Cryo-Electron Microscopy to Study Bax Pores and MOMP

  • Tomomi KuwanaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1877)


During apoptosis, mitochondria permeabilize the outer membranes to release apoptogenic proteins from the intermembrane space. This process termed mitochondrial outer membrane permeabilization (MOMP) is regulated by Bcl-2 family proteins. Bax is an effector proapoptotic molecule that permeabilizes the lipid membranes when it is activated by activator BH3-only proteins. We investigated this critical event by developing simple but faithful vesicle systems—outer membrane vesicles (OMVs) and liposomes—to visualize the pores in the membrane by using cryo-electron microscopy (cryo-EM). We have revealed the morphology of the pore, determined the localization of Bax labeled with nanogold and have performed image analysis to help understand the mechanisms of pore formation induced by Bax.

Key words

Apoptosis Mitochondria Bax Liposomes Cryo-electron microscopy Nanogold 



The work described here was supported by NIH grants R21AG024157, R21AG024478, and R01GM086389 and funds from Department of Pathology, University of Iowa Carver College of Medicine to T.K. I thank my collaborators, Donald Newmeyer, Bjoern Peters, and William B. Kiosses (La Jolla Institute for Allergy and Immunology); Joel Quispe, Clint Potter, and Bridget Carragher (National Resources for Automated Molecular Microscopy at The Scripps Research Institute); and Norman Olson, James Bouwer, and Timothy Booth (Cryo-EM Core Facility at University of California San Diego) for their support and excellent technical help. The Cryo-EM Core Facility at UCSD was supported by NIH grants to Dr. Timothy Baker and a gift from Agouron Institute to UCSD.


  1. 1.
    Chipuk JE, Moldoveanu T, Llambi F, Parsons MJ, Green DR (2010) The BCL-2 family reunion. Mol Cell 37:299–310CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    Gillies LA, Kuwana T (2014) Apoptosis regulation at the mitochondrial outer membrane. J Cell Biochem 115:632–640CrossRefPubMedCentralPubMedGoogle Scholar
  3. 3.
    Kuwana T, Mackey MR, Perkins G, Ellisman MH, Latterich M, Schneiter R, Green DR, Newmeyer DD (2002) Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane. Cell 111:331–342CrossRefPubMedGoogle Scholar
  4. 4.
    Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X (1997) Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91:479–489CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Verhaegen M, Bauer JA, Martin de la Vega C, Wang G, Wolter KG, Brenner JC, Nikolovska-Coleska Z, Bengtson A, Nair R, Elder JT, Van Brocklin M, Carey TE, Bradford CR, Wang S, Soengas MS (2006) A novel BH3 mimetic reveals a mitogen-activated protein kinase-dependent mechanism of melanoma cell death controlled by p53 and reactive oxygen species. Cancer Res 66:11348–11359CrossRefPubMedGoogle Scholar
  6. 6.
    Du C, Fang M, Li Y, Li L, Wang X (2000) Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 102:33–42CrossRefPubMedGoogle Scholar
  7. 7.
    Hegde R, Srinivasula SM, Zhang Z, Wassell R, Mukattash R, Cilenti L, DuBois G, Lazebnik Y, Zervos AS, Fernandes-Alnemri T, Alnemri ES (2002) Identification of Omi/HtrA2 as a mitochondrial apoptotic serine protease that disrupts inhibitor of apoptosis protein-caspase interaction. J Biol Chem 277:432–438CrossRefPubMedGoogle Scholar
  8. 8.
    Kuwana T, Bouchier-Hayes L, Chipuk JE, Bonzon C, Sullivan BA, Green DR, Newmeyer DD (2005) BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly. Mol Cell 17:525–535CrossRefPubMedGoogle Scholar
  9. 9.
    Schafer B, Quispe J, Choudhary V, Chipuk JE, Ajero TG, Du H, Schneiter R, Kuwana T (2009) Mitochondrial outer membrane proteins assist Bid in Bax-mediated lipidic pore formation. Mol Biol Cell 20:2276–2285CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Gillies LA, Du H, Peters B, Knudson CM, Newmeyer DD, Kuwana T (2015) Visual and functional demonstration of growing Bax-induced pores in mitochondrial outer membranes. Mol Biol Cell 26:339–349CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Kuwana T, Olson NH, Kiosses WB, Peters B, Newmeyer DD (2016) Pro-apoptotic Bax molecules densely populate the edges of membrane pores. Sci Rep 6:27299CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Grosse L, Wurm CA, Bruser C, Neumann D, Jans DC, Jakobs S (2016) Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in apoptosis. EMBO J 35:402–413CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Salvador-Gallego R, Mund M, Cosentino K, Schneider J, Unsay J, Schraermeyer U, Engelhardt J, Ries J, Garcia-Saez AJ (2016) Bax assembly into rings and arcs in apoptotic mitochondria is linked to membrane pores. EMBO J 35:389–401CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Bleicken S, Jeschke G, Stegmueller C, Salvador-Gallego R, Garcia-Saez AJ, Bordignon E (2014) Structural model of active bax at the membrane. Mol Cell 56:496–505CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Czabotar PE, Lessene G, Strasser A, Adams JM (2014) Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nat Rev Mol Cell Biol 15:49–63CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Dewson G, Kluck RM (2009) Mechanisms by which Bak and Bax permeabilise mitochondria during apoptosis. J Cell Sci 122:2801–2808CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Gavathiotis E, Walensky LD (2011) Tracking BAX once its trigger is pulled. Cell Cycle 10:868–870CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Subburaj Y, Cosentino K, Axmann M, Pedrueza-Villalmanzo E, Hermann E, Bleicken S, Spatz J, Garcia-Saez AJ (2015) Bax monomers form dimer units in the membrane that further self-assemble into multiple oligomeric species. Nat Commun 6:8042CrossRefPubMedCentralPubMedGoogle Scholar
  19. 19.
    Uren RT, O’Hely M, Iyer S, Bartolo R, Shi MX, Brouwer JM, Alsop AE, Dewson G, Kluck RM (2017) Disordered clusters of Bak dimers rupture mitochondria during apoptosis. Elife 6:e19944CrossRefPubMedCentralPubMedGoogle Scholar
  20. 20.
    Zhang Z, Subramaniam S, Kale J, Liao C, Huang B, Brahmbhatt H, Condon SG, Lapolla SM, Hays FA, Ding J, He F, Zhang XC, Li J, Senes A, Andrews DW, Lin J (2015) BH3-in-groove dimerization initiates and helix 9 dimerization expands Bax pore assembly in membranes. EMBO J 35:208–236CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    Suzuki M, Youle RJ, Tjandra N (2000) Structure of bax. Coregulation of dimer formation and intracellular localization. Cell 103:645–654CrossRefPubMedGoogle Scholar
  22. 22.
    Dobro MJ, Melanson LA, Jensen GJ, McDowall AW (2010) Plunge freezing for electron cryomicroscopy. Methods Enzymol 481:63–82CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.La Jolla Institute for Allergy and ImmunologyLa JollaUSA

Personalised recommendations