title: Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis.
abstract: The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.
authors.affiliation: Department of Anesthesiology and Perioperative Care, University of California Irvine; Department of Biomedical and Molecular Sciences, Queen's University; edmund.ong@uci.edu.
authors.forename: Edmund
authors.initials: E
authors.lastname: Ong
authors.affiliation: Department of Anesthesiology and Perioperative Care, University of California Irvine; Department of Biomedical and Molecular Sciences, Queen's University; Department of Pharmacology, University of California Irvine.
authors.forename: Catherine
authors.initials: C
authors.lastname: Cahill
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D011992:Endosomes / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D008024:Ligands; D008081:Liposomes / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods*; D010784:Photomicrography / Q000379:methods*; D061251:Primary Cell Culture; D021381:Protein Transport / Q000187:drug effects; D043562:Receptors, G-Protein-Coupled / Q000032:analysis / Q000378:metabolism*; D011984:Sensory Receptor Cells / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D008024:Ligands; D008081:Liposomes; D043562:Receptors, G-Protein-Coupled