A guide to measuring phagosomal dynamics

Abstract

Phagocytosis is an essential mechanism for immunity and homeostasis, performed by a subset of cells known as phagocytes. Upon target engulfment, de novo formation of specialized compartments termed phagosomes takes place. Phagosomes then undergo a series of fusion and fission events as they interact with the endolysosomal system and other organelles, in a dynamic process known as phagosome maturation. Because phagocytes play a key role in tissue patrolling and immune surveillance, phagosome maturation is associated with signaling pathways that link phagocytosis to antigen presentation and the development of adaptive immune responses. In addition, and depending on the nature of the cargo, phagosome integrity may be compromised, triggering additional cellular mechanisms including inflammation and autophagy. Upon completion of maturation, phagosomes enter a recently described phase: phagosome resolution, where catabolites from degraded cargo are metabolized, phagosomes are resorbed, and vesicles of phagosomal origin are recycled. Finally, phagocytes return to homeostasis and become ready for a new round of phagocytosis. Altogether, phagosome maturation and resolution encompass a series of dynamic events and organelle crosstalk that can be measured by biochemical, imaging, photoluminescence, cytometric, and immune-based assays that will be described in this guide.

Cross-Modal Imaging Reveals Nanoparticle Uptake Dynamics in Hematopoietic Bone Marrow during Inflammation

“…Our research began with validating and measuring the effective labeling of CLIO-AF647 nanoparticles by macrophages in vitro. In accordance with previous studies, 30 we found that CLIO-AF647 nanoparticles fuse with lysosomes when internalized by macrophages. We further optimized the detection capabilities of the MRI and fluorescence imaging modalities.…”

Irregular particle morphology and membrane rupture facilitate ion gradients in the lumen of phagosomes

“…2 A and S2 A). To improve the uptake efficiency, the particles were opsonized with rabbit immunoglobulin (Ig) G raised against streptavidin, which facilitates Fc-receptor-mediated uptake by human macrophages and DCs ( 15 ) ( Figs. 2 A and S2 A).…”

VAMP5 promotes Fcγ receptor-mediated phagocytosis and regulates phagosome maturation in macrophages

“…The disappearance of Myc-VAMP5 from the phagosome could be due to pinching off by tubulation/scission or vesiculation and (or) internalization within the phagosome by inward budding via intraluminal vesicle for degradation ( Fairn and Grinstein, 2012 ; Lancaster et al. , 2021 ; Levin-Konigsberg and Mantegazza, 2021 ). Our microscopic analysis failed to detect any Myc-VAMP5 signal within phagosomes where the surface Myc-VAMP5 signal had already disappeared ( Figure 4B ; Supplemental Figure S6).…”