Open AccessProbing Endoplasmic Reticulum Dynamics using Fluorescence Imaging and Photobleaching Techniques
Author(s) -
Costantini Lindsey,
Snapp Erik
Publication year - 2013
Publication title -
current protocols in cell biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.149
H-Index - 38
eISSN - 1934-2616
pISSN - 1934-2500
DOI - 10.1002/0471143030.cb2107s60
Subject(s) - endoplasmic reticulum , fluorescence recovery after photobleaching , photobleaching , fluorescence , biophysics , dynamics (music) , chemistry , fluorescence lifetime imaging microscopy , physics , biology , biochemistry , optics , acoustics
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.