In Vitro Dissection of Autophagy

Min Zhang1, Dawei Liu2, Liang Ge1

1 State Key Laboratory of Membrane Biology, Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, 2 Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 11.23
DOI:  10.1002/cpcb.33
Online Posting Date:  December, 2017
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Autophagy is an essential cellular process for bulk degradation of cytoplasmic components through the lysosome. Underlying this process is an intricate interaction between protein factors and the cell endomembrane system, leading to a gradual maturation of the autophagic membrane. This structure sequesters a portion of the cytoplasm by the formation of a double‐membrane compartment called the autophagosome. The autophagosome then delivers the cargo to the lysosome to complete degradation. The molecular mechanism accounting for the generation of the autophagic membrane is a longstanding question. Here, a cell‐free approach that has been established to understand the mechanism of early autophagic membrane generation is described. This system has provided insight into the membrane source of the autophagosome, the early protein‐membrane associations, and the membrane remodeling that generates the autophagosomal precursors. The cell‐free assay, in combination with other established approaches (e.g., cell imaging), will facilitate a deeper understanding of the mechanism of autophagy. © 2017 by John Wiley & Sons, Inc.

Keywords: autophagy; autophagosome; cell‐free reconstitution; LC3 lipidation

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Table of Contents

  • Introduction
  • Basic Protocol 1: Cell‐Free LC3 Lipidation
  • Support Protocol 1: Three‐Step Membrane Fractionation and Lipidation Reaction
  • Basic Protocol 2: Membrane Recruitment Assay
  • Basic Protocol 3: Small Autophagosomal Precursor Formation Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Cell‐Free LC3 Lipidation

  • Atg5 knockout (KO) mouse embryonic fibroblast (MEF) cells (Dr. Noboru Mizushima)
  • Dulbecco's modified Eagle medium (DMEM) plus 10% FBS (fetal bovine serum)
  • PBS (see recipe), room temperature and ice cold
  • 0.05% trypsin‐EDTA (1×; Gibco)
  • B88 lysis buffer (see recipe)
  • Trypan blue
  • Human HEK293T (human embryonic kidney) cells (Berkeley Tissue Culture Center)
  • E. coli BL21 cells (NEB)
  • pET28a‐LC3 (Ge et al., )
  • IPTG
  • Imidazole
  • Protease inhibitors cocktail (Roche)
  • Lysozyme
  • Triton X‐100
  • Ni‐Sepharose (packed beads) (GE)
  • 0.2% Tween‐20
  • NAP‐10 columns (GE)
  • Thrombin (Roche)
  • 4‐(2‐Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF, Sigma)
  • Coomassie blue
  • 10× ATP regeneration system (see recipe)
  • 10 mM GTP stock (10 µl/tube, store at least 2 years at −80°C)
  • B88 buffer (see recipe)
  • SDS‐PAGE loading buffer
  • Earle's balanced salt solution (EBSS; Thermo)
  • Phospholipids C reagent (Wako)
  • 10‐cm cell culture dishes
  • Centrifuge tubes (1.5‐, 15‐, and 50‐ml)
  • Refrigerated benchtop swinging bucket centrifuge (e.g., Sorvall ST 16R, Thermo)
  • 22‐G needles and syringes (1‐, 3‐, and 5‐ml)
  • Glass slides and coverslips
  • Microscope
  • 2.5‐, 3.5‐, and 5‐ml ultracentrifuge tubes
  • Benchtop ultracentrifuges (>150,000 × g, e.g., Optima Max‐XP, Beckman or e.g., Optima XE‐90, Beckman)
  • Microplate spectrophotometer (e.g., xMark™ Microplate Absorbance Spectrophotometer, Bio‐Rad)
  • 37°C shaker
  • Spectrophotometer (with cuvettes, e.g., Spectronic Genesys 5, Thermo)
  • Centrifuge for large volumes (e.g., Sorvall RC6+)
  • Sonicator (e.g., Branson Sonifier 450)
  • Rotator, 4ºC
  • Protein purification columns
  • Centrifugal filter (10‐kD cutoff)
  • 30°C water bath
  • Additional reagents and equipment for the Bradford assay, SDS‐PAGE

Support Protocol 1: Three‐Step Membrane Fractionation and Lipidation Reaction

  Additional Materials (also see protocol 1)
  • Atg5 KO MEF (Dr. Noboru Mizushima)
  • HB1 lysis buffer (see recipe)
  • 1.25 M, 1.1 M, and 0.25 M sucrose buffers (Golgi isolation kit, Sigma)
  • HB1 buffer (see recipe)
  • 50% Optiprep solution (see recipe)
  • Optiprep dilution buffer (see recipe)

Basic Protocol 2: Membrane Recruitment Assay

  Additional Materials (also see protocol 1)
  • X‐tremeGene HP cell transfection reagent (Roche)
  • 50% Optiprep in B88 buffer (see reciperecipes)

Basic Protocol 3: Small Autophagosomal Precursor Formation Assay

  Additional Materials (also see protocol 1 and protocol 2Support Protocol)
  • Refrigerated bench‐top microcentrifuge (e.g., Eppendorf Centrifuge 5430R)
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Literature Cited

Literature Cited
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