Research ArticleCell Migration

Dynamic regulation of neutrophil polarity and migration by the heterotrimeric G protein subunits Gαi-GTP and Gβγ

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Science Signaling  23 Feb 2016:
Vol. 9, Issue 416, pp. ra22
DOI: 10.1126/scisignal.aad8163
  • Fig. 1 Gβγ activation alone reduces neutrophil motility.

    (A) Diagram of canonical G protein regulation by GPCRs, including chemoattractant receptors. Pi, inorganic phosphate. (B) Mechanism of action of 12155, which binds directly to Gβγ subunits and results in the release of free Gβγ subunits from Gα-GDP without activating the Gα subunit. (C) Gβγ activation reduces the basal motility of neutrophils. Primary mouse neutrophils were treated with vehicle (DMSO), 10 μM 12155, or 1 μM fMLP, and then were tracked for 25 min by microscopy and analyzed by ImageJ software. Tracks of individual neutrophils for each treatment are shown for a single experiment and are representative of three experiments. (D) Data from three experiments as represented in (C) were analyzed with the Chemotaxis and Migration tool from ibidi to determine the velocity (left) and the distance traveled (right) by the indicated cells. Each point represents an individual cell from three separate experiments that were pooled and analyzed as indicated below. Data from 20 cells under each condition were analyzed for statistical significance by one-way analysis of variance (ANOVA) with Bonferroni posttest. *P < 0.05 and ***P < 0.001. (E) 12155 causes a concentration-dependent decrease in basal migration. Mouse neutrophils were stimulated with the indicated concentrations of 12155 and then were tracked and analyzed as described in (C) and (D). Each point represents the average ± SEM for 20 cells from the data shown in fig. S3 (C and D), which were pooled from three independent experiments.

  • Fig. 2 Uniform Gβγ activation promotes the formation of nonpolarized lamellipodia and increases cell adhesion.

    (A) Uniform Gβγ activation stimulates the formation of nonpolarized circular lamellipodia. Mouse neutrophils were treated with vehicle (DMSO), 10 μM 12155, or 1 μM fMLP, and then were fixed and imaged by DIC microscopy. Images are representative of multiple cells from four independent experiments. Scale bars, 5 μm. (B) Gβγ activation reduces basal cell polarity. Mouse neutrophils were treated with the indicated reagents as described in (A), fixed, stained for actin, and imaged by epifluorescence microscopy. Cells were scored (between 20 and 50) for each independent experiment in a blinded manner to determine the number of polarized cells. Data are means ± SEM of the percentages of cells that were polarized in three independent experiments. (C) Mouse neutrophils were stimulated with the indicated concentrations of 12155 and were analyzed as described in (B) to determine the percentage of cells that exhibited nonpolarized lamellipodia formation. Data are means ± SEM of three independent experiments. (D) Gβγ activation increases cell adhesion to an ICAM-1–coated substrate. Mouse neutrophils were uniformly stimulated as described in (A), and the numbers of cells that became detached from the surface were counted. Adhesion was calculated as inversely proportional to the number of detached cells. Data are means ± SEM of three independent experiments. All data were analyzed by one-way ANOVA with Bonferroni posttest. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 3 The 12155-dependent effects on cell migration are blocked by GRK2ct.

    (A and B) HL-60 cells were transfected by nucleofection with plasmid encoding YFP alone or in the presence of plasmid encoding GRK2ct. Transfected cells were selected on the basis of the abundance of YFP. The cells were further segregated on the basis of whether the cells showed high or low fluorescence, which was interpreted as evidence of increased or decreased amounts of GRK2ct, respectively. (A) Representative individual YFP-expressing cells are shown before and after treatment with 12155. (B) Quantitation of data from the experiments shown in (A). After treatment with 12155, YFP-expressing cells were identified and scored (in a blinded manner) for the appearance of the characteristic flattened fried egg or “normal” morphologies. Cells were imaged at ×60 magnification. Experiments are from four separate sets of transfections, with multiple cells examined in each experiment. Data are means ± SEM of pooled data from three independent experiments and analyzed by one-way ANOVA with Bonferroni posttest. ****P < 0.0001 compared to control cells transfected with YFP alone. The proportions of cells that showed a characteristic flattened symmetrical morphology after treatment with 12155 are as follows:YFP alone, 35 of 41 cells; YFP + GRK2ct (high), 4 of 41 cells; and YFP + GRK2ct (low), 15 of 17 cells.

  • Fig. 4 Direct activation of Gi heterotrimers is sufficient to stimulate cell migration, induce polarization, and reduce adhesion.

    (A) Diagram of the mechanism of action of Mas7, a mastoparan derivative. Mas7 directly interacts with Gi heterotrimers and catalyzes nucleotide exchange on Gαi, which leads to the receptor-independent activation of signaling by both Gαi and Gβγ. (B) Activation of Gi heterotrimers stimulates neutrophil migration. Mouse neutrophils were treated with vehicle (DMSO), 10 μM 12155, 2 μM Mas7, or 1 μM fMLP, and then were tracked and analyzed as described in Fig. 1A. Data are from a single experiment and are representative of four experiments. (C) Activation of Gi heterotrimers increases both the speed of neutrophil migration (top) and the distance traveled (bottom). Mouse neutrophils were treated, tracked, and analyzed as described in Fig. 1A. Data are means ± SEM of 20 cells under each condition from three experiments. (D) Activation of Gi heterotrimers induces cell polarization. Mouse neutrophils were treated with the indicated compounds, fixed, and imaged by DIC microscopy. Images are representative of multiple cells from four individual experiments. Scale bars, 5 μm. (E) Mouse neutrophils were treated with the indicated compounds, fixed, stained for actin, and imaged for fluorescence. Images were analyzed in a blinded manner as described in Fig. 2B to determine the number of polarized cells. Data are means ± SEM of the percentages of polarized cells from three independent experiments. All data were analyzed by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 5 Treatment of Gαi1Q204L-expressing HL-60 cells with 12155 results in cell polarization and migration.

    (A and B) HL-60 cells were cotransfected with plasmid encoding YFP and with plasmid encoding either wild-type (WT) Gαi1 or the Gαi1(Q204L) mutant. Transfected cells were selected on the basis of the abundance of YFP. (A) Representative individual YFP-expressing cells are shown before and after a 30-min treatment with either 12155 or fMLP. (B) Quantitation of data from the experiments shown in (A) for 12155 treatment. After treatment, the YFP-expressing cells were identified and scored (in a blinded manner) for the presence of the characteristic flattened fried egg or normal morphologies. Cells were imaged at ×60 magnification. Data are means ± SEM of pooled data from three independent experiments and were analyzed by a Student’s t test. ****P < 0.0001 compared to control transfected cells expressing YFP alone. The proportions of cells that cells showed a characteristic flattened symmetrical morphology after treatment with 12155 are as follows: YFP + WTGαi1, 22 of 29 cells; YFP + Gαi1(Q204L), 2 of 20 cells. Fluor, fluorescent cells.

  • Fig. 6 Stimulation of Gβγ alone results in enhanced and sustained increases in cAMP abundance compared to activation of Gi protein heterotrimers.

    (A) Human neutrophils were stimulated for the indicated times with 10 μM 12155, 2 μM Mas7, or 1 μM fMLP. The amount of cAMP generated under each condition was measured as described in Materials and Methods and is presented as the amount of cAMP (pmol) produced per 75,000 cells. Data are means ± SEM of three independent experiments and were analyzed by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. (B) 12155 results in a concentration-dependent increase in cAMP. Mouse neutrophils were stimulated with the indicated concentrations of 12155 for 30 min and were analyzed as described in (A). Data are presented as the amount of cAMP (pmol) produced per 100,000 cells. Data are means ± SEM of three independent experiments.

  • Fig. 7 Inhibition of PKA restores polarity to 12155-treated cells.

    (A) Mouse neutrophils were preincubated with myr-PKI or DMSO before being treated with the indicated compounds, fixed, and imaged by DIC microscopy. Images are representative of multiple cells from three independent experiments. Scale bars, 5 μm. (B) Mouse neutrophils were preincubated with the indicated inhibitors, treated with the indicated compounds, fixed, and then imaged by DIC microscopy. The images were analyzed in a blinded manner as described in Fig. 2B to determine the numbers of cells with circular uniform pseudopodia. Data are means ± SEM of three independent experiments and were analyzed by one-way ANOVA. ***P < 0.001 compared to cells treated with 12155 alone. (C) Mouse neutrophils were preincubated with inhibitor, treated with the indicated compounds, fixed, stained for actin, and imaged by epifluorescence microscopy. The images were analyzed in a blinded manner as described in Fig. 2B to determine the numbers of actin-polarized cells. Data are means ± SEM of the percentages of actin-polarized cells from three independent experiments and were analyzed by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. (D) PMNs were preincubated with myr-PKI or DMSO, treated with the indicated compounds, fixed, stained for actin with Acti-stain 555 phalloidin, and imaged by confocal fluorescence microscopy or DIC. Images are representative of multiple cells from three independent experiments. Scale bars, 5 μm.

  • Fig. 8 Inhibition of PKA enhances basal migration but does not restore migration or reduce adhesion in 12155-treated cells.

    (A) Inhibition of PKA enhances basal migration, but not in the presence of 12155. Mouse neutrophils were preincubated with 1 μM myr-PKI (m-PKI) or 300 μM Rp-cAMPs, treated with DMSO or 10 μM 12155, and then tracked for 25 min and analyzed by ImageJ software. Tracks of neutrophils treated with the indicated compounds are depicted. Data are from a single experiment and are representative of three experiments. (B) Pooled data from experiments performed as described in (A). The tracked neutrophils were analyzed with the Chemotaxis and Migration tool from ibidi. Data are means ± SEM of 20 cells for each condition from three independent experiments and were analyzed by one-way ANOVA. *P < 0.05, ***P < 0.001. (C) Inhibition of PKA is unable to reduce Gβγ-stimulated adhesion. Mouse neutrophils were uniformly stimulated with DMSO, 1 μM myr-PKI, 300 μM Rp-cAMPs, 10 μM 12155, or 2 μM Sp-cAMPs, as indicated, and the numbers of cells that detached from the bottom of ICAM-1–coated wells were counted. Adhesion was calculated as inversely proportional to the number of detached cells. Data are means ± SEM of three independent experiments and were analyzed by one-way ANOVA. *P < 0.05 and ***P < 0.001 compared to DMSO-treated control cells. (D) Model depicting the role of Gi proteins in neutrophil migration. When neutrophils are stimulated with the chemoattractant fMLP or with Mas7, both Gαi and Gβγ are activated. Gβγ activation stimulates the production of cAMP. In this model, cAMP inhibits cell polarization by activating PKA, but the concurrent activation of Gαi keeps the concentration of cAMP relatively low, perhaps maintaining a cAMP gradient. In parallel, Gβγ stimulates cell adhesion, which is opposed by Gαi-GTP in a cAMP-independent manner.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/416/ra22/DC1

    Fig. S1. Basal polarization of mouse neutrophils is dependent on ICAM-1.

    Fig. S2. Activation of Gβγ subunits alone reduces neutrophil motility on an ICAM-1–coated surface.

    Fig. S3. Uniform activation of Gβγ subunits prevents neutrophil polarity.

    Fig. S4. GRK2ct blocks the effects of 12155 on cell migration and polarity.

    Fig. S5. Activation of Gi heterotrimers stimulates neutrophil polarity.

    Fig. S6. The Gαi1Q204L mutant, but not wild-type Gαi1, prevents cell flattening and enables cell polarization and migration.

    Fig. S7. Inhibition of PKA restores the polarity of Gβγ-activated cells.

    Movie S1. Activation of Gβγ subunits alone reduces neutrophil motility on an ICAM-1–coated surface.

    Movie S2. Activation of Gi heterotrimers stimulates neutrophil polarity and migration.

    Movie S3. Activated Gαi does not affect the basal or fMLP-stimulated behavior of HL-60 cells.

    Movies S4 to S6. Treatment of cells containing activated Gαi with 12155 results in polarized extension of pseudopods, cell movement, and decreased adhesion.

    Movie S7. Treatment of cells containing wild-type Gαi with 12155 results in cell flattening and depolarization similar to that of untransfected cells.

    Movie S8. Inhibition of PKA restores the polarity of Gβγ-activated cells.

    Movie S9. Inhibition of PKA enhances the basal migration of mouse neutrophils in the absence of 12155.

    Movie S10. Inhibition of PKA does not restore migration in the presence of 12155.

  • Supplementary Materials for:

    Dynamic regulation of neutrophil polarity and migration by the heterotrimeric G protein subunits Gαi-GTP and Gβγ

    Chinmay R. Surve, Jesi Y. To, Sundeep Malik, Minsoo Kim, Alan V. Smrcka*

    *Corresponding author. E-mail: alan_smrcka{at}URMC.rochester.edu

    This PDF file includes:

    • Fig. S1. Basal polarization of mouse neutrophils is dependent on ICAM-1.
    • Fig. S2. Activation of Gβγ subunits alone reduces neutrophil motility on an ICAM-1–coated surface.
    • Fig. S3. Uniform activation of Gβγ subunits prevents neutrophil polarity.
    • Fig. S4. GRK2ct blocks the effects of 12155 on cell migration and polarity.
    • Fig. S5. Activation of Gi heterotrimers stimulates neutrophil polarity.
    • Fig. S6. The Gαi1Q204L mutant, but not wild-type Gαi1, prevents cell flattening and enables cell polarization and migration.
    • Fig. S7. Inhibition of PKA restores the polarity of Gβγ-activated cells.
    • Legends for movies S1 to S10

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 1 MB

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Activation of Gβγ subunits alone reduces neutrophil motility on an ICAM-1–coated surface.
    • Movie S2 (.mp4 format). Activation of Gi heterotrimers stimulates neutrophil polarity and migration.
    • Movie S3 (.avi format). Activated Gαi does not affect the basal or fMLP-stimulated behavior of HL-60 cells.
    • Movies S4 to S6 (.avi format). Treatment of cells containing activated Gαi with 12155 results in polarized extension of pseudopods, cell movement, and decreased adhesion.
    • Movie S7 (.avi format). Treatment of cells containing wild-type Gαi with 12155 results in cell flattening and depolarization similar to that of untransfected cells.
    • Movie S8 (.mp4 format). Inhibition of PKA restores the polarity of Gβγ-activated cells.
    • Movie S9 (.mp4 format). Inhibition of PKA enhances the basal migration of mouse neutrophils in the absence of 12155.
    • Movie S10 (.mp4 format). Inhibition of PKA does not restore migration in the presence of 12155.

    [Download Movies S1 to S10]


    Citation: C. R. Surve, J. Y. To, S. Malik, M. Kim, A. V. Smrcka, Dynamic regulation of neutrophil polarity and migration by the heterotrimeric G protein subunits Gαi-GTP and Gβγ. Sci. Signal. 9, ra22 (2016).

    © 2016 American Association for the Advancement of Science

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