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Mammalian target of rapamycin and Rictor control neutrophil chemotaxis by regulating Rac/Cdc42 activity and the actin cytoskeleton.

He Y, Li D, Cook SL, Yoon MS, Kapoor A, Rao CV, Kenis PJ, Chen J, Wang F - Mol. Biol. Cell (2013)

Bottom Line: By using neutrophil-like HL-60 cells, we describe a pivotal role for Rictor, a component of mammalian target of rapamycin complex 2 (mTORC2), in regulating assembly of the actin cytoskeleton during neutrophil chemotaxis.In addition, experiments with chemical inhibition and kinase-dead mutants indicate that mTOR kinase activity and AKT phosphorylation are dispensable for chemotaxis.Instead, our results suggest that the small Rho GTPases Rac and Cdc42 serve as downstream effectors of Rictor to regulate actin assembly and organization in neutrophils.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801 Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801 Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801.

ABSTRACT
Chemotaxis allows neutrophils to seek out sites of infection and inflammation. The asymmetric accumulation of filamentous actin (F-actin) at the leading edge provides the driving force for protrusion and is essential for the development and maintenance of neutrophil polarity. The mechanism that governs actin cytoskeleton dynamics and assembly in neutrophils has been extensively explored and is still not fully understood. By using neutrophil-like HL-60 cells, we describe a pivotal role for Rictor, a component of mammalian target of rapamycin complex 2 (mTORC2), in regulating assembly of the actin cytoskeleton during neutrophil chemotaxis. Depletion of mTOR and Rictor, but not Raptor, impairs actin polymerization, leading-edge establishment, and directional migration in neutrophils stimulated with chemoattractants. Of interest, depletion of mSin1, an integral component of mTORC2, causes no detectable defects in neutrophil polarity and chemotaxis. In addition, experiments with chemical inhibition and kinase-dead mutants indicate that mTOR kinase activity and AKT phosphorylation are dispensable for chemotaxis. Instead, our results suggest that the small Rho GTPases Rac and Cdc42 serve as downstream effectors of Rictor to regulate actin assembly and organization in neutrophils. Together our findings reveal an mTORC2- and mTOR kinase-independent function and mechanism of Rictor in the regulation of neutrophil chemotaxis.

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mTOR kinase activity is dispensable for chemotaxis. (A) Western blotting of AKT (p-S473) and S6K1 (p-T389) phosphorylation in cells pretreated with vehicle (DMSO), rapamycin (100 nM), or Torin1 (250 nM) for 30 min and stimulated with fMLP (100 nM) for indicated time points. AKT is the loading control. (B) Chemotaxis of dHL-60 cells with or without Torin1 treatment (250 nM, 30 min) in a microfluidic gradient device. Phase-contrast images of cells 10 and 480 s after fMLP stimulation. Bar, 50 μm. (C) Trajectory of dHL-60 cells migrating in the microfluidic chamber. Cell migration was recorded using time-lapse microscope, and the migration path of individual cells was analyzed and plotted using ImageJ (National Institutes of Health, Bethesda, MD). (D) Western blotting of mTOR in control and mTOR-depleted dHL-60 cells with or without rescue. mTOR-depleted cells were differentiated and transfected with WT mTOR or a kinase-dead mutant of mTOR. mTOR was depleted with shRNA-3, which targets the 3′-UTR of mTOR and thus exerts no effect on ectopically expressed mTOR. GAPDH is the loading control. (E) Both wild-type mTOR and the kinase-dead mTOR mutant rescue the migratory defects of mTOR-depleted cells revealed with the micropipette assay. Time-lapse images of representative cells for various conditions. The two images in each column show the positions of individual cells (identified with a superimposed letter) after exposure to fMLP. Bar, 10 μm. (F) Speeds of cell migration for control, mTOR-depleted, and rescued cells revealed with the micropipette assay. Values are means ± SEM (n = 18 for control, 19 for mTOR shRNA alone, 17 for mTOR shRNA plus wild-type mTOR, and 15 for mTOR shRNA plus mTOR kinase-dead mutant). Asterisks indicate that the cells differ statistically from the mTOR-depleted cells (*p < 0.001).
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Figure 3: mTOR kinase activity is dispensable for chemotaxis. (A) Western blotting of AKT (p-S473) and S6K1 (p-T389) phosphorylation in cells pretreated with vehicle (DMSO), rapamycin (100 nM), or Torin1 (250 nM) for 30 min and stimulated with fMLP (100 nM) for indicated time points. AKT is the loading control. (B) Chemotaxis of dHL-60 cells with or without Torin1 treatment (250 nM, 30 min) in a microfluidic gradient device. Phase-contrast images of cells 10 and 480 s after fMLP stimulation. Bar, 50 μm. (C) Trajectory of dHL-60 cells migrating in the microfluidic chamber. Cell migration was recorded using time-lapse microscope, and the migration path of individual cells was analyzed and plotted using ImageJ (National Institutes of Health, Bethesda, MD). (D) Western blotting of mTOR in control and mTOR-depleted dHL-60 cells with or without rescue. mTOR-depleted cells were differentiated and transfected with WT mTOR or a kinase-dead mutant of mTOR. mTOR was depleted with shRNA-3, which targets the 3′-UTR of mTOR and thus exerts no effect on ectopically expressed mTOR. GAPDH is the loading control. (E) Both wild-type mTOR and the kinase-dead mTOR mutant rescue the migratory defects of mTOR-depleted cells revealed with the micropipette assay. Time-lapse images of representative cells for various conditions. The two images in each column show the positions of individual cells (identified with a superimposed letter) after exposure to fMLP. Bar, 10 μm. (F) Speeds of cell migration for control, mTOR-depleted, and rescued cells revealed with the micropipette assay. Values are means ± SEM (n = 18 for control, 19 for mTOR shRNA alone, 17 for mTOR shRNA plus wild-type mTOR, and 15 for mTOR shRNA plus mTOR kinase-dead mutant). Asterisks indicate that the cells differ statistically from the mTOR-depleted cells (*p < 0.001).

Mentions: We found that Torin1 treatment markedly reduced the levels of both phosphorylated S6K1 (at threonine 389) and AKT (by 84 and 88%, respectively; Figure 3A and Supplemental Figure S4, A and B). By contrast, rapamycin treatment reduced S6K1 phosphorylation by 79% but had little effect on AKT phosphorylation (Figure 3A and Supplemental Figure S4, A and B). Despite its ability to inhibit S6K1 and AKT phosphorylation, Torin1 treatment had no detectable effects on chemotaxis of dHL-60 cells and primary neutrophils (Figure 3, B and C, Table 1, and data not shown).


Mammalian target of rapamycin and Rictor control neutrophil chemotaxis by regulating Rac/Cdc42 activity and the actin cytoskeleton.

He Y, Li D, Cook SL, Yoon MS, Kapoor A, Rao CV, Kenis PJ, Chen J, Wang F - Mol. Biol. Cell (2013)

mTOR kinase activity is dispensable for chemotaxis. (A) Western blotting of AKT (p-S473) and S6K1 (p-T389) phosphorylation in cells pretreated with vehicle (DMSO), rapamycin (100 nM), or Torin1 (250 nM) for 30 min and stimulated with fMLP (100 nM) for indicated time points. AKT is the loading control. (B) Chemotaxis of dHL-60 cells with or without Torin1 treatment (250 nM, 30 min) in a microfluidic gradient device. Phase-contrast images of cells 10 and 480 s after fMLP stimulation. Bar, 50 μm. (C) Trajectory of dHL-60 cells migrating in the microfluidic chamber. Cell migration was recorded using time-lapse microscope, and the migration path of individual cells was analyzed and plotted using ImageJ (National Institutes of Health, Bethesda, MD). (D) Western blotting of mTOR in control and mTOR-depleted dHL-60 cells with or without rescue. mTOR-depleted cells were differentiated and transfected with WT mTOR or a kinase-dead mutant of mTOR. mTOR was depleted with shRNA-3, which targets the 3′-UTR of mTOR and thus exerts no effect on ectopically expressed mTOR. GAPDH is the loading control. (E) Both wild-type mTOR and the kinase-dead mTOR mutant rescue the migratory defects of mTOR-depleted cells revealed with the micropipette assay. Time-lapse images of representative cells for various conditions. The two images in each column show the positions of individual cells (identified with a superimposed letter) after exposure to fMLP. Bar, 10 μm. (F) Speeds of cell migration for control, mTOR-depleted, and rescued cells revealed with the micropipette assay. Values are means ± SEM (n = 18 for control, 19 for mTOR shRNA alone, 17 for mTOR shRNA plus wild-type mTOR, and 15 for mTOR shRNA plus mTOR kinase-dead mutant). Asterisks indicate that the cells differ statistically from the mTOR-depleted cells (*p < 0.001).
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Related In: Results  -  Collection

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Figure 3: mTOR kinase activity is dispensable for chemotaxis. (A) Western blotting of AKT (p-S473) and S6K1 (p-T389) phosphorylation in cells pretreated with vehicle (DMSO), rapamycin (100 nM), or Torin1 (250 nM) for 30 min and stimulated with fMLP (100 nM) for indicated time points. AKT is the loading control. (B) Chemotaxis of dHL-60 cells with or without Torin1 treatment (250 nM, 30 min) in a microfluidic gradient device. Phase-contrast images of cells 10 and 480 s after fMLP stimulation. Bar, 50 μm. (C) Trajectory of dHL-60 cells migrating in the microfluidic chamber. Cell migration was recorded using time-lapse microscope, and the migration path of individual cells was analyzed and plotted using ImageJ (National Institutes of Health, Bethesda, MD). (D) Western blotting of mTOR in control and mTOR-depleted dHL-60 cells with or without rescue. mTOR-depleted cells were differentiated and transfected with WT mTOR or a kinase-dead mutant of mTOR. mTOR was depleted with shRNA-3, which targets the 3′-UTR of mTOR and thus exerts no effect on ectopically expressed mTOR. GAPDH is the loading control. (E) Both wild-type mTOR and the kinase-dead mTOR mutant rescue the migratory defects of mTOR-depleted cells revealed with the micropipette assay. Time-lapse images of representative cells for various conditions. The two images in each column show the positions of individual cells (identified with a superimposed letter) after exposure to fMLP. Bar, 10 μm. (F) Speeds of cell migration for control, mTOR-depleted, and rescued cells revealed with the micropipette assay. Values are means ± SEM (n = 18 for control, 19 for mTOR shRNA alone, 17 for mTOR shRNA plus wild-type mTOR, and 15 for mTOR shRNA plus mTOR kinase-dead mutant). Asterisks indicate that the cells differ statistically from the mTOR-depleted cells (*p < 0.001).
Mentions: We found that Torin1 treatment markedly reduced the levels of both phosphorylated S6K1 (at threonine 389) and AKT (by 84 and 88%, respectively; Figure 3A and Supplemental Figure S4, A and B). By contrast, rapamycin treatment reduced S6K1 phosphorylation by 79% but had little effect on AKT phosphorylation (Figure 3A and Supplemental Figure S4, A and B). Despite its ability to inhibit S6K1 and AKT phosphorylation, Torin1 treatment had no detectable effects on chemotaxis of dHL-60 cells and primary neutrophils (Figure 3, B and C, Table 1, and data not shown).

Bottom Line: By using neutrophil-like HL-60 cells, we describe a pivotal role for Rictor, a component of mammalian target of rapamycin complex 2 (mTORC2), in regulating assembly of the actin cytoskeleton during neutrophil chemotaxis.In addition, experiments with chemical inhibition and kinase-dead mutants indicate that mTOR kinase activity and AKT phosphorylation are dispensable for chemotaxis.Instead, our results suggest that the small Rho GTPases Rac and Cdc42 serve as downstream effectors of Rictor to regulate actin assembly and organization in neutrophils.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801 Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801 Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801.

ABSTRACT
Chemotaxis allows neutrophils to seek out sites of infection and inflammation. The asymmetric accumulation of filamentous actin (F-actin) at the leading edge provides the driving force for protrusion and is essential for the development and maintenance of neutrophil polarity. The mechanism that governs actin cytoskeleton dynamics and assembly in neutrophils has been extensively explored and is still not fully understood. By using neutrophil-like HL-60 cells, we describe a pivotal role for Rictor, a component of mammalian target of rapamycin complex 2 (mTORC2), in regulating assembly of the actin cytoskeleton during neutrophil chemotaxis. Depletion of mTOR and Rictor, but not Raptor, impairs actin polymerization, leading-edge establishment, and directional migration in neutrophils stimulated with chemoattractants. Of interest, depletion of mSin1, an integral component of mTORC2, causes no detectable defects in neutrophil polarity and chemotaxis. In addition, experiments with chemical inhibition and kinase-dead mutants indicate that mTOR kinase activity and AKT phosphorylation are dispensable for chemotaxis. Instead, our results suggest that the small Rho GTPases Rac and Cdc42 serve as downstream effectors of Rictor to regulate actin assembly and organization in neutrophils. Together our findings reveal an mTORC2- and mTOR kinase-independent function and mechanism of Rictor in the regulation of neutrophil chemotaxis.

Show MeSH
Related in: MedlinePlus