title: Live cell imaging of Argonaute proteins in mammalian cells.
abstract: The central effector of mammalian RNA interference (RNAi) is the RNA-induced silencing complex (RISC). Proteins of the Argonaute family are the core components of RISC. Recent work from multiple laboratories has shown that Argonaute family members are associated with at least two types of cytoplasmic RNA granules: GW/Processing bodies and stress granules. These Argonaute-containing granules harbor proteins that function in mRNA degradation and translational repression in response to stress. The known role of Argonaute proteins in miRNA-mediated translational repression and siRNA-directed mRNA cleavage (i.e., Argonaute 2) has prompted speculation that the association of Argonautes with these granules may reflect the activity of RNAi in vivo. Accordingly, studying the dynamic association between Argonautes and RNA granules in living cells will undoubtedly provide insight into the regulatory mechanisms of RNA-based silencing. This chapter describes a method for imaging fluorescently tagged Argonaute proteins in living mammalian cells using spinning disk confocal microscopy.
authors.affiliation: Department of Cell Biology, Li Ka Shing Institute of Virology, University of Alberta, Edmonton, AB, Canada
authors.forename: Justin M
authors.initials: JM
authors.lastname: Pare
authors.forename: Joaquin
authors.initials: J
authors.lastname: Lopez-Orozco
authors.forename: Tom C
authors.initials: TC
authors.lastname: Hobman
mesh_terms: D060565:Argonaute Proteins; D018053:Arsenites / Q000494:pharmacology; D002460:Cell Line; D004249:DNA Damage / Q000187:drug effects; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D057054:Molecular Imaging / Q000295:instrumentation; D014176:Protein Biosynthesis / Q000187:drug effects / Q000235:genetics; D013723:Teratogens / Q000494:pharmacology; D014162:Transfection
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; D018053:Arsenites; D015852:Eukaryotic Initiation Factor-2; D013723:Teratogens; C015001:arsenite