title: Visual and functional demonstration of growing Bax-induced pores in mitochondrial outer membranes.
abstract: Bax induces mitochondrial outer membrane permeabilization (MOMP), a critical step in apoptosis in which proteins are released into the cytoplasm. To resolve aspects of the mechanism, we used cryo-electron microscopy (cryo-EM) to visualize Bax-induced pores in purified mitochondrial outer membranes (MOMs). We observed solitary pores that exhibited negative curvature at their edges. Over time, the pores grew to ∼ 100-160 nm in diameter after 60-90 min, with some pores measuring more than 300 nm. We confirmed these results using flow cytometry, which we used to monitor the release of fluorescent dextrans from isolated MOM vesicles. The dextran molecules were released gradually, in a manner constrained by pore size. However, the release rates were consistent over a range of dextran sizes (10-500 kDa). We concluded that the pores were not static but widened dramatically to release molecules of different sizes. Taken together, the data from cryo-EM and flow cytometry argue that Bax promotes MOMP by inducing the formation of large, growing pores through a mechanism involving membrane-curvature stress.
authors.affiliation: La Jolla Institute, La Jolla, CA 92037.
authors.forename: Laura A
authors.initials: LA
authors.lastname: Gillies
authors.affiliation: Department of Pathology, University of Iowa, Iowa City, IA 52242.
authors.forename: Han
authors.initials: H
authors.lastname: Du
authors.affiliation: La Jolla Institute, La Jolla, CA 92037.
authors.forename: Bjoern
authors.initials: B
authors.lastname: Peters
authors.affiliation: Department of Pathology, University of Iowa, Iowa City, IA 52242.
authors.forename: C Michael
authors.initials: CM
authors.lastname: Knudson
authors.affiliation: La Jolla Institute, La Jolla, CA 92037.
authors.forename: Donald D
authors.initials: DD
authors.lastname: Newmeyer
authors.affiliation: La Jolla Institute, La Jolla, CA 92037 tomomi@lji.org.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Kuwana
mesh_terms: D000818:Animals; D002850:Chromatography, Gel; D020285:Cryoelectron Microscopy; D022162:Cytoplasmic Vesicles / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D003911:Dextrans / Q000737:chemistry / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D051336:Mitochondrial Membranes / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D009865:Oocytes / Q000378:metabolism; D010539:Permeability / Q000187:drug effects; D016062:Porosity; D011994:Recombinant Proteins / Q000494:pharmacology; D014981:Xenopus; D029867:Xenopus Proteins / Q000235:genetics / Q000494:pharmacology*; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000494:pharmacology*
chemical_list: D003911:Dextrans; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D051028:bcl-2-Associated X Protein; D016650:Fluorescein-5-isothiocyanate