title: Live-Cell Imaging of the Estrogen Receptor by Total Internal Reflection Fluorescence Microscopy.
abstract: Trafficking studies of plasma membrane-localized intracellular estrogen receptors have mainly relied on biochemical and histological techniques to locate the receptor before and after estradiol stimulation. More often than not these experiments were performed using postmortem, lysed, or fixed tissue samples, whose tissue or cellular structure is typically severely altered or at times completely lost, making the definitive localization of estrogen receptors difficult to ascertain. To overcome this limitation we began using total internal reflection fluorescence microscopy (TIRFM) to study the trafficking of plasma membrane estrogen receptors. This real-time imaging approach, described in this chapter, permits observation of live, intact cells while allowing visualization of the steps (in time and spatial distribution) involved in receptor activation by estradiol and movements on and near the membrane. TIRFM yields high-contrast real-time images of fluorescently labeled E6BSA molecules on and just below the cell surface and is ideal for studying estrogen receptor trafficking in living cells.
authors.affiliation: Department of Physiology and Biophysics, and the Zilkha Neurogenetic Institute, Keck School of Medicine of the University of Southern California, 1501 San Pablo St., ZNI 323, Los Angeles, CA, 90033, USA.
authors.forename: Kassandra
authors.initials: K
authors.lastname: Kisler
authors.affiliation: Department of Physiology and Biophysics, Keck Schoolof Medicine of the University of Southern California, Los Angeles, CA, USA. reymundd@usc.edu.|The Zilkha Neurogenetic Institute, Keck School of Medicine of the University of Southern California, Los Angeles, CA, USA. reymundd@usc.edu.
authors.forename: Reymundo
authors.initials: R
authors.lastname: Dominguez
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D004958:Estradiol / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000819:agonists / Q000378:metabolism*; D004967:Estrogens / Q000494:pharmacology; D007090:Image Interpretation, Computer-Assisted; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D021381:Protein Transport; D013997:Time Factors; D057188:Workflow
chemical_list: D047628:Estrogen Receptor alpha; D004967:Estrogens; D004958:Estradiol
keywords: E6BSA-FITC; ERα; Endocytosis; Estrogen action; Estrogen receptor alpha; Membrane-initiated estradiol signal ing; Plasma membrane estrogen receptor s; TIRFM; Total internal reflection fluorescence microscopy; Trafficking