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 is recruited to the leading edge of polarized dHL-60 cells. (A) Immuno­fluorescence of Rictor (red) and F-actin (green) in cells plated on fibrinogen without (top) or with (middle) fMLP stimulation (100 nM, 2 min). Fluorescence images of Rictor (red), F-actin (green), Rictor/F-actin merged images, and differential interference contrast (DIC) images of cells. The Rictor immunofluorescence is specific because incubation with the Rictor peptide completely abolishes the immunofluorescence (bottom). Bar, 10 μm. (B) Fluorescence line profiles of Rictor (Red) and F-actin (green) in dHL-60 cells with or without fMLP stimulation. The graph below the fluorescence image plots the fluorescence intensity of each probe (y-axis) vs. distance (x-axis) for the corresponding cell. (C) Ratios of mean fluorescence intensity of Rictor and GFP between the front and the back of cells. Left, representative Rictor and GFP images; right, quantification of 41 cells (for Rictor) and 38 cells (for GFP) collected from four independent experiments. The front of the cell is defined as the area within the first 3 μm of the cell (indicated by the yellow line), as depicted earlier (Shin et al., 2010), and the rest of the cell is defined as the back. Student's t test was performed. The asterisk indicates that the ratio for Rictor differs statistically from that of GFP (*p < 0.01).
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3814157&req=5

Figure 1: Rictor is recruited to the leading edge of polarized dHL-60 cells. (A) Immuno­fluorescence of Rictor (red) and F-actin (green) in cells plated on fibrinogen without (top) or with (middle) fMLP stimulation (100 nM, 2 min). Fluorescence images of Rictor (red), F-actin (green), Rictor/F-actin merged images, and differential interference contrast (DIC) images of cells. The Rictor immunofluorescence is specific because incubation with the Rictor peptide completely abolishes the immunofluorescence (bottom). Bar, 10 μm. (B) Fluorescence line profiles of Rictor (Red) and F-actin (green) in dHL-60 cells with or without fMLP stimulation. The graph below the fluorescence image plots the fluorescence intensity of each probe (y-axis) vs. distance (x-axis) for the corresponding cell. (C) Ratios of mean fluorescence intensity of Rictor and GFP between the front and the back of cells. Left, representative Rictor and GFP images; right, quantification of 41 cells (for Rictor) and 38 cells (for GFP) collected from four independent experiments. The front of the cell is defined as the area within the first 3 μm of the cell (indicated by the yellow line), as depicted earlier (Shin et al., 2010), and the rest of the cell is defined as the back. Student's t test was performed. The asterisk indicates that the ratio for Rictor differs statistically from that of GFP (*p < 0.01).

Mentions: We assessed the expression of mTOR, Rictor, and Raptor in neutrophils. Undifferentiated HL-60 cells (uHL-60), dHL-60, and primary human neutrophils expressed mTOR, Raptor, and Rictor (Supplemental Figure S1A). Raptor is the major component of the mTORC1 (Wullschleger et al., 2006; Bhaskar and Hay, 2007). Immunofluorescence of Rictor in nonstimulated dHL-60 cells was diffusely distributed, with some cortical localization around the plasma membranes, but was preferentially distributed to the leading edge of 71% of cells (of 126 examined) treated with the bacteria-derived chemoattractant formyl-Met-Leu-Phe (fMLP), colocalizing with F-actin (Figure 1A and data not shown). The immunofluorescence was specific for Rictor, as verified by competition experiments with a blocking peptide (Figure 1A; bottom). The asymmetric accumulation of Rictor was confirmed by quantitative analysis of fluorescence intensity across the cell, revealing a steep gradient of fluorescence signals (86 ± 15% decrease; Figure 1B). Moreover, the ratio of mean intensity of Rictor immunofluorescence between the leading and the trailing edges was 1.89 ± 0.09 (mean ± SEM; Figure 1C). By contrast, the ratio for green fluorescence protein (GFP) in polarized dHL-60 cells was 1.12 ± 0.11 (Figure 1C), indicating that the asymmetric distribution of Rictor was not due to overall morphological changes during neutrophil polarization.


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 is recruited to the leading edge of polarized dHL-60 cells. (A) Immuno­fluorescence of Rictor (red) and F-actin (green) in cells plated on fibrinogen without (top) or with (middle) fMLP stimulation (100 nM, 2 min). Fluorescence images of Rictor (red), F-actin (green), Rictor/F-actin merged images, and differential interference contrast (DIC) images of cells. The Rictor immunofluorescence is specific because incubation with the Rictor peptide completely abolishes the immunofluorescence (bottom). Bar, 10 μm. (B) Fluorescence line profiles of Rictor (Red) and F-actin (green) in dHL-60 cells with or without fMLP stimulation. The graph below the fluorescence image plots the fluorescence intensity of each probe (y-axis) vs. distance (x-axis) for the corresponding cell. (C) Ratios of mean fluorescence intensity of Rictor and GFP between the front and the back of cells. Left, representative Rictor and GFP images; right, quantification of 41 cells (for Rictor) and 38 cells (for GFP) collected from four independent experiments. The front of the cell is defined as the area within the first 3 μm of the cell (indicated by the yellow line), as depicted earlier (Shin et al., 2010), and the rest of the cell is defined as the back. Student's t test was performed. The asterisk indicates that the ratio for Rictor differs statistically from that of GFP (*p < 0.01).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Rictor is recruited to the leading edge of polarized dHL-60 cells. (A) Immuno­fluorescence of Rictor (red) and F-actin (green) in cells plated on fibrinogen without (top) or with (middle) fMLP stimulation (100 nM, 2 min). Fluorescence images of Rictor (red), F-actin (green), Rictor/F-actin merged images, and differential interference contrast (DIC) images of cells. The Rictor immunofluorescence is specific because incubation with the Rictor peptide completely abolishes the immunofluorescence (bottom). Bar, 10 μm. (B) Fluorescence line profiles of Rictor (Red) and F-actin (green) in dHL-60 cells with or without fMLP stimulation. The graph below the fluorescence image plots the fluorescence intensity of each probe (y-axis) vs. distance (x-axis) for the corresponding cell. (C) Ratios of mean fluorescence intensity of Rictor and GFP between the front and the back of cells. Left, representative Rictor and GFP images; right, quantification of 41 cells (for Rictor) and 38 cells (for GFP) collected from four independent experiments. The front of the cell is defined as the area within the first 3 μm of the cell (indicated by the yellow line), as depicted earlier (Shin et al., 2010), and the rest of the cell is defined as the back. Student's t test was performed. The asterisk indicates that the ratio for Rictor differs statistically from that of GFP (*p < 0.01).
Mentions: We assessed the expression of mTOR, Rictor, and Raptor in neutrophils. Undifferentiated HL-60 cells (uHL-60), dHL-60, and primary human neutrophils expressed mTOR, Raptor, and Rictor (Supplemental Figure S1A). Raptor is the major component of the mTORC1 (Wullschleger et al., 2006; Bhaskar and Hay, 2007). Immunofluorescence of Rictor in nonstimulated dHL-60 cells was diffusely distributed, with some cortical localization around the plasma membranes, but was preferentially distributed to the leading edge of 71% of cells (of 126 examined) treated with the bacteria-derived chemoattractant formyl-Met-Leu-Phe (fMLP), colocalizing with F-actin (Figure 1A and data not shown). The immunofluorescence was specific for Rictor, as verified by competition experiments with a blocking peptide (Figure 1A; bottom). The asymmetric accumulation of Rictor was confirmed by quantitative analysis of fluorescence intensity across the cell, revealing a steep gradient of fluorescence signals (86 ± 15% decrease; Figure 1B). Moreover, the ratio of mean intensity of Rictor immunofluorescence between the leading and the trailing edges was 1.89 ± 0.09 (mean ± SEM; Figure 1C). By contrast, the ratio for green fluorescence protein (GFP) in polarized dHL-60 cells was 1.12 ± 0.11 (Figure 1C), indicating that the asymmetric distribution of Rictor was not due to overall morphological changes during neutrophil polarization.

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