The small Rho GTPase Cdc42 regulates neutrophil polarity via CD11b integrin signaling - PubMed (original) (raw)

The small Rho GTPase Cdc42 regulates neutrophil polarity via CD11b integrin signaling

Kathleen Szczur et al. Blood. 2009.

Abstract

Neutrophil migration to sites of infection is the first line of cellular defense. A key event of migration is the maintenance of a polarized morphology, which is characterized by a single leading edge of filamentous actin and a contractile uropod devoid of filamentous actin protrusions. Using a mouse model of high Cdc42 activity, we previously demonstrated the importance of Cdc42 activity in neutrophil migration. However, the specific functions of Cdc42 in this process remain to be understood. Using neutrophils genetically deficient in Cdc42, we show that Cdc42 regulates directed migration by maintaining neutrophil polarity. Although it is known to be activated at the front, Cdc42 suppresses protrusions at the uropod. Interestingly, Cdc42 makes use of the integrin CD11b during this process. Cdc42 determines the redistribution of CD11b at the uropod. In turn, using CD11b-null cells and CD11b crosslinking experiments, we show that CD11b modulates myosin light chain phosphorylation to suppress lateral protrusions. Our results uncover a new mechanism in which Cdc42 regulates the uropod through CD11b signaling to maintain polarity in migrating neutrophils. It also reveals new functions for CD11b in neutrophil polarity.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Cdc42 is critical to maintain front/back polarity of neutrophils during migration. (A) Expression of Cdc42 in WT and Cdc42−/− neutrophils analyzed by immunoblot. (B) Neutrophil migration using a Boyden chamber in uniform concentration or in a gradient of 1 μM fMLP in which fMLP is placed only in the lower chamber to measure chemokinesis or chemotaxis, respectively. The histogram represents the number of migrated neutrophils per field (mean ± SD, representative experiment in triplicate of 3 independent experiments). (C) Neutrophil migration using transwells coated with fibrinogen in uniform concentration or in a gradient of 10μM fMLP. The histogram represents the total number of migrated neutrophils recovered from the bottom well (mean ± SD, representative experiment in triplicate of 3 independent experiments). (D) Neutrophil migration was examined by time-lapse video microscopy in a gradient of 10μM fMLP and on surface coated with fibrinogen, in a Zigmond chamber. Representative images (1 minute between each frame) of migrating cells, fMLP concentration increases from left to right. Arrows point to inappropriate protrusions. Cell trajectory analysis: the schema represents the migration trajectory of cells moving up fMLP gradient for 25 minutes. Trajectories were tracked with Volocity software and realigned to the same horizontal axis. The black circle represents the starting position. Cdc42-deficient neutrophils exhibited overall displacement toward the fMLP gradient. Speed (sp, μm/min) and straightness (st) of migration are indicated on the right. Data are mean ± SEM; n = 100. The histogram represents the percentage of cells exhibiting change in direction arising from inappropriate lateral protrusions (mean ± SD; n = 3 independent videos); at least 50 cells per video were analyzed. Images were captured at 37°C using a Zeiss Axiovert 200 microscope at 10× objective, NA 0.3, with ORCA-ER C4742-95 camera driven by Openlab software (supplemental Videos). *Results that are significantly different from WT (P < .05).

Figure 2

Figure 2

Dynamic analysis of actin distribution using eGFP-actin reporter. Sequential images showing the distribution of eGFP-actin reporter in WT and Cdc42−/− neutrophils in response to a local source of fMLP (10μM, indicated by X) (30 seconds between each frame). Arrows point to actin protrusions. Note that eGFP-actin remains at one pole of the cell facing the source of fMLP in WT but not in Cdc42−/− cells. Each set of images are representative of a set of 10. Scale bar represents 5 μm. Fluorescence images were captured at 37°C using a Zeiss Axiovert 200 fluorescence microscope at 40× objective, NA 0.6, with ORCA-ER C4742-95 camera driven Openlab software. The histograms are speed, straightness of migration, and percentage of cells with multiple protrusions and turns of WT and Cdc42−/− cells expressing eGFP-actin, which were analyzed in a Zigmond chamber assay as in Figure 1 and showed similar results as WT and Cdc42−/− cells (Figure 1).

Figure 3

Figure 3

Cdc42 regulates CD11b distribution. (A) WT and Cdc42−/− neutrophils were stimulated or not stimulated with fMLP and on Fg-coated slides for 10 minutes. The cells were fixed and stained with anti-CD45 (in red) or anti-CD44 (in red) and anti-CD11b (in green). The black-and-white pictures are the phase-contrast images. The images are one x-y view of the z-series analyzed by deconvolution in Volocity. Two-dimensional representation of mean intensity of fluorescence of CD11b and CD45 or CD44 of the region of interest indicated by the box, analyzed in ImageJ. Arrows point to the cell front. Histogram is ratio of mean intensity of fluorescence of CD11b along the sides of the cells to the front normalized to CD45 or CD44 (mean ± SD; n = 55). Scale bar represents 5 μm. (B) The average of fluorescence intensity at the substrate contact surface of CD11b and CD44 was measured along the lateral sides of the cells in at least 5 sections per cell (supplemental data). Histogram shows mean ± SEM of all measurements per cell and of n = 20 cells, representative of 3 independent experiments. The slides were mounted with Slowfade Gold antifade reagent. Fluorescence images were captured at room temperature using a Leica DMI6000 fluorescence microscope at 63×/1.3 NA objective, with ORCA-ER C4742-95 camera driven by Openlab software. Scale bar represents 5 μm.

Figure 4

Figure 4

Cdc42 regulates CD11b functioning. (A) Neutrophils were stimulated or not stimulated with fMLP in suspension at 37°C. The reaction was stopped by adding paraformaldehyde, and the cells were stained for CD11b. The arrows indicate small clusters and patches of CD11b in WT cells. CD11b clusters were analyzed in ImageJ software. A 3-dimensional representation of fluorescence intensity, which was generated in ImageJ, is shown. Histogram represents the number of CD11b clusters per cell (mean ± SD; n = 40 cells from 3 independent experiments). Scale bar represents 5 μm. The slides were mounted with Slowfade Gold antifade reagent. Fluorescence images were captured at room temperature using a Leica DMIRB fluorescence microscope at 63× objective, NA 1.3, with ORCA-ER C4742-95 camera driven and analyzed with Openlab software. (B) Adhesion and deadhesion of neutrophils. Neutrophils, stimulated with fMLP in the presence of Ca2+ and Mg2+, were allowed to adhere to fibrinogen at the indicated time. The nonadherent fraction was removed. The wells were carefully washed with phosphate-buffered saline, and the adherent fraction was immediately enumerated at the light microscope. Deadhesion: 30 minutes after adhesion to fibrinogen, the nonadherent fraction was removed and fMLP was replaced with phosphate-buffered saline without Ca2+ and Mg2+. The remaining adherent fraction was enumerated at the light microscope 10 minutes after fMLP removal. The histograms represent the number of adherent cells per field (mean ± SD; n = 3 independent experiments).

Figure 5

Figure 5

CD11b is critical for polarity of neutrophils during migration. (A) F-actin (rhodamine-phalloidin) analysis of WT, CD11b−/−, and Cdc42−/− neutrophils 10 minutes after stimulation with fMLP and on glass. The black-and-white pictures are the phase-contrast images. The images are one x-y view of the z-series analyzed by deconvolution in Volocity. Histogram represents total surface area and area of lamellipodia (mean ± SD of n = 30 cells) and number of cells with more than one protrusion (mean ± SD; 3 independent experiments). Arrows indicate F-actin protrusions. Scale bar represents 5 μm. (B) WT cells were treated with functional anti-CD11b blocking antibody or isotype control for 20 minutes at room temperature and stimulated with fMLP and on fibrinogen-coated slides in the presence of antibody. The cells were analyzed for F-actin structures with rhodamine-phalloidin (mean ± SD; n = 30 from 3 independent experiments). The slides were mounted with Slowfade Gold antifade reagent. Z series of fluorescence images were captured using a Leica DMIRB or Leica DMI6000 fluorescence microscope at 63×/1.3 NA objective, with ORCA-ER C4742-95 camera driven by Openlab software and analyzed by deconvolution with Volocity software. (C) Migration of cells analyzed by time lapse video microscopy in a Zigmond chamber in gradient of fMLP on glass, as in Figure 1. Schema of cell trajectory is shown. Straightness of migration is indicated as mean ± SEM. *Results that are significantly different from WT (P < .001). Histogram represents percentage of cells that developed lateral protrusions during the course of migration and changed direction (mean ± SD; n = 3 independent videos). Only cells that had moved more than 20 μm were analyzed. Images were captured at 37°C using a Zeiss Axiovert 200 microscope at 10×/0.3 NA objective, with ORCA-ER C4742-95 camera driven by Openlab software.

Figure 6

Figure 6

Cdc42 regulates neutrophil polarity via CD11b. (A) Deadhesion. WT and Cdc42−/− neutrophils were stimulated with fMLP and on slides coated with anti-CD11b to enforce CD11b activation. The stimulus was removed 30 minutes after adhesion, and the remaining adherent fraction was enumerated 10 minutes after stimulus removal at the light microscope (mean ± SD; n = 3 independent experiments, as in Figure 3). (B) WT and Cdc42−/− neutrophils were stimulated with fMLP and on Fg-coated slides or on slides coated with anti-CD11b to enforce CD11b activation, compared with isotype control. The cells were then fixed and stained with rhodamine-phalloidin (in red) to analyze F-actin structures. The black-and-white pictures represent the phase-contrast images. The images are one x-y view of the z-series analyzed by deconvolution in Volocity. Histograms are number of cells with more than one protrusion (percentage, mean ± SD; n = 3 independent experiments); at least 30 cells per experiment were analyzed. Surface area of F-actin (square microns) and total cell spreading (square microns) are shown as mean ± SD of at least n = 30 cells from at least 2 independent experiments. Arrows point to actin protrusions. Scale bar represents 5 μm. The slides were mounted with Slowfade Gold antifade reagent. Z series of fluorescence images were captured at room temperature using a Leica DMI6000 fluorescence microscope at 63×/1.3 NA objective, with ORCA-ER C4742-95 camera driven by Openlab software and analyzed by deconvolution with Volocity software. (C) Migration responses of cells plated on anti-CD11b–coated surface was analyzed in a Zigmond chamber in fMLP gradient as in Figure 1. The schema represents the migration trajectory of cells moving up fMLP gradient for 25 minutes. Straightness of migration is indicated as mean ± SEM; n = 70 cells from 3 independent videos. Histogram represents percentage of cells with change in direction arising from inappropriate lateral protrusions (mean ± SD; n = 3 independent videos).

Figure 7

Figure 7

Cdc42 suppresses actin through CD11b-induced p-MLC signaling. (A) WT cells were crosslinked with CD11b or isotype controls for 20 minutes and were analyzed for RhoA activity using the pulldown assay with Rhotekine beads. Total RhoA of cell lysates is used as loading control. The cells were also analyzed for phosphorylated MLC at Ser19 and total p38MAPK as loading control (one blot representative of 3 independent experiments). (B) WT and Cdc42−/− neutrophils with or without Y-27632 treatment were stimulated with fMLP and seeded on fibrinogen or on an anti-CD11b–coated plate for 10 minutes. In addition, WT cells treated with functional anti-CD11b blocking antibody were stimulated with fMLP and on Fg-coated slides. The cells were lysed on plate and analyzed for p-MLC. Histograms represent densitometry analysis compared with WT after normalization to loading (mean ± SD; n = 3 independent experiments). Of note, the experiments with and without Y-27632 were performed independently such that the absolute level of p-MLC is not comparable. (C) Cells, treated or not with Y-27632, were stimulated or not on fMLP and on Fg or glass or anti-CD11b–coated slides for 10 minutes. The cells were stained with anti-p-MLC (in green) and rhodamine-phalloidin (in red). In addition, WT cells treated with anti-CD11b or isotype were plated on fibrinogen and analyzed for F-actin and p-MLC distribution. These later experiments were performed separately. WT cells treated with isotype are not different from nontreated WT cells, and only one image of WT control is shown. The black-and-white pictures represent the phase-contrast images. The images colored in red and green are one x-y view of the z-series analyzed by deconvolution in Volocity. Percentage of cells with p-MLC staining along the lateral sides of the cells is enumerated (mean ± SD; n = 3 independent experiments). The average intensity of fluorescence per area of p-MLC along the sides of the cells was analyzed in Openlab, and the data are shown as histogram (mean ± SD of n = 30-50 cells, from at least 2 independent experiments). Percentage of cells with more than one protrusion (mean ± SD; n = 3 independent experiments); at least 30 cells per experiments were analyzed. *Results that are significantly different from WT (P < .05). Arrows point to actin protrusions. Arrowheads point to p-MLC lateral distribution. Scale bar represents 5 μm. The slides were mounted with Slowfade Gold antifade reagent. Z series of fluorescence images were captured at room temperature using a Leica DMI6000 fluorescence microscope at 63×/1.3 NA objective, with ORCA-ER C4742-95 camera driven by Openlab software and analyzed by deconvolution with Volocity software.

References

    1. Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell. 1994;76:301–314. - PubMed
    1. Schenkel AR, Mamdouh Z, Muller WA. Locomotion of monocytes on endothelium is a critical step during extravasation. Nat Immunol. 2004;5(4):393–400. - PubMed
    1. Ley K, Laudanna C, Cybulsky MI, Nourshargh S. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat Rev Immunol. 2007;7(9):678–689. - PubMed
    1. Ridley AJ, Schwartz MA, Burridge K, et al. Cell migration: integrating signals from front to back. Science. 2003;302(5651):1704–1709. - PubMed
    1. Van Haastert PJ, Devreotes PN. Chemotaxis: signalling the way forward. Nat Rev Mol Cell Biol. 2004;5(48):626–634. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources