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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 dHL-60 chemotaxis. (A) Western blotting of mTOR, Raptor, and Rictor in dHL-60 cells transfected with specific or NT shRNAs. GAPDH was a loading control. HL-60 cells were infected with lentiviruses containing the various shRNAs and were differentiated for 5 d in the presence of DMSO. (B) Relative levels of mTOR, Raptor, and Rictor in cells with or without depletion. Values are normalized to the level in control cells (with the NT shRNA, 100%) and are means ± SEM (n = 4). (C) Chemotaxis of dHL-60 cells with various treatments in a microfluidic gradient device. After adhering to the fibrinogen-coated surface of the microfluidic chamber, cells (2 × 106) were exposed to an fMLP gradient for 20 min. Phase-contrast images of cells 10 and 910 s after fMLP stimulation. Bar, 50 μm. Supplemental Movies S1–S3 are of cells with or without mTOR and Rictor depletion. (D) Images with higher magnification of cells with NT (I) and Rictor shRNA (II) treatment, 910 s after exposure to fMLP gradients. Bar, 10 μm. (E) Western blotting of Rictor in control and Rictor-depleted dHL-60 cells with or without rescue. Rictor-depleted cells were differentiated and transfected with wild-type (WT) Rictor. α-Tubulin was a loading control. (F) Wild-type Rictor rescues the migratory defects of Rictor-depleted cells revealed with the micropipette assay. Time-lapse images of representative cells for various conditions. The three images in each column show the positions of individual cells (identified with a superimposed letter) after exposure to fMLP. Bar, 10 μm. (G) Speeds of cell migration for control, Rictor-depleted, and rescued cells revealed with the micropipette assay. Values are means ± SEM (n = 21 for control, 16 for Rictor shRNA alone, and 15 for Rictor shRNA plus wild-type Rictor). The cells with Rictor rescue differ statistically from the Rictor-depleted cells (p < 0.01).
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Figure 2: Rictor depletion impairs dHL-60 chemotaxis. (A) Western blotting of mTOR, Raptor, and Rictor in dHL-60 cells transfected with specific or NT shRNAs. GAPDH was a loading control. HL-60 cells were infected with lentiviruses containing the various shRNAs and were differentiated for 5 d in the presence of DMSO. (B) Relative levels of mTOR, Raptor, and Rictor in cells with or without depletion. Values are normalized to the level in control cells (with the NT shRNA, 100%) and are means ± SEM (n = 4). (C) Chemotaxis of dHL-60 cells with various treatments in a microfluidic gradient device. After adhering to the fibrinogen-coated surface of the microfluidic chamber, cells (2 × 106) were exposed to an fMLP gradient for 20 min. Phase-contrast images of cells 10 and 910 s after fMLP stimulation. Bar, 50 μm. Supplemental Movies S1–S3 are of cells with or without mTOR and Rictor depletion. (D) Images with higher magnification of cells with NT (I) and Rictor shRNA (II) treatment, 910 s after exposure to fMLP gradients. Bar, 10 μm. (E) Western blotting of Rictor in control and Rictor-depleted dHL-60 cells with or without rescue. Rictor-depleted cells were differentiated and transfected with wild-type (WT) Rictor. α-Tubulin was a loading control. (F) Wild-type Rictor rescues the migratory defects of Rictor-depleted cells revealed with the micropipette assay. Time-lapse images of representative cells for various conditions. The three images in each column show the positions of individual cells (identified with a superimposed letter) after exposure to fMLP. Bar, 10 μm. (G) Speeds of cell migration for control, Rictor-depleted, and rescued cells revealed with the micropipette assay. Values are means ± SEM (n = 21 for control, 16 for Rictor shRNA alone, and 15 for Rictor shRNA plus wild-type Rictor). The cells with Rictor rescue differ statistically from the Rictor-depleted cells (p < 0.01).

Mentions: Rictor's subcellular localization in polarized neutrophils suggested a potential role in controlling leading-edge protrusion and chemotaxis. To test this possibility, we used a lentivirus-based system to stably express small hairpin RNAs (shRNAs) that efficiently depleted mTOR, Rictor, and Raptor in dHL-60 cells (Figure 2, A and B). To ensure specificity, we used at least two shRNAs that deplete the same target genes. The chemotactic behaviors of mTOR-, Raptor-, or Rictor-depleted cells were examined by using a microfluidic gradient device, which enabled us to watch populations of cells moving in a highly stable gradient over a long distance (Herzmark et al., 2007; He et al., 2011). In this assay, cells containing nontarget (NT) shRNA polarized and moved rapidly in a concentration gradient of fMLP (1.4 nM/μm; Figure 2, C and D, Supplemental Figure S1B, and Supplemental Movie S1), as described earlier (He et al., 2011). Of interest, depletion of Raptor or treatment with rapamycin, a potent and specific inhibitor for mTORC1, had little effect on cell polarization and migration, suggesting that mTORC1 is dispensable for neutrophil chemotaxis (Figure 2C and Supplemental Figure S1B). By contrast, cells with mTOR or Rictor depletion exhibited severe chemotactic defects (Figure 2, C and D, Supplemental Figure S1B, and Supplemental Movies S2 and S3). These cells failed to polarize properly and instead formed small, poorly developed and unstable leading edges (Figure 2, C and D, Supplemental Figure S1B, and Supplemental Movies S2 and S3). Only 9% of Rictor-depleted cells exhibited stable and persistent polarity during migration, in sharp contrast to control cells (89%). Furthermore, Rictor-depleted cells migrated much more slowly (Figure 2, C and D, and Supplemental Figure S1B), resulting in a large fraction of cells in the lower end of the fMLP gradient (Figure 2C). In addition, the chemotactic indices, defined as the ratio of migration in the correct direction to the actual length of the migration path (Xu et al., 2005; He et al., 2011), were 0.81 and 0.63 for control and Rictor-depleted cells, respectively, suggesting defects in directional migration upon Rictor depletion (data not shown). The chemotactic behaviors of mTOR and Rictor-depleted cells were also examined by using the micropipette chemotaxis assay. In this assay, mTOR- or Rictor-depleted cells showed polarization and migration defects highly reminiscent of those in the microfluidic assay (Supplemental Figure S1C; see Supplemental Movies S4 and S5, which show the migratory behaviors of cells in gradients created by the micropipette). To further examine the specificity of Rictor depletion, we ectopically expressed wild-type Rictor in dHL-60 cells with Rictor depleted (Figure 2E). We used shRNAs targeting 3′-untranslated region (UTR) of Rictor, which specifically deplete endogenous Rictor, not the ectopic form. The endogenous gene products were depleted, whereas the exogenously expressed mRNAs devoid of the 3′-UTR were resistant. The ectopic expression of wild-type Rictor largely rescued the polarization and migratory defects caused by Rictor depletion (Figure 2, F and G, and data not shown). Taken together, these results suggest that mTOR and Rictor, but not Raptor, are required for neutrophil 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)

Rictor depletion impairs dHL-60 chemotaxis. (A) Western blotting of mTOR, Raptor, and Rictor in dHL-60 cells transfected with specific or NT shRNAs. GAPDH was a loading control. HL-60 cells were infected with lentiviruses containing the various shRNAs and were differentiated for 5 d in the presence of DMSO. (B) Relative levels of mTOR, Raptor, and Rictor in cells with or without depletion. Values are normalized to the level in control cells (with the NT shRNA, 100%) and are means ± SEM (n = 4). (C) Chemotaxis of dHL-60 cells with various treatments in a microfluidic gradient device. After adhering to the fibrinogen-coated surface of the microfluidic chamber, cells (2 × 106) were exposed to an fMLP gradient for 20 min. Phase-contrast images of cells 10 and 910 s after fMLP stimulation. Bar, 50 μm. Supplemental Movies S1–S3 are of cells with or without mTOR and Rictor depletion. (D) Images with higher magnification of cells with NT (I) and Rictor shRNA (II) treatment, 910 s after exposure to fMLP gradients. Bar, 10 μm. (E) Western blotting of Rictor in control and Rictor-depleted dHL-60 cells with or without rescue. Rictor-depleted cells were differentiated and transfected with wild-type (WT) Rictor. α-Tubulin was a loading control. (F) Wild-type Rictor rescues the migratory defects of Rictor-depleted cells revealed with the micropipette assay. Time-lapse images of representative cells for various conditions. The three images in each column show the positions of individual cells (identified with a superimposed letter) after exposure to fMLP. Bar, 10 μm. (G) Speeds of cell migration for control, Rictor-depleted, and rescued cells revealed with the micropipette assay. Values are means ± SEM (n = 21 for control, 16 for Rictor shRNA alone, and 15 for Rictor shRNA plus wild-type Rictor). The cells with Rictor rescue differ statistically from the Rictor-depleted cells (p < 0.01).
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Related In: Results  -  Collection

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Figure 2: Rictor depletion impairs dHL-60 chemotaxis. (A) Western blotting of mTOR, Raptor, and Rictor in dHL-60 cells transfected with specific or NT shRNAs. GAPDH was a loading control. HL-60 cells were infected with lentiviruses containing the various shRNAs and were differentiated for 5 d in the presence of DMSO. (B) Relative levels of mTOR, Raptor, and Rictor in cells with or without depletion. Values are normalized to the level in control cells (with the NT shRNA, 100%) and are means ± SEM (n = 4). (C) Chemotaxis of dHL-60 cells with various treatments in a microfluidic gradient device. After adhering to the fibrinogen-coated surface of the microfluidic chamber, cells (2 × 106) were exposed to an fMLP gradient for 20 min. Phase-contrast images of cells 10 and 910 s after fMLP stimulation. Bar, 50 μm. Supplemental Movies S1–S3 are of cells with or without mTOR and Rictor depletion. (D) Images with higher magnification of cells with NT (I) and Rictor shRNA (II) treatment, 910 s after exposure to fMLP gradients. Bar, 10 μm. (E) Western blotting of Rictor in control and Rictor-depleted dHL-60 cells with or without rescue. Rictor-depleted cells were differentiated and transfected with wild-type (WT) Rictor. α-Tubulin was a loading control. (F) Wild-type Rictor rescues the migratory defects of Rictor-depleted cells revealed with the micropipette assay. Time-lapse images of representative cells for various conditions. The three images in each column show the positions of individual cells (identified with a superimposed letter) after exposure to fMLP. Bar, 10 μm. (G) Speeds of cell migration for control, Rictor-depleted, and rescued cells revealed with the micropipette assay. Values are means ± SEM (n = 21 for control, 16 for Rictor shRNA alone, and 15 for Rictor shRNA plus wild-type Rictor). The cells with Rictor rescue differ statistically from the Rictor-depleted cells (p < 0.01).
Mentions: Rictor's subcellular localization in polarized neutrophils suggested a potential role in controlling leading-edge protrusion and chemotaxis. To test this possibility, we used a lentivirus-based system to stably express small hairpin RNAs (shRNAs) that efficiently depleted mTOR, Rictor, and Raptor in dHL-60 cells (Figure 2, A and B). To ensure specificity, we used at least two shRNAs that deplete the same target genes. The chemotactic behaviors of mTOR-, Raptor-, or Rictor-depleted cells were examined by using a microfluidic gradient device, which enabled us to watch populations of cells moving in a highly stable gradient over a long distance (Herzmark et al., 2007; He et al., 2011). In this assay, cells containing nontarget (NT) shRNA polarized and moved rapidly in a concentration gradient of fMLP (1.4 nM/μm; Figure 2, C and D, Supplemental Figure S1B, and Supplemental Movie S1), as described earlier (He et al., 2011). Of interest, depletion of Raptor or treatment with rapamycin, a potent and specific inhibitor for mTORC1, had little effect on cell polarization and migration, suggesting that mTORC1 is dispensable for neutrophil chemotaxis (Figure 2C and Supplemental Figure S1B). By contrast, cells with mTOR or Rictor depletion exhibited severe chemotactic defects (Figure 2, C and D, Supplemental Figure S1B, and Supplemental Movies S2 and S3). These cells failed to polarize properly and instead formed small, poorly developed and unstable leading edges (Figure 2, C and D, Supplemental Figure S1B, and Supplemental Movies S2 and S3). Only 9% of Rictor-depleted cells exhibited stable and persistent polarity during migration, in sharp contrast to control cells (89%). Furthermore, Rictor-depleted cells migrated much more slowly (Figure 2, C and D, and Supplemental Figure S1B), resulting in a large fraction of cells in the lower end of the fMLP gradient (Figure 2C). In addition, the chemotactic indices, defined as the ratio of migration in the correct direction to the actual length of the migration path (Xu et al., 2005; He et al., 2011), were 0.81 and 0.63 for control and Rictor-depleted cells, respectively, suggesting defects in directional migration upon Rictor depletion (data not shown). The chemotactic behaviors of mTOR and Rictor-depleted cells were also examined by using the micropipette chemotaxis assay. In this assay, mTOR- or Rictor-depleted cells showed polarization and migration defects highly reminiscent of those in the microfluidic assay (Supplemental Figure S1C; see Supplemental Movies S4 and S5, which show the migratory behaviors of cells in gradients created by the micropipette). To further examine the specificity of Rictor depletion, we ectopically expressed wild-type Rictor in dHL-60 cells with Rictor depleted (Figure 2E). We used shRNAs targeting 3′-untranslated region (UTR) of Rictor, which specifically deplete endogenous Rictor, not the ectopic form. The endogenous gene products were depleted, whereas the exogenously expressed mRNAs devoid of the 3′-UTR were resistant. The ectopic expression of wild-type Rictor largely rescued the polarization and migratory defects caused by Rictor depletion (Figure 2, F and G, and data not shown). Taken together, these results suggest that mTOR and Rictor, but not Raptor, are required for neutrophil 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