Actin cytoskeleton reorganization by Syk regulates Fcγ receptor responsiveness by increasing its lateral mobility and clustering

Abstract

Clustering of immunoreceptors upon association with multivalent ligands triggers important responses including phagocytosis, secretion of cytokines, and production of immunoglobulins. We applied single-molecule detection and tracking methods to study the factors that control the mobility and clustering of phagocytic Fcγ receptors (FcγR). While the receptors exist as monomers in resting macrophages, two distinct populations were discernible based on their mobility: some diffuse by apparent free motion, while others are confined within submicron boundaries that reduce the frequency of spontaneous collisions. Src-family and Syk kinases determine the structure of the actin cytoskeleton, which is fenestrated, accounting for the heterogeneous diffusion of the FcγR. Stimulation of these kinases during phagocytosis induces reorganization of the cytoskeleton both locally and distally in a manner that alters receptor mobility and clustering, generating a feedback loop that facilitates engagement of FcγR at the tip of pseudopods, directing the progression of phagocytosis.

Figure 1. Single-molecule imaging of FcγRIIA reveals heterogeneous mobility in human primary macrophages

A. Low density labelling of FcγRIIA with Q-dots (red) at the surface of primary monocyte-derived macrophages. Representative image of more than 100 cells, from more than 10 independent experiments. B. Tracks obtained by SPT of FcγRIIA in one representative field of view. Confined motion is represented in navy blue and pointed by arrowheads; free motion is represented in cyan and pointed by arrows. Representative field of more than 100 cells, from more than 10 independent experiments. C. Percentage of confined receptors (circles) and free receptors (triangles), labelled with Cy3 (purple) or Q-dot (orange). Each dot represents one individual cell. Mean (black bar) and standard error to the mean (red bars) are displayed for each population. 18756 tracks from n = 40 cells and 27506 tracks from n = 130 cells were analysed for Cy3 and Q-dot labelled cells, respectively. D. Median diffusion coefficients of confined receptors (diamonds) and free receptors (squares), labelled with Cy3 (purple) or Q-dot (orange). E. Ratio of merge frequency between free receptors and confined receptors, labelled with Cy3. Each dot represents one individual cell. 18756 tracks from n = 40 cells were analysed. F. Fluorescence intensity histograms of detected Cy3-labelled FcγRIIA with different primary Fab concentrations (in μg/mL) in fixed cells. Intensity modes were analysed with a Gaussian fit (red line) and their mean intensity value is reported in the top right boxes. G. Average fluorescence intensity of detected particles determined by Gaussian fitting (blue), and number of detected particles (green), measured over time during photobleaching experiments with Cy3-labelled FcγRIIA in fixed cells. H. Representative measurements of the fluorescence intensity of individual features over time, illustrating photobleaching events.

Figure 2. FcγRIIA mobility is not confined by cholesterol-rich microdomains

A. Filipin staining of cholesterol in primary human macrophages. Representative images of more than 30 fields, from 3 independent experiments. B. Quantification of filipin intensity at the plasma membrane on individual primary macrophages. Measurements of each separated experiment were normalized to the mean intensity of the control cells. C. Median diffusion coefficient (for all particles regardless of motion type) in control (purple) and methyl-β-cyclodextrin treated cells (orange). 8790 tracks from 35 cells and 5640 tracks from 26 cells were analysed for control and methyl-β-cyclodextrin-treated cells, respectively. D. Percentage of confined receptors (circles) and free receptors (triangles), in control (purple) and methyl-β-cyclodextrin treated cells (orange).

Figure 3. FcγRIIA mobility is regulated by Syk and Src-family kinases

A. Endogenous Src activity revealed by anti-phospho-Src Y416 immunoblotting in resting untreated or PP1 treated primary human macrophages. Representative western blot from 3 independent experiments. B. Endogenous Syk activity revealed by anti-phospho-Syk Y525/526 immunoblotting in resting untreated or piceatannol treated primary human macrophages. Representative western blot from 3 independent experiments. C. Percentage of confined FcγRIIA (circles) and free FcγRIIA (triangles), in control (purple), PP1 (orange) and piceatannol-treated (blue) primary human macrophages. 27506 tracks from n = 130 cells, 7768 tracks from n = 50 cells and 7027 tracks from n = 51 cells were analysed for control, PP1 and piceatannol treated cells, respectively. D. Median diffusion coefficients of FcγRIIA in control (purple), PP1 (orange) and piceatannol (blue) treated primary human macrophages. E. Percentage of confined FcγR (circle) and free FcγR (triangle), in wild type (purple) and syk^−/− (blue) mouse bone marrow-derived macrophages. 9747 tracks from n = 16 cells and 18955 tracks from n = 20 cells were analysed for wild type and syk^−/− macrophages, respectively. F. Median diffusion coefficients of FcγR in wild type (purple) and syk^−/− (blue) mouse bone marrow-derived macrophages.

Figure 4. Receptor tyrosine phosphorylation does not account for FcγRIIA confinement in resting macrophages

A. Amino acid sequence of FcγRIIA cytosolic tail. The three tyrosines (blue) in the wild type protein were replaced by phenylalanines (red) in the 3Y-F mutant construct. B. Percentage of confined receptors (circles) and free receptors (triangles), observed for wild type or 3Y-F mutant receptors in control (orange and blue, respectively), or piceatannol treated (purple and green, respectively) RAW 264.7 macrophages. 24367 tracks from n = 37 cells, 41272 tracks from n = 40 cells, 11184 tracks from n = 37 cells and 15053 tracks from n = 34 cells were analysed for wild type, wild type + piceatannol, mutant and mutant + piceatannol cells, respectively. C. Median diffusion coefficients observed for wild type or 3Y-F mutant receptors in control (orange and blue, respectively), or piceatannol-treated (purple and green, respectively) RAW 264.7 macrophages.

Figure 5. FcγRIIA mobility is restricted by the actin cytoskeleton

A. Median diffusion coefficient in control (purple) and latrunculin B-treated human primary macrophages (orange). 6005 tracks from n = 50 cells and 5959 tracks from n = 40 cells were analysed for control and latrunculin-treated cells, respectively. B. Percentage of confined receptors (circles) and free receptors (triangles), in control (purple) and latrunculin B-treated cells (orange). C. Confocal image of bleb formation in RAW 264.7 macrophages upon jasplakinolide treatment; FcγRIIA-GFP (green) and LifeAct-mRFP (red). Representative image of more than 20 cells from 3 independent experiments. D. Diffusion of FcγRIIA-GFP determined by FRAP at the plasma membrane of untreated RAW macrophages (diamonds) or in the blebs of jasplakinolide treated RAW macrophages (circles), in presence (orange) or absence (purple) of piceatannol.

Figure 6. Syk inhibition leads to a large redistribution of the actin cytoskeleton in human primary macrophages

A. Confocal images of phalloidin-labelled F-actin distribution in human primary macrophages. Top panel: transversal slice, bottom panel: Z projection of maximal intensities. Representative images of more than 50 cells from more than 10 independent experiments. Blue arrows indicate podosomes. B. Ratio of phalloidin intensity between the dorsal and ventral surface of individual cells. Measurements of each separated experiment were normalized by the mean intensity of the control cells. C. Quantification of total phalloidin intensity on individual cells. Measurements of each separated experiment were normalized by the mean intensity of the control cells. D. High resolution scanning electron microscopy of the cortical cytoskeleton of human primary macrophages after detergent-based plasma membrane removal. Representative images of at least 12 cells from 3 independent experiments.

Figure 7. Syk-mediated actin reorganization of the actin cytoskeleton dictates FcγR mobility during phagocytosis

A. Confocal images of F-actin localization Fc-mediated during phagocytosis in RAW 264.7 macrophages in absence (left) or presence (right) of piceatannol. LifeAct-GFP (green), IgG-coated 5 μm polystyrene beads (red). Representative image of at least 12 cells from 3 independent experiments. B. Schematic of the frustrated phagocytosis model on IgG-coated coverslips. C. Time series of confocal images of actin-GFP distribution during frustrated phagocytosis, at the surface in contact with the coverslip, in untreated (top panel) or piceatannol treated (bottom panel) RAW macrophages. Representative images of more than 30 cells from 5 independent experiments. D. FcγR mobility within the phagocytic cup in RAW 264.7 macrophages. Tracks obtained by SPT of Fcγ during 5 sec (beige) are overplayed on the image of Actin-GFP (grey). Representative image of more than 50 cells from more than 5 independent experiments. E. Percentage of confined receptors (circles) and free receptors (triangles), observed in the actin-poor leading edge (pink) of the actin-rich area (green) during frustrated phagocytosis. 398 tracks from n = 27 cells were analysed. F. Percentage of confined receptors (circles) and free receptors (triangles), observed during frustrated phagocytosis in untreated (purple) or piceatannol treated (orange) RAW macrophages. 11 tracks from n = 11 cells and 55 tracks from n = 15 cells were analysed for control and piceatannol-treated cells, respectively.