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Protocol

Investigating Bax Subcellular Localization and Membrane Integration Grant Dewson1 Cell Signalling and Cell Death Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne, Victoria 3052, Australia; Department of Medical Biology, The University of Melbourne, Melbourne, Victoria 3010, Australia

Bax is a pivotal effector of apoptosis responsible for permeabilization of the mitochondrial outer membrane (MOM). A key event in mitochondrial damage is the translocation of Bax from the cytosol to the MOM. A simple and effective method for assessing the cytosol vs. mitochondrial localization of Bax is digitonin fractionation, which uses a low concentration of detergent to permeabilize the plasma membrane without damaging intracellular membranes. This allows separation of the cytosol (light membranes) from the heavy membranes (with mitochondria and nuclei) by centrifugation. Localization of Bax can then be assessed by immunoblotting. To further differentiate membrane-integrated Bax from that which is peripherally associated, carbonate extraction of the membrane fraction can be performed before immunoblotting. Treatment of membranes at high pH disrupts protein–protein interactions, whereas protein–lipid interactions are largely retained, although membrane integrity is lost.

MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.

Reagents

Apoptotic stimulus Carbonate extraction buffer (0.1 M Na2CO3, pH 11.5) Digitonin Immediately before use, prepare a fresh solution of 10% (w/v) digitonin in H2O and boil to dissolve. Alternatively, resuspend in DMSO for long-term storage.

DNase I (Roche) HCl (0.1 M) Mouse embryonic fibroblasts (MEF) or cell line of interest, pretreated with a broad-range caspase inhibitor 1

Correspondence: [email protected]

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G. Dewson This protocol was validated in HeLa cells, but has been optimized for MEF. The number of cells required will depend on their expression of Bax, but a minimum of 1 × 10 6 cells is recommended as a starting point. In cells that will be treated with a death stimulus, caspases should first be blocked with a broad-range caspase inhibitor, such as qVD-OPh or z-VAD-fmk (Enzyme Systems).

Nuclease buffer (10×) Permeabilization buffer Phosphate-buffered saline (PBS) (1×; Ca2+- and Mg2+-free) (ice-cold) Protease inhibitor cocktail tablets, without EDTA (complete; Roche) (1 tablet/50 mL) SDS–PAGE and immunoblotting reagents, including primary antibodies for Bax, Bak, and/or cytochrome c Bax translocates from the cytosol to mitochondria during apoptosis, whereas Bak is constitutively integrated into the MOM (Wolter et al. 1997; Griffiths et al. 1999); thus, immunoblotting for Bak serves as a useful positive control for membrane integration. To correlate Bax translocation with mitochondrial damage, subcellular fractions may also be immunoblotted for cytochrome c (Waterhouse et al. 2004).

SDS–PAGE sample buffer (reducing) (2×) Trypan blue (BioRad) Equipment

Centrifuge (benchtop) at 4˚C Centrifuge tubes Heat blocks at 37˚C and 95˚C Hemocytometer Microscope (transmitted light) SDS–PAGE apparatus Western transfer apparatus

METHOD An overview of digitonin fractionation is provided in Figure 1. Depending on the number of samples, this protocol can be completed in  45 min.

Inducing Apoptosis and Permeabilizing Cells

1. Treat cells with an apoptotic stimulus as required. 2. Harvest the cells by centrifugation at 2500g for 5 min. Wash once in 1 mL of ice-cold 1× PBS. Perform a cell count using a hemocytometer. 3. Centrifuge the cells at 2500g for 5 min at 4˚C. Discard the supernatant and gently resuspend the cell pellet in permeabilization buffer supplemented with 0.025% digitonin and protease inhibitors at 1 × 107 cells per mL. Cells should be resuspended gently but thoroughly by pipetting up and down several times with a P1000 pipette, avoiding generation of bubbles. Disaggregation of “clumpy” cells is crucial for the efficiency of digitonin permeabilization.

4. Incubate the cells on ice for 10 min. 5. Verify permeabilization by trypan blue uptake. i. Remove 5 µL of permeabilized cells and add an equal volume of trypan blue. ii. Assess trypan blue uptake by light microscopy. 95%–100% of cells should appear blue. If processing a large number of samples it is not necessary to confirm permeabilization of all samples, although verification of both untreated cells and cells treated with an apoptotic stimulus is recommended.

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Investigating Bax Subcellular Localization

Harvest cells, wash in PBS, centrifuge (2500g, 5 min, 4°C)

Treat cells with apoptotic stimulus

Confirm permeabilization by trypan blue uptake Centrifuge (13,000g, 5 min, 4°C)

“Cytosol” (incl. light membranes)

Immunoblot for Bax, Bak, cytochrome c

Resuspend cell pellet to permeabilize cells incubate (10 min, ice) Heavy membranes (incl. mitochondria)

Investigate Bak/Bax oligomerization and conformation change by cysteine linkage and/or BN-PAGE

Perform carbonate extraction and immunoblot for Bax, Bak, cytochrome c

FIGURE 1. Digitonin fractionation for investigation of Bax subcellular localization and membrane integration. Recent evidence indicates there is a reversible association of Bax with mitochondria, and prosurvival proteins also show differential distribution and relocalize during apoptosis (Hsu et al. 1997; Kaufmann et al. 2003; Edlich et al. 2011). Bax subcellular localization and membrane integration in apoptotic cells can be assessed using digitonin fractionation followed by carbonate extraction of the membrane fraction and immunoblotting, as described here. The heavy membrane fraction can also be used for investigation of Bak and Bax oligomerization and/or conformation change; see Protocol: Investigating the Oligomerization of Bak and Bax during Apoptosis by Cysteine Linkage (Dewson 2015a) and Protocol: Blue Native PAGE and Antibody Gel Shift to Assess Bak and Bax Conformation Change and Oligomerization (Dewson 2015b).

6. Centrifuge the cells at 13,000g for 5 min at 4˚C to separate the supernatant (cytosol) from the pellet (heavy membranes including mitochondria). 7. Transfer the supernatant (cytosolic fraction) to a new tube and add an equal volume of 2× reducing SDS–PAGE sample buffer. Proceed with the membrane fraction pellet as follows.

• •

If membrane integration is not to be assessed, resuspend the pellet in the same total volume of 1× reducing SDS–PAGE sample buffer as used for the cytosolic fraction and proceed directly to Step 13. To assess membrane integration using carbonate extraction, proceed directly to Step 8.

Performing Carbonate Extraction Carbonate extraction of the membrane fraction of digitonin-permeabilized cells (with nuclei) is very messy due to rupture of the nuclear membrane, which causes samples to become viscous. To avoid this problem, mitochondria can be further purified from nuclei by manual cell disruption and differential centrifugation. However, the ease and final yield depends on cell type. A more straightforward method is incorporation of a DNase step before separation of the fractions, as described here.

8. Resuspend the membrane pellet in carbonate extraction buffer at 2 × 107 cells per mL. Incubate on ice for 20 min. 9. Neutralize the solution with 1/3 volume of 0.1 M HCl. Incubate for 5 min at room temperature. 10. Add 1/10 volume of 10× nuclease buffer and 1 unit of DNase I. Incubate for 10 min at 37˚C.

11. Centrifuge the sample at 13,000g for 10 min. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot086447

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G. Dewson

12. Transfer the supernatant (carbonate-sensitive fraction) to a new tube. Add an equal volume of 2× reducing SDS–PAGE sample buffer. Resuspend the pellet (carbonate-resistant fraction) in the same total volume of 1× reducing SDS–PAGE sample buffer. If the pellet sample is too viscous, it can be resuspended in a greater volume of sample buffer. Be sure to adjust the volume of the cytosolic fraction accordingly.

Immunoblotting

13. Heat all samples for 5 min at 95˚C.

14. Load the same volume of each fraction for SDS–PAGE and perform immunoblotting for Bax, Bak, and cytochrome c as needed. Volumes should be adjusted for dilution of the supernatant and membrane samples by HCl. See Troubleshooting.

TROUBLESHOOTING Problem (Step 14): It is difficult to detect the protein of interest in apoptotic samples by immuno-

blotting. Solution: It is important to block activated caspases during apoptosis, as they will lead to cellular

destruction and loss of cells and proteins (including release of cytochrome c to the cytosol). qVDOPh and z-VAD-fmk are two broad range caspase inhibitors commonly used in cell culture. Alternatively, a shorter time-point for apoptosis induction can be tested. Problem (Step 14): Cytochrome c is released in nonapoptotic samples. Solution: To maintain the integrity of the mitochondria during the fractionation process, adjust the

sugar and salt concentrations of the permeabilization buffer to ensure it is norm-osmotic (300 mOsm).

RELATED TECHNIQUES

Membrane fractions can be treated with chemical cross-linkers or exogenous oxidant to monitor Bak/Bax oligomerization in the MOM during apoptosis by cysteine linkage; see Protocol: Investigating the Oligomerization of Bak and Bax during Apoptosis by Cysteine Linkage (Dewson 2015a).

RECIPES Nuclease Buffer (10×)

100 mM Tris–HCl, pH 7.5 25 mM MgCl2 1 mM CaCl2 Permeabilization Buffer

20 mM HEPES/KOH, pH 7.5 250 mM sucrose 50 mM KCl 2.5 mM MgCl2 470

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Investigating Bax Subcellular Localization

Phosphate-Buffered Saline (PBS)

Amount to add (for 1× solution)

Final concentration (1×)

Amount to add (for 10× stock)

Final concentration (10×)

8g 0.2 g 1.44 g 0.24 g

137 mM 2.7 mM 10 mM 1.8 mM

80 g 2g 14.4 g 2.4 g

1.37 M 27 mM 100 mM 18 mM

If necessary, PBS may be supplemented with the following: 0.133 g 1 mM 1.33 g CaCl2•2H2O MgCl2•6H2O 0.10 g 0.5 mM 1.0 g

10 mM 5 mM

Reagent NaCl KCl Na2HPO4 KH2PO4

PBS can be made as a 1× solution or as a 10× stock. To prepare 1 L of either 1× or 10× PBS, dissolve the reagents listed above in 800 mL of H2O. Adjust the pH to 7.4 (or 7.2, if required) with HCl, and then add H2O to 1 L. Dispense the solution into aliquots and sterilize them by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle or by filter sterilization. Store PBS at room temperature.

SDS–PAGE Sample Buffer (Reducing) (2×)

125 mM Tris–HCl, pH 6.8 20% glycerol 4% SDS 0.1% bromophenol blue 5% β-mercaptoethanol

ACKNOWLEDGMENTS

G.D. is supported by the National Health and Medical Research Council of Australia (637335), Australian Research Council (FT100100791), and the Association for International Cancer Research (10–230). The present work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS. REFERENCES Dewson G. 2015a. Investigating the oligomerization of Bak and Bax during apoptosis by cysteine linkage. Cold Spring Harb Protoc doi: 10.1101/pdb .prot086470. Dewson G. 2015b. Blue native PAGE and antibody gel shift to assess Bak and Bax conformation change and oligomerization. Cold Spring Harb Protoc doi: 10.1101/pdb.prot086488. Edlich F, Banerjee S, Suzuki M, Cleland MM, Arnoult D, Wang C, Neutzner A, Tjandra N, Youle RJ. 2011. Bcl-xL Retrotranslocates Bax from the mitochondria into the cytosol. Cell 145: 104–116. Griffiths GJ, Dubrez L, Morgan CP, Jones NA, Whitehouse J, Corfe BM, Dive C, Hickman JA. 1999. Cell damage-induced conformational changes of the pro-apoptotic protein Bak in vivo precede the onset of apoptosis. J Cell Biol 144: 903–914.

Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot086447

Hsu Y-T, Wolter KG, Youle RJ. 1997. Cytosol-to-membrane redistribution of Bax and Bcl-XL during apoptosis. Proc Natl Acad Sci 94: 3668–3672. Kaufmann T, Schlipf S, Sanz J, Neubert K, Stein R, Borner C. 2003. Characterization of the signal that directs Bcl-xL, but not Bcl-2, to the mitochondrial outer membrane. J Cell Biol 160: 53–64. Waterhouse NJ, Steel R, Kluck R, Trapani JA. 2004. Assaying cytochrome C translocation during apoptosis. Methods Mol Biol 284: 307–313. Wolter KG, Hsu YT, Smith CL, Nechushtan A, Xi XG, Youle RJ. 1997. Movement of Bax from the cytosol to mitochondria during apoptosis. J Cell Biol 139: 1281–1292.

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Investigating Bax subcellular localization and membrane integration.

Bax is a pivotal effector of apoptosis responsible for permeabilization of the mitochondrial outer membrane (MOM). A key event in mitochondrial damage...
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