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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 inhibiton blocks neutrophil chemotaxis.Human neutrophils were treated with vehicle (0.1% DMSO), EHT1864 (Rac1/2 inhibitor, 50 µM), wortmannin (PI3K inhibitor, 100 nM), Y27632 (ROCK inhibitor, 10 µM), or PF3758309 (PAK inhibitor, 10 µM) for 15 min and chemotaxis was induced by adding fMLP (10 nM) in the outer well of an Insall chamber. Time-lapse images were obtained every 10 s for 20 min at 40× magnification using DIC microscopy. (A) The migration paths of individual cells were quantified as spider plots using MtrackJ (Image J) and MATLAB. (B) Average migration speeds of neutrophils treated as indicated in (A). (C) Representative time-lapse images of DMSO- or PF3758309-treated cells at the first 0, 5, 10, 15 min with cell paths (white). (D) Representative kinetics of cell migration was plotted against time. (E) A dose-dependent inhibitory effect of PF3758309 on average migration speed. Results were obtained from 3 independent experiments and data quantification was performed for at least 30 cells per treatment. * P<0.05 compared to the average migration speed of DMSO-treated cells. Scale bar = 20 µm.
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pone-0073063-g004: PAK inhibiton blocks neutrophil chemotaxis.Human neutrophils were treated with vehicle (0.1% DMSO), EHT1864 (Rac1/2 inhibitor, 50 µM), wortmannin (PI3K inhibitor, 100 nM), Y27632 (ROCK inhibitor, 10 µM), or PF3758309 (PAK inhibitor, 10 µM) for 15 min and chemotaxis was induced by adding fMLP (10 nM) in the outer well of an Insall chamber. Time-lapse images were obtained every 10 s for 20 min at 40× magnification using DIC microscopy. (A) The migration paths of individual cells were quantified as spider plots using MtrackJ (Image J) and MATLAB. (B) Average migration speeds of neutrophils treated as indicated in (A). (C) Representative time-lapse images of DMSO- or PF3758309-treated cells at the first 0, 5, 10, 15 min with cell paths (white). (D) Representative kinetics of cell migration was plotted against time. (E) A dose-dependent inhibitory effect of PF3758309 on average migration speed. Results were obtained from 3 independent experiments and data quantification was performed for at least 30 cells per treatment. * P<0.05 compared to the average migration speed of DMSO-treated cells. Scale bar = 20 µm.

Mentions: The above described PAK-mediated regulation of actomyosin contractility and surface adhesion led us to further examine the role of PAK in neutrophil chemotaxis. The determining components of efficient chemotaxis include directional sensing, morphological polarization and motility. Our data show that upon exposure to an fMLP gradient, neutrophils first uniformly spread and then immediately developed morphological polarity, in which the leading edge formed distinct lamellipodia to differentiate from the uropod (Fig. 4C and Movie S1). Within 20 min, neutrophils migrated toward the fMLP gradient at the average speed of 7.2±0.5 µm/min (Fig. 4A and 4B).


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 inhibiton blocks neutrophil chemotaxis.Human neutrophils were treated with vehicle (0.1% DMSO), EHT1864 (Rac1/2 inhibitor, 50 µM), wortmannin (PI3K inhibitor, 100 nM), Y27632 (ROCK inhibitor, 10 µM), or PF3758309 (PAK inhibitor, 10 µM) for 15 min and chemotaxis was induced by adding fMLP (10 nM) in the outer well of an Insall chamber. Time-lapse images were obtained every 10 s for 20 min at 40× magnification using DIC microscopy. (A) The migration paths of individual cells were quantified as spider plots using MtrackJ (Image J) and MATLAB. (B) Average migration speeds of neutrophils treated as indicated in (A). (C) Representative time-lapse images of DMSO- or PF3758309-treated cells at the first 0, 5, 10, 15 min with cell paths (white). (D) Representative kinetics of cell migration was plotted against time. (E) A dose-dependent inhibitory effect of PF3758309 on average migration speed. Results were obtained from 3 independent experiments and data quantification was performed for at least 30 cells per treatment. * P<0.05 compared to the average migration speed of DMSO-treated cells. Scale bar = 20 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760889&req=5

pone-0073063-g004: PAK inhibiton blocks neutrophil chemotaxis.Human neutrophils were treated with vehicle (0.1% DMSO), EHT1864 (Rac1/2 inhibitor, 50 µM), wortmannin (PI3K inhibitor, 100 nM), Y27632 (ROCK inhibitor, 10 µM), or PF3758309 (PAK inhibitor, 10 µM) for 15 min and chemotaxis was induced by adding fMLP (10 nM) in the outer well of an Insall chamber. Time-lapse images were obtained every 10 s for 20 min at 40× magnification using DIC microscopy. (A) The migration paths of individual cells were quantified as spider plots using MtrackJ (Image J) and MATLAB. (B) Average migration speeds of neutrophils treated as indicated in (A). (C) Representative time-lapse images of DMSO- or PF3758309-treated cells at the first 0, 5, 10, 15 min with cell paths (white). (D) Representative kinetics of cell migration was plotted against time. (E) A dose-dependent inhibitory effect of PF3758309 on average migration speed. Results were obtained from 3 independent experiments and data quantification was performed for at least 30 cells per treatment. * P<0.05 compared to the average migration speed of DMSO-treated cells. Scale bar = 20 µm.
Mentions: The above described PAK-mediated regulation of actomyosin contractility and surface adhesion led us to further examine the role of PAK in neutrophil chemotaxis. The determining components of efficient chemotaxis include directional sensing, morphological polarization and motility. Our data show that upon exposure to an fMLP gradient, neutrophils first uniformly spread and then immediately developed morphological polarity, in which the leading edge formed distinct lamellipodia to differentiate from the uropod (Fig. 4C and Movie S1). Within 20 min, neutrophils migrated toward the fMLP gradient at the average speed of 7.2±0.5 µm/min (Fig. 4A and 4B).

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