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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 alters neutrophil calcium signaling.Human neutrophils were loaded with the intracellular Ca2+ dye fluo-4 (2 µM) and plated on fibronectin-coated surfaces. After treatment with DMSO or PF3758309 (10 µM), neutrophil chemotaxis was induced by the addition of fMLP in an Insall chamber. Intracellular Ca2+ release was monitored every 5 s for 15 min by fluorescent microscopy. (A) Time-lapse images after the peak Ca2+ spike were shown. (B) Ca2+ spikes in cells treated with 0.1% DMSO (black line) or PF3758309 (PF; PAK inhibitor, dotted line) were quantified and presented as mean intensity of at least 5 neutrophils in a field of view. Data shown is representative from 3 independent experiments. Scale ba = 20 µm.
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pone-0073063-g005: PAK inhibition alters neutrophil calcium signaling.Human neutrophils were loaded with the intracellular Ca2+ dye fluo-4 (2 µM) and plated on fibronectin-coated surfaces. After treatment with DMSO or PF3758309 (10 µM), neutrophil chemotaxis was induced by the addition of fMLP in an Insall chamber. Intracellular Ca2+ release was monitored every 5 s for 15 min by fluorescent microscopy. (A) Time-lapse images after the peak Ca2+ spike were shown. (B) Ca2+ spikes in cells treated with 0.1% DMSO (black line) or PF3758309 (PF; PAK inhibitor, dotted line) were quantified and presented as mean intensity of at least 5 neutrophils in a field of view. Data shown is representative from 3 independent experiments. Scale ba = 20 µm.

Mentions: fMLP receptor signaling induces intracellular Ca2+ transients via the activation of the Gβγ complex and phospholipase C, triggering Ca2+-dependent downstream pathways such as protein kinase C [2], [39]. These pathways have been shown to collectively play a role in cytoskeletal dynamics in neutrophils [40]. PAK1-deficient bone marrow-derived mast cells have been shown to exhibit diminished intracellular calcium mobilization [9], [13]. However, a role for the PAK pathway in the coupling of calcium signaling during neutrophil chemotaxis has not been explored. Next, to characterize the role of PAK in intracellular Ca2+ mobilization, neutrophils were loaded with the Ca2+-sensitive reporter dye fluo-4 AM and allowed to migrate under a fMLP gradient while intracellular Ca2+ release was measured for 15 min. As shown in Fig. 5, neutrophils exhibited a single spike of Ca2+ release upon exposure to fMLP, followed by a gradually decreasing level of Ca2+ during chemotaxis. In contrast, neutrophils treated with the PAK inhibitor, PF3758309, displayed multiple peaks of Ca2+ release and failed to develop polarized morphology or migrate toward the fMLP gradient (Fig. 5). These data imply that PAK activity contributes to the regulation of intracellular Ca2+ transients during neutrophil chemotaxis.


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 alters neutrophil calcium signaling.Human neutrophils were loaded with the intracellular Ca2+ dye fluo-4 (2 µM) and plated on fibronectin-coated surfaces. After treatment with DMSO or PF3758309 (10 µM), neutrophil chemotaxis was induced by the addition of fMLP in an Insall chamber. Intracellular Ca2+ release was monitored every 5 s for 15 min by fluorescent microscopy. (A) Time-lapse images after the peak Ca2+ spike were shown. (B) Ca2+ spikes in cells treated with 0.1% DMSO (black line) or PF3758309 (PF; PAK inhibitor, dotted line) were quantified and presented as mean intensity of at least 5 neutrophils in a field of view. Data shown is representative from 3 independent experiments. Scale ba = 20 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760889&req=5

pone-0073063-g005: PAK inhibition alters neutrophil calcium signaling.Human neutrophils were loaded with the intracellular Ca2+ dye fluo-4 (2 µM) and plated on fibronectin-coated surfaces. After treatment with DMSO or PF3758309 (10 µM), neutrophil chemotaxis was induced by the addition of fMLP in an Insall chamber. Intracellular Ca2+ release was monitored every 5 s for 15 min by fluorescent microscopy. (A) Time-lapse images after the peak Ca2+ spike were shown. (B) Ca2+ spikes in cells treated with 0.1% DMSO (black line) or PF3758309 (PF; PAK inhibitor, dotted line) were quantified and presented as mean intensity of at least 5 neutrophils in a field of view. Data shown is representative from 3 independent experiments. Scale ba = 20 µm.
Mentions: fMLP receptor signaling induces intracellular Ca2+ transients via the activation of the Gβγ complex and phospholipase C, triggering Ca2+-dependent downstream pathways such as protein kinase C [2], [39]. These pathways have been shown to collectively play a role in cytoskeletal dynamics in neutrophils [40]. PAK1-deficient bone marrow-derived mast cells have been shown to exhibit diminished intracellular calcium mobilization [9], [13]. However, a role for the PAK pathway in the coupling of calcium signaling during neutrophil chemotaxis has not been explored. Next, to characterize the role of PAK in intracellular Ca2+ mobilization, neutrophils were loaded with the Ca2+-sensitive reporter dye fluo-4 AM and allowed to migrate under a fMLP gradient while intracellular Ca2+ release was measured for 15 min. As shown in Fig. 5, neutrophils exhibited a single spike of Ca2+ release upon exposure to fMLP, followed by a gradually decreasing level of Ca2+ during chemotaxis. In contrast, neutrophils treated with the PAK inhibitor, PF3758309, displayed multiple peaks of Ca2+ release and failed to develop polarized morphology or migrate toward the fMLP gradient (Fig. 5). These data imply that PAK activity contributes to the regulation of intracellular Ca2+ transients during neutrophil chemotaxis.

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