FcγR-mediated phagocytosis is a cellular event that is evolutionary conserved to digest IgG-opsonized pathogens. Pseudopod formation during phagocytosis is a limiting step in managing the uptake of particles, and in this paper, we show that the conventional kinesin is involved in both receptor and membrane delivery to the phagocytic cup. Expression of a mutant kinesin isoform (GFP dominant negative mutant of kinesin H chain [EGFP-Kif5B-DN]) in RAW264.7 cells significantly reduced binding of IgG–sheep RBCs when macrophages were faced with multiple encounters with opsonized particles. Scanning electron microscopy analysis of EGFP-Kif5B-DN–expressing cells challenged with two rounds of IgG–sheep RBCs showed sparse, extremely thin pseudopods. We saw disrupted Rab11 trafficking to the phagocytic cup in EGFP-Kif5B-DN–transfected cells. Our particle overload assays also implicated phagosome membrane recycling in pseudopod formation. We observed reduced phagosome fission and trafficking in mutant kinesin-expressing cells, as well as reduced cell surface expression of FcγRs and Mac-1 receptors. In conclusion, anterograde trafficking via kinesin is essential for both receptor recycling from the phagosome and delivery of Rab11-containing membrane stores to effect broad and functional pseudopods during FcγR-mediated phagocytosis.
The coordinated effort of cells in the immune system relies heavily on surface receptor interactions. Immune receptor mobility provides vital information on the function and responses of immune cells, and these measurements shed light on their interactions with other membrane, cytosolic, and extracellular matrix proteins. These measurements can be obtained using the fluorescence recovery after photobleaching (FRAP) technique in living cells. We describe here general approaches for FRAP using green fluorescent protein fusion proteins.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.