Christian Renken - Academia.edu (original) (raw)

Papers by Christian Renken

Research paper thumbnail of Implications of the generation of reactive oxygen species by photoactivated calcein for mitochondrial studies

European Journal of Biochemistry, 2000

Calcein is a fluorescent probe that is widely used in studies of cell viability and mitochondrial... more Calcein is a fluorescent probe that is widely used in studies of cell viability and mitochondrial function by microscopy fluorescence imaging. It was found to have a strong photosensitizing action that prevalently involves the generation of reactive oxygen species (ROS). The photooxidation properties of calcein in solution were studied in the presence of histidine and tryptophan as oxidizable substrates. The photodegradation of histidine was mainly mediated by singlet oxygen ( 1 O 2 ), as shown by the inhibitory effect of sodium azide, a specific 1 O 2 scavenger. On the other hand, mixed photosensitization mechanisms were present when tryptophan was used as the target of the calcein-stimulated photoprocess. In addition to 1 O 2 , hydroxyl radicals and hydrogen peroxide were involved as reactive species, as shown by using mannitol and catalase as scavengers.

Research paper thumbnail of Regulation of CovR expression in Group B Streptococcus impacts blood-brain barrier penetration

Molecular Microbiology, 2010

The pathogen encodes a number of virulence factors including the pluripotent b-haemolysin/cytolys... more The pathogen encodes a number of virulence factors including the pluripotent b-haemolysin/cytolysin (b-H/C). As GBS has the disposition of both a commensal organism and an invasive pathogen, it is important for the organism to appropriately regulate b-H/C and other virulence factors in response to the environment. GBS can repress transcription of b-H/C using the two-component system, CovR/CovS. Recently, we described that the serine/ threonine kinase Stk1 can phosphorylate CovR at threonine 65 to relieve repression of b-H/C. In this study, we show that infection with CovR-deficient GBS strains resulted in increased sepsis. Although CovRdeficient GBS showed decreased ability to invade the brain endothelium in vitro, they were more proficient in induction of permeability and pro-inflammatory signalling pathways in brain endothelium and penetration of the blood-brain barrier (BBB) in vivo. Microarray analysis revealed that CovR positively regulates its own expression and regulates the expression of 153 genes. Collectively, our results suggest that the positive feedback loop which regulates CovR transcription modulates host cell interaction and immune defence and may facilitate the transition of GBS from a commensal organism to a virulent meningeal pathogen.

Research paper thumbnail of Electron Tomography of Neuronal Mitochondria: Three-Dimensional Structure and Organization of Cristae and Membrane Contacts

Journal of Structural Biology, 1997

The structure of neuronal mitochondria from chick and rat was examined using electron microscope ... more The structure of neuronal mitochondria from chick and rat was examined using electron microscope tomography of chemically fixed tissue embedded in plastic and sliced in D500-nm-thick sections. Three-dimensional reconstructions of representative mitochondria were made from single-axis tilt series acquired with an intermediate voltage electron microscope (400 kV). The tilt increment was either 1°or 2°ranging from 260°to 160°. The mitochondrial ultrastructure was similar across species and neuronal regions. The outer and inner membranes were each D7 nm thick. The inner boundary membrane was found to lie close to the outer membrane, with a total thickness across both membranes of D22 nm. We discovered that the inner membrane invaginates to form cristae only through narrow, tubular openings, which we call crista junctions. Sometimes the cristae remain tubular throughout their length, but often multiple tubular cristae merge to form lamellar compartments. Punctate regions, D14 nm in diameter, were observed in which the inner and outer membranes appeared in contact (total thickness of both membranes D14 nm). These contact sites are known to a play a key role in the transport of proteins into the mitochondrion. It has been hypothesized that contact sites may be proximal to crista junctions to facilitate transport of proteins destined for the cristae. How-ever, our statistical analyses indicated that contact sites are randomly located with respect to these junctions. In addition, a close association was observed between endoplasmic reticulum membranes and the outer mitochondrial membrane, consistent with the reported mechanism of transport of certain lipids into the mitochondrion. r 1997 Academic Press

Research paper thumbnail of Electron tomography of mitochondria after the arrest of protein import associated with Tom19 depletion

European Journal of Cell Biology, 2001

In a mutant form of Neurospora crassa, in which sheltered RIP (repeat induced point mutation) was... more In a mutant form of Neurospora crassa, in which sheltered RIP (repeat induced point mutation) was used to deplete Tom19, protein transport through the TOM/TIM pathway is arrested by the addition of p-fluorophenylalanine (FPA). Using intermediate-voltage electron tomography, we have generated threedimensional reconstructions of 28 FPA-treated mitochondria at four time points (0 ± 32 h) after the addition of FPA. We determined that the cristae surface area and volume were lost in a roughly linear manner. A decrease in mitochondrial volume was not observed until after 16 h of FPA treatment. The inner boundary membrane did not appear to shrink or contract away from the outer membrane. Interestingly, the close apposition of these membranes remained over the entire periphery, even after all of the cristae had disappeared. The different dynamics of the shrinkage of cristae membrane and inner boundary membrane has implications for compartmentalization of electron transport proteins. Two structurally distinct types of contact sites were observed, consistent with recently published work. We determined that the cristae in the untreated (control) mitochondria are all lamellar. The cristae of FPA-treated mitochondria retain the lamellar morphology as they reduce in size and do not adopt tubular shapes. Importantly, the crista junctions exhibit tubular as well as slot-like connections to the inner boundary membrane, persisting until the cristae disappear, indicating that their stability is not dependent on continuous protein import through the complex containing Tom19.

Research paper thumbnail of In Search of the Structure of MAC in the Mitochondrial Outer Membrane

Biophysical Journal, 2010

mechanisms through which upregulation of Bcl2 affects earlier steps of BAXmediated apoptosis are ... more mechanisms through which upregulation of Bcl2 affects earlier steps of BAXmediated apoptosis are not fully understood. We found that BAX insertion into the MOM was the earliest apoptotic step inhibited by Bcl2 overexpression. Paradoxically, we also found that BAX translocation to the mitochondria was not inhibited but rather spontaneously increased in this same genetic context. This increase in mitochondrial associated BAX required a physical interaction between BAX and Bcl2. We therefore propose that, at least when upregulated, Bcl2 behaves as a 'decoy receptor' which sequestrates BAX at the mitochondria but inhibits its insertion into the MOM, committing the cell to survive. Cancer is defined by a pronounced inhibition of cell death. The BCL2 family of proteins tightly regulates the delicate balance between life and death. One method of regulation is the compartmentalization of antagonistic members. For example, Bax, a pro-apoptotic member of this family, acts as the penultimate factor in the apoptotic cascade by releasing apoptogenic factors such as Cytochrome C from the mitochondrial lumen. The normally cytosolic protein translocates from one internal compartment to another through an elusive mechanism. Individual organelles are defined not only by function (mediated by specific membrane bound proteins), but by the unique composition of their phospholipid membranes. In this work, we have evaluated the contribution of organelle lipids to the localization of of BCL2 proteins.

Research paper thumbnail of In Search of the Structure of MAC in the Mitochondrial Outer Membrane

Biophysical Journal, 2010

mechanisms through which upregulation of Bcl2 affects earlier steps of BAXmediated apoptosis are ... more mechanisms through which upregulation of Bcl2 affects earlier steps of BAXmediated apoptosis are not fully understood. We found that BAX insertion into the MOM was the earliest apoptotic step inhibited by Bcl2 overexpression. Paradoxically, we also found that BAX translocation to the mitochondria was not inhibited but rather spontaneously increased in this same genetic context. This increase in mitochondrial associated BAX required a physical interaction between BAX and Bcl2. We therefore propose that, at least when upregulated, Bcl2 behaves as a 'decoy receptor' which sequestrates BAX at the mitochondria but inhibits its insertion into the MOM, committing the cell to survive. Cancer is defined by a pronounced inhibition of cell death. The BCL2 family of proteins tightly regulates the delicate balance between life and death. One method of regulation is the compartmentalization of antagonistic members. For example, Bax, a pro-apoptotic member of this family, acts as the penultimate factor in the apoptotic cascade by releasing apoptogenic factors such as Cytochrome C from the mitochondrial lumen. The normally cytosolic protein translocates from one internal compartment to another through an elusive mechanism. Individual organelles are defined not only by function (mediated by specific membrane bound proteins), but by the unique composition of their phospholipid membranes. In this work, we have evaluated the contribution of organelle lipids to the localization of of BCL2 proteins.

Research paper thumbnail of Apoptosis in the Skeletal Muscle of Rats with Heart Failure is Associated with Increased Serum Levels of TNF α and Sphingosine

Journal of Molecular and Cellular Cardiology, 2001

Research paper thumbnail of How Mitochondrial Structure Can Affect Energy Metabolism: Insights From Electron Microscopic Tomography

Biophysical Journal, 2009

Research paper thumbnail of Chapter 6 Principles and Practice in Electron Tomography

Methods in Cell Biology, 2008

... Koster and Barcena, 2006] , [Lucic et al., 2005] and [McIntosh et al., 2005] ). Protocols for... more ... Koster and Barcena, 2006] , [Lucic et al., 2005] and [McIntosh et al., 2005] ). Protocols for carrying out electron tomography have also been published in recent volumes of Methods in Cell Biology and Methods in Molecular Biology ( [Hoog and Antony, 2007] , [Marko and Hsieh ...

Research paper thumbnail of Cryo-Electron Tomography Elucidates the Molecular Architecture of Treponema pallidum, the Syphilis Spirochete

Journal of Bacteriology, 2009

Cryo-electron tomography (CET) was used to examine the native cellular organization of Treponema ... more Cryo-electron tomography (CET) was used to examine the native cellular organization of Treponema pallidum, the syphilis spirochete. T. pallidum cells appeared to form flat waves, did not contain an outer coat and, except for bulges over the basal bodies and widening in the vicinity of flagellar filaments, displayed a uniform periplasmic space. Although the outer membrane (OM) generally was smooth in contour, OM extrusions and blebs frequently were observed, highlighting the structure's fluidity and lack of attachment to underlying periplasmic constituents. Cytoplasmic filaments converged from their attachment points opposite the basal bodies to form arrays that ran roughly parallel to the flagellar filaments along the inner surface of the cytoplasmic membrane (CM). Motile treponemes stably attached to rabbit epithelial cells predominantly via their tips. CET revealed that T. pallidum cell ends have a complex morphology and assume at least four distinct morphotypes. Images of dividing treponemes and organisms shedding cell envelope-derived blebs provided evidence for the spirochete's complex membrane biology. In the regions without flagellar filaments, peptidoglycan (PG) was visualized as a thin layer that divided the periplasmic space into zones of higher and lower electron densities adjacent to the CM and OM, respectively. Flagellar filaments were observed overlying the PG layer, while image modeling placed the PG-basal body contact site in the vicinity of the stator-P-collar junction. Bioinformatics and homology modeling indicated that the MotB proteins of T. pallidum, Treponema denticola, and Borrelia burgdorferi have membrane topologies and PG binding sites highly similar to those of their well-characterized Escherichia coli and Helicobacter pylori orthologs. Collectively, our results help to clarify fundamental differences in cell envelope ultrastructure between spirochetes and gram-negative bacteria. They also confirm that PG stabilizes the flagellar motor and enable us to propose that in most spirochetes motility results from rotation of the flagellar filaments against the PG.

Research paper thumbnail of Structure of frozen–hydrated triad junctions: A case study in motif searching inside tomograms

Journal of Structural Biology, 2009

We used tomographic reconstructions of frozen-hydrated triad junctions to determine the structure... more We used tomographic reconstructions of frozen-hydrated triad junctions to determine the structure of the macromolecular complex associated with calcium release from the sarcoplasmic reticulum (SR), during excitation-contraction coupling. Using a rapid motif search algorithm with a reference motif of the ryanodine receptor (RyR) provided by single-particle cryo-electron microscopy, fortynine receptors were located in five tomograms. Following co-alignment of the receptors and division into quadrants centered on the 4-fold symmetry axis, the receptors were classified using multivariate statistics. Global and class averages reveal that the SR membrane in the vicinity of the receptor is highly curved, creating an open vestibule with a gap of 4 nm between the receptor pore and the calsequestrin layer in the SR lumen. The in-plane densities in the calsequestrin layer have paracrystalline order, consistent with the packing of calsequestrin dimers in the three-dimensional crystal structure. Faint densities ("tethers") extend to the calsequestrin layer from densities in the SR membrane located 15 nm from the symmetry axis of the RyR. In a class average of RyRs with proximal transverse tubules (TT), a cytoplasmic density is observed near the receptor that could represent the most consistent location of tethers observed in tomograms between the SR and TT membranes.

Research paper thumbnail of Structural and functional features and significance of the physical linkage between ER and mitochondria

Journal of Cell Biology, 2006

Research paper thumbnail of The Mitochondrial Inner Membrane Protein Mitofilin Controls Cristae Morphology

Molecular Biology of The Cell, 2005

Mitochondria are complex organelles with a highly dynamic distribution and internal organization.... more Mitochondria are complex organelles with a highly dynamic distribution and internal organization. Here, we demonstrate that mitofilin, a previously identified mitochondrial protein of unknown function, controls mitochondrial cristae morphology. Mitofilin is enriched in the narrow space between the inner boundary and the outer membranes, where it forms a homotypic interaction and assembles into a large multimeric protein complex. Down-regulation of mitofilin in HeLa cells by using specific small interfering RNA lead to decreased cellular proliferation and increased apoptosis, suggesting abnormal mitochondrial function. Although gross mitochondrial fission and fusion seemed normal, ultrastructural studies revealed disorganized mitochondrial inner membrane. Inner membranes failed to form tubular or vesicular cristae and showed as closely packed stacks of membrane sheets that fused intermittently, resulting in a complex maze of membranous network. Electron microscopic tomography estimated a substantial increase in inner:outer membrane ratio, whereas no cristae junctions were detected. In addition, mitochondria subsequently exhibited increased reactive oxygen species production and membrane potential. Although metabolic flux increased due to mitofilin deficiency, mitochondrial oxidative phosphorylation was not increased accordingly. We propose that mitofilin is a critical organizer of the mitochondrial cristae morphology and thus indispensable for normal mitochondrial function.

Research paper thumbnail of The bactericidal effect of a complement-independent antibody is osmolytic and specific to Borrelia

Proceedings of The National Academy of Sciences, 2009

A complement-independent bactericidal IgG1 against the OspB of Borrelia burgdorferi increased the... more A complement-independent bactericidal IgG1 against the OspB of Borrelia burgdorferi increased the permeability of the outer membrane through the creation of openings of 2.8 -4.4 nm, resulting in its osmotic lysis. Cryo-electron microscopy and tomography demonstrated that exposure to the antibody causes the formation of outer membrane projections and large breaks which may precede the increase in permeability of the outer membrane. The bactericidal effect of this antibody is not transferable to Escherichia coli expressing rOspB on its outer membrane. Additionally, the porin P66, the only protein that coprecipitated with OspB, is dispensable for the bactericidal mechanism.

Research paper thumbnail of Electron Tomographic Study of the Microdomain between Mitochondria and the Endoplasmic Reticulum

Microscopy and Microanalysis, 2004

ABSTRACT Export Date: 3 May 2012, Source: Scopus

Research paper thumbnail of Preservation of Mitochondrial Structure and Function after Bid or Bax-Mediated Cytochrome c Release

Journal of Cell Biology, 2000

Proapoptotic members of the Bcl-2 protein family, including Bid and Bax, can activate apoptosis b... more Proapoptotic members of the Bcl-2 protein family, including Bid and Bax, can activate apoptosis by directly interacting with mitochondria to cause cytochrome c translocation from the intermembrane space into the cytoplasm, thereby triggering Apaf-1-mediated caspase activation. Under some circumstances, when caspase activation is blocked, cells can recover from cytochrome c translocation; this suggests that apoptotic mitochondria may not always suffer catastrophic damage arising from the process of cytochrome c release. We now show that recombinant Bid and Bax cause complete cytochrome c loss from isolated mitochondria in vitro, but preserve the ultrastructure and protein im-port function of mitochondria, which depend on inner membrane polarization. We also demonstrate that, if caspases are inhibited, mitochondrial protein import function is retained in UV-irradiated or staurosporinetreated cells, despite the complete translocation of cytochrome c . Thus, Bid and Bax act only on the outer membrane, and lesions in the inner membrane occurring during apoptosis are shown to be secondary caspase-dependent events.

Research paper thumbnail of A thermodynamic model describing the nature of the crista junction: a structural motif in the mitochondrion

Journal of Structural Biology, 2002

The use of electron tomography has allowed the three-dimensional membrane topography of the mitoc... more The use of electron tomography has allowed the three-dimensional membrane topography of the mitochondrion to be better understood. The most striking feature of this topology is the crista junction, a structure that may serve to divide functionally the inner-membrane and inter-membrane spaces. In situ these junctions seem to have a preferred size and shape independent of the source of the mitochondrion with few exceptions. When mitochondria are isolated and have a condensed matrix the crista junctions enlarge and become non-discrete. Upon permeation of the inner membrane and subsequent swelling of the matrix space, the uniform circular nature of the crista junction reappears. We examine the distribution of shapes and sizes of crista junctions and suggest a thermodynamic model that explains the distribution based on current theories of bilayer membrane shapes. The theory of spontaneous curvature shows the circular junction to be a thermodynamically stable structure whose size and shape is influenced by the relative volume of the matrix. We conclude that the crista junction exists predominantly as a circular junction with other shapes as exceptions made possible by specific characteristics of the lipid bilayer.

Research paper thumbnail of The Pro-apoptotic Proteins, Bid and Bax, Cause a Limited Permeabilization of the Mitochondrial Outer Membrane that Is Enhanced by Cytosol

Journal of Cell Biology, 1999

During apoptosis, an important pathway leading to caspase activation involves the release of cyto... more During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, argu-ing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c . However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.

Research paper thumbnail of Structure of frozen–hydrated triad junctions: A case study in motif searching inside tomograms

We used tomographic reconstructions of frozen-hydrated triad junctions to determine the structure... more We used tomographic reconstructions of frozen-hydrated triad junctions to determine the structure of the macromolecular complex associated with calcium release from the sarcoplasmic reticulum (SR), during excitation-contraction coupling. Using a rapid motif search algorithm with a reference motif of the ryanodine receptor (RyR) provided by single-particle cryo-electron microscopy, 49 receptors were located in five tomograms. Following co-alignment of the receptors and division into quadrants centered on the 4-fold symmetry axis, the receptors were classified using multivariate statistics. Global and class averages reveal that the SR membrane in the vicinity of the receptor is highly curved, creating an open vestibule with a gap of 4 nm between the receptor pore and the calsequestrin layer in the SR lumen. The in-plane densities in the calsequestrin layer have paracrystalline order, consistent with the packing of calsequestrin dimers in the three-dimensional crystal structure. Faint densities (''tethers") extend to the calsequestrin layer from densities in the SR membrane located 15 nm from the symmetry axis of the RyR. In a class average of RyRs with proximal transverse tubules (TT), a cytoplasmic density is observed near the receptor that could represent the most consistent location of tethers observed in tomograms between the SR and TT membranes.

Research paper thumbnail of Implications of the generation of reactive oxygen species by photoactivated calcein for mitochondrial studies

European Journal of Biochemistry, 2000

Calcein is a fluorescent probe that is widely used in studies of cell viability and mitochondrial... more Calcein is a fluorescent probe that is widely used in studies of cell viability and mitochondrial function by microscopy fluorescence imaging. It was found to have a strong photosensitizing action that prevalently involves the generation of reactive oxygen species (ROS). The photooxidation properties of calcein in solution were studied in the presence of histidine and tryptophan as oxidizable substrates. The photodegradation of histidine was mainly mediated by singlet oxygen ( 1 O 2 ), as shown by the inhibitory effect of sodium azide, a specific 1 O 2 scavenger. On the other hand, mixed photosensitization mechanisms were present when tryptophan was used as the target of the calcein-stimulated photoprocess. In addition to 1 O 2 , hydroxyl radicals and hydrogen peroxide were involved as reactive species, as shown by using mannitol and catalase as scavengers.

Research paper thumbnail of Regulation of CovR expression in Group B Streptococcus impacts blood-brain barrier penetration

Molecular Microbiology, 2010

The pathogen encodes a number of virulence factors including the pluripotent b-haemolysin/cytolys... more The pathogen encodes a number of virulence factors including the pluripotent b-haemolysin/cytolysin (b-H/C). As GBS has the disposition of both a commensal organism and an invasive pathogen, it is important for the organism to appropriately regulate b-H/C and other virulence factors in response to the environment. GBS can repress transcription of b-H/C using the two-component system, CovR/CovS. Recently, we described that the serine/ threonine kinase Stk1 can phosphorylate CovR at threonine 65 to relieve repression of b-H/C. In this study, we show that infection with CovR-deficient GBS strains resulted in increased sepsis. Although CovRdeficient GBS showed decreased ability to invade the brain endothelium in vitro, they were more proficient in induction of permeability and pro-inflammatory signalling pathways in brain endothelium and penetration of the blood-brain barrier (BBB) in vivo. Microarray analysis revealed that CovR positively regulates its own expression and regulates the expression of 153 genes. Collectively, our results suggest that the positive feedback loop which regulates CovR transcription modulates host cell interaction and immune defence and may facilitate the transition of GBS from a commensal organism to a virulent meningeal pathogen.

Research paper thumbnail of Electron Tomography of Neuronal Mitochondria: Three-Dimensional Structure and Organization of Cristae and Membrane Contacts

Journal of Structural Biology, 1997

The structure of neuronal mitochondria from chick and rat was examined using electron microscope ... more The structure of neuronal mitochondria from chick and rat was examined using electron microscope tomography of chemically fixed tissue embedded in plastic and sliced in D500-nm-thick sections. Three-dimensional reconstructions of representative mitochondria were made from single-axis tilt series acquired with an intermediate voltage electron microscope (400 kV). The tilt increment was either 1°or 2°ranging from 260°to 160°. The mitochondrial ultrastructure was similar across species and neuronal regions. The outer and inner membranes were each D7 nm thick. The inner boundary membrane was found to lie close to the outer membrane, with a total thickness across both membranes of D22 nm. We discovered that the inner membrane invaginates to form cristae only through narrow, tubular openings, which we call crista junctions. Sometimes the cristae remain tubular throughout their length, but often multiple tubular cristae merge to form lamellar compartments. Punctate regions, D14 nm in diameter, were observed in which the inner and outer membranes appeared in contact (total thickness of both membranes D14 nm). These contact sites are known to a play a key role in the transport of proteins into the mitochondrion. It has been hypothesized that contact sites may be proximal to crista junctions to facilitate transport of proteins destined for the cristae. How-ever, our statistical analyses indicated that contact sites are randomly located with respect to these junctions. In addition, a close association was observed between endoplasmic reticulum membranes and the outer mitochondrial membrane, consistent with the reported mechanism of transport of certain lipids into the mitochondrion. r 1997 Academic Press

Research paper thumbnail of Electron tomography of mitochondria after the arrest of protein import associated with Tom19 depletion

European Journal of Cell Biology, 2001

In a mutant form of Neurospora crassa, in which sheltered RIP (repeat induced point mutation) was... more In a mutant form of Neurospora crassa, in which sheltered RIP (repeat induced point mutation) was used to deplete Tom19, protein transport through the TOM/TIM pathway is arrested by the addition of p-fluorophenylalanine (FPA). Using intermediate-voltage electron tomography, we have generated threedimensional reconstructions of 28 FPA-treated mitochondria at four time points (0 ± 32 h) after the addition of FPA. We determined that the cristae surface area and volume were lost in a roughly linear manner. A decrease in mitochondrial volume was not observed until after 16 h of FPA treatment. The inner boundary membrane did not appear to shrink or contract away from the outer membrane. Interestingly, the close apposition of these membranes remained over the entire periphery, even after all of the cristae had disappeared. The different dynamics of the shrinkage of cristae membrane and inner boundary membrane has implications for compartmentalization of electron transport proteins. Two structurally distinct types of contact sites were observed, consistent with recently published work. We determined that the cristae in the untreated (control) mitochondria are all lamellar. The cristae of FPA-treated mitochondria retain the lamellar morphology as they reduce in size and do not adopt tubular shapes. Importantly, the crista junctions exhibit tubular as well as slot-like connections to the inner boundary membrane, persisting until the cristae disappear, indicating that their stability is not dependent on continuous protein import through the complex containing Tom19.

Research paper thumbnail of In Search of the Structure of MAC in the Mitochondrial Outer Membrane

Biophysical Journal, 2010

mechanisms through which upregulation of Bcl2 affects earlier steps of BAXmediated apoptosis are ... more mechanisms through which upregulation of Bcl2 affects earlier steps of BAXmediated apoptosis are not fully understood. We found that BAX insertion into the MOM was the earliest apoptotic step inhibited by Bcl2 overexpression. Paradoxically, we also found that BAX translocation to the mitochondria was not inhibited but rather spontaneously increased in this same genetic context. This increase in mitochondrial associated BAX required a physical interaction between BAX and Bcl2. We therefore propose that, at least when upregulated, Bcl2 behaves as a 'decoy receptor' which sequestrates BAX at the mitochondria but inhibits its insertion into the MOM, committing the cell to survive. Cancer is defined by a pronounced inhibition of cell death. The BCL2 family of proteins tightly regulates the delicate balance between life and death. One method of regulation is the compartmentalization of antagonistic members. For example, Bax, a pro-apoptotic member of this family, acts as the penultimate factor in the apoptotic cascade by releasing apoptogenic factors such as Cytochrome C from the mitochondrial lumen. The normally cytosolic protein translocates from one internal compartment to another through an elusive mechanism. Individual organelles are defined not only by function (mediated by specific membrane bound proteins), but by the unique composition of their phospholipid membranes. In this work, we have evaluated the contribution of organelle lipids to the localization of of BCL2 proteins.

Research paper thumbnail of In Search of the Structure of MAC in the Mitochondrial Outer Membrane

Biophysical Journal, 2010

mechanisms through which upregulation of Bcl2 affects earlier steps of BAXmediated apoptosis are ... more mechanisms through which upregulation of Bcl2 affects earlier steps of BAXmediated apoptosis are not fully understood. We found that BAX insertion into the MOM was the earliest apoptotic step inhibited by Bcl2 overexpression. Paradoxically, we also found that BAX translocation to the mitochondria was not inhibited but rather spontaneously increased in this same genetic context. This increase in mitochondrial associated BAX required a physical interaction between BAX and Bcl2. We therefore propose that, at least when upregulated, Bcl2 behaves as a 'decoy receptor' which sequestrates BAX at the mitochondria but inhibits its insertion into the MOM, committing the cell to survive. Cancer is defined by a pronounced inhibition of cell death. The BCL2 family of proteins tightly regulates the delicate balance between life and death. One method of regulation is the compartmentalization of antagonistic members. For example, Bax, a pro-apoptotic member of this family, acts as the penultimate factor in the apoptotic cascade by releasing apoptogenic factors such as Cytochrome C from the mitochondrial lumen. The normally cytosolic protein translocates from one internal compartment to another through an elusive mechanism. Individual organelles are defined not only by function (mediated by specific membrane bound proteins), but by the unique composition of their phospholipid membranes. In this work, we have evaluated the contribution of organelle lipids to the localization of of BCL2 proteins.

Research paper thumbnail of Apoptosis in the Skeletal Muscle of Rats with Heart Failure is Associated with Increased Serum Levels of TNF α and Sphingosine

Journal of Molecular and Cellular Cardiology, 2001

Research paper thumbnail of How Mitochondrial Structure Can Affect Energy Metabolism: Insights From Electron Microscopic Tomography

Biophysical Journal, 2009

Research paper thumbnail of Chapter 6 Principles and Practice in Electron Tomography

Methods in Cell Biology, 2008

... Koster and Barcena, 2006] , [Lucic et al., 2005] and [McIntosh et al., 2005] ). Protocols for... more ... Koster and Barcena, 2006] , [Lucic et al., 2005] and [McIntosh et al., 2005] ). Protocols for carrying out electron tomography have also been published in recent volumes of Methods in Cell Biology and Methods in Molecular Biology ( [Hoog and Antony, 2007] , [Marko and Hsieh ...

Research paper thumbnail of Cryo-Electron Tomography Elucidates the Molecular Architecture of Treponema pallidum, the Syphilis Spirochete

Journal of Bacteriology, 2009

Cryo-electron tomography (CET) was used to examine the native cellular organization of Treponema ... more Cryo-electron tomography (CET) was used to examine the native cellular organization of Treponema pallidum, the syphilis spirochete. T. pallidum cells appeared to form flat waves, did not contain an outer coat and, except for bulges over the basal bodies and widening in the vicinity of flagellar filaments, displayed a uniform periplasmic space. Although the outer membrane (OM) generally was smooth in contour, OM extrusions and blebs frequently were observed, highlighting the structure's fluidity and lack of attachment to underlying periplasmic constituents. Cytoplasmic filaments converged from their attachment points opposite the basal bodies to form arrays that ran roughly parallel to the flagellar filaments along the inner surface of the cytoplasmic membrane (CM). Motile treponemes stably attached to rabbit epithelial cells predominantly via their tips. CET revealed that T. pallidum cell ends have a complex morphology and assume at least four distinct morphotypes. Images of dividing treponemes and organisms shedding cell envelope-derived blebs provided evidence for the spirochete's complex membrane biology. In the regions without flagellar filaments, peptidoglycan (PG) was visualized as a thin layer that divided the periplasmic space into zones of higher and lower electron densities adjacent to the CM and OM, respectively. Flagellar filaments were observed overlying the PG layer, while image modeling placed the PG-basal body contact site in the vicinity of the stator-P-collar junction. Bioinformatics and homology modeling indicated that the MotB proteins of T. pallidum, Treponema denticola, and Borrelia burgdorferi have membrane topologies and PG binding sites highly similar to those of their well-characterized Escherichia coli and Helicobacter pylori orthologs. Collectively, our results help to clarify fundamental differences in cell envelope ultrastructure between spirochetes and gram-negative bacteria. They also confirm that PG stabilizes the flagellar motor and enable us to propose that in most spirochetes motility results from rotation of the flagellar filaments against the PG.

Research paper thumbnail of Structure of frozen–hydrated triad junctions: A case study in motif searching inside tomograms

Journal of Structural Biology, 2009

We used tomographic reconstructions of frozen-hydrated triad junctions to determine the structure... more We used tomographic reconstructions of frozen-hydrated triad junctions to determine the structure of the macromolecular complex associated with calcium release from the sarcoplasmic reticulum (SR), during excitation-contraction coupling. Using a rapid motif search algorithm with a reference motif of the ryanodine receptor (RyR) provided by single-particle cryo-electron microscopy, fortynine receptors were located in five tomograms. Following co-alignment of the receptors and division into quadrants centered on the 4-fold symmetry axis, the receptors were classified using multivariate statistics. Global and class averages reveal that the SR membrane in the vicinity of the receptor is highly curved, creating an open vestibule with a gap of 4 nm between the receptor pore and the calsequestrin layer in the SR lumen. The in-plane densities in the calsequestrin layer have paracrystalline order, consistent with the packing of calsequestrin dimers in the three-dimensional crystal structure. Faint densities ("tethers") extend to the calsequestrin layer from densities in the SR membrane located 15 nm from the symmetry axis of the RyR. In a class average of RyRs with proximal transverse tubules (TT), a cytoplasmic density is observed near the receptor that could represent the most consistent location of tethers observed in tomograms between the SR and TT membranes.

Research paper thumbnail of Structural and functional features and significance of the physical linkage between ER and mitochondria

Journal of Cell Biology, 2006

Research paper thumbnail of The Mitochondrial Inner Membrane Protein Mitofilin Controls Cristae Morphology

Molecular Biology of The Cell, 2005

Mitochondria are complex organelles with a highly dynamic distribution and internal organization.... more Mitochondria are complex organelles with a highly dynamic distribution and internal organization. Here, we demonstrate that mitofilin, a previously identified mitochondrial protein of unknown function, controls mitochondrial cristae morphology. Mitofilin is enriched in the narrow space between the inner boundary and the outer membranes, where it forms a homotypic interaction and assembles into a large multimeric protein complex. Down-regulation of mitofilin in HeLa cells by using specific small interfering RNA lead to decreased cellular proliferation and increased apoptosis, suggesting abnormal mitochondrial function. Although gross mitochondrial fission and fusion seemed normal, ultrastructural studies revealed disorganized mitochondrial inner membrane. Inner membranes failed to form tubular or vesicular cristae and showed as closely packed stacks of membrane sheets that fused intermittently, resulting in a complex maze of membranous network. Electron microscopic tomography estimated a substantial increase in inner:outer membrane ratio, whereas no cristae junctions were detected. In addition, mitochondria subsequently exhibited increased reactive oxygen species production and membrane potential. Although metabolic flux increased due to mitofilin deficiency, mitochondrial oxidative phosphorylation was not increased accordingly. We propose that mitofilin is a critical organizer of the mitochondrial cristae morphology and thus indispensable for normal mitochondrial function.

Research paper thumbnail of The bactericidal effect of a complement-independent antibody is osmolytic and specific to Borrelia

Proceedings of The National Academy of Sciences, 2009

A complement-independent bactericidal IgG1 against the OspB of Borrelia burgdorferi increased the... more A complement-independent bactericidal IgG1 against the OspB of Borrelia burgdorferi increased the permeability of the outer membrane through the creation of openings of 2.8 -4.4 nm, resulting in its osmotic lysis. Cryo-electron microscopy and tomography demonstrated that exposure to the antibody causes the formation of outer membrane projections and large breaks which may precede the increase in permeability of the outer membrane. The bactericidal effect of this antibody is not transferable to Escherichia coli expressing rOspB on its outer membrane. Additionally, the porin P66, the only protein that coprecipitated with OspB, is dispensable for the bactericidal mechanism.

Research paper thumbnail of Electron Tomographic Study of the Microdomain between Mitochondria and the Endoplasmic Reticulum

Microscopy and Microanalysis, 2004

ABSTRACT Export Date: 3 May 2012, Source: Scopus

Research paper thumbnail of Preservation of Mitochondrial Structure and Function after Bid or Bax-Mediated Cytochrome c Release

Journal of Cell Biology, 2000

Proapoptotic members of the Bcl-2 protein family, including Bid and Bax, can activate apoptosis b... more Proapoptotic members of the Bcl-2 protein family, including Bid and Bax, can activate apoptosis by directly interacting with mitochondria to cause cytochrome c translocation from the intermembrane space into the cytoplasm, thereby triggering Apaf-1-mediated caspase activation. Under some circumstances, when caspase activation is blocked, cells can recover from cytochrome c translocation; this suggests that apoptotic mitochondria may not always suffer catastrophic damage arising from the process of cytochrome c release. We now show that recombinant Bid and Bax cause complete cytochrome c loss from isolated mitochondria in vitro, but preserve the ultrastructure and protein im-port function of mitochondria, which depend on inner membrane polarization. We also demonstrate that, if caspases are inhibited, mitochondrial protein import function is retained in UV-irradiated or staurosporinetreated cells, despite the complete translocation of cytochrome c . Thus, Bid and Bax act only on the outer membrane, and lesions in the inner membrane occurring during apoptosis are shown to be secondary caspase-dependent events.

Research paper thumbnail of A thermodynamic model describing the nature of the crista junction: a structural motif in the mitochondrion

Journal of Structural Biology, 2002

The use of electron tomography has allowed the three-dimensional membrane topography of the mitoc... more The use of electron tomography has allowed the three-dimensional membrane topography of the mitochondrion to be better understood. The most striking feature of this topology is the crista junction, a structure that may serve to divide functionally the inner-membrane and inter-membrane spaces. In situ these junctions seem to have a preferred size and shape independent of the source of the mitochondrion with few exceptions. When mitochondria are isolated and have a condensed matrix the crista junctions enlarge and become non-discrete. Upon permeation of the inner membrane and subsequent swelling of the matrix space, the uniform circular nature of the crista junction reappears. We examine the distribution of shapes and sizes of crista junctions and suggest a thermodynamic model that explains the distribution based on current theories of bilayer membrane shapes. The theory of spontaneous curvature shows the circular junction to be a thermodynamically stable structure whose size and shape is influenced by the relative volume of the matrix. We conclude that the crista junction exists predominantly as a circular junction with other shapes as exceptions made possible by specific characteristics of the lipid bilayer.

Research paper thumbnail of The Pro-apoptotic Proteins, Bid and Bax, Cause a Limited Permeabilization of the Mitochondrial Outer Membrane that Is Enhanced by Cytosol

Journal of Cell Biology, 1999

During apoptosis, an important pathway leading to caspase activation involves the release of cyto... more During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, argu-ing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c . However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.

Research paper thumbnail of Structure of frozen–hydrated triad junctions: A case study in motif searching inside tomograms

We used tomographic reconstructions of frozen-hydrated triad junctions to determine the structure... more We used tomographic reconstructions of frozen-hydrated triad junctions to determine the structure of the macromolecular complex associated with calcium release from the sarcoplasmic reticulum (SR), during excitation-contraction coupling. Using a rapid motif search algorithm with a reference motif of the ryanodine receptor (RyR) provided by single-particle cryo-electron microscopy, 49 receptors were located in five tomograms. Following co-alignment of the receptors and division into quadrants centered on the 4-fold symmetry axis, the receptors were classified using multivariate statistics. Global and class averages reveal that the SR membrane in the vicinity of the receptor is highly curved, creating an open vestibule with a gap of 4 nm between the receptor pore and the calsequestrin layer in the SR lumen. The in-plane densities in the calsequestrin layer have paracrystalline order, consistent with the packing of calsequestrin dimers in the three-dimensional crystal structure. Faint densities (''tethers") extend to the calsequestrin layer from densities in the SR membrane located 15 nm from the symmetry axis of the RyR. In a class average of RyRs with proximal transverse tubules (TT), a cytoplasmic density is observed near the receptor that could represent the most consistent location of tethers observed in tomograms between the SR and TT membranes.