Jcb_201702028 871..874

871 The Rockefeller University Press $30.00 J. Cell Biol. Vol. 216 No. 4 871–873 https://doi.org/10.1083/jcb.201702028 When leucocytes encounter ligand-bearing targets, tens to hundreds of receptors of different types are triggered, leading to downstream signaling. How receptor triggering occurs is uncertain (van der Merwe and Dushek, 2011), and even less is known about how the distinct signals the receptors produce are integrated, allowing “go/no-go” response choices to be made. What is clear is that many of the receptors have antagonistic effects, i.e., some receptors are directly activating, whereas others are tasked with suppressing the activators. In new work appearing in this issue, Lopes et al. make the case that signal integration, in macrophages at least, relies on the nanometer-scale (or nanoscopic) reorganization of local groups or nanoclusters (NCs) of receptors (Lopes et al., 2017). Cleverly, Lopes et al. (2017) reduce the problem of signal integration to its simplest form. They study the interplay between an activating receptor (Fcγ receptor I [FcγRI]) and an inhibitory regulator, signal reduction protein α (SIRPα); together, these molecules play a major role in controlling macrophage activation and phagocytosis (Barclay and van den Berg, 2014; Getahun and Cambier, 2015). FcγRI binds to pathogen-immobilized antibodies, leading to receptor phosphorylation by, for example, the Src-family kinases (Fig. 1 A), and the recruitment of activating downstream signaling effectors. In contrast, the SIRPα receptor binds not to antibodies but to a “don’t eat me” signaling ligand expressed by most human cells called CD47 (Barclay and van den Berg, 2014). The SIRPα/CD47 axis is such an important modulator of macrophage function that it is now a promising target for cancer immunotherapy (Ngo et al., 2016). CD47 engagement also results in SIRPα phosphorylation, but in this case SIRPα recruits a phosphatase, SHP-1, that reduces FcγRI-dependent signaling, perhaps by acting directly on the receptor (Barclay and van den Berg, 2014). Using this system, Lopes et al. (2017) set out to observe first how the receptors are organized in the nonactivated state, and then how this changes when one or both receptors are bound to ligands. Dissecting the complex interplay of these receptors required observational methods that were up to the task. The direct imaging of signaling responses in immune cells is challenging, however. Typical problems are: adequate labeling of the proteins of interest; controlling the initiation of responses so that imaging can be performed in good time; achieving the time and spatial resolution necessary for unraveling molecular reorganization; and obtaining good statistics. Lopes et al. (2017) sought to control the triggering status of their human macrophages by plating them onto either poly-l-lysine– (nonactivating) or human IgG (activating)–coated microscope cover glass surfaces and fixing the cells after a predetermined interval (10–30 min). Although the signaling effects of glass substrates (Chang et al., 2016) might otherwise have prompted some degree of skepticism, Lopes et al. (2017) were able to reprise their main findings using macrophages settled onto nonactivating and ligand-presenting supported lipid bilayers. The use of specific fluorescent primary antibodies in combination with multicolor superresolution optical microscopy (direct stochastic optical reconstruction microscopy [dSTO RM]) and careful image analysis allowed molecular reorganization to be observed down to 50-nm spatial resolution with high statistical confidence. The authors also took care to avoid the overcounting of blinking fluorophores, a very important control when using dSTO RM (Baumgart et al., 2016). Although some of the organizational changes observed were rather subtle, the use of rigorous controls and state-of-the-art image analyses coupled with data simulation allowed Lopes et al. (2017) to conclude that the changes were real. What Lopes et al. (2017) found first is that FcγRI and SIRPα form NCs under both nonactivating and activating conditions that were ∼40–70 nm in diameter, engaged 75–80% of all the receptors, and were distributed at a density of 3–6 NCs/ μm2. Only minor changes were observed for activating versus nonactivating conditions, however. NC sizes slightly decreased (SIRPα) or remained constant (FcγRI), with the fraction of NC-associated molecules increasing by a small amount and the density slightly decreasing. Although the change in NC density was attributed to increased internalization of both receptors (as measured by flow cytometry), no functional sequelae were attributed to this or the other changes. The main finding, therefore, was that nanoclustering of FcγRI (and SIRPα) is constitutive, in the manner claimed for many other receptors, but contrary to previous work on Fc receptors (Jaumouillé et al., 2014). How cells integrate antagonistic receptor signaling events is enigmatic. Using superresolution optical microscopy, Lopes et al. (2017. J. Cell Biol. https ://doi .org /10 .1083 /jcb .201608094) demonstrate the nanometer-scale molecular reorganization of antagonistic signaling receptors in macrophages, after engagement by the receptors of activating and inhibitory ligands. They propose that large-scale rearrangements of this type underpin decision-making by these cells. Macrophages: micromanagers of antagonistic signaling nanoclusters