Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria‐Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus‐Infected Primary Fibroblasts

Chad D. Williamson1, Daniel S. Wong2, Petros Bozidis3, Aiping Zhang4, Anamaris M. Colberg‐Poley5

1 Laboratory of Cell Biology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, 2 Cellular and Molecular Physiology Program, Sackler School for Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts, 3 Laboratory of Microbiology, Department of Medicine, School of Health Sciences, University of Ioannina, Ioannina, Greece, 4 Center for Genetic Medicine Research, Children's Research Institute, Washington, D.C., 5 Department of Pediatrics, George Washington University School of Medicine and Health Sciences, Washington, D.C.
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.27
DOI:  10.1002/0471143030.cb0327s68
Online Posting Date:  September, 2015
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Increasingly mechanistic virology studies require dependable and sensitive methods for isolating purified organelles containing functional cellular sub‐domains. The mitochondrial network is, in part, closely apposed to the endoplasmic reticulum (ER). The mitochondria‐associated membrane (MAM) fraction provides direct physical contact between the ER and mitochondria. Characterization of the dual localization and trafficking of human cytomegalovirus (HCMV) UL37 proteins required establishing protocols in which the ER and mitochondria could be reliably separated. Because of its documented role in lipid and ceramide transfer from the ER to mitochondria, a method to purify MAM from infected cells was also developed. Two robust procedures were developed to efficiently isolate mitochondria, ER, and MAM fractions while providing substantial protein yields from HCMV‐infected primary fibroblasts and from transfected HeLa cells. Furthermore, this unit includes protocols for isolation of detergent resistant membranes from subcellular fractions as well as techniques that allow visualization of the mitochondrial network disruption that occurs in permissively infected cells by their optimal resolution in Percoll gradients. © 2015 by John Wiley & Sons, Inc.

Keywords: subcellular fractionation; human fibroblasts; ER; mitochondria; MAM; HCMV; protein localization; sucrose gradient; Percoll gradient; differential centrifugation

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Table of Contents

  • Introduction
  • Basic Protocol 1: Differential Sucrose Gradient Isolation of ER and Mitochondria
  • Basic Protocol 2: Separation of Mitochondria and Mitochondria‐Associated Membrane Fraction
  • Basic Protocol 3: Flotation of Detergent‐Resistant MAM Membranes from Crude ER or Mitochondrial Fractions
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Differential Sucrose Gradient Isolation of ER and Mitochondria

  • Human foreskin fibroblasts (HFFs; Viromed SF cells)
  • HeLa cells (ATCC CCL‐2)
  • HCMV (desired strain) or DNA for transfection
  • 2% and 10% (v/v) FBS
  • Lipofectamine 2000 (Invitrogen; unit 20.6; Hawley‐Nelson and Ciccarone, )
  • Opti‐MEM I (Invitrogen)
  • 1.0, 1.3, 1.5, 1.7, and 2.0 M sucrose solutions (see reciperecipes), sterile
  • Phosphate‐buffered saline (PBS), pH 7.4 ( appendix 2A)
  • 0.25% trypsin/EDTA (Life Technologies, cat. no. 25200114)
  • 1× mannitol/Tris/EDTA (1× MTE) buffer (see recipe)
  • 100 mM PMSF stock (see recipe)
  • Ultrapure water
  • 70% ethanol
  • 175‐cm2 flasks (∼0.8–2 × 107 cells/flask)
  • 37°C CO 2 incubator
  • Pre‐sterilized (autoclaved) Beckman polyallomer centrifuge tubes: 14 × 89–mm (cat. no. 331372) or 11 × 60–mm (cat. no. 328874)
  • 5‐ml serological pipets
  • Aspirator
  • Sterile 15‐ml conical tubes
  • Beckman GS‐6R centrifuge with GH‐3.8 swinging‐bucket rotor
  • Analog sonicator with 1/8‐in. microtip (Branson Ultrasonics model 250)
  • 250‐ml glass beakers
  • 1.5‐ml microcentrifuge tubes
  • 14‐ml polypropylene, round‐bottom, snap‐cap tubes (17 × 100–mm; Falcon cat. no. 2059)
  • Beckman J2‐MI centrifuge with JA20.1 rotor
  • Beckman XL‐90 ultracentrifuge with SW60 Ti and SW41 Ti rotors
  • 1‐ml syringes and 20‐G needles
  • Parafilm
  • Beckman GS‐15R centrifuge with F2402 rotor

Basic Protocol 2: Separation of Mitochondria and Mitochondria‐Associated Membrane Fraction

  • Untransfected or transfected HeLa cells (3 × 107 cells, ten 100 × 20–mm tissue culture dishes, 100% confluent); uninfected or HCMV‐infected HFF cells (5 × 107 cells, four 850‐cm2 roller bottles, 90% confluent); or twenty 100 × 20‐mm tissue culture dishes, 70% to 90% confluent), and appropriate complete medium for the cell line
  • Phosphate‐buffered saline (PBS), pH 7.4 ( appendix 2A)
  • 0.25% trypsin/EDTA (e.g., Life Technologies)
  • Sucrose homogenization medium (SHM; see recipe), ice cold
  • MSF (optional but recommended, see recipe)
  • 70% ethanol
  • Mannitol buffer A (see recipe), ice cold
  • 30% (v/v) Percoll solution (see recipe), ice cold
  • Mannitol buffer B (see recipe), ice cold
  • Cell lifter, sterile
  • 50‐ and 15‐ml conical tubes (e.g., Corning Falcon)
  • Bright‐field microscope
  • Beckman GS‐15R tabletop centrifuge with swinging‐bucket rotor (e.g., S4180) and fixed‐angle rotor (e.g., F2402)
  • 12 × 75‐mm polypropylene tubes (e.g., Corning Falcon)
  • 2‐ml Potter‐Elvehjem plastic‐coasted tissue grinder with PTFE pestle (e.g., Wheaton safe‐grind type, cat. no. 358003)
  • Overhead stirrer for tissue grinder (Wheaton, cat no. 903475)
  • Phase‐contrast microscope
  • 16 × 76–mm Ultraclear ultracentrifuge tubes (Beckman, cat. no. 344085)
  • Sorvall Legend RT centrifuge or equivalent
  • Millipore Amicon Ultra‐15 Centrifugal Filter Units with Ultracel‐3 membrane (3‐Da cutoff; 24‐pk: Millipore cat. no. 900324)
  • Beckman XL‐90 ultracentrifuge with Ti 70 fixed‐angle rotor
  • 14 × 89–mm Ultraclear ultracentrifuge tubes (Beckman, cat. no. 344059)
  • 50‐ml beaker
  • 1‐ml syringes and 20‐G needles

Basic Protocol 3: Flotation of Detergent‐Resistant MAM Membranes from Crude ER or Mitochondrial Fractions

  • MBST buffer, ice cold (see recipe)
  • 5%, 30%, and 90% sucrose in MBS buffer, ice cold (see reciperecipes)
  • Analog sonicator with 1/8‐in. microtip (optional; Branson Ultrasonics model 250)
  • Polyallomer ultracentrifuge tubes, 11 × 60–mm (Beckman, cat. no. 328874), sterilized by autoclaving and then pre‐chilled
  • Beckman XL‐90 ultracentrifuge with SW60 Ti rotor
  • Additional reagents and equipment for isolation of ER/MAM pellet ( protocol 1, step 24) or a MAM/mitochondria pellet ( protocol 2, step 20)
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