title: Rapamycin-based inducible translocation systems for studying phagocytosis.
abstract: Phagocytosis is an immune receptor-mediated process whereby cells engulf large particles. The process is dynamic and requires several localized factors acting in concert with and sequentially after the engagement of immune receptors to envelope the particle. Once the particle is internalized, the nascent -phagosome undergoes a series of events leading to its maturation to the microbicidal phagolysosome. Investigating these dynamic and temporally controlled series of events in live cells requires noninvasive methods. The ability to rapidly recruit the proteins of interest to the sites of phagocytosis or to nascent phagosomes would help dissect the regulatory mechanisms involved during phagocytosis. Here, we describe a general approach to express in RAW264.7 murine macrophages, a genetically encoded rapamycin--induced heterodimerization system. In the presence of rapamycin, tight association between FK506-binding protein (FKBP) and FKBP rapamycin-binding protein (FRB) is observed. Based on this principle, a synthetic system consisting of a targeting domain attached to FKBP can recruit a protein of interest fused to FRB upon the addition of rapamycin. Previously, this technique has been used to target lipid-modifying enzymes and small GTPases to the phagosome or plasma membrane. The recruitment of the FRB module can be monitored by fluorescent microscopy if a fluorescent protein is fused to the FRB sequence. While the focus of this chapter is on phagocytic events, this method can be employed to study any organelle of interest when the appropriate targeting sequence is used.
authors.affiliation: Program in Cell Biology, The Hospital for Sick Children,Toronto, Ontario, Canada.
authors.forename: Michal
authors.initials: M
authors.lastname: Bohdanowicz
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Fairn
mesh_terms: D000818:Animals; D002460:Cell Line; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010587:Phagocytosis / Q000235:genetics / Q000502:physiology*; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D012756:Sheep; D020123:Sirolimus / Q000494:pharmacology*; D022021:Tacrolimus Binding Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D022021:Tacrolimus Binding Proteins; D020123:Sirolimus