Heterogeneous but not random: Cargo degradation in phagosomes is kinetically coupled to their intracellular mobility

Abstract

Phagosome maturation, in which ingested pathogens are degraded through a complex sequence of biochemical changes, is an essential process in infection control and tissue homeostasis. The maturation of phagosomes requires their intracellular transport. However, mechanisms underlying this dynamical-biochemical interdependence is poorly understood due to the lack of methods to independently manipulate either process. Here we present a study to magnetically manipulate the transport of single phagosomes and simultaneously measure their maturation and degradative functions. We reveal that the transport velocity of phagosomes acts as a maturation clock to regulate the timing of biochemical signaling activities in phagosomes. We show that velocity of phagosomes on microtubules determines their rate of fusion with lysosomes and consequently the rate of lumen acidification, following a linear relationship. Meanwhile, the timing of phagosome transport from actin cortex to microtubules controls the kinetics of early phagosome assembly and rate of transition to late phagosomes.

Anisotropic presentation of ligands on cargos modulates degradative function of phagosomes

“…S1 ). The SAv serves as a cushion layer to separate the pHrodo Red dye from the particle surface, as we observed that pHrodo Red becomes insensitive to pH changes when directly conjugated on particles ( 25 ). From fluorescence images, we confirmed the anisotropic presentation of SAv and IgG on pHMoon particles ( Fig.…”

“…At each pH, the fluorescence emission of pHrodo Red and CF640R was acquired using epifluorescecnce microscopy. Intracellular pH calibration of pH sensors was done following a previously published protocol ( 25 , 26 ). RAW 264.7 cells stably expressing actin-GFP were seeded as described above.…”

“…Fluorescence emission of pHrodo Red and reference dye CF640R was obtained at each pH using epifluorescecnce microscopy. Fluorescence intensity of individual pHMoon Sensors was analyzed using single-particle tracking Matlab algorithm ( 25 , 27 ) with some modifications. In live-cell acidification measurements, an internal calibration curve was generated for individual pH sensors based on their I pHrodoRed / I CF640R at pH 7.2 in cell and at pH 4.5 after potassium-rich buffer calibration.…”

“…In live-cell acidification measurements, an internal calibration curve was generated for individual pH sensors based on their I pHrodoRed / I CF640R at pH 7.2 in cell and at pH 4.5 after potassium-rich buffer calibration. This helped to eliminate the potential effect of particle-to-particle variation in pH calibration ( 25 , 26 ). To measure pHMoon intensity as a function of particle orientation, pHMoon Sensors were suspended in a mixture of glycerol and PBS buffer (1:1 v/v).…”