Unit

UNIT 21.7 Probing Endoplasmic Reticulum Dynamics using Fluorescence Imaging and Photobleaching Techniques

  1. Lindsey Costantini,
  2. Erik Snapp

Published Online: 24 SEP 2013

DOI: 10.1002/0471143030.cb2107s60

Current Protocols in Cell Biology

Current Protocols in Cell Biology

How to Cite

Costantini, L. and Snapp, E. 2013. Probing Endoplasmic Reticulum Dynamics using Fluorescence Imaging and Photobleaching Techniques. Current Protocols in Cell Biology. 60:21.7:21.7.1–21.7.29.

Author Information

  1. Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York

Publication History

  1. Published Online: 24 SEP 2013

Abstract

This unit describes approaches and tools for studying the dynamics and organization of endoplasmic reticulum (ER) membranes and proteins in living cells using fluorescence microscopy. The ER plays a key role in secretory protein biogenesis, calcium regulation, and lipid synthesis. However, study of these processes has often been restricted to biochemical assays that average millions of lysed cells or imaging of static fixed cells. With new fluorescent protein (FP) reporter tools, sensitive commercial microscopes, and photobleaching techniques, investigators can interrogate the behaviors of ER proteins, membranes, and stress pathways in single live cells. Solutions are described for imaging challenges relevant to the ER, including the mobility of ER membranes, a range of ER structures, and the influence of post-translational modifications on FP reporters. Considerations for performing photobleaching assays for ER proteins are discussed. Finally, reporters and drugs for studying misfolded secretory protein stress and the unfolded protein response are described. Curr. Protoc. Cell Biol. 60:21.7.1-21.7.29. © 2013 by John Wiley & Sons, Inc.

Keywords:

  • FRAP;
  • FLIP;
  • confocal microscopy;
  • live cell imaging;
  • superfolder green fluorescent protein;
  • diffusion;
  • membrane;
  • tubule;
  • microtubule