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Detection of the Mitochondrial Apoptosis‐Induced Channel (MAC) and Its Regulation by Bcl‐2 Family Proteins

Kathleen W. Kinnally1,  Sonia Martinez‐Caballero1,  Laurent M. Dejean1

1New York University, College of Dentistry, New York, New York

Publication Name: 
Current Protocols in Toxicology
Unit Number: 
Unit 2.12
DOI: 
10.1002/0471140856.tx0212s30
Online Posting Date: 
December, 2006
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Abstract

Apoptosis is a phenomenon fundamental to higher eukaryotes that is integral to such diverse cellular processes as tissue homeostasis, organogenesis, and response to toxins. The release from mitochondria of apoptotic factors such as cytochrome c is a key step during apoptosis of most cells. Cytochrome c release occurs through the MAC (mitochondrial apoptosis-induced channel), a pore which forms in the mitochondrial outer membrane during early apoptosis and is exquisitely regulated by the Bcl-2 family of proteins. This unit presents basic and advanced tools for detecting MAC and defining its regulation by Bcl-2 family proteins and pharmacological agents. Protocols include the use of time-lapse video-microscopy to monitor the onset of apoptosis in living cells and patch-clamp techniques for mitochondria or proteoliposomes containing mitochondrial proteins, which allow direct detection of MAC. These approaches enable an evaluation of the role of MAC and mitochondria in apoptosis of a variety of cell types by many inducers.

Keywords: MAC; mitochondrial apoptosis-induced channel; apoptosis; patch-clamp; Bcl-2; Bax.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Detection of Apoptotic Events Using Time-Lapse Microscopy
  • Support Protocol 1: Cleaning Coverslips for Time-Lapse Videomicroscopy Experiments
  • Support Protocol 2: Cleaning Rose Chambers
  • Preparation of Isolated Mitochondria and Proteoliposomes Containing Mitochondrial Outer Membranes
  • Basic Protocol 2: Isolation of Large Amounts of Mitochondria from Cultured Cells
  • Alternate Protocol 1: Quick Isolation of Small Amounts of Mitochondria from Cultured Cells
  • Basic Protocol 3: Purification of the Inner and Outer Membranes from Isolated Mitochondria
  • Support Protocol 3: Immunoblot Analysis of Mitochondrial Proteins
  • Basic Protocol 4: Preparation of Proteoliposomes Containing Mitochondrial Outer Membranes
  • Support Protocol 4: Preparation of Small Liposomes of Azolectin
  • Detecting MAC in Isolated Mitochondria and Proteoliposomes
  • Basic Protocol 5: Patch Clamping Isolated Mitochondria
  • Alternate Protocol 2: Patch Clamping Proteoliposomes
  • Alternate Protocol 3: Detection of Cytochrome c Release from Proteoliposomes Containing Control and Apoptotic Outer Membranes
  • Basic Protocol 6: Modulation of Bax Content by Immunoprecipitation
  • Basic Protocol 7: Preparation of Proteoliposomes Containing Solubilized Mitochondrial Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Detection of Apoptotic Events Using Time-Lapse Microscopy

 Materials
  • 90% (v/v) ethanol solution
  • Cells for inoculating (e.g., HeLa, MEFs; see appendix 3B)
  • 10 ml appropriate cell culture medium (e.g., Leibowitz's medium; US Biological) containing 0.011 g/liter phenol red (appendix 3B; optional) and apoptotic inducer (e.g., 0.5 to 1 µM staurosporine; optional)
  • 1 M HEPES (pH 7.4) solution (see recipe), sterile (optional)
  • Forceps, sterile
  • 25-mm2 square or round glass coverslip, clean (Support Protocol 1)
  • 5-ml petri dish
  • Rose chamber (~50 × 37–mm with a 23-mm circular cutout in the center): composed of a 2 to 3 mm thick square sylastic spacer, two aluminum holders, and four screws (see Khodjakov and Rieder, 2006)
  • Screwdriver
  • 28-G 1-in. needles (two)
  • 5-ml syringe
  • Heat block (e.g., WPI; or see Rieder and Cole, 1998)
  • Microscope equipped with epifluorescence plus phase contrast (PC) or differential interface contrast (DIC) optics (e.g., Nikon Eclipse TE300 phase contrast/differential interference contrast microscope)
  • Two shutters (e.g., Uniblitz) and controlling software (e.g., ImageJ, freely available at http://rsb.info.nih.gov/ij)
  • Charge-coupled device (CCD) camera, e.g., Spot RT Monochrome CCD camera (Diagnostics Instruments)
  • Video conversion software (e.g., AVS video converter version 3 from NCTsoft, http://www.nctsoft.com)
  • Additional reagents and equipment for growing mammalian cells (appendix 3B)

NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique should be used accordingly.

NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.


Support Protocol 1: Cleaning Coverslips for Time-Lapse Videomicroscopy Experiments

 Materials
  • 1% (v/v) regular detergent solution: 10 ml of regular detergent (e.g., Liqui Nox; Alconox) and 990 ml distilled water; store up to 1 year at room temperature
  • 90% (v/v) ethanol
  • 3.8% (v/v) HCl solution: add 100 ml of concentrated HCl (38% v/v) to 900 ml of distilled water (in that order); store up to 1 year at room temperature
  • 0.1 M EDTA, pH 8.0: mix 200 ml of 0.5 M EDTA, pH 8.0, (appendix 2A) with 800 ml distilled water; store up to 6 months at 4°C
  • Disposable gloves
  • 600-ml beakers
  • 100 to 200 square (Corning 25 mm, no. 1.5) or circular (Fisher 25 mm, no.1 circ.) glass coverslips
  • Bath sonicator (e.g., Branson Model 1210)
  • Screw capped jar with a wide mouth

NOTE: For best results always transfer the coverslips into each new solution individually, wearing disposable gloves.

Support Protocol 2: Cleaning Rose Chambers

 Materials
  • 1% (v/v) regular detergent solution: 10 ml of regular detergent (e.g., Liqui Nox; Alconox) and 990 ml distilled water; store up to 1 year at room temperature
  • 0.1 M EDTA, pH 8.0: 200 ml of 0.5 M EDTA, pH 8.0, (appendix 2A) and 800 ml distilled water; store up to 6 months at 4°C
  • 90% (v/v) ethanol solution
  • Rose chamber (see Basic Protocol 1)
  • 600-ml beakers
  • Bath sonicator (e.g., Branson Model 1210)
  • Microwave
  • Whatman no. 1 filter paper
  • Pyrex jar

Basic Protocol 2: Isolation of Large Amounts of Mitochondria from Cultured Cells

 Materials
  • Tissue culture cells (e.g., FL5.12, MEFs, HeLa; appendix 3B)
  • Appropriate complete tissue culture medium (appendix 3B)
  • Phosphate-buffered saline (PBS; appendix 2A)
  • 1× mitochondrial isolation (MI) buffer (see recipe)
  • Protease inhibitor cocktail for mammalian tissue (Sigma)
  • 0.1 M PMSF stock solution: dissolve 1.74 g phenylmethylsulfonylfluoride (PMSF; Sigma) in 10 ml propanol; store up to 3 months at room temperature
  • 50 mg/ml digitonin stock solution: dissolve 50 mg digitonin (e.g., Calbiochem) in 1 ml of dimethylsulfoxide (DMSO; Sigma); store up to 6 months at –20°C
  • Ethanol-precipitated bovine serum albumin (BSA; e.g., Sigma)
  • 15-, 50-, and 500-ml centrifuge tubes or bottles with caps
  • Refrigerated centrifuge with fixed-angle rotor
  • Rugged rotator (e.g., Fisher Scientific)
  • 25-ml glass homogenizer and tight motor-driven Teflon pestle (e.g., Thomas)
  • –80°C freezer
  • Additional reagents and equipment for determining protein concentration (appendix 3G)

NOTE: Perform all operations on ice and use ice-cold buffers and instruments.

Alternate Protocol 1: Quick Isolation of Small Amounts of Mitochondria from Cultured Cells

 Additional Materials (also see Basic Protocol 2)
  • Cells growing in an appropriate complete culture medium (see Basic Protocol 2)
  • 2× mitochondrial isolation (MI) buffer (see recipe)
  • Swinging-bucket rotor
  • Manual glass homogenizer with tight Teflon pestle

NOTE: Perform all operations on ice and use ice-cold buffers and instruments.

Basic Protocol 3: Purification of the Inner and Outer Membranes from Isolated Mitochondria

 Materials
  • Isolated mitochondria (Basic Protocol 2)
  • 1× and 2× mitochondrial isolation (MI) buffer (see recipes)
  • Lysis buffer (see recipe)
  • 35% (w/v) sucrose solution (see recipe)
  • French press
  • Refrigerated centrifuge and 15-ml centrifuge tubes
  • 10-, 20-, or 50-ml beaker
  • Small magnetic stirrer
  • Glass homogenizers (e.g., Thomas) with Teflon pestles
  • 10-cm stainless steel 12-G cannula on a 10-ml syringe
  • Refrigerated ultracentrifuge with hanging-bucket rotor and 5-ml collapsible ultracentrifuge tubes
  • Additional reagents and equipment for determining protein concentration (appendix 3G)

NOTE: All operations should be done on ice; using ice-cold buffers and instruments.

Support Protocol 3: Immunoblot Analysis of Mitochondrial Proteins

 Materials
  • Purified sample of inner and outer membranes or solubilized mitochondrial proteins (Basic Protocol 3 or Basic Protocol 6)
  • 2× Laemmli loading buffer (e.g., Bio-Rad or see SDS sample buffer, 2× in appendix 3F)
  • 13% or 15% (w/v) SDS-polyacrylamide gel (appendix 3F)
  • Methanol, HPLC grade (e.g., Fischer)
  • Electrotransfer buffer (see recipe)
  • PBS/Tween: PBS (appendix 2A) containing 0.2% (w/v) Tween 20
  • PBS/Tween/5% milk: PBS-Tween supplemented with 5% (w/v) nonfat milk
  • Primary antibody against VDAC (e.g., Calbiochem), COXIV (e.g., Molecular Probes), cytochrome c (e.g., Pharmingen), or Bax (e.g., Santa Cruz)
  • Horseradish peroxidase–conjugated secondary antibody (e.g., Jackson Immunoresearch)
  • ECL Western Blotting Detection Reagents kit (Amersham Biosciences)
  • Semi-dry transfer cell (e.g., Trans-Blot SD, Bio-Rad) with power source
  • PVDF membranes and thick filter papers (e.g., Bio-Rad)
  • Additional reagents and equipment for performing SDS-PAGE (appendix 3F)

Basic Protocol 4: Preparation of Proteoliposomes Containing Mitochondrial Outer Membranes

 Materials
  • Purified mitochondrial outer membrane (Basic Protocol 3)
  • Small azolectin liposomes (Support Protocol 4)
  • 5 mM HEPES solution (see recipe)
  • Anhydrous calcium sulfate (e.g., Drierite)
  • 0.15 M KCl solution (see recipe)
  • 35 × 60–mm cover glasses (e.g., Gold Seal)
  • 10-cm petri dishes (e.g., Fisher)

Support Protocol 4: Preparation of Small Liposomes of Azolectin

 Materials
  • Azolectin (l--phosphatidylcholine from soybean Type IVs; Sigma): store at –20°C, in the dark and under vacuum to avoid oxidation of the lipids
  • Chloroform (e.g., Fischer)
  • 0.15 M KCl solution (see recipe)
  • 30-ml Corex tubes, washed sequentially with sulfuric acid and distilled water, and dried before use
  • Nitrogen gas tank
  • Microtip sonicator (e.g., Sonicator 60 Sonic Dismembrator with microtip; Fischer)
  • Refrigerated ultracentrifuge and fixed-angle rotor
  • 10-ml ultracentrifuge tubes

Basic Protocol 5: Patch Clamping Isolated Mitochondria

 Materials
  • Isolated mitochondria (Alternate Protocol 1)
  • Patching media (see recipe)
  • Gradient media (see recipe)
  • 20 mM dibucaine stock solution: dissolve 760 mg dibucaine (e.g., Sigma) in 1 ml distilled water; protect from light and store up to 2 to 3 months at 4°C
  • Horse cytochrome c (e.g., Sigma)
  • Agar bridges (see recipe)
  • 1.0-mm Borosilicate glass capillaries with filaments (World Precision Instruments)
  • Microelectrode puller (Sutter Instruments Model P-87) with a 2-mm box filament and pressure set in position 500; a two-line program can make suitable micropipets (see Table 2.12.2)
  • Ag/AgCl2 electrode (Warner Instruments)
  • Micromanipulator
  • 35 × 60–mm glass coverslips and Lucite holder
  • Phase-contrast microscope with 40× lens and 15× oculars mounted on an antivibration table.
  • Electrophysiology setup including a patch-clamp amplifier, low pass filter, computer and software, e.g., purchase Axon pClamp (Axon Instruments) or download Strathclyde Electrophysiological package at http://spider.science.strath.ac.uk/physpharm/index.php pageName=software
     
    Table 2.12.2 Two-Step Program to Pull Microelectrodes Suitable for Patch Clamping Isolated Mitochondria or Proteoliposomes Using a Sutter Micropipet Puller Model P-87a
    HeatPullVelocityTime
    Ramp +20°—20150
    Ramp +15°7040200
     aSuggested modifications to optimize the program for small tips and low resistance can be obtained by running glass.exe, a program available for free from the Sutter website at http://www.sutter.com/news/software_downloads.html.

Alternate Protocol 2: Patch Clamping Proteoliposomes

 Additional Materials (see also Basic Protocol 5)
  • Proteoliposomes containing mitochondrial outer membranes (see Basic Protocol 6)

Alternate Protocol 3: Detection of Cytochrome c Release from Proteoliposomes Containing Control and Apoptotic Outer Membranes

 Materials
  • Purified mitochondrial outer membrane (Basic Protocol 3)
  • Small liposomes of azolectin (Support Protocol 4)
  • 3 mg/ml horse cytochrome c stock solution : dissolve 3 mg of horse cytochrome c (e.g., Sigma) in 1 ml of PBS (appendix 2A); store at 4°C for up to 2 to 3 weeks
  • 5 mM HEPES solution (see recipe)
  • Anhydrous calcium sulfate (e.g., Drierite)
  • 0.15 M KCl solution (see recipe)
  • 0.5 M NaCl solution (see recipe)
  • 10 mM Tris solution (see recipe)
  • 35 × 60–mm coverslips (e.g., Gold Seal)
  • 10-cm petri dishes (e.g., Fischer)
  • 1.5-ml polyallomer microcentrifuge tubes
  • Light microscope with 20× or 40× magnification
  • Refrigerated ultracentrifuge with fixed-angle rotor and 1.5-ml ultracentrifuge tubes

Basic Protocol 6: Modulation of Bax Content by Immunoprecipitation

 Materials
  • Isolated mitochondria (Basic Protocol 2)
  • 1× mitochondrial isolation (MI) buffer (see recipe)
  • 20% (w/v) CHAPS solution: dissolve 200 mg of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in 1 ml of 1× MI buffer (see recipe); store up to 6 months at –20°C
  • Immunoprecipitation (IP) buffer (see recipe) supplemented with 2% (w/v) CHAPS
  • Primary antibody against Bax N-terminus (e.g., Santa Cruz)
  • 1 mg/ml IgG from rabbit or mouse serum (e.g., Sigma) solution: dilute 5 mg of IgG solution in 5 ml of PBS; divide into 100-µl aliquots and store up to 3 months at –20°C
  • Protein G-agarose (Calbiochem)
  • Phosphate-buffered saline (PBS; appendix 2A)
  • 2× Laemmli loading buffer (e.g., Bio-Rad)
  • Microtip sonicator (e.g., Sonicator 60 Sonic Dismembrator with microtip; Fischer)
  • Refrigerated microcentrifuge
  • Rugged rotator (e.g., Fisher Scientific)
  • Refrigerated ultracentrifuge, fixed-angle rotor and 1.5-ml ultracentrifuge tubes
  • Additional equipment and reagents for determining protein concentrations (appendix 3G)

Basic Protocol 7: Preparation of Proteoliposomes Containing Solubilized Mitochondrial Proteins

 Materials
  • Total mitochondrial lysates or supernatants from immunoprecipitation assays (Basic Protocol 6)
  • IP buffer (see recipe)/2% (w/v) CHAPS
  • Small azolectin liposomes (Support Protocol 4)
  • IP buffer (see recipe)
  • IP buffer (see recipe)/2% detergent-removing gel (Pierce)
  • 5 mM HEPES solution (see recipe)
  • 0.15 M KCl solution (see recipe)
  • Anhydrous calcium sulfate (e.g., Drierite)
  • Light microscope with 40× magnification
  • 50 µl-dialysis chamber and cellulose acetate membranes with a 10-kDa MWCO (Harvard Apparatus)
  • 500-ml beaker
  • Magnetic stirrer
  • 35 × 60–mm cover glasses (e.g., Gold Seal)
  • 10-cm petri dishes (e.g., Fischer)
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Figures

  • Figure 2.12.1
    Procedures used to detect the mitochondrial apoptosis-induced channel (MAC) from apoptotic cells. Numbers refer to the corresponding basic protocols. Apoptosis can be monitored in living cells using time-lapse videomicroscopy (1). Patch-clamp techniques (5) allow a direct detection of MAC from isolated mitochondria (2) or purified mitochondrial outer membranes (3) reconstituted into proteoliposomes (4). Solubilization and immunoprecipitation (6) can be used to modulate the level of mitochondrial proteins, like pro-apoptotic Bax, and examine the effects on MAC activity by patch clamping proteoliposomes containing those detergent-solubilized proteins (7).

    View Image
  • Figure 2.12.2
    Fibroblasts transiently transfected with a plasmid carrying GFP-Bax (8 µg) using lipofectamine (4 µl) so that ~20% of the cells express GFP-Bax. After GFP fluorescence was initially detected, DIC and fluorescence images were taken every 15 min for 25 hr. DIC (A,C) and fluorescence (B,D) images show diffuse GFP-BAX in cells 1 and 2 that becomes punctuate with time as the cells undergo apoptosis. Phase-contrast and fluorescence images show mitochondria isolated from HeLa cells that stably express low levels of GFP-Bax before (E, F) and after 5 hr of apoptosis induction with 1 µM staurosporine (G, H). The mean (± SE) conductance of patches shown in (E) and (G) was measured by patch clamping these isolated mitochondria. Scale bar is 5 µm. Adapted from Dejean et al. (2005) by permission from the American Society for Cell Biology.

    View Image
  • Figure 2.12.3
    Immunotblot assessment of purified mitochondrial outer membrane proteins from apoptotic FL5.12 cells. Immunoblots show the presence of the outer membrane protein voltage dependent anion channel (VDAC) but not the inner membrane protein cytochrome oxidase subunit IV (COXIV) in the outer membranes (OM, 2 µg). Inner membranes (IM, 2 µg) are the positive control for COXIV. Reprinted by permission of Federation of the European Biochemical Societies from Martinez-Caballero, S., Dejean, L.M., and Kinnally, K.W. Some amphiphilic cations block the mitochondrial apoptosis-induced channel, MAC, FEBS Letters, Vol. 56, pp. 835-38, 2004.

    View Image
  • Figure 2.12.4
    Single channel behavior of MAC. MAC activity can be monitored by directly patch clamping of mitochondria isolated from apoptotic cells or of proteoliposomes containing purified mitochondrial outer membranes. (A) Phase-contrast images show seal formation between the microelectrode and a mitochondrion (top) and a proteoliposome (bottom). (B) Typical current traces of the single channel behavior of MAC activity in mitochondria (top) and in a proteoliposome containing mitochondrial outer membranes from apoptotic cells (bottom). (C) A representative current trace of reconstituted MAC shows a fast blockade after perfusion of the bath with 50 µM dibucaine. (D,E) Cytochrome c (cyt c) induces Type 1 and 2 effects on MAC activity. Current traces of MAC in the absence (control) and presence of 100 µM cytochrome c (cyt c) show a decrease in current and increase in noise corresponding to a Type 1 effect (D). A current trace of MAC (E) illustrates that while hemoglobin (Hb) has no effect, cytochrome c (cyt c) induces a rapid reduction in MAC conductance. Reproduced from Pavlov et al., The Journal of Cell Biology, 2001, Vol. 155, pages 725 to 732 by copyright permission of The Rockefeller University Press; reprinted from Int. Rev. Cytol., Vol. 238, Grigoriev, S.M., Muro, C., Dejean, L.M., Campo, M.L., Martinez-Caballero, S., and Kinnally, K.W., Electrophysiological approaches to the study of protein translocation in mitochondria, pages 227 to 274, Copyright 2004 with permission from Elsevier; reproduced from Guo et al. (2004) with permission from the American Physiological Society; reprinted by permission of Federation of the European Biochemical Societies from Martinez-Caballero, S., Dejean, L.M., and Kinnally, K.W. Some amphiphilic cations block the mitochondrial apoptosis-induced channel, MAC, FEBS Letters, Vol. 56, pages 835 to 38, 2004.

    View Image
  • Figure 2.12.5
    (A) Total mitochondrial lysates of control and apoptotic HeLa cells immunoprecipitated with anti-Bax antibodies (Bax Ab) or total rabbit IgG (control Ab). The pellets containing the immunoprecipitated proteins (P) and their respective supernatants (S) were subjected to SDS-PAGE, and the presence of Bax was assessed by immunoblotting. (B) The supernatants from the immunoprecipitation assays described above and corresponding to immunoprecipitates with anti-Bax antibodies (Bax Ab, black) or with total rabbit IgG (control Ab, open) were reconstituted into proteoliposomes. MAC detection frequency was determined by patch clamping (n = 20 to 23 independent patches/condition). p values shown were calculated using Fisher's exact test. Reprinted from Dejean et al. (2005) with permission from the American Society for Cell Biology.

    View Image

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