Biogenesis of phagolysosomes proceeds through a sequential series of interactions with the endocytic apparatus

TLDR

The authors isolated low‑density latex‑bead phagosomes by sucrose gradient flotation and characterized their composition over time using two‑dimensional gel electrophoresis, immunocytochemistry, and biochemical assays to track interactions with endocytic organelles. They found that phagosomes sequentially acquire and lose markers—including Rab GTPases and Lamp proteins—during maturation, with Lamp‑2 transfer being microtubule‑dependent and rapid contacts with late endocytic components indicating a highly dynamic, regulated phagolysosome formation process.

Abstract

We have examined the modifications occurring during the transformation of phagosomes into phagolysosomes in J-774 macrophages. The use of low density latex beads as markers of phagosomes (latex bead compartments, LBC) allowed the isolation of these organelles by flotation on a simple sucrose gradient. Two-dimensional gel electrophoresis, immunocytochemistry, and biochemical assays have been used to characterize the composition of LBC at different time points after their formation, as well as their interactions with the organelles of the endocytic pathway. Our results show that LBC acquire and lose various markers during their transformation into phagolysosomes. Among these are members of the rab family of small GTPases as well as proteins of the lamp family. The transfer of the LBC of lamp 2, a membrane protein associated with late endocytic structures, was shown to be microtubule dependent. Video-microscopy showed that newly formed phagosomes were involved in rapid multiple contacts with late components of the endocytic pathway. Collectively, these observations suggest that phagolysosome formation is a highly dynamic process that involves the gradual and regulated acquisition of markers from endocytic organelles.