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T G Frey

Publications and source records attributed to T G Frey.

At least 19 recordsLinked to original sources
PubMed2002

Insight into mitochondrial structure and function from electron tomography.

In recent years, electron tomography has provided detailed three-dimensional models of mitochondria that have redefined our concept of mitochondrial structure. The models reveal an inner membrane consisting of two components, the inner boundary membrane (IBM) closely apposed to the outer membrane and the cristae membrane that projects into the matrix compartment. These two components are connected by tubular structures of relatively uniform size called crista junctions. The distribution of crista junction sizes and shapes is predicted by a thermodynamic model based upon the energy of membrane bending, but proteins likely also play a role in determining the conformation of the inner membrane. Results of structural studies of mitochondria during apoptosis demonstrate that cytochrome c is released without detectable disruption of the outer membrane or extensive swelling of the mitochondrial matrix, suggesting the formation of an outer membrane pore large enough to allow passage of holo-cytochrome c. The possible compartmentation of inner membrane function between the IBM and the cristae membrane is also discussed.

Animals↗
PubMed2001

Membrane architecture of mitochondria in neurons of the central nervous system.

Electron tomography was used to help redefine the membrane architecture of mitochondria in neurons of the brain. Investigations were conducted on unexplored questions of structural homogeneity between mitochondria in the four intensely studied regions of the brain and in the functionally distinct neuronal sub-compartments. These mitochondria have the majority of cristae composed of both tubular and lamellar segments with the tubes arranged more peripherally and the lamellae more centrally located. Cristae that are entirely tubular were not commonly seen and those that are entirely lamellar were rare. It was determined that cristae connect through narrow, sometimes very long tubular regions to the peripheral surface of the inner membrane. A structurally distinct type of contact site was revealed in brain mitochondria, which we named the bridge contact site. These bridges may play a role in the structural integrity of the outer and inner membrane systems. It was found that the membrane architecture in the various brain regions and neuronal compartments was strikingly uniform, including consistently tubular crista junctions. The functional consequences of this junctional architecture are discussed in relation to the segregation of proteins between the inner boundary membrane and the cristae membranes, and in relation to the model of microcompartmentation of macromolecules inside cristae.

Animals↗
PubMed2001

Electron tomography of mitochondria after the arrest of protein import associated with Tom19 depletion.

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 three-dimensional 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.

Fungal Proteins↗
PubMed2000

The internal structure of mitochondria.

Electron microscopic (EM) tomography is providing important new insights into the internal organization of mitochondria. The standard baffle model for cristae structure, called into question years ago, has now clearly been shown to be inaccurate. Depending on source and conformational state, cristae can vary from simple tubular structures to more complex lamellar structures merging with the inner boundary membrane through tubular structures 28 nm in diameter. The structural information provided by EM tomography has important implications for mitochondrial bioenergetics, biogenesis and the role of mitochondria in apoptosis. The structural paradigm defined by EM tomography is helping in the design of new experimental approaches to mitochondrial function.

Animals↗
PubMed2000

Recent structural insight into mitochondria gained by microscopy.

Novel applications of microscopy have recently provided new insights into mitochondrial structures. Diverse techniques such as high resolution scanning electron microscopy, transmission electron microscopy, electron microscope tomography and light microscopy have contributed a better understanding of mitochondrial compartmentalization, dynamic networks of mitochondria, intermembrane bridges, segregation of mitochondrial DNA and contacts with the endoplasmic reticulum among other aspects. This review focuses on advances reported in the last five years concerning aspects of mitochondrial substructure or dynamics gained through new techniques, whether they be novel microscope methods or new ways to prepare or label specimens. Sometimes these advances have produced surprising results and more often than not, they have challenged current conceptions of how mitochondria work.

Animals↗
PubMed1998

Electron tomography of mitochondria from brown adipocytes reveals crista junctions.

Electron microscope tomography was used to examine the membrane topology of brown adipose tissue (BAT) mitochondria prepared by cryofixation or chemical fixation techniques. These mitochondria contain an uncoupling protein which results in the conversion of energy from electron transport into heat. The three-dimensional reconstructions of BAT mitochondria provided a view of the inner mitochondrial membrane different in important features from descriptions found in the literature. The work reported here provides new insight into BAT mitochondria architecture by identifying crista junctions, including multiple junctions connecting a crista to the same side of the inner boundary membrane, in a class of mitochondria that have no tubular cristae, but only lamellar cristae. Crista junctions were defined previously as the tubular membranes of relatively uniform diameter that connect a crista membrane with the inner boundary membrane. We have also found that the cristae architecture of cryofixed mitochondria, including crista junctions, is similar to that found in chemically fixed mitochondria, suggesting that this architecture is not a fixation artifact. The stacks of lamellar cristae extended through more of the BAT mitochondrial volume than did the cristae we observed in neuronal mitochondria. Hence, the inner membrane surface area was larger in the former. In chemically fixed mitochondria, contact sites were easily visualized because the outer and inner boundary membranes were separated by an 8 nm space. However, in cryofixed mitochondria almost all the outer membrane was observed to be in close contact with the inner boundary membrane.

Adipocytes↗
PubMed1997

Electron tomography of large, multicomponent biological structures.

Electron tomography is an extremely useful method for deriving three-dimensional structure from electron microscope images. The application of this technique to the reconstruction of large, complex structures such as mitochondria is described in conjunction with several tools for segmentation, measurement, classification, and visualization. In addition, the use of massively parallel computers to perform the tomographic reconstruction efficiently using R-weighted backprojection or iterative techniques is described.

Algorithms↗
PubMed1994

Electron microscopy of cytochrome c oxidase crystals: labeling of subunit III with a monomaleimide undecagold cluster compound.

Two-dimensional crystals of beef heart mitochondrial cytochrome c oxidase dimers were labeled at Cys-115 of subunit III with a monomaleimide derivative of an undecagold cluster compound. The binding site of the gold cluster compound and hence the site of subunit III were identified by image processing of cryoelectron micrographs of the crystals preserved in a mixture of glucose and uranyl acetate. The shape of the cytochrome oxidase dimer can be approximated as a parallelogram which is 44 by 82 A with an included angle of 80 degrees oriented with its long dimension along the a axis of the crystal. Labeling of subunit III was confirmed by a shift in the mobility of approximately 50% of subunit III molecules upon electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate. Averaged images of undecagold cluster labeled crystals and of unlabeled crystals were calculated; each image represents an average of approximately 17,000 molecules of either labeled or unlabeled cytochrome oxidase. On the basis of a statistical analysis of the differences between the two images, the gold cluster binds along a line 30 degrees from the a axis and 29 A from the center of the dimer. This result is interpreted in the context of other structural studies including the site of cytochrome c binding which Frey and Murray found to be near the a axis and 18 A from the center of the dimer [Frey, T. G., & Murray, J. M. (1994) J. Mol. Biol. 237, 275-297].(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Electron microscopy of cytochrome c oxidase crystals. Monomer-dimer relationship and cytochrome c binding site.

Cytochrome c oxidase was isolated from beef heart mitochondria by detergent extraction yielding two different crystal forms. Extraction with Triton detergents produced vesicular crystals with two-dimensional crystalline arrays of cytochrome c oxidase dimers while extraction with sodium deoxycholate produced crystalline sheets of cytochrome c oxidase monomers. The structures of both crystal forms were determined in two-dimensional projection along an axis normal to the plane of the membrane by cryoelectron microscopy of crystals embedded in vitreous ice (frozen-hydrated). The projection structures of unstained frozen hydrated monomers and of dimers are similar to the structures of the crystals in negative stain. The molecular outline of dimers can be approximated by a parallelogram 44 A by 82 A with an included angle of 80 degrees. Monomers are less regular consisting of two large domains with a smaller domain at one end and a total length of approximately 82 A. Comparison of the two structures reveals the orientation of cytochrome c oxidase monomers within dimers, an orientation which is different from earlier models of monomer-monomer interaction, and suggests a very close interaction between monomers when they associate to form dimers. The crystalline sheets of cytochrome c oxidase monomers bind tightly the small peripheral membrane protein substrate, cytochrome c, and this binding accentuates a tendency of these crystals to stack upon one another. Images of crystals of the cytochrome c oxidase/cytochrome c complex were analyzed by crosscorrelation analysis versus the monomer crystal image. Two types of two-layer crystals have been identified. Both types have one layer rotated by 180 degrees with respect to the other, but they differ in the shifts of origin along crystal axes of the two layers. Difference images formed by subtracting simulated multilayered crystal images (which have no bound cytochrome c) from the complex crystals (cytochrome c oxidase plus cytochrome c) contain one positive difference peak for each cytochrome oxidase monomer within a unit cell. Comparison of the difference peak loci among the different crystal forms is interpreted based upon a consensus cytochrome c binding site in the single layer cytochrome oxidase monomer crystal image.

Animals↗
PubMed1994

Cytochrome c oxidase: structural studies by electron microscopy of two-dimensional crystals.

Cytochrome c oxidase is a complex integral membrane protein consisting of 13 different polypeptide chains and four metal centers having a total molecular weight of approximately 200,000 daltons. It can be isolated in two 2-dimensional crystalline forms differing in aggregation state of the enzyme. One crystal form consists of cytochrome oxidase dimers (approximately 400,000 daltons) embedded unidirectionally in the lipid bilayer of a collapsed vesicle while the other form consists of crystalline sheets of cytochrome oxidase monomers. Both crystal forms have been studied by electron microscopy during the past two decades, and this paper summarizes the results of early structural studies as well as more recent results applying techniques of cryoelectron microscopy and digital image processing. The structure of frozen-hydrated cytochrome oxidase dimers at 20 A resolution is discussed as well as the packing of monomers within dimers and the site of cytochrome c binding.

Animals↗
PubMed1993

X-ray absorption spectroscopy of oriented cytochrome oxidase.

The polarized X-ray absorption spectra of the copper, iron and zinc sites of mitochondrial cytochrome oxidase in oriented membrane multilayers have been examined. The copper X-ray absorption edge spectra indicate the presence of a tetragonal copper, which we assign as CuB, oriented with the long axis approximately orthogonal to the membrane normal. We have also detected the presence of a relatively long (2.6 A) Cu-S or Cu-Cl interaction, which we assign to a copper-thioether (probably Met210) coordination at the CuA site, with the bond oriented along the membrane normal. The coordination of the zinc, the iron and the CuB heme a3 binuclear site are discussed.

Animals↗
PubMed1990

Electron cryo-microscopic analysis of crystalline cytochrome oxidase.

The structure of cytochrome oxidase from beef heart mitochondria has been analysed by cryo-electron microscopy of vesicle crystals of the space group p22(1)2(1), with cell dimensions a = 102 A, b = 123 A, gamma = 90 degrees. Several methods of specimen preparation were applied to the vesicular two-dimensional crystals in the electron microscope, to ensure that the structure was preserved to the maximum resolution. The two most informative density maps were from specimens embedded in ice and from negative staining in a 1:1 mixture of glucose and uranyl acetate. The three-dimensional structure of the ice-embedded molecule shows a single, well resolved, but convoluted density, which represents in size and shape one cytochrome oxidase dimer. At the bottom of the molecule, a substantial part of the protein is embedded in the lipid bilayer of the vesicle. The molecule then extends upwards, out of the bilayer, into the internal space within the vesicle. Here, the structure first passes through a region within the molecule containing a hollow cavity that lies roughly at the centre of mass of the dimer, and then branches into two well-resolved halves at some distance from the membrane. The negatively stained structure, in contrast, shows a stain-excluding region in the centre of the vesicle at the level of the cavity in the ice-embedded structure, but otherwise has a similar overall external shape. In addition, there is a small rotation of the whole molecule by approximately 25 degrees relative to the orientation of ice-embedded specimens. We interpret these differences to mean that the central cavity seen in the ice-embedded structure is too small to allow the stain to penetrate during the drying process and that the drying process causes the rotation. The structures described here are consistent with one another and allow an interpretation at higher resolution than from previous work.

Animals↗
PubMed1987

A new procedure for the purification of monodisperse highly active cytochrome c oxidase from bovine heart.

A simple and rapid method for the isolation of a large quantity of cytochrome c oxidase from bovine heart mitochondria was developed, based on selective solubilization of mitochondrial protein with first Triton and then lauryl maltoside. Gel filtration shows that the lauryl maltoside-solubilized oxidase preparation is in a hydrodynamically homogeneous state with a Stokes radius of 7.5 +/- 0.2 nm. It contains 8.0 mumol of haem (with an a/a3 ratio of 1)/g of protein. The catalytic constant (maximum turnover number) with respect to cytochrome c approaches 600 S-1. After further purification of the solubilized enzyme on a sucrose-gradient centrifugation, the purified enzyme has a haem content of 10.3 mumol/g of protein and eight major polypeptide bands shown on SDS/polyacrylamide-gel electrophoresis.

Animals↗
PubMed1987

Electron density profile of two-dimensionally crystalline membranous cytochrome c oxidase at low resolution.

Unilamellar vesicles of membranous cytochrome c oxidase have been isolated whose distribution of protein in the membrane plane was predominantly crystalline. The vesicles were collapsed via controlled partial dehydration, resulting, at first, in the formation of unoriented, mostly unstacked, membrane pairs. Further controlled partial dehydration resulted in the formation of oriented multilayers of stacks of membrane pairs, retaining the in-plane crystallinity. The above were monitored by electron microscopy and x-ray diffraction. Analysis of the x-ray diffraction from unoriented, unstacked membrane pairs by two independent methods provided the membrane electron density profile to 30 A resolution.

Animals↗
PubMed1986

Ferromagnetic contrast agents: a new approach.

Most contrast agents used in NMR imaging studies to date have been paramagnetic. However, it is also possible to obtain selective contrast with a ferromagnetic agent, and these agents are potentially more sensitive than paramagnetic compounds because of their large magnetic moments. The water relaxation ability of ferromagnetic, albumin-coated magnetite (Fe3O4) particles has been investigated. These particles are quite effective at reducing both T1 and T2 at relatively low particle concentrations. The potential applications of these particles include improved visualization of the liver, gastrointestinal tract, and genitourinary tract, as well as specific targeting and detection of small tumors or other cells with unique surface receptors.

Animals↗
PubMed1984

Selective contrast in electron microscopy of crystalline cytochrome oxidase.

We have used various techniques for preparation of specimens for electron microscopy in order to selectively contrast different regions of vesicle crystals of cytochrome c oxidase dimers. The results are consistent with a dimer composed of two y-shaped monomers [Fuller et al., J. Mol. Biol. 134 (1979) 305] aligned along one pair of arms with the other pair of arms approximately 70 A apart. The four arms of the monomers lie within and perpendicular to the lipid bilayer in which the dimer is embedded, and the arms protrude approximately 25 A from the lipid bilayer on the matrix side of the membrane. The cytoplasmic side domains of the two monomers split away from one another forming a large cleft in the dimer. Monovalent antibodies (Fab fragments) to subunit IV appear to bind to the two monomer arms which are closely apposed across the two-fold axis of the dimer.

Crystallography↗