title: Visualizing and quantitating sequence-dependent GPCR recycling.
abstract: Recent advances in direct imaging have given us a new appreciation of the spatial and temporal dynamics of membrane trafficking processes, and have allowed us to ask questions that were difficult to address with traditional methods. A relevant example of this is protein sorting in the endosome, which serves as the primary sorting station for proteins internalized from the cell surface. In this chapter, we discuss fluorescence imaging protocols to directly visualize and quantitate the recycling of G protein-coupled receptors (GPCRs)-a highly physiologically relevant family of signaling receptors-in real time in living cells. The protocols allow direct visualization and quantitation of both GPCR exit from the endosome and GPCR delivery to the cell surface. The methods may be extended to study the endolysosomal sorting of many proteins that undergoes endocytic cycling, and may be adapted to other organelles and systems where proteins are sorted.
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA.
authors.forename: Shanna L
authors.initials: SL
authors.lastname: Bowman
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA.
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Soohoo
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA.
authors.forename: Manojkumar A
authors.initials: MA
authors.lastname: Puthenveedu
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals; D021381:Protein Transport; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism*; D059008:Time-Lapse Imaging
chemical_list: D021382:Protein Sorting Signals; D043562:Receptors, G-Protein-Coupled
keywords: Endosome; G protein-coupled receptor; Live cell imaging; Recycling; Total internal reflection fluorescence microscopy