JCB_201608094 1123..1142

Macrophages play a key role in immune defenses by phagocytosis of pathogens and other harmful foreign particles. Phagocytosis can be initiated by the binding of Fcγ receptors (FcγRs) to the constant Fc domain of IgG molecules on opsonized particles. This interaction induces lateral clustering of FcγRs (Jaumouillé and Grinstein, 2011) and phosphorylation by Src-family kinases (SFKs) of the immunoreceptor tyrosine-based activation motif (ITAM) present in the receptor’s cytosolic domain or on an associated common γ-chain (Ghazizadeh et al., 1994; Wang et al., 1994). Phosphorylation of the ITAM induces reorganization of the actin cytoskeleton, formation of a phagocytic synapse, and engulfment of the target particle (Flannagan et al., 2012). FcγRs are presumed to exist as monomers and be evenly distributed on the cell surface (Jaumouillé et al., 2014). Upon interaction with their ligand, FcγRs are thought to diffuse through the surface membrane to accumulate around a particle by a zipper-like mechanism (Griffin et al., 1975). Serial binding of Fc receptors to ligands extends a pseudopod along the particle, and integrins aid its progression and closure of the phagocytic cup (Freeman et al., 2016). Triggering of this process is dependent on the balance of signals from specific activating and inhibitory receptors. Signal regulatory protein α (SIRPα) negatively regulates macrophage phagocytosis (Fujioka et al., 1996; Veillette et al., 1998; Jiang et al., 1999; Oldenborg et al., 2001; Okazawa et al., 2005) by interacting with CD47, essentially a “marker of self” (Oldenborg et al., 2000). Ligation of the commonly expressed form of CD47 results in phosphorylation of the immunoreceptor tyrosine-based inhibition motif (ITIM) on the cytoplasmic tail of SIRPα, which leads to the recruitment of the tyrosine phosphatase SHP-1. This blocks phagocytosis, at least in part by preventing the accumulation of myosin-II A at the phagocytic synapse (Tsai and Discher, 2008). Recently, superresolution microscopy has established that many receptors are clustered at the plasma membrane on a nanometer scale (Garcia-Parajo et al., 2014) and that the nanoscale organization of T cell, natural killer cell, or B cell surfaces changes when cells are activated (Lillemeier et al., 2010; Mattila et al., 2013; Pageon et al., 2013; Kläsener et al., 2014; Oszmiana et al., 2016). In macrophages, confocal fluorescence microscopy has established that both the inhibitory receptor SIRPα and the activating FcγRI accumulate at the phagocytic Signal integration between activating Fc receptors and inhibitory signal regulatory protein α (SIRPα) controls macrophage phagocytosis. Here, using dual-color direct stochastic optical reconstruction microscopy, we report that Fcγ receptor I (FcγRI), FcγRII, and SIRPα are not homogeneously distributed at macrophage surfaces but are organized in discrete nanoclusters, with a mean radius of 71 ± 11 nm, 60 ± 6 nm, and 48 ± 3 nm, respectively. Nanoclusters of FcγRI, but not FcγRII, are constitutively associated with nanoclusters of SIRPα, within 62 ± 5 nm, mediated by the actin cytoskeleton. Upon Fc receptor activation, Src-family kinase signaling leads to segregation of FcγRI and SIRPα nanoclusters to be 197 ± 3 nm apart. Co-ligation of SIRPα with CD47 abrogates nanocluster segregation. If the balance of signals favors activation, FcγRI nanoclusters reorganize into periodically spaced concentric rings. Thus, a nanometerand micron-scale reorganization of activating and inhibitory receptors occurs at the surface of human macrophages concurrent with signal integration. Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages