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Live-Cell Imaging of the Estrogen Receptor by Total Internal Reflection Fluorescence Microscopy

  • Kassandra Kisler
  • Reymundo Dominguez
Part of the Methods in Molecular Biology book series (MIMB, volume 1366)

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.

Key words

Estrogenaction Estrogenreceptoralpha ERα Membrane-initiated estradiol signaling Plasma membrane estrogen receptors Total internal reflection fluorescence microscopy TIRFM Trafficking Endocytosis E6BSA-FITC 

Notes

Acknowledgements

This work was supported by a University of Southern California Provost’s Award to RD. Thanks to Dr. Robert H. Chow for support during the experimental studies described.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Physiology and Biophysics, and the Zilkha Neurogenetic InstituteKeck School of Medicine of the University of Southern CaliforniaLos AngelesUSA
  2. 2.Department of Physiology and BiophysicsKeck Schoolof Medicine of the University of Southern CaliforniaLos AngelesUSA
  3. 3.The Zilkha Neurogenetic InstituteKeck School of Medicine of the University of Southern CaliforniaLos AngelesUSA

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