Biochemically Reconstituted Fusion of Phagosomes with Endosomes and Lysosomes

Abstract

Professional phagocytic cells, such as macrophages, ingest large particles into a specialized endocytic compartment, the phagosome, which eventually turns into a phagolysosome and degrades its contents. This phagosome "maturation" is governed by successive fusion of the phagosome with early sorting endosomes, late endosomes, and lysosomes. Further changes occur by fission of vesicles from the maturing phagosome and by on-and-off cycling of cytosolic proteins. We present here a detailed protocol which allows to reconstitute in a cell-free system the fusion events between phagosomes and the different endocytic compartments. This reconstitution can be used to define the identity of, and interplay between, key players of the fusion events.

Keywords: Macrophages; Membrane fusion; Phagocytes; Phagocytosis; Phosphoinositide lipids; Rab GTPases; SNARE proteins; Tethering factors.