Arp2/3 Complex Is Required for Macrophage Integrin Functions but Is Dispensable for FcR Phagocytosis and In Vivo Motility

Abstract

The Arp2/3 complex nucleates branched actin, forming networks involved in lamellipodial protrusion, phagocytosis, and cell adhesion. We derived primary bone marrow macrophages lacking Arp2/3 complex (Arpc2^-/-) and directly tested its role in macrophage functions. Despite protrusion and actin assembly defects, Arpc2^-/- macrophages competently phagocytose via FcR and chemotax toward CSF and CX3CL1. However, CR3 phagocytosis and fibronectin haptotaxis, both integrin-dependent processes, are disrupted. Integrin-responsive actin assembly and αM/β2 integrin localization are compromised in Arpc2^-/- cells. Using an in vivo system to observe endogenous monocytes migrating toward full-thickness ear wounds we found that Arpc2^-/- monocytes maintain cell speeds and directionality similar to control. Our work reveals that the Arp2/3 complex is not a general requirement for phagocytosis or chemotaxis but is a critical driver of integrin-dependent processes. We demonstrate further that cells lacking Arp2/3 complex function in vivo remain capable of executing important physiological responses that require rapid directional motility.

Keywords: Arp2/3; actin; directed migration; integrin; macrophage; phagocytosis.

Figure 1. Arpc2−/− macrophage characterization and phenotypes

A) Expression of Arp2/3 complex subunits in WT and Arpc2−/− macrophages. GAPDH is presented for loading comparison. B) Phalloidin (F-actin) and Arpc2 staining in WT and Arpc2−/− macrophages. Scale (image and inset) = 10 µm. C) Vinculin staining (grey) and merged staining of vinculin (red) and actin (cyan) in WT and Arpc2−/− macrophages. Scale = 3 µm. D) F-actin staining intensity of WT (black bars) and Arpc2−/− (KO, grey bars) macrophages on a range of fibronectin concentrations. Integrated pixel density plotted as mean ± SEM. ***p < 0.0001. A.U., Arbitrary Units. Blot panels display total actin level. N = at least 113 cells/condition, pooled from three separate experiments. E) Spread cell area in µm², WT (black bars) or Arpc2−/− (KO, grey bars) macrophages on a range of fibronectin concentrations. Plotted as mean ± SEM. ***p < 0.0001. N = at least 113 cells/condition, pooled from three separate experiments. F) WT (black bars) and Arpc2−/− (KO, grey bars) macrophage cell shape on a range of fibronectin concentrations, plotted as mean ± SEM. A.U., Arbitrary Units. N = at least 77 cells/condition, pooled from three separate experiments. G) Scanning electron micrographs of WT and Arpc2−/− macrophages. Scale = 2 µm. See also Figure S1, Movie S1.

Figure 2. The Arp2/3 Complex is dispensable for FcR phagocytosis

A) Scanning electron micrograph of WT and Arpc2−/− macrophages with 10 micron IgG-coated beads. Scale = 1 µm. B) WT and Arpc2−/− macrophages at early (20 min.) and later (60 min.) time points bound to 6 micron beads. Staining (L to R): F-actin, external beads, all beads. Arrows denote internalized beads. Scale = 5 µm. C) Phagocytic index (internalized beads/total beads) of WT (black bars) or Arpc2−/− (KO, grey bars) macrophages at early (20 min.) or later (60 min.) time points with 2, 6, or 10 micron IgG-opsonized beads. Data is plotted as mean ± SEM. **p ≤ 0.0014, *p ≤ 0.03. Means represent compiled data from three separate experiments. D) Phagocytic index (internalized beads/total beads) of WT macrophages acutely treated with DMSO (−, black bars) or 150 µM CK-666 (+, grey bars) at early (20 min.) or later (60 min.) time points with 2, 6, or 10 micron IgG-opsonized beads. Data is plotted as mean ± SEM. None of the p-values under these conditions were statistically significant. Means represent compiled data from three separate experiments. E) Phagocytic index (internalized beads/total beads) of WT (black bars) or Arpc2−/− (KO, grey bars) macrophages in the absence (−) or presence (0.5, 1) of 0.5 or 1 µM Cytochalasin D (Cyto.D) after 60 minutes with 2, 6, or 10 micron IgG-opsonized beads. Data is plotted as mean ± SEM. ***p ≤ 0.001, **p = 0.0019, *p ≤ 0.0282. Means represent compiled data from three separate experiments. See also Figure S1, Movie S2.

Figure 3. The Arp2/3 complex is critical for CR3 phagocytosis

A) Scanning electron micrograph of WT and Arpc2−/− macrophages with 10 micron C3bi-coated beads. Scale = 1 µm. B) WT and Arpc2−/− macrophages at early (20 min.) and later (60 min.) time points bound to 6 micron beads. Staining (L to R): F-actin, external beads, all beads. Arrows denote internalized beads. Scale = 5 µm. C) Phagocytic index (internalized beads/total beads) of WT (black bars) or Arpc2−/− (KO, grey bars) macrophages at early (20 min.) or later (60 min.) time points with 2, 6, or 10 micron C3bi-opsonized beads. Data is plotted as mean ± SEM. **p = 0.0063, *p ≤ 0.0258. Means represent compiled data from three separate experiments. D) Phagocytic index (internalized beads/total beads) of WT macrophages acutely treated with DMSO (−, black bars) or 150 µM CK-666 (+, grey bars) at early (20 min.) or later (60 min.) time points with 2, 6, or 10 micron C3bi-opsonized beads. Data is plotted as mean ± SEM. ***p = 0.0005, **p = 0.0028, *p < 0.05. Means represent compiled data from three separate experiments. E) Flow cytometry analysis of αM and β2 integrin surface levels in WT (black bars) and Arpc2−/− (KO, grey bars) macrophages. Average Mean Fluorescence Intensity (MFI) of αM and β2 integrin on cultured macrophages were plotted ± SEM. Data were combined from three independent datasets. Negative control (−) cells were incubated with secondary antibody only. No statistically significant p-values were detected. A.U., Arbitrary Units. F) αM and β2 integrin surface levels on spread WT (black bars) and Arpc2−/− (KO, grey bars) macrophages in culture plated on 1 µg/mL collagen. Mean integrated pixel densities are plotted ± SEM. No statistically significant p-values were detected. A.U., Arbitrary Units. N = at least 132 cells. G) Confocal images (L to R, with insets) of αM integrin, F-actin and p34 in WT (top row) or Arpc2−/− macrophages (bottom row) plated on 1 µg/mL collagen. Scale = 20 µm. H) Scanning electron micrograph of WT or Arpc2−/− macrophages with bound C3bi-opsonized 2 micron beads. Scale = 500 nm. See also Figure S2.

Figure 4. The Arp2/3 complex promotes actin assembly and coordinates cellular signaling at CR3 phagocytic cups

A) Airyscan images of F-actin stains during FcR (left column, 20 min. time point) or CR3 phagocytosis (right column, 60 min. time point) of 10 micron beads. WT cells are in top row, Arpc2−/− in bottom row. Circle drawn on Arpc2−/− CR3 image depicts the diameter of the bead bound to this cell. Scale = 3 µm. B) F-actin staining intensity at WT (black bars) and Arpc2−/− (KO, grey bars) FcR phagocytic cups (left column) or CR3 phagocytic cups (right column). Data with 2 micron (top row), 6 micron (middle row) or 10 micron (bottom row) beads were gathered at 20 or 60 minute time points. Data is plotted as mean integrated pixel density ± SEM. ***p ≤ 0.0004, **p ≤ 0.0068, *p ≤ 0.024. A.U., Arbitrary Units. N = at least 277 (2 micron), 107 (6 micron) or 52 (10 micron) beads analyzed for F-actin, pooled from at least 4 unique experiments. C) WT (top) or Arpc2−/− (bottom) macrophages stably expressing Lifeact-GFP bound to 2 micron pHrodo red-IgG conjugated latex beads. First time point (t = 0) corresponds to initial binding of first bead in panel to the cell surface. Color-coded arrowheads indicate the same bead from frame to frame. Scale = 5 µm. D) Summary table of pHrodo-IgG and pHrodo-C3 experiments. pHrodo-IgG experiments encompassed seven (WT) or eight (Arpc2−/−) experimental repetitions. pHrodo-C3 experiments encompassed four (WT) or three (Arpc2−/−) experimental repetitions. Data are presented as percent of total analyzed beads. E) WT (top) or Arpc2−/− (bottom) macrophages stably expressing Lifeact-GFP were bound to 2 micron pHrodo red-C3 conjugated latex beads. First time point (t = 0) corresponds to initial binding of first bead in panel to the cell surface. Scale = 5 µm. F) Length of time after pHrodo bead binding required for WT or Arpc2−/− (KO) macrophages to produce a shell of F-actin in FcR or CR3 phagocytosis assays. Data is plotted as mean (in minutes)± SEM. **p = 0.0033, ***p < 0.0001. G) Length of time between F-actin peak (t_actin) and bead acidification (i.e. phagosome maturation) in WT or Arpc2−/− (KO) macrophages in FcR or CR3 phagocytosis assays. See also Figure S3, Figure S4, Movie S2.

Figure 5. Enhanced Arpc2−/− macrophage motility requires myosin II

A) Random migration speed of WT (black bars) or Arpc2−/− macrophages on a range of fibronectin concentrations (KO, grey bars) plotted as mean ± SEM. ****p < 0.0001, ***p = 0.0007. N = at least 89 cells, from 3 separate experiments. B) WT (top) and Arpc2−/− macrophages (bottom) migrating randomly on 1 µg/mL fibronectin. Scale = 10 µm. C) Random migration speed of macrophages (WT and KO, black bars) and fibroblasts (WT and KO, grey bars) in the presence or absence of myosin II-disrupting drugs (15 µM Blebbistatin and 10 µM Y-27632) on 1 µg/mL fibronectin. DMSO = negative control. ***p < 0.0001, **P = 0.0075. N = at least 114 cells, pooled from three separate experiments. D) WT (top) and Arpc2−/− macrophages (bottom) migrating randomly on 1 µg/mL fibronectin in the presence of 15 µM Blebbistatin. Scale = 10 µm. E) Scanning electron micrographs of WT and Arpc2−/− macrophages treated with 15 µM Blebbistatin. Scale = 2 µm. F) Phagocytic index (internalized beads/total beads) of WT (black bars) or Arpc2−/− macrophages (KO, grey bars) in the absence (−) or presence (+) of 15 µM Blebbistatin, after 60 minutes with 2, 6, or 10 micron IgG-opsonized beads. Data is plotted as mean ± SEM. *p = 0.0494. Means represent compiled data from three separate experiments. G) Confocal images, with insets, of F-actin (left) or myosin IIA (right) in WT (top) or Arpc2−/− macrophages (bottom) plated on 1 µg/mL fibronectin. Scale = 10 µm. See also Figure S5, Movie S3.

Figure 6. The Arp2/3 complex is dispensable for chemotaxis to CSF and CX3CL1, but is required for fibronectin haptotaxis

A, C, E) Wind rose plots generated from WT or Arpc2−/− macrophages migrating in a CSF gradient (A), CX3CL1 gradient (C) or FN gradient (E). Each plot is from one representative experiment of mixed WT and Arpc2−/− cells. 0°on the rose plot is the direction of the gradient. B, D, F) Forward Migration Index (FMI) of WT and Arpc2−/− macrophages plotted as mean ± 95% CI in CSF (B), CX3CL1 (D), or FN gradients (F). Data tables relate mean ± SEM of cell speed (V), FMI, d/T and total cells tracked (N). Quantitative measurements were pooled from multiple experiments using identical chamber conditions. G) WT and Arpc2−/− macrophages plated on 1 µg/mL collagen stained for αM integrin, vinculin and p-Tyr (4G10), β1 integrin, WAVE2 and F-actin at bound 6 micron Cy5-fibronectin (Cy5-FN) beads after 15 minute incubation. Arrowheads denote bound beads on Arpc2−/− macrophages. Scale = 10 µm. H) Localization of β1 integrin, p-Tyr and F-actin to Cy5-FN beads 5, 15 or 30 minutes after bead binding by WT or Arpc2−/− macrophages plotted as mean integrated pixel density (A.U.) ± SEM. **p = 0.0011, ***p < 0.0001. I) Schematic depiction of integrin-Arp2/3 complex coordination. Upon ligation integrin initiates an outside-in signal (black arrows) that induces the Arp2/3 complex to nucleate a branched actin network (an inside-out response). The branched actin network then reinforces the ‘upstream’ pathway (red arrows), perhaps by coordinating or concentrating these factors at new sites of integrin engagement. Without the Arp2/3 complex, macrophages still produce an outside-in signal but lack the ability to reinforce the initial signal, leading to loss of ECM sensing and inhibition of CR3 phagocytosis. See also Figure S6, Movies S4 and S5.

Figure 7. Monocyte motility and directionality in vivo do not require the Arp2/3 complex

A) Allele combination used for in vivo experimentation. CX3CR1-Cre-ER^T2 produced from the endogenous CX3CR1 promoter. Lox-Stop-Lox tdTomato expressed from the endogenous Rosa26 promoter. The Arpc2 allele as previously described. Tamoxifen induces LoxP recombination. B) Control (Arpc2+/−) or Arpc2−/− blood monocytes harvested after tamoxifen treatment. Scale = 10 µm. C) 0.3 mm diameter wounds were induced on day 0. tdTom+ monocytes were imaged the next day as they were recruited to the wound edge (arrow). D) Representative fields demonstrating tdTom+ Control and Arpc2−/− monocytes moving in situ toward a one day old wound over 15 minutes. Arrow denotes the direction of the wound. Individual cells are highlighted across the image series with colored arrows. Scale = 20 µm. E) Wind rose plots generated from endogenous Control (Arpc2+/−) or Arpc2−/− monocytes migrating toward one day old wounds in vivo. Data is from one representative experiment. 0°on the rose plot is the direction of the gradient. F) Forward Migration Index (FMI) of Control (Arpc2+/−) and Arpc2−/− monocytes in vivo plotted as mean ± 95% CI. Data table relates mean ± SEM of cell speed (V), FMI, d/T and total cells tracked (N). Quantitative measurements were pooled from 5 Control or 6 Arpc2−/− adult mice imaged one day after wounding. G) Velocity distribution of Control and Arpc2−/− monocytes from in vivo migration tracks (N = 234 or 382, respectively). Each point on the x-axis corresponds to a range of 50 microns per hour (0–50, 51–100, etc.). Y-axis: fraction of Control (cyan) or Arpc2−/− monocytes (red) migrating within given range. X-axis labels correspond to the upper bound of the 50 micron per hour range. H) FMI distribution of Control and Arpc2−/− monocytes (N = 234 or 382, respectively) from in vivo migration tracks. Each x-axis division corresponds to a range of 0.14, while the y-axis is the fraction of each population that falls within given range. The dotted vertical line divides monocytes with a positive FMI (to the right) that are capable of migrating directionally from those to the left (FMI ≤ 0) that do not. See also Figure S7, Movie S6.