-rich leading edge of stimulated neutrophils, suggesting a role for PAK in Rac-dependent actin remodeling.Treatment with the pharmacological PAK inhibitor, PF3758309, abrogated the integrity of RhoA-mediated actomyosin contractility and surface adhesion.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Oregon Health and Science University, Portland, Oregon, United States of America.

ABSTRACT
Neutrophils serve as a first line of defense in innate immunity owing in part to their ability to rapidly migrate towards chemotactic factors derived from invading pathogens. As a migratory function, neutrophil chemotaxis is regulated by the Rho family of small GTPases. However, the mechanisms by which Rho GTPases orchestrate cytoskeletal dynamics in migrating neutrophils remain ill-defined. In this study, we characterized the role of p21-activated kinase (PAK) downstream of Rho GTPases in cytoskeletal remodeling and chemotactic processes of human neutrophils. We found that PAK activation occurred upon stimulation of neutrophils with f-Met-Leu-Phe (fMLP), and PAK accumulated at the actin-rich leading edge of stimulated neutrophils, suggesting a role for PAK in Rac-dependent actin remodeling. Treatment with the pharmacological PAK inhibitor, PF3758309, abrogated the integrity of RhoA-mediated actomyosin contractility and surface adhesion. Moreover, inhibition of PAK activity impaired neutrophil morphological polarization and directional migration under a gradient of fMLP, and was associated with dysregulated Ca(2+) signaling. These results suggest that PAK serves as an important effector of Rho-family GTPases in neutrophil cytoskeletal reorganization, and plays a key role in driving efficient directional migration of human neutrophils.

Show MeSH
Related in: MedlinePlus