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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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Rictor depletion impairs Rac and Cdc42 activities in dHL-60 cells. (A) The levels of Rac-GTP in suspended dHL-60 cells. dHL-60 cells with or without Rictor depletion were stimulated with 100 nM of fMLP for various time points in suspension and lysed for the pull-down assay. Levels of total Rac were used to show cell lysate input. (B) Quantification of relative levels of Rac-GTP level in suspended dHL-60 cells with and without Rictor depletion 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 Rictor depletion (*p < 0.001). (C) The levels of Rac-GTP in adherent dHL-60 cells. Control cells or Rictor-depleted cells plated on fibrinogen-coated plates were unstimulated or stimulated for 1 or 5 min with a uniform concentration of fMLP (100 nM) and lysed for pull-down assay. Levels of total Rac were used to show cell lysate input. (D) Quantification of relative levels of Rac-GTP in adherent dHL-60 cells with and without Rictor depletion. Each bar represents the mean ± SEM (n = 4). Values are normalized to the level of Rac-GTP (= 100%) in cells without Rictor depletion before fMLP stimulation (*p < 0.001). (E) The levels of Cdc42-GTP in dHL-60 cells with or without Rictor depletion. dHL-60 cells with or without Rictor depletion were stimulated with 100 nM of fMLP for 30 s in suspension and lysed for the pull-down assay. Levels of total Cdc42 were used to show cell lysate input. (F) Quantification of relative levels of Cdc42-GTP in suspended dHL-60 cells with and without Rictor depletion. Each bar represents the mean ± SEM (n = 4). Values are normalized to the level of Cdc42-GTP (= 100%) in cells without Rictor depletion before fMLP stimulation (*p < 0.001).
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Figure 5: Rictor depletion impairs Rac and Cdc42 activities in dHL-60 cells. (A) The levels of Rac-GTP in suspended dHL-60 cells. dHL-60 cells with or without Rictor depletion were stimulated with 100 nM of fMLP for various time points in suspension and lysed for the pull-down assay. Levels of total Rac were used to show cell lysate input. (B) Quantification of relative levels of Rac-GTP level in suspended dHL-60 cells with and without Rictor depletion 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 Rictor depletion (*p < 0.001). (C) The levels of Rac-GTP in adherent dHL-60 cells. Control cells or Rictor-depleted cells plated on fibrinogen-coated plates were unstimulated or stimulated for 1 or 5 min with a uniform concentration of fMLP (100 nM) and lysed for pull-down assay. Levels of total Rac were used to show cell lysate input. (D) Quantification of relative levels of Rac-GTP in adherent dHL-60 cells with and without Rictor depletion. Each bar represents the mean ± SEM (n = 4). Values are normalized to the level of Rac-GTP (= 100%) in cells without Rictor depletion before fMLP stimulation (*p < 0.001). (E) The levels of Cdc42-GTP in dHL-60 cells with or without Rictor depletion. dHL-60 cells with or without Rictor depletion were stimulated with 100 nM of fMLP for 30 s in suspension and lysed for the pull-down assay. Levels of total Cdc42 were used to show cell lysate input. (F) Quantification of relative levels of Cdc42-GTP in suspended dHL-60 cells with and without Rictor depletion. Each bar represents the mean ± SEM (n = 4). Values are normalized to the level of Cdc42-GTP (= 100%) in cells without Rictor depletion before fMLP stimulation (*p < 0.001).

Mentions: If AKT does not mediate Rictor's function in neutrophils, how might Rictor regulate actin polymerization and organization during neutrophil chemotaxis? Rac and Cdc42 are members of the Rho GTPase family, which have well established roles in the regulation of actin dynamics in various cell types (Sanchez-Madrid and del Pozo, 1999; Etienne-Manneville and Hall, 2002). Rac2 deletion in mouse neutrophils or expression of dominant-negative Rac mutants in human neutrophil cell lines markedly prevents cell polarization and actin polymerization (Roberts et al., 1999; Williams et al., 2000; Srinivasan et al., 2003). Inhibition of Cdc42 in neutrophils with dominant-negative mutants impairs leading-edge stability and persistent motility (Srinivasan et al., 2003). We therefore asked whether Rac and/or Cdc42 might serve as downstream targets to mediate the function of Rictor in neutrophils (Figure 5).


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)

Rictor depletion impairs Rac and Cdc42 activities in dHL-60 cells. (A) The levels of Rac-GTP in suspended dHL-60 cells. dHL-60 cells with or without Rictor depletion were stimulated with 100 nM of fMLP for various time points in suspension and lysed for the pull-down assay. Levels of total Rac were used to show cell lysate input. (B) Quantification of relative levels of Rac-GTP level in suspended dHL-60 cells with and without Rictor depletion 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 Rictor depletion (*p < 0.001). (C) The levels of Rac-GTP in adherent dHL-60 cells. Control cells or Rictor-depleted cells plated on fibrinogen-coated plates were unstimulated or stimulated for 1 or 5 min with a uniform concentration of fMLP (100 nM) and lysed for pull-down assay. Levels of total Rac were used to show cell lysate input. (D) Quantification of relative levels of Rac-GTP in adherent dHL-60 cells with and without Rictor depletion. Each bar represents the mean ± SEM (n = 4). Values are normalized to the level of Rac-GTP (= 100%) in cells without Rictor depletion before fMLP stimulation (*p < 0.001). (E) The levels of Cdc42-GTP in dHL-60 cells with or without Rictor depletion. dHL-60 cells with or without Rictor depletion were stimulated with 100 nM of fMLP for 30 s in suspension and lysed for the pull-down assay. Levels of total Cdc42 were used to show cell lysate input. (F) Quantification of relative levels of Cdc42-GTP in suspended dHL-60 cells with and without Rictor depletion. Each bar represents the mean ± SEM (n = 4). Values are normalized to the level of Cdc42-GTP (= 100%) in cells without Rictor depletion before fMLP stimulation (*p < 0.001).
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Figure 5: Rictor depletion impairs Rac and Cdc42 activities in dHL-60 cells. (A) The levels of Rac-GTP in suspended dHL-60 cells. dHL-60 cells with or without Rictor depletion were stimulated with 100 nM of fMLP for various time points in suspension and lysed for the pull-down assay. Levels of total Rac were used to show cell lysate input. (B) Quantification of relative levels of Rac-GTP level in suspended dHL-60 cells with and without Rictor depletion 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 Rictor depletion (*p < 0.001). (C) The levels of Rac-GTP in adherent dHL-60 cells. Control cells or Rictor-depleted cells plated on fibrinogen-coated plates were unstimulated or stimulated for 1 or 5 min with a uniform concentration of fMLP (100 nM) and lysed for pull-down assay. Levels of total Rac were used to show cell lysate input. (D) Quantification of relative levels of Rac-GTP in adherent dHL-60 cells with and without Rictor depletion. Each bar represents the mean ± SEM (n = 4). Values are normalized to the level of Rac-GTP (= 100%) in cells without Rictor depletion before fMLP stimulation (*p < 0.001). (E) The levels of Cdc42-GTP in dHL-60 cells with or without Rictor depletion. dHL-60 cells with or without Rictor depletion were stimulated with 100 nM of fMLP for 30 s in suspension and lysed for the pull-down assay. Levels of total Cdc42 were used to show cell lysate input. (F) Quantification of relative levels of Cdc42-GTP in suspended dHL-60 cells with and without Rictor depletion. Each bar represents the mean ± SEM (n = 4). Values are normalized to the level of Cdc42-GTP (= 100%) in cells without Rictor depletion before fMLP stimulation (*p < 0.001).
Mentions: If AKT does not mediate Rictor's function in neutrophils, how might Rictor regulate actin polymerization and organization during neutrophil chemotaxis? Rac and Cdc42 are members of the Rho GTPase family, which have well established roles in the regulation of actin dynamics in various cell types (Sanchez-Madrid and del Pozo, 1999; Etienne-Manneville and Hall, 2002). Rac2 deletion in mouse neutrophils or expression of dominant-negative Rac mutants in human neutrophil cell lines markedly prevents cell polarization and actin polymerization (Roberts et al., 1999; Williams et al., 2000; Srinivasan et al., 2003). Inhibition of Cdc42 in neutrophils with dominant-negative mutants impairs leading-edge stability and persistent motility (Srinivasan et al., 2003). We therefore asked whether Rac and/or Cdc42 might serve as downstream targets to mediate the function of Rictor in neutrophils (Figure 5).

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