Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

Signal integration between activating Fc receptors and inhibitory signal regulatory protein alpha (SIRP alpha) controls macrophage phagocytosis. Here, using dual-color direct stochastic optical reconstruction microscopy, we report that Fc gamma receptor I (Fc gamma RI), Fc gamma RII, and SIRP alpha 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 Fc gamma RI, but not Fc gamma RII, are constitutively associated with nanoclusters of SIRPa, within 62 +/- 5 nm, mediated by the actin cytoskeleton. Upon Fc receptor activation, Src-family kinase signaling leads to segregation of Fc gamma RI and SIRPa nanoclusters to be 197 +/- 3 nm apart. Co-ligation of SIRPa with CD47 abrogates nanocluster segregation. If the balance of signals favors activation, Fc gamma RI nanoclusters reorganize into periodically spaced concentric rings. Thus, a nanometer- and micron-scale reorganization of activating and inhibitory receptors occurs at the surface of human macrophages concurrent with signal integration.