Immune cells degrade internalized pathogens in phagosomes through sequential biochemical changes. The degradation must be fast enough for effective infection control. The presumption is that each phagosome degrades cargos autonomously with a distinct but stochastic kinetic rate. However, here we show that the degradation kinetics of individual phagosomes is not stochastic but coupled to their intracellular motility. By engineering RotSensors that are optically anisotropic, magnetic responsive, and fluorogenic in response to degradation activities in phagosomes, we monitored cargo degradation kinetics in single phagosomes simultaneously with their translational and rotational dynamics. We show that phagosomes that move faster centripetally are more likely to encounter and fuse with lysosomes, thereby acidifying faster and degrading cargos more efficiently. The degradation rates increase nearly linearly with the translational and rotational velocities of phagosomes. Our results indicate that the centripetal motion of phagosomes functions as a clock for controlling the progression of cargo degradation.

The digestion of pathogens inside phagosomes by immune cells occurs through asequence of reactions including acidification and proteolysis,b ut howt he reactions are orchestrated in the right order is unclear due to al acko f methods to simultaneously measure more than one reaction in phagosomes.H ere we report ab ifunctional Janus-particle probe to simultaneously monitor acidification and proteolysis in single phagosomes in live cells.Eachprobe consists of apH reporter and aproteolysis reporter that are spatially separated but function concurrently.U sing the Janus probes,w ef ound the acidic pH needed to initiate and maintain proteolysis, revealing the mechanism for the sequential occurrence of both reactions during pathogen digestion. We showed howb acterium-derived lipopolysaccharides alter the acidification and proteolysis in phagosomes.T his study showcases Janusparticle probes as ag enerally applicable tool for monitoring multiple reactions in intracellular vesicles.

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