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Significance Phagocytic receptors collaboratively regulate innate immune responses against infections. It has been proposed that receptor synergy depends on their spatial distribution on phagosomes. However, studies to reveal the relationship between phagocytic receptor organization and their collaborative signaling have been impeded by the lack of methods to access receptors on the intracellular membranes of organelles. Here, we report a technique to directly manipulate the spatial organization of receptors and signaling proteins on phagosomes inside living cells. We provide direct evidence demonstrating that Dectin-1 and TLR2 must be within nanoscale proximity on phagosomes to elicit synergistic antifungal responses in macrophages. This approach delineates receptor interactions and signaling through physical manipulation and is applicable to other phagocytic receptors of innate immune cells.

Geometrical reorganization of Dectin-1 and TLR2 on single phagosomes alters their synergistic immune signaling