Measurement of Autophagic Activity in Mammalian Cells

Fiona M. Menzies1, Kevin Moreau1, Claudia Puri1, Maurizio Renna1, David C. Rubinsztein1

1 Cambridge Institute for Medical Research, Cambridge, United Kingdom
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 15.16
DOI:  10.1002/0471143030.cb1516s54
Online Posting Date:  March, 2012
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Abstract

Macroautophagy (referred to herein as autophagy) is a process in which cells engulf portions of cytoplasm in double‐membrane vesicles called autophagosomes. These autophagosomes can also capture protein oligomers associated with neurodegenerative diseases, infectious agents (like bacteria), and even organelles (like mitochondria). Autophagosomes are transported along microtubules towards the microtubule organizing center of cells, where the lysosomes are clustered. After fusion and content exchange with lysosomes, the autophagosome cargo is degraded by lysosomal hydrolases. This unit describes some of the core autophagy assays that are in common use, including LC3 immunoblotting, light microscopy analyses of different stages of autophagy, electron microscopy, and an assay of autophagy substrate accumulation. Autophagy assays should generally not be performed alone, but should be accompanied by complementary assays to enable robust interpretations. Curr. Protoc. Cell Biol. 54:15.16.1‐15.16.25. © 2012 by John Wiley & Sons, Inc.

Keywords: autophagy; LC3; microscopy; electron microscopy; Huntington's disease

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Assessment of Autophagic Flux by Measuring LC3‐II Levels in the Presence of Bafilomycin A1
  • Basic Protocol 2: Monitoring the Number of Autophagosomes and Autophagic Precursors by Microscopy
  • Basic Protocol 3: Identification of Autophagic Structures by Electron Microscopy
  • Basic Protocol 4: Use of Aggregate Number as a Measure of Protein Clearance
  • Support Protocol 1: Cell Culture
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Assessment of Autophagic Flux by Measuring LC3‐II Levels in the Presence of Bafilomycin A1

  Materials
  • Cultured cell line (e.g., HeLa cells) or primary culture of interest
  • Putative autophagy modulator to be tested
  • Bafilomycin A 1 (see recipe)
  • Phosphate‐buffered saline (PBS, appendix 2A), ice cold, with and without protease inhibitors (Complete EDTA‐free Protease Inhibitor Cocktail tablets, Roche Diagnostics, catalog no. 11 873 580)
  • Lysis buffer (see recipe)
  • Protein concentration assay kit (e.g., DC Protein Assay Kit, Bio‐Rad)
  • 3× sample loading buffer (see recipe)
  • Prestained molecular weight marker (e.g., SeeBlue Plus2, Invitrogen)
  • 12% SDS polyacrylamide gel (see recipe)
  • 1× running/transfer buffer (see recipe)
  • Blocking buffer: 6% (w/v) nonfat dry milk, 0.1% (v/v) Tween‐20 in 1× PBS
  • Primary antibodies: rabbit anti‐LC3 (Novus Biologicals), rabbit anti‐actin (Sigma‐Aldrich)
  • PBS‐T: PBS containing 0.1% (v/v) Tween 20
  • Secondary antibody: horseradish peroxidise (HRP)−conjugated anti−rabbit IgG (GE Healthcare)
  • ECL immunoblotting detection reagents (e.g., GE Healthcare)
  • 6‐well culture plates
  • Cell scraper
  • 1.5‐ml microcentrifuge tubes
  • PVDF membrane (Immobilon‐P; Millipore)
  • Extra‐thick filter paper (Bio‐Rad)
  • Protein transfer apparatus (e.g., Trans‐Blot SD semidry transfer cell, Bio‐Rad)
  • Plastic wrap (e.g., Saran Wrap)
  • Film cassette (e.g., Hypercassette, GE Healthcare)
  • High‐performance chemiluminescence film (e.g., Hyperfilm ECL, GE Healthcare)
  • Automated film developer (e.g., RP X‐OMAT Processor, Model M6B, Kodak)
  • Densitometer
  • Image J software (http://rsbweb.nih.gov/ij/applet/)
  • StatView 4.3 software (Abacus Concepts)
  • Additional reagents and equipment for SDS‐PAGE and immunoblotting (units 6.1& 6.2)

Basic Protocol 2: Monitoring the Number of Autophagosomes and Autophagic Precursors by Microscopy

  Materials
  • Cells to be tested: e.g., untransfected HeLa cells, HeLa cells stably expressing GFP‐LC3 (Bampton et al., ) or mRFP‐GFP‐LC3 (Kimura et al., ), or HEK cells stably expressing GFP‐DFCP1 (Axe et al., )
  • Overexpression plasmid: e.g., GFP‐Atg16L1 (Mizushima et al., ) or GFP‐LC3 (Kabeya et al., )
  • Transfection reagent (e.g., Lipofectamine 2000, Invitrogen, or TransIT2020; Miras Bio)
  • Hank's balanced salt solution (HBSS, Sigma)
  • Phosphate‐buffered saline (PBS, appendix 2A)
  • Fixative: 4% (w/v) paraformaldehyde in PBS (see recipe) or ice‐cold methanol
  • 0.1% Triton X‐100 in PBS
  • 10% fetal bovine serum (FBS) in PBS
  • Primary antibody:
    • Rabbit anti‐Atg16L1 (Cosmo Bio, catalog no. CAC‐TMD‐PH‐AT16L, or MBL International, catalog no. PM040, both at 1:100 dilution)
    • Rabbit anti‐Atg12 (Cell Signaling, catalog no. 2010, 1:200 dilution)
    • Mouse anti‐LC3 (NanoTools, 1:1000 dilution)
  • Secondary antibody: e.g., goat anti‐rabbit or anti‐mouse conjugated to Alexa Fluor 488 or 594 (Invitrogen)
  • ProLong Gold antifade reagent with DAPI (Invitrogen)
  • 13 × 13−mm coverslips
  • 6‐well culture plates
  • Cellomics ArrayScan VTI HCS Reader (ThermoScientific) or LSM 710 confocal microscope (Zeiss) and ImageJ software

Basic Protocol 3: Identification of Autophagic Structures by Electron Microscopy

  Materials
  • HeLa cells at 70% confluency in 10‐cm Petri dishes
  • Overexpression plasmid: e.g., GFP‐Atg16L1 (Mizushima et al., ), FLAG‐Atg16 (Cadwell et al., ), or GFP‐LC3 (Kabeya et al., )
  • Transfection reagent (e.g., Lipofectamine 2000, Invitrogen, or TransIT2020)
  • Hank's balance salt solution (HBSS, Sigma)
  • 4% paraformaldehyde/0.4% glutaraldehyde (see recipe)
  • Phosphate‐buffered saline (PBS, appendix 2A)
  • 0.2% (w/v) glycine in PBS
  • 12% (w/v) gelatin solution (see recipe)
  • 2.3 M sucrose solution (see recipe)
  • 15% PVP in 1.7 M sucrose (optional, see recipe)
  • Liquid nitrogen
  • 1% methylcellulose/1.15 M sucrose (see recipe)
  • 0.1% and 1% (w/v) bovine serum albumin (BSA) in PBS
  • Primary antibody: anti‐GFP (Invitrogen, whole serum, A‐6455, 1:100), anti‐FLAG (Sigma M2, 1:100), or anti‐Atg16L1 (Cosmo Bio, 1:25)
  • Protein A−gold (CMC‐Utrecht, NL)
  • 1% (w/v) glutaraldehyde in PBS
  • MilliQ‐purified water
  • 2% uranyl acetate solution, pH 7.0 (see recipe)
  • 1.8% methylcellulose/0.4% uranyl acetate (see recipe)
  • Cell scrapers
  • 1.5‐ and 2‐ml microcentrifuge tubes
  • Benchtop centrifuge with swinging rotor for microcentrifuge tubes
  • Tube rotator
  • Toothpicks
  • Cryoultramicrotome (e.g., Leica ultracut UCT with EM FCS cryochamber)
  • Dry diamond knife, cryo, 45°, 3 mm (Diatome or Drukker International)
  • 200‐mesh carbon‐coated Formvar copper grids (Agar Scientific)
  • Filter paper

Basic Protocol 4: Use of Aggregate Number as a Measure of Protein Clearance

  Materials
  • Atg5 knockout mouse embryonic fibroblasts (MEFs) and matched wildtype control MEFs
  • htt‐exon1‐Q74‐GFP expression plasmid
  • Drug of interest or expression plasmid for gene of interest (optional)
  • Transfection reagent (e.g., Lipofectamine 2000, Invitrogen, or TransIT2020)
  • Rapamycin (positive control)
  • Phosphate‐buffered saline (PBS, appendix 2A)
  • 4% paraformaldehyde (see recipe)
  • 70% glycerol containing 1 µg/ml DAPI
  • 13 × 13−mm coverslips
  • 6‐well plates
  • Fluorescence microscope with 60× objective

Support Protocol 1: Cell Culture

  Materials
  • Cells of interest:
    • HeLa cells, HEK cells, or MEFs
    • Stable cell lines, e.g., HeLa cells expressing GFP‐LC3 or mRFP‐GFP‐LC3, HEK cells expressing GFP‐DFCP1
  • Dulbecco's modified Eagle medium (DMEM, Sigma‐Aldrich D6546)
  • Fetal bovine serum (FBS)
  • Penicillin/streptomycin solution
  • L‐Glutamine
  • Phosphate‐buffered saline (PBS)
  • 1× trypsin/EDTA solution
  • 37°C humidified 5% CO 2 incubator
  • 75‐cm2 tissue culture flasks
  • Cell scrapers
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Figures

Videos

Literature Cited

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