Bax assembly into rings and arcs in apoptotic mitochondria is linked to membrane pores

Bax is a key regulator of apoptosis that, under cell stress, accumulates at mitochondria, where it oligomerizes to mediate the permeabilization of the mitochondrial outer membrane leading to cytochrome c release and cell death. However, the underlying mechanism behind Bax function remains poorly understood. Here, we studied the spatial organization of Bax in apoptotic cells using dual‐color single‐molecule localization‐based super‐resolution microscopy. We show that active Bax clustered into a broad distribution of distinct architectures, including full rings, as well as linear and arc‐shaped oligomeric assemblies that localized in discrete foci along mitochondria. Remarkably, both rings and arcs assemblies of Bax perforated the membrane, as revealed by atomic force microscopy in lipid bilayers. Our data identify the supramolecular organization of Bax during apoptosis and support a molecular mechanism in which Bax fully or partially delineates pores of different sizes to permeabilize the mitochondrial outer membrane.

[1]  M. Rehm,et al.  Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation , 2010, Cell Death and Differentiation.

[2]  Y. Tan,et al.  Biophysical characterization of the oligomeric state of Bax and its complex formation with Bcl-XL. , 1999, Biochemical and biophysical research communications.

[3]  J. Plitzko,et al.  Incomplete pneumolysin oligomers form membrane pores , 2014, Open Biology.

[4]  E. Conte,et al.  Dynamic Interaction of cBid with Detergents, Liposomes and Mitochondria , 2012, PloS one.

[5]  B. Antonsson.  Bax and other pro-apoptotic Bcl-2 family "killer-proteins" and their victim the mitochondrion , 2001, Cell and Tissue Research.

[6]  Yi-Te Hsu,et al.  Movement of Bax from the Cytosol to Mitochondria during Apoptosis , 1997, The Journal of cell biology.

[7]  Orlando L. Sánchez-Muñoz,et al.  Pores formed by Baxα5 relax to a smaller size and keep at equilibrium. , 2010, Biophysical journal.

[8]  Heinrich Leonhardt,et al.  Targeting and tracing antigens in live cells with fluorescent nanobodies , 2006, Nature Methods.

[9]  S. Hell,et al.  Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. , 1994, Optics letters.

[10]  S. Hell,et al.  Nanoscale resolution in GFP-based microscopy , 2006, Nature Methods.

[11]  Stefan Jakobs,et al.  Bax assembles into large ring‐like structures remodeling the mitochondrial outer membrane in apoptosis , 2016, The EMBO journal.

[12]  S. Korsmeyer,et al.  Proapoptotic BAX and BAK: A Requisite Gateway to Mitochondrial Dysfunction and Death , 2001, Science.

[13]  E. Pérez-Payá,et al.  Membrane-insertion fragments of Bcl-xL, Bax, and Bid. , 2004, Biochemistry.

[14]  Arthur E. Johnson,et al.  Bax Forms an Oligomer via Separate, Yet Interdependent, Surfaces* , 2010, The Journal of Biological Chemistry.

[15]  Cristina Muñoz-Pinedo,et al.  Different mitochondrial intermembrane space proteins are released during apoptosis in a manner that is coordinately initiated but can vary in duration. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[16]  D. Chang,et al.  Dynamics and structure of the Bax-Bak complex responsible for releasing mitochondrial proteins during apoptosis , 2008, Journal of Cell Science.

[17]  G. Anderluh,et al.  Membrane pore formation at protein-lipid interfaces. , 2014, Trends in biochemical sciences.

[18]  K. J. Oh,et al.  Organization of the Mitochondrial Apoptotic BAK Pore , 2013, The Journal of Biological Chemistry.

[19]  J. Zimmerberg,et al.  Bax-type Apoptotic Proteins Porate Pure Lipid Bilayers through a Mechanism Sensitive to Intrinsic Monolayer Curvature* , 2002, The Journal of Biological Chemistry.

[20]  I. Mingarro,et al.  Peptides derived from apoptotic Bax and Bid reproduce the poration activity of the parent full-length proteins. , 2005, Biophysical journal.

[21]  P. Schwille,et al.  Pore formation by a Bax-derived peptide: effect on the line tension of the membrane probed by AFM. , 2007, Biophysical journal.

[22]  Erinna F. Lee,et al.  Bax Crystal Structures Reveal How BH3 Domains Activate Bax and Nucleate Its Oligomerization to Induce Apoptosis , 2013, Cell.

[23]  A. García-Sáez.  The secrets of the Bcl-2 family , 2012, Cell Death and Differentiation.

[24]  Mason R. Mackey,et al.  Bid, Bax, and Lipids Cooperate to Form Supramolecular Openings in the Outer Mitochondrial Membrane , 2002, Cell.

[25]  D. Andrews,et al.  Membrane Binding by tBid Initiates an Ordered Series of Events Culminating in Membrane Permeabilization by Bax , 2008, Cell.

[26]  P. Colman,et al.  To trigger apoptosis, Bak exposes its BH3 domain and homodimerizes via BH3:groove interactions. , 2008, Molecular cell.

[27]  H. Ewers,et al.  A simple, versatile method for GFP-based super-resolution microscopy via nanobodies , 2012, Nature Methods.

[28]  A. García-Sáez,et al.  Proapoptotic Bax and Bak Proteins Form Stable Protein-permeable Pores of Tunable Size , 2013, The Journal of Biological Chemistry.

[29]  N. Volkmann,et al.  Three-dimensional structure of Bax-mediated pores in membrane bilayers , 2013, Cell Death and Disease.

[30]  Maya Topf,et al.  Stepwise visualization of membrane pore formation by suilysin, a bacterial cholesterol-dependent cytolysin , 2014, eLife.

[31]  G. Jeschke,et al.  Structural model of active Bax at the membrane. , 2014, Molecular cell.

[32]  S. Gaudet,et al.  Cell-to-cell variability in cell death: can systems biology help us make sense of it all? , 2014, Cell Death and Disease.

[33]  S. E. Stewart,et al.  Assembly of streptolysin O pores assessed by quartz crystal microbalance and atomic force microscopy provides evidence for the formation of anchored but incomplete oligomers. , 2015, Biochimica et biophysica acta.

[34]  J. Martinou,et al.  Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria. , 2000, The Biochemical journal.

[35]  A. García-Sáez,et al.  More Than a Pore: The Interplay of Pore-Forming Proteins and Lipid Membranes , 2015, The Journal of Membrane Biology.

[36]  Viola Vogel,et al.  Binding-activated localization microscopy of DNA structures. , 2011, Nano letters.

[37]  Gael Moneron,et al.  Nanoscopy in a living multicellular organism expressing GFP. , 2011, Biophysical journal.

[38]  J. Mears,et al.  Conformational changes in Dnm1 support a contractile mechanism for mitochondrial fission , 2010, Nature Structural &Molecular Biology.

[39]  Nico Tjandra,et al.  Bcl-xL Retrotranslocates Bax from the Mitochondria into the Cytosol , 2011, Cell.

[40]  Nico Tjandra,et al.  Structure of Bax Coregulation of Dimer Formation and Intracellular Localization , 2000, Cell.

[41]  R. Gilbert,et al.  Perforin oligomers form arcs in cellular membranes: a locus for intracellular delivery of granzymes , 2014, Cell Death and Differentiation.

[42]  Olivier Sandre,et al.  Cascades of transient pores in giant vesicles: line tension and transport. , 2003, Biophysical journal.

[43]  D. Andrews,et al.  Bax forms multispanning monomers that oligomerize to permeabilize membranes during apoptosis , 2005, The EMBO journal.

[44]  G. Dewson,et al.  Bax dimerizes via a symmetric BH3:groove interface during apoptosis , 2011, Cell Death and Differentiation.

[45]  A. García-Sáez,et al.  Cardiolipin effects on membrane structure and dynamics. , 2013, Langmuir : the ACS journal of surfaces and colloids.

[46]  H. Steinhoff,et al.  Molecular Details of Bax Activation, Oligomerization, and Membrane Insertion* , 2009, The Journal of Biological Chemistry.

[47]  A. García-Sáez,et al.  Mechanistic differences in the membrane activity of Bax and Bcl-xL correlate with their opposing roles in apoptosis. , 2013, Biophysical journal.

[48]  S. Korsmeyer,et al.  The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues. , 2000, Molecular cell.

[49]  J. Spatz,et al.  Bax monomers form dimer units in the membrane that further self-assemble into multiple oligomeric species , 2015, Nature Communications.