Background

In vitro reconstitution of biological processes is important to understand the molecular components and mechanisms underlying them. One such process is phagosome maturation, which is involved in degradation of pathogens taken up by macrophage cells of the immune system, and is also used as a process of nutrition in lower eukaryotes (Vieira et al., 2002). The transport of phagosomes on microtubules is intimately connected to their maturation (Blocker et al., 1997; Vieira et al., 2002). Notably, several intracellular pathogens disrupt phagosome transport to survive in a latent form inside cells (Harrison et al., 2004; Harrison and Grinstein, 2002; Rai et al., 2016; Sun et al., 2007). Therefore, reconstitution of phagosome transport might help to understand the strategy used by pathogens for immune evasion. Here, we describe a detailed protocol to prepare LBPs from cell extracts of the social amoeba Dictyostelium discoideum. This protocol has been adapted and modified from the work by Gotthard et al. (2006). Briefly, the cells are pulsed with latex beads and chased for different time durations to enable either early or late phagosome formation. Such phagosomes are buoyant in nature and float away from other endogenous vesicles when spun at high speeds. These phagosomes, collected along with the cytosol, show robust motion on in vitro polymerized microtubules. A detailed version of this protocol has also been published elsewhere (Barak et al., 2014). Using this method, we have recently shown cholesterol as a key regulator of phagosome transport and maturation (Rai et al., 2016). Furthermore, this assay has helped us to elucidate the mechanism of disruption of phagosome transport by lipophosphoglycan (LPG) from the parasite Leishmania donovani. A description of the phagosome extract preparation from Dictyostelium cells is detailed below. This protocol describes only the purification of LBPs. The in vitro motility assay has been described elsewhere (Barak et al., 2014).