Probing Endoplasmic Reticulum Dynamics using Fluorescence Imaging and Photobleaching Techniques

Lindsey Costantini1, Erik Snapp1

1 Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 21.7
DOI:  10.1002/0471143030.cb2107s60
Online Posting Date:  September, 2013
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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

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Imaging Endoplasmic Reticulum Structures in Fixed Cells
  • Alternate Protocol 1: Imaging Endoplasmic Reticulum Structures in Live Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Imaging Endoplasmic Reticulum Structures in Fixed Cells

  Materials
  • Cultures of adherent cells or cells in suspension expressing fluorescent protein reporter or ER‐specific antigen (see Strategic Planning)
  • Imaging medium (see recipe)
  • Drug treatment stock solutions
    • 10 mg/ml A23187 (Sigma‐Aldrich, cat. no. C7522)/dimethyl sulfoxide (DMSO) or ethanol: store up to 6 months at −20°C
    • 5 mg/ml brefeldin A (Sigma‐Aldrich, cat. no. B7651)/ethanol: store up to 1 year at −20°C
    • 1 M dithiothreitol (DTT; Sigma‐Aldrich, cat. no. D0632)/deionized water: store up to 6 months at −20°C; avoid repeated freeze‐thawing
    • 5 mg/ml nocodazole (Sigma‐Aldrich, cat. no. M1404)/DMSO: store up to 1 year at −20°C
    • 1 M thapsigargin (Sigma‐Aldrich, cat. no. T9033)/DMSO: store up to 6 months at −20°C
    • 5 mg/ml tunicamycin (Tm; Sigma‐Aldrich, cat. no. T7765)/DMSO: store up to 1 year at −20°C; avoid repeated freeze‐thawing
  • Phosphate‐buffered saline (PBS, appendix 2A), warmed to the appropriate cell growth temperature
  • Fixative solution (see recipe)
  • Permeabilization solution (see recipe)
  • Blocking solution: 10% (v/v) fetal bovine serum (FBS)/PBS ( appendix 2A)
  • Primary antibody against ER antigen, e.g., rabbit anti‐PDI (ADI‐SPA‐890‐D; Assay Designs or Thermo Fisher Scientific) and secondary antibody labeled with fluorescent dye
  • Mattek glass‐bottom dishes or Labtek coverglass chambers (Nunc), or similar (treated with concentrated poly‐L‐lysine if using cells in suspension; see receipe)
  • 63× NA 1.4 oil plan apochromat objective and immersion oil supplied by the lens manufacturer
  • Confocal laser scanning (e.g., Carl Zeiss 710, Leica SP8, Olympus Fluoview 1000, or Nikon A1+), high‐speed confocal (e.g., Nikon C2+, Carl Zeiss Live, or Live Duoscan), spinning disk (e.g., Carl Zeiss Cell Observer SD or Olympus DSU), or wide‐field fluorescence microscope adapted for deconvolution (e.g., Deltavision, Applied Precision), with appropriate filter sets and illumination sources.
  • Microscope software, e.g., ImageJ (http://rsbweb.nih.gov/ij) or Volocity (Perkin Elmer) to create a three‐dimensional reconstructions
  • Additional reagents and equipment for carrying out confocal microscopy (unit 4.5), wide‐field fluorescence microscopy (unit 4.2), and fluorescence recovery after photobleaching (FRAP; unit 21.1)
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Figures

Videos

Literature Cited

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