An In Vitro System to Analyze Generation and Degradation of Phagosomal Phosphatidylinositol Phosphates.

Abstract

Phagosomes are formed when phagocytic cells take up large particles, and they develop into phagolysosomes where the particles are degraded. The transformation of nascent phagosomes into phagolysosomes is a complex multi-step process, and the precise timing of these steps depends at least in part on phosphatidylinositol phosphates (PIPs). Some such-called "intracellular pathogens" are not delivered to microbicidal phagolysosomes and manipulate the PIP composition of the phagosomes they reside in. Studying the dynamic changes of the PIP composition of inert-particle phagosomes will help to understand why the pathogens' manipulations reprogram phagosome maturation.We here describe a method to detect and to follow generation and degradation of PIPs on purified phagosomes. To this end, phagosomes formed around inert latex beads are purified from J774E macrophages and incubated in vitro with PIP-binding protein domains or PIP-binding antibodies. Binding of such PIP sensors to phagosomes indicates presence of the cognate PIP and is quantified by immunofluorescence microscopy. When phagosomes are incubated with PIP sensors and ATP at a physiological temperature, the generation and degradation of PIPs can be followed, and PIP-metabolizing enzymes can be identified using specific inhibitory agents.