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Cryo-Electron Microscopy to Study Bax Pores and MOMP

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

Abstract

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 

Notes

Acknowledgments

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.

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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

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