Limits...
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.

Show MeSH

Related in: MedlinePlus

Rictor depletion impairs actin polymerization in dHL-60 cells. (A) Quantification of F-actin levels in suspended cells. Cells with or without Rictor depletion were stimulated with fMLP (100 nM) for various times in suspension and fixed for staining with fluorescently labeled phalloidin. Fluorescence in stained cells was determined by flow cytometry. Values are mean ± SEM (n = 4). (B) F-actin staining of dHL-60 cells with or without Rictor or mTOR depletion after fMLP stimulation. Cells were plated on fibrinogen-coated coverslips for 20 min and stimulated with uniform fMLP (100 nM) for 2 min. Images of F-actin and DIC. Bar, 10 μm. (C) Quantification of the number of dHL-60 cells with polarized actin polymerization with and without Rictor depletion. Cells were stimulated with uniform fMLP (100 nM) for 2 min, as described in B. Each bar represents the mean ± SEM (n = 4). (D) The dynamics of actin-YFP in dHL-60 cells exposed to an fMLP gradient. HL-60 cells stably expressing actin-YFP were infected with lentivirus containing NT shRNA or Rictor shRNA (shRNA-1) and subsequently differentiated for 5 d. Cells were plated on fibrinogen-coated coverslips and stimulated with a chemotactic gradient delivered by a micropipette containing 10 μM fMLP for the times indicated. Fluorescence images of actin-YFP and the corresponding DIC images. Bar, 10 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3814157&req=5

Figure 4: Rictor depletion impairs actin polymerization in dHL-60 cells. (A) Quantification of F-actin levels in suspended cells. Cells with or without Rictor depletion were stimulated with fMLP (100 nM) for various times in suspension and fixed for staining with fluorescently labeled phalloidin. Fluorescence in stained cells was determined by flow cytometry. Values are mean ± SEM (n = 4). (B) F-actin staining of dHL-60 cells with or without Rictor or mTOR depletion after fMLP stimulation. Cells were plated on fibrinogen-coated coverslips for 20 min and stimulated with uniform fMLP (100 nM) for 2 min. Images of F-actin and DIC. Bar, 10 μm. (C) Quantification of the number of dHL-60 cells with polarized actin polymerization with and without Rictor depletion. Cells were stimulated with uniform fMLP (100 nM) for 2 min, as described in B. Each bar represents the mean ± SEM (n = 4). (D) The dynamics of actin-YFP in dHL-60 cells exposed to an fMLP gradient. HL-60 cells stably expressing actin-YFP were infected with lentivirus containing NT shRNA or Rictor shRNA (shRNA-1) and subsequently differentiated for 5 d. Cells were plated on fibrinogen-coated coverslips and stimulated with a chemotactic gradient delivered by a micropipette containing 10 μM fMLP for the times indicated. Fluorescence images of actin-YFP and the corresponding DIC images. Bar, 10 μm.

Mentions: We measured the level of F-actin in both suspended and adherent cells with or without Rictor depletion (Figure 4). As described previously (Wang et al., 2002; Weiner et al., 2006; Shin et al., 2010), exposure of suspended control dHL-60 cells (i.e., containing NT shRNAs) induced rapid and transient actin polymerization, which peaked at around 1 min (Figure 4A). Rictor depletion moderately reduced the level of F-actin in cells under resting conditions (by ∼20%) and substantially abolished the transient accumulation of F-actin in suspended cells after exposure to chemoattractant (Figure 4A). Similarly, mTOR depletion also substantially decreased the level of actin polymerization in suspended dHL-60 cells (Supplemental Figure S5A). In addition, when plated on the fibrinogen substrate and exposed to a uniform concentration of fMLP, control cells developed a polarized morphology with F-actin concentrated at the leading edge, as revealed by phalloidin staining (Figure 4B, top). Rictor depletion in the adherent cells nearly completely prevented polarized morphology and F-actin accumulation (Figure 4B). Quantitative analysis revealed that only 12% of Rictor-depleted cells showed asymmetric accumulation of F-actin at the leading edge, in sharp contrast to control cells (78%; Figure 4C). Similar to Rictor depletion, mTOR depletion markedly impaired F-actin accumulation in adherent cells, in keeping with its effect on cell polarization and migration (Figure 4B).


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 actin polymerization in dHL-60 cells. (A) Quantification of F-actin levels in suspended cells. Cells with or without Rictor depletion were stimulated with fMLP (100 nM) for various times in suspension and fixed for staining with fluorescently labeled phalloidin. Fluorescence in stained cells was determined by flow cytometry. Values are mean ± SEM (n = 4). (B) F-actin staining of dHL-60 cells with or without Rictor or mTOR depletion after fMLP stimulation. Cells were plated on fibrinogen-coated coverslips for 20 min and stimulated with uniform fMLP (100 nM) for 2 min. Images of F-actin and DIC. Bar, 10 μm. (C) Quantification of the number of dHL-60 cells with polarized actin polymerization with and without Rictor depletion. Cells were stimulated with uniform fMLP (100 nM) for 2 min, as described in B. Each bar represents the mean ± SEM (n = 4). (D) The dynamics of actin-YFP in dHL-60 cells exposed to an fMLP gradient. HL-60 cells stably expressing actin-YFP were infected with lentivirus containing NT shRNA or Rictor shRNA (shRNA-1) and subsequently differentiated for 5 d. Cells were plated on fibrinogen-coated coverslips and stimulated with a chemotactic gradient delivered by a micropipette containing 10 μM fMLP for the times indicated. Fluorescence images of actin-YFP and the corresponding DIC images. Bar, 10 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814157&req=5

Figure 4: Rictor depletion impairs actin polymerization in dHL-60 cells. (A) Quantification of F-actin levels in suspended cells. Cells with or without Rictor depletion were stimulated with fMLP (100 nM) for various times in suspension and fixed for staining with fluorescently labeled phalloidin. Fluorescence in stained cells was determined by flow cytometry. Values are mean ± SEM (n = 4). (B) F-actin staining of dHL-60 cells with or without Rictor or mTOR depletion after fMLP stimulation. Cells were plated on fibrinogen-coated coverslips for 20 min and stimulated with uniform fMLP (100 nM) for 2 min. Images of F-actin and DIC. Bar, 10 μm. (C) Quantification of the number of dHL-60 cells with polarized actin polymerization with and without Rictor depletion. Cells were stimulated with uniform fMLP (100 nM) for 2 min, as described in B. Each bar represents the mean ± SEM (n = 4). (D) The dynamics of actin-YFP in dHL-60 cells exposed to an fMLP gradient. HL-60 cells stably expressing actin-YFP were infected with lentivirus containing NT shRNA or Rictor shRNA (shRNA-1) and subsequently differentiated for 5 d. Cells were plated on fibrinogen-coated coverslips and stimulated with a chemotactic gradient delivered by a micropipette containing 10 μM fMLP for the times indicated. Fluorescence images of actin-YFP and the corresponding DIC images. Bar, 10 μm.
Mentions: We measured the level of F-actin in both suspended and adherent cells with or without Rictor depletion (Figure 4). As described previously (Wang et al., 2002; Weiner et al., 2006; Shin et al., 2010), exposure of suspended control dHL-60 cells (i.e., containing NT shRNAs) induced rapid and transient actin polymerization, which peaked at around 1 min (Figure 4A). Rictor depletion moderately reduced the level of F-actin in cells under resting conditions (by ∼20%) and substantially abolished the transient accumulation of F-actin in suspended cells after exposure to chemoattractant (Figure 4A). Similarly, mTOR depletion also substantially decreased the level of actin polymerization in suspended dHL-60 cells (Supplemental Figure S5A). In addition, when plated on the fibrinogen substrate and exposed to a uniform concentration of fMLP, control cells developed a polarized morphology with F-actin concentrated at the leading edge, as revealed by phalloidin staining (Figure 4B, top). Rictor depletion in the adherent cells nearly completely prevented polarized morphology and F-actin accumulation (Figure 4B). Quantitative analysis revealed that only 12% of Rictor-depleted cells showed asymmetric accumulation of F-actin at the leading edge, in sharp contrast to control cells (78%; Figure 4C). Similar to Rictor depletion, mTOR depletion markedly impaired F-actin accumulation in adherent cells, in keeping with its effect on cell polarization and migration (Figure 4B).

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