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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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Actin polymers are not responsible for Rictor regulation of Rac activity. (A) Quantification of F-actin levels in suspended cells. Cells with or without pretreatment of latrunculin B (10 μg/ml, 10 min) were stimulated with fMLP (100 nM) for various times in suspension and fixed for staining with fluorescently labeled phalloidin. (B) The levels of Rac-GTP in suspended dHL-60 cells with or without latrunculin B pretreatment (10 min; 10 μg/ml, top; 20 μg/ml, bottom) were stimulated with 100 nM of fMLP (30 s) and lysed for the pull-down assay. Levels of total Rac were used to show cell lysate input. (C) Quantification of relative levels of Rac-GTP level in suspended dHL-60 cells with or without latrunculin B pretreatment 30 s after fMLP stimulation. Each bar represents the mean ± SEM (error bars). Values are normalized to the level of Rac-GTP (= 100%) in cells without latrunculin B pretreatment (*p < 0.001). (D) The fluorescence image of Rictor (green), F-actin (red), and merged Rictor and F-actin image and DIC image of dHL-60 cells pretreated or not pretreated with latrunculin B (10 μg/ml, 10 min) and stimulated with a uniform concentration of fMLP (100 nM, 1 min). The white arrows point to the areas with Rictor cortical localization. Bar, 10 μm.
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Figure 6: Actin polymers are not responsible for Rictor regulation of Rac activity. (A) Quantification of F-actin levels in suspended cells. Cells with or without pretreatment of latrunculin B (10 μg/ml, 10 min) were stimulated with fMLP (100 nM) for various times in suspension and fixed for staining with fluorescently labeled phalloidin. (B) The levels of Rac-GTP in suspended dHL-60 cells with or without latrunculin B pretreatment (10 min; 10 μg/ml, top; 20 μg/ml, bottom) were stimulated with 100 nM of fMLP (30 s) and lysed for the pull-down assay. Levels of total Rac were used to show cell lysate input. (C) Quantification of relative levels of Rac-GTP level in suspended dHL-60 cells with or without latrunculin B pretreatment 30 s after fMLP stimulation. Each bar represents the mean ± SEM (error bars). Values are normalized to the level of Rac-GTP (= 100%) in cells without latrunculin B pretreatment (*p < 0.001). (D) The fluorescence image of Rictor (green), F-actin (red), and merged Rictor and F-actin image and DIC image of dHL-60 cells pretreated or not pretreated with latrunculin B (10 μg/ml, 10 min) and stimulated with a uniform concentration of fMLP (100 nM, 1 min). The white arrows point to the areas with Rictor cortical localization. Bar, 10 μm.

Mentions: It was demonstrated that actin polymers serve as a component of a feedback loop containing PI(3,4,5)P3 and Rac (Xu et al., 2003) in chemotaxing neutrophils and that inhibition of actin polymerization impairs chemoattractant-induced Rac activation (Van Keymeulen et al., 2006). Because Rictor depletion markedly reduced the level of actin polymers (Figure 4A), these findings raise the possibility that Rictor might regulate Rac activity via actin polymerization. To test this, we treated dHL-60 cells with latrunculin B, a widely used inhibitor of actin polymerization, and assessed the effect on fMLP-induced Rac activation (Figure 6, A–C). As shown in Figure 6, B and 6C, treatment of cells with an intermediate concentration of latrunculin B (10 μg/ml, 10-min pretreatment) slightly reduced fMLP-induced Rac activation (∼9% reduction compared with control cells), whereas a higher latrunculin B concentration (20 μg/ml) caused a stronger effect (∼26% reduction). Of note, even at the concentration of 10 μg/ml, latrunculin B had a stronger inhibitory effect on actin polymerization than Rictor depletion (Figure 6A; compare with Figure 4A). Because Rictor depletion led to >60% reduction in Rac-GTP (Figure 5, A and B), the decrease in actin polymerization alone cannot account for Rictor's effect on Rac activities. Therefore actin polymerization might contribute to but is not responsible for Rictor regulation of Rac activities in neutrophils during chemotaxis.


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)

Actin polymers are not responsible for Rictor regulation of Rac activity. (A) Quantification of F-actin levels in suspended cells. Cells with or without pretreatment of latrunculin B (10 μg/ml, 10 min) were stimulated with fMLP (100 nM) for various times in suspension and fixed for staining with fluorescently labeled phalloidin. (B) The levels of Rac-GTP in suspended dHL-60 cells with or without latrunculin B pretreatment (10 min; 10 μg/ml, top; 20 μg/ml, bottom) were stimulated with 100 nM of fMLP (30 s) and lysed for the pull-down assay. Levels of total Rac were used to show cell lysate input. (C) Quantification of relative levels of Rac-GTP level in suspended dHL-60 cells with or without latrunculin B pretreatment 30 s after fMLP stimulation. Each bar represents the mean ± SEM (error bars). Values are normalized to the level of Rac-GTP (= 100%) in cells without latrunculin B pretreatment (*p < 0.001). (D) The fluorescence image of Rictor (green), F-actin (red), and merged Rictor and F-actin image and DIC image of dHL-60 cells pretreated or not pretreated with latrunculin B (10 μg/ml, 10 min) and stimulated with a uniform concentration of fMLP (100 nM, 1 min). The white arrows point to the areas with Rictor cortical localization. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 6: Actin polymers are not responsible for Rictor regulation of Rac activity. (A) Quantification of F-actin levels in suspended cells. Cells with or without pretreatment of latrunculin B (10 μg/ml, 10 min) were stimulated with fMLP (100 nM) for various times in suspension and fixed for staining with fluorescently labeled phalloidin. (B) The levels of Rac-GTP in suspended dHL-60 cells with or without latrunculin B pretreatment (10 min; 10 μg/ml, top; 20 μg/ml, bottom) were stimulated with 100 nM of fMLP (30 s) and lysed for the pull-down assay. Levels of total Rac were used to show cell lysate input. (C) Quantification of relative levels of Rac-GTP level in suspended dHL-60 cells with or without latrunculin B pretreatment 30 s after fMLP stimulation. Each bar represents the mean ± SEM (error bars). Values are normalized to the level of Rac-GTP (= 100%) in cells without latrunculin B pretreatment (*p < 0.001). (D) The fluorescence image of Rictor (green), F-actin (red), and merged Rictor and F-actin image and DIC image of dHL-60 cells pretreated or not pretreated with latrunculin B (10 μg/ml, 10 min) and stimulated with a uniform concentration of fMLP (100 nM, 1 min). The white arrows point to the areas with Rictor cortical localization. Bar, 10 μm.
Mentions: It was demonstrated that actin polymers serve as a component of a feedback loop containing PI(3,4,5)P3 and Rac (Xu et al., 2003) in chemotaxing neutrophils and that inhibition of actin polymerization impairs chemoattractant-induced Rac activation (Van Keymeulen et al., 2006). Because Rictor depletion markedly reduced the level of actin polymers (Figure 4A), these findings raise the possibility that Rictor might regulate Rac activity via actin polymerization. To test this, we treated dHL-60 cells with latrunculin B, a widely used inhibitor of actin polymerization, and assessed the effect on fMLP-induced Rac activation (Figure 6, A–C). As shown in Figure 6, B and 6C, treatment of cells with an intermediate concentration of latrunculin B (10 μg/ml, 10-min pretreatment) slightly reduced fMLP-induced Rac activation (∼9% reduction compared with control cells), whereas a higher latrunculin B concentration (20 μg/ml) caused a stronger effect (∼26% reduction). Of note, even at the concentration of 10 μg/ml, latrunculin B had a stronger inhibitory effect on actin polymerization than Rictor depletion (Figure 6A; compare with Figure 4A). Because Rictor depletion led to >60% reduction in Rac-GTP (Figure 5, A and B), the decrease in actin polymerization alone cannot account for Rictor's effect on Rac activities. Therefore actin polymerization might contribute to but is not responsible for Rictor regulation of Rac activities in neutrophils during chemotaxis.

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