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Research into phagocytosis and host-pathogen interactions in the lower eukaryote Dictyostelium discoideum has flourished in recent years. This chapter presents a glimpse of where this research stands, with emphasis on the cell biology of the phagocytic process and on the wealth of molecular genetic data that have been gathered. The basic mechanistic machinery and most of the underlying genes appear to be evolutionarily conserved, reflecting the fact that phagocytosis arose as an efficient way to ingest food in single protozoan cells devoid of a rigid cell wall. In spite of some differences, the signal transduction pathways regulating phagosome biogenesis are also emerging as ultimately similar between Dictyostelium and macrophages. Both cell types are hosts for many pathogenic invasive bacteria, which exploit phagocytosis to grow intracellularly. We present an overwiew, based on the analysis of mutants, on how Dictyostelium contributes as a genetic model system to decipher the complexity of host-pathogen interactions.

Rab proteins are master regulators of vesicular membrane traffic of endocytic and exocytic pathways. They basically serve to recruit proteins and lipids required for vesicle formation, docking, and fusion. Each Rab protein is able to recruit one or more effectors, and, through the action of effectors, it drives its specific downstream functions. The Rab interacting lysosomal protein (RILP) is a common effector of Rab7 and Rab34, two Rab proteins implicated in the biogenesis of lysosomes. RILP is recruited onto late endosomal/lysosomal membranes by Rab7-GTP where it induces the recruitment of the dynein-dynactin motor complexes. Therefore, through the timed and selective dynein motor recruitment onto late endosomes and lysosomes, Rab7 and RILP control transport to endocytic degradative compartments. A similar role for Rab7 and RILP has been demonstrated also for phagosomes. Indeed, RILP recruits dynein-dynactin motors on Rab7-GTP-positive phagosomes and the recruitment not only displaces phagosomes centripetally, but also promotes the extension of phagosomal tubules toward late endocytic compartments. RILP is therefore a key protein for the biogenesis of lysosomes and phagolysosomes. This chapter describes how to express wild-type or mutated RILP in mammalian cells, and how to test the effects caused by RILP dysfunction. In particular, we report assays to monitor the interaction between RILP and Rab7, morphology and distribution of endosomes, and to measure degradation of endocytic markers.

Publisher Summary Rab5 is a 25-kDa GTP-binding protein localized to the plasma membrane, clathrin-coated vesicles, and early endosomes, and it functions as a regulatory factor of endocytosis. As for other Rab proteins, Rab5 is geranylgeranylated at its C terminus, and this modification is essential for its function. To obtain Rab5 in the isoprenylated form, the chapter makes use of a baculovirus expression system. This chapter describes a method to purify both posttranslationally modified and unmodified Rab5 from Spodoptera frugiperda (Sf9) insect cells overexpressing the protein. Purified posttranslationally modified and unmodified Rab5 protein efficiently binds GTP and GDP. However, as expected, Rab-GDI is active only on modified Rab5. When modified Rab5 complexed with Rab-GDI is introduced into permeabilized cells, Rab5 is localized to its correct site of function and induces the formation of enlarged early endosomes as previously observed in vivo , indicating that it is functionally active.

Publisher Summary Two different kinds of recombinant vaccinia viruses (VVs) have been used to express Rab GTPases. The first one contains the DNA coding for the Rab GTPase under the control of an early VV promoter. The second kind of recombinant VV that is being used contains the gene for phage T7 RNA polymerase under the control of a promoter that is active both at early and late stages of the viral infection. To explore the function of Rab GTPases, it is useful to express wild-type and mutant proteins in tissue culture cells and then investigate their effects on cell morphology and intracellular traffic. For example, overexpression of Rab5 alters the morphology of early endosomes and increases the rate of endocytosis, whereas the expression of negative mutants of Rab1 inhibits vesicular transport between the endoplasmic reticulum and the Golgi apparatus. VV, a large DNA virus, has become popular as a vector for transient expression of Rab proteins in mammalian cells because it infects a broad range of cells and results in high-level and rapid expression.