TIRF imaging of Fc gamma receptor microclusters dynamics and signaling on macrophages during frustrated phagocytosis

Abstract

BackgroundRecent evidence indicates that in addition to the T-cell receptor, microclustering is an important mechanism for the activation of the B-cell receptor and the mast cell Fcε-receptor. In macrophages and neutrophils, particles opsonized with immunoglobulin G (IgG) antibodies activate the phagocytic Fcγ-receptor (FcγR) leading to rearrangements of the actin cytoskeleton. The purpose of this study was to establish a system for high-resolution imaging of FcγR microclustering dynamics and the recruitment of the downstream signaling machinery to these microclusters.MethodsWe developed a supported lipid bilayer platform with incorporated antibodies on its surface to study the formation and maturation of FcγR signaling complexes in macrophages. Time-lapse multicolor total internal reflection microscopy was used to capture the formation of FcγR-IgG microclusters and their assembly into signaling complexes on the plasma membrane of murine bone marrow derived macrophages.ResultsUpon antibody binding, macrophages formed FcγR-IgG complexes at the leading edge of advancing pseudopods. These complexes then moved toward the center of the cell to form a structure reminiscent of the supramolecular complex observed in the T-cell/antigen presenting cell immune synapse. Colocalization of signaling protein Syk with nascent clusters of antibodies indicated that phosphorylated receptor complexes underwent maturation as they trafficked toward the center of the cell. Additionally, imaging of fluorescent BtkPH domains indicated that 3′-phosphoinositides propagated laterally away from the FcγR microclusters.ConclusionWe demonstrate that surface-associated but mobile IgG induces the formation of FcγR microclusters at the pseudopod leading edge. These clusters recruit Syk and drive the production of diffusing PI(3,4,5)P3 that is coordinated with lamellar actin polymerization. Upon reaching maximal extension, FcγR microclusters depart from the leading edge and are transported to the center of the cellular contact region to form a synapse-like structure, analogous to the process observed for T-cell receptors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-016-0143-2) contains supplementary material, which is available to authorized users.

Membrane-cytoskeleton mechanical feedback mediated by myosin-I controls phagocytic efficiency

“…We tested whether these myosin 1-actin puncta were associated with FcR clusters by transfecting cells with EGFP-FcgRIIA. As has been previously observed, macrophages spreading on IgG formed discrete FcR clusters 41,42 . We found that myo1f and FcgRIIA distinctly colocalized at these structures ( Fig.…”

HEV ORF3 downregulatesCD14 and CD64 to impair macrophages phagocytosis through inhibiting JAK/STAT pathway

“…CD14 is one of the most common phagocytic receptors on the surface of macrophages, which is the coreceptor for LPS and can mediate the innate immune response to LPS via MyD88, TIRAP, and TRAF6 . CD64 is one member of the Fc gamma receptor (FcγR) family, which can activate the phagocytosis through recognizing bacteria and viruses opsonized by IgG …”

The “Phagocytic Synapse” and Clearance of Apoptotic Cells

“…Following phagocyte contact with IgG-coated surfaces or on supported lipid bilayers, formation of FcγRII nanoclusters suggests activation-driven organization of receptor redistribution ( 75 ). FcγR clusters are localized in front of ruffles on extending pseudopods, with rapid recruitment of Syk to advancing pseudopods and subsequent retrograde movement toward the cell center ( 76 ). PI3K co-localized with actin around FcγR clusters, suggestive of signal propagation from FcγR and consistent with PI3K-dependent control of actin cytoskeletal rearrangements ( 76 ).…”

“…FcγR clusters are localized in front of ruffles on extending pseudopods, with rapid recruitment of Syk to advancing pseudopods and subsequent retrograde movement toward the cell center ( 76 ). PI3K co-localized with actin around FcγR clusters, suggestive of signal propagation from FcγR and consistent with PI3K-dependent control of actin cytoskeletal rearrangements ( 76 ).…”