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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 enhances vinculin-mediated surface contacts.Human neutrophils adherent on fibronectin surfaces were treated in the presence or absence of fMLP (10 nM) for 3 min. In selected experiments, neutrophils were pretreated with PF3758309 (PF; PAK inhibitor, 10 µM), EHT1864 (Rac1/2 inhibitor, 50 µM) or wortmannin (PI3K inhibitor, 100 nM). (A) Cells were stained for vinculin (green) and F-actin (red). Arrows indicate filopodia-like vinculin clusters shown in the insets. (B) Pearson’s coefficient for vinculin and actin immunofluorescence in cells treated as indicated in (A). (C) Neutrophil surface vinculin was visualized using TIRF microscopy. Arbitrary units (a.u.) for vinculin signal intensity are shown. (D) The mean TIRF signals of vinculin immunofluorescence were quantified from at least 5 cells per treatment. * P<0.05 compared to the basal vinculin signal in DMSO-treated cells. Scale ba = (A) 10 µm; (C) 2 µm.
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pone-0073063-g003: PAK inhibition enhances vinculin-mediated surface contacts.Human neutrophils adherent on fibronectin surfaces were treated in the presence or absence of fMLP (10 nM) for 3 min. In selected experiments, neutrophils were pretreated with PF3758309 (PF; PAK inhibitor, 10 µM), EHT1864 (Rac1/2 inhibitor, 50 µM) or wortmannin (PI3K inhibitor, 100 nM). (A) Cells were stained for vinculin (green) and F-actin (red). Arrows indicate filopodia-like vinculin clusters shown in the insets. (B) Pearson’s coefficient for vinculin and actin immunofluorescence in cells treated as indicated in (A). (C) Neutrophil surface vinculin was visualized using TIRF microscopy. Arbitrary units (a.u.) for vinculin signal intensity are shown. (D) The mean TIRF signals of vinculin immunofluorescence were quantified from at least 5 cells per treatment. * P<0.05 compared to the basal vinculin signal in DMSO-treated cells. Scale ba = (A) 10 µm; (C) 2 µm.

Mentions: To stabilize the lamellipodium, small adhesion structures, known as focal complexes, are formed at the leading edge via Rac/Cdc42 activity which link integrins to surface and cytoskeletal proteins [37]. To determine the contribution of PAK activity to focal complex assembly in neutrophils, we examined the localization of vinculin, a marker of focal complex assembly, under control and pharmacologically-inhibited conditions. As seen in Fig. 3A and B, vinculin localized to the cytosol of unstimulated neutrophils and accumulated at the actin-rich leading edge as well as the uropod after fMLP stimulation, indicating the presence of vinculin-containing cell adhesions. Neutrophils treated with the Rac1/2 inhibitor EHT1864 displayed a similar pattern of vinculin localization as compared to control cells, whereas treatment with the PI3K inhibitor wortmannin led to the less polarized localization of vinculin immunofluorescence (Fig. 3A and 3B). Interestingly, PAK-inhibited neutrophils formed vinculin-positive, filopodia-like structures at the cell periphery in response to fMLP (Fig. 3A). Similar vinculin clusters have been found in neutrophils treated with TNF-α [38], which confers inhibitory signals to neutrophil motility by promoting firm adhesion and limiting polarization. Accordingly, we next aimed to examine whether PAK inhibition enhanced neutrophil adhesion strength by increasing surface contacts. Neutrophil adhesion complexes were studied by total internal reflection fluorescence (TIRF) microscopy, by which immunofluorescence signals adjacent (∼150 nm) to the interface between a surface and a specimen are exclusively detected with a high spatial resolution as an indicator of surface contact area. As seen in Fig. 3C and 3D, TIRF microscopy for vinculin immunofluorescence revealed that PF3758309-treatment dramatically enhanced the level of vinculin-mediated surface contact as compared to vehicle, both in the presence and absence of fMLP. Together, these results demonstrate that PAK may negatively regulate surface adhesion during neutrophil polarization.


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 enhances vinculin-mediated surface contacts.Human neutrophils adherent on fibronectin surfaces were treated in the presence or absence of fMLP (10 nM) for 3 min. In selected experiments, neutrophils were pretreated with PF3758309 (PF; PAK inhibitor, 10 µM), EHT1864 (Rac1/2 inhibitor, 50 µM) or wortmannin (PI3K inhibitor, 100 nM). (A) Cells were stained for vinculin (green) and F-actin (red). Arrows indicate filopodia-like vinculin clusters shown in the insets. (B) Pearson’s coefficient for vinculin and actin immunofluorescence in cells treated as indicated in (A). (C) Neutrophil surface vinculin was visualized using TIRF microscopy. Arbitrary units (a.u.) for vinculin signal intensity are shown. (D) The mean TIRF signals of vinculin immunofluorescence were quantified from at least 5 cells per treatment. * P<0.05 compared to the basal vinculin signal in DMSO-treated cells. Scale ba = (A) 10 µm; (C) 2 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760889&req=5

pone-0073063-g003: PAK inhibition enhances vinculin-mediated surface contacts.Human neutrophils adherent on fibronectin surfaces were treated in the presence or absence of fMLP (10 nM) for 3 min. In selected experiments, neutrophils were pretreated with PF3758309 (PF; PAK inhibitor, 10 µM), EHT1864 (Rac1/2 inhibitor, 50 µM) or wortmannin (PI3K inhibitor, 100 nM). (A) Cells were stained for vinculin (green) and F-actin (red). Arrows indicate filopodia-like vinculin clusters shown in the insets. (B) Pearson’s coefficient for vinculin and actin immunofluorescence in cells treated as indicated in (A). (C) Neutrophil surface vinculin was visualized using TIRF microscopy. Arbitrary units (a.u.) for vinculin signal intensity are shown. (D) The mean TIRF signals of vinculin immunofluorescence were quantified from at least 5 cells per treatment. * P<0.05 compared to the basal vinculin signal in DMSO-treated cells. Scale ba = (A) 10 µm; (C) 2 µm.
Mentions: To stabilize the lamellipodium, small adhesion structures, known as focal complexes, are formed at the leading edge via Rac/Cdc42 activity which link integrins to surface and cytoskeletal proteins [37]. To determine the contribution of PAK activity to focal complex assembly in neutrophils, we examined the localization of vinculin, a marker of focal complex assembly, under control and pharmacologically-inhibited conditions. As seen in Fig. 3A and B, vinculin localized to the cytosol of unstimulated neutrophils and accumulated at the actin-rich leading edge as well as the uropod after fMLP stimulation, indicating the presence of vinculin-containing cell adhesions. Neutrophils treated with the Rac1/2 inhibitor EHT1864 displayed a similar pattern of vinculin localization as compared to control cells, whereas treatment with the PI3K inhibitor wortmannin led to the less polarized localization of vinculin immunofluorescence (Fig. 3A and 3B). Interestingly, PAK-inhibited neutrophils formed vinculin-positive, filopodia-like structures at the cell periphery in response to fMLP (Fig. 3A). Similar vinculin clusters have been found in neutrophils treated with TNF-α [38], which confers inhibitory signals to neutrophil motility by promoting firm adhesion and limiting polarization. Accordingly, we next aimed to examine whether PAK inhibition enhanced neutrophil adhesion strength by increasing surface contacts. Neutrophil adhesion complexes were studied by total internal reflection fluorescence (TIRF) microscopy, by which immunofluorescence signals adjacent (∼150 nm) to the interface between a surface and a specimen are exclusively detected with a high spatial resolution as an indicator of surface contact area. As seen in Fig. 3C and 3D, TIRF microscopy for vinculin immunofluorescence revealed that PF3758309-treatment dramatically enhanced the level of vinculin-mediated surface contact as compared to vehicle, both in the presence and absence of fMLP. Together, these results demonstrate that PAK may negatively regulate surface adhesion during neutrophil polarization.

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