Cubic Membrane Structure in Amoeba (Chaos carolinensis) Mitochondria Determined by Electron Microscopic Tomography

Cubic membranes occur in a variety of membrane-bound organelles in many cell types. By transmission electron microscopy (TEM) these membrane systems appear to consist of highly curved periodic surfaces that fit mathematical models analogous to those used to describe lipidic cubic phases. For the first time, a naturally occurring cubic membrane system has been reconstructed in three dimensions by electron microscopic tomography, and its periodicity directly characterized. Double-tilt tomographic reconstruction of mitochondria in the amoeba, Chaos carolinensis, confirms that their cristae (inner membrane infoldings) have the cubic structure suggested by modeling studies based on thin-section TEM images. Analysis of the membrane surfaces in the reconstruction reveals the connectivity of the internal compartments within the mitochondria. In the cubic regions, the matrix is highly condensed and confined to a continuous, small space between adjacent cristal membranes. The cristae form large, undulating cisternae that communicate with the peripheral (inner membrane) compartment through narrow tubular segments as seen in other types of mitochondria. The cubic periodicity of these mitochondrial membranes provides an ideal specimen for measuring geometrical distortions in biological electron tomography. It may also prove to be a useful model system for studies of the correlation of cristae–matrix organization with mitochondrial activity.     

Three-dimensional periodic cubic membrane structure in the mitochondria of amoebaeChaos carolinensis

Summary Through computer simulation of images produced by the transmission electron microscope (TEM), we have identified three-dimensional periodic cubic membrane structures in giant amoebae (Chaos carolinensis) mitochondria. The cubic membranes are based on the highly curved three-dimensional periodic cubic surfaces, sharing the same geometry of mathematically defined periodic minimal surfaces. The double-membrane structures identified here divide space into three separate and convoluted subspaces. Specimen preparation, specifically the tendency to cut oblique sections, of this membrane crystal has added to the complexity of the resulting TEM projections and until now prevented researchers from recognizing them. It is the added complexity of the oblique sections, though, that allows us to match the TEM projection to a computer simulation of the same with confidence. In this study, formation of cubic membrane structures in amoeba mitochondria was found to be dependent on diet. The cubic structures only occurred in the absence of food, and disappeared in the presence of food, suggesting a structural adaptation and possible advantages for amoeba's survival in nature. The verification of mathematically well-defined structures in unfed amoeba mitochondria is also important to the understanding of the mitochondrial bioenergetics in relation to the topology of the inner membrane, where major cellular energy production as well as free-radical generation are taking place. This understanding may carry great impact upon human health as far as aging and age-related degenerative diseases are concerned, especially as mitochondrial disorders have been implicated in these processes.Abbreviations G gyroid - D double diamond - P primitive - TEM transmission electron microscopy - PCS periodic cubic surface     

The mitochondrial inner membrane protein mitofilin controls cristae morphology

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.     

The relevance of mitochondrial membrane topology to mitochondrial function

This review summarizes recent findings from electron tomography about the three-dimensional shape of mitochondrial membranes and its possible influence on a range of mitochondrial functions. The inner membrane invaginations called cristae are pleomorphic, typically connected by narrow tubular junctions of variable length to the inner boundary membrane. This design may restrict intra-mitochondrial diffusion of metabolites such as ADP, and of soluble proteins such as cytochrome c. Tomographic images of a variety of mitochondria suggest that inner membrane topology reflects a balance between membrane fusion and fission. Proteins that can affect cristae morphology include tBid, which triggers cytochrome c release in apoptosis, and the dynamin-like protein Mgm1, involved in inter-mitochondrial membrane fusion. In frozen-hydrated rat-liver mitochondria, the space between the inner and outer membranes contains 10-15 nm particles that may represent macromolecular complexes involved in activities that span the two membranes.     

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.     

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.     

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

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 ≈500-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 −60° to +60°. The mitochondrial ultrastructure was similar across species and neuronal regions. The outer and inner membranes were each ≈7 nm thick. The inner boundary membrane was found to lie close to the outer membrane, with a total thickness across both membranes of ≈22 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, ≈14 nm in diameter, were observed in which the inner and outer membranes appeared in contact (total thickness of both membranes ≈14 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. However, 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.     

Trial comparison of mitochondrial cristae craters images obtained by the freeze-fracturing and ultrathin sections method

In two-folded lamina of the mitochondrial cristae occurs in mitochondria of spermatocytes large areas of the inner and outer halves in freeze-fracturing technique morphological observations suggest that in mitochondrial membrane there exist "crater-like' structures with internal diameter of approximately 18 nm. A question has come up why no mention has so far been found in the literature of the appearance of similar structure in mitochondrial cristae in specimens in transmission electron microscope (TEM) observed. Thus comparison of our findings obtained by the freeze-fracturing (FF) method with those achieved by TEM was made.     

Structure and dynamics of the mitochondrial inner membrane cristae

Three-dimensional images of mitochondria provided by electron tomography reveal that the micro-compartments (cristae) defined by the inner membrane are connected to the periphery of this membrane by narrow tubular junctions, which likely restrict diffusion. The tomograms also strongly suggest that inner membrane topology represents a balance between membrane fusion and fission processes. The hypothesis being developed is that inner membrane topology is a regulated property of mitochondria. This review summarizes the evidence about how inner membrane shape influences mitochondrial function and, conversely, what is known about the factors that determine this membrane's topology.     

Role of membrane contact sites in protein import into mitochondria

Mitochondria import more than 1,000 different proteins from the cytosol. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated by protein transport machineries of the mitochondrial membranes. Five main pathways for protein import into mitochondria have been identified. Most pathways use the translocase of the outer mitochondrial membrane (TOM) as the entry gate into mitochondria. Depending on specific signals contained in the precursors, the proteins are subsequently transferred to different intramitochondrial translocases. In this article, we discuss the connection between protein import and mitochondrial membrane architecture. Mitochondria possess two membranes. It is a long‐standing question how contact sites between outer and inner membranes are formed and which role the contact sites play in the translocation of precursor proteins. A major translocation contact site is formed between the TOM complex and the presequence translocase of the inner membrane (TIM23 complex), promoting transfer of presequence‐carrying preproteins to the mitochondrial inner membrane and matrix. Recent findings led to the identification of contact sites that involve the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. MICOS plays a dual role. It is crucial for maintaining the inner membrane cristae architecture and forms contacts sites to the outer membrane that promote translocation of precursor proteins into the intermembrane space and outer membrane of mitochondria. The view is emerging that the mitochondrial protein translocases do not function as independent units, but are embedded in a network of interactions with machineries that control mitochondrial activity and architecture.     

Differential protein distributions define two sub-compartments of the mitochondrial inner membrane in yeast

The mitochondrial inner membrane exhibits a complex topology. Its infolds, the cristae membranes, are contiguous with the inner boundary membrane (IBM), which runs parallel to the outer membrane. Using live cells co-expressing functional fluorescent fusion proteins, we report on the distribution of inner membrane proteins in budding yeast. To this end we introduce the enlarged mitochondria of Deltamdm10, Deltamdm31, Deltamdm32, and Deltammm1 cells as a versatile model system to study sub-mitochondrial protein localizations. Proteins of the F(1)F(0) ATP synthase and of the respiratory chain complexes III and IV were visualized in the cristae-containing interior of the mitochondria. In contrast, proteins of the TIM23 complex and of the presequence translocase-associated motor were strongly enriched at the IBM. The different protein distributions shown here demonstrate that the cristae membranes and the IBM are functionally distinct sub-compartments.     

Biochemical and stereological analysis of rat liver mitochondria in different thyroid states

The concentrations of the inner mitochondrial membrane markers cardiolipin and cytochrome alpha have been measured in liver homogenates and in purified mitochondria after thyroxine administration to thyroidectomized and normal rats. The biochemical results have been correlated with stereological electron micrographic analyses of hepatocytes in liver sections, and of isolated mitochondrial pellets. There were progressive and parallel increases in homogenate and mitochondrial cardiolipin concentration, and in mitochondrial cytochrome alpha concentration, after administration of 20 microgram of thyroxine on alternate days to thyroidectomized rats, and of 300 microgram on alternate days to normal rats. Electron microscope measurements showed marked differences in the shape of the mitochondria and in the number of cristae in different thyroid states. Hypothyroid mitochondria were shorter and wider than controls, and hyperthyroid mitochondria longer but of similar width. Mitochondrial volume per unit cell volume was virtually unchanged in hypo- and hyperthyroid animals. The most striking changes were a decrease in the area of the inner membrane plus cristae in thyroidectomized rats, and a substantial increase in membrane area after thyroxine administration. The biochemical and electron micrographic results indicate that, in rat liver, thyroid hormone administration leads to a selective increase in the relative amount of mitochondrial inner membranes, with little or no change in the mitochondrial volume per unit cell volume, or in total mitochondrial protein per unit total cell protein.     

Pathways shaping the mitochondrial inner membrane

Mitochondria are complex organelles with two membranes. Their architecture is determined by characteristic folds of the inner membrane, termed cristae. Recent studies in yeast and other organisms led to the identification of four major pathways that cooperate to shape cristae membranes. These include dimer formation of the mitochondrial ATP synthase, assembly of the mitochondrial contact site and cristae organizing system (MICOS), inner membrane remodelling by a dynamin-related GTPase (Mgm1/OPA1), and modulation of the mitochondrial lipid composition. In this review, we describe the function of the evolutionarily conserved machineries involved in mitochondrial cristae biogenesis with a focus on yeast and present current models to explain how their coordinated activities establish mitochondrial membrane architecture.     

The inner membrane protein Mdm33 controls mitochondrial morphology in yeast

Mitochondrial distribution and morphology depend on MDM33, a Saccharomyces cerevisiae gene encoding a novel protein of the mitochondrial inner membrane. Cells lacking Mdm33 contain ring-shaped, mostly interconnected mitochondria, which are able to form large hollow spheres. On the ultrastructural level, these aberrant organelles display extremely elongated stretches of outer and inner membranes enclosing a very narrow matrix space. Dilated parts of Delta mdm33 mitochondria contain well-developed cristae. Overexpression of Mdm33 leads to growth arrest, aggregation of mitochondria, and generation of aberrant inner membrane structures, including septa, inner membrane fragments, and loss of inner membrane cristae. The MDM33 gene is required for the formation of net-like mitochondria in mutants lacking components of the outer membrane fission machinery, and mitochondrial fusion is required for the formation of extended ring-like mitochondria in cells lacking the MDM33 gene. The Mdm33 protein assembles into an oligomeric complex in the inner membrane where it performs homotypic protein-protein interactions. Our results indicate that Mdm33 plays a distinct role in the mitochondrial inner membrane to control mitochondrial morphology. We propose that Mdm33 is involved in fission of the mitochondrial inner membrane.     

New findings on 3-D microanatomy of cellular structures in human tissues and organs. An HRSEM study

We present here findings obtained on a large number of human tissues over a period of more than ten years, by our modification of the Osmium maceration method for high resolution scanning electron microscopy (HRSEM). Data are documented by original pictures which illustrate both some 3-D intracellular features not previously shown in human tissues, and results obtained in our current studies on mitochondrial morphology and on the secretory process of salivary glands. We have demonstrated that mitochondria of cells of practically all human tissues and organs have usually tubular cristae, and that even the cristae that look lamellar are joined to the inner mitochondrial membrane by tubular connexions similar to the crista junctions later seen by electron tomography. Concerning salivary glands an important result is the development of a morphometric method that allows the quantitative evaluation of the secretory events.     

Intramitochondrial filamentous bodies in the thick limb of henle of the rat kidney

"Intramitochondrial filamentous bodies" (IMFB) were occasionally found within the matrix of some mitochondria of the thick limb of Henle of the rat kidney, but not elsewhere in the tubular system. Three types were recognized: type I, an accumulation of filaments 55 A thick; type II, a bundle of parallel filaments having the same thickness as those of type I and regular spacing, 87 A apart, from center to center; and type III, consisting of type II with regular light bands of 280 A periodicity and a helical border of prismatic tubular cristae. In addition to these, electron-opaque masses showing variable and faint substructures were found in the matrix of mitochondria. It is suggested that all these IMFB may originate from mitochondrial cristae and that type II IMFB may be an intermediate developmental form between type I and type III. After uranyl acetate staining, IMFB and the membranes of prismatic tubular cristae showed highly increased electron opacity. The literature has been reviewed for reports of intramitochondrial filamentous inclusions in various types of cells. These inclusions have been classified according to their structural characteristics and the localization in the mitochondria and compared with IMFB reported herein.     

Characterization of dimeric ATP synthase and cristae membrane ultrastructure from Saccharomyces and Polytomella mitochondria

There is increasing evidence now that F(1)F(0) ATP synthase is arranged in dimers in the inner mitochondrial membrane of several organisms. The dimers are also considered to be the building blocks of oligomers. It was recently found that the monomers in beef and the alga Polytomella ATP synthase dimer make an angle of approximately 40 degrees and approximately 70 degrees, respectively. This arrangement is considered to induce a strong local bending of the membrane. To further understand the packing of dimers into oligomers we performed an electron microscopy analysis of ATP synthase dimers purified from Saccharomyces cerevisiae. Two types of dimers were found in which the angle between the monomers is either approximately 90 degrees or approximately 35 degrees. According to our interpretation, the wide-angle dimers (70-90 degrees) are "true-dimers" whereas the small-angle dimers (35-40 degrees) rather are "pseudo-dimers", which represent breakdown products of two adjacent true dimers in the oligomer. Ultrathin sectioning of intact Polytomella mitochondria indicates that the inner mitochondrial or cristae membrane is folded into lamellae and tubuli. Oligomers of ATP synthase can arrange in a helical fashion in tubular-shaped cristae membranes. These results strongly support the hypothesized role of ATP synthase oligomers in structural determination of the mitochondrial inner membrane.     

A morphologic and morphometric study of the mitochondria in several hepatoma cell lines and in isolated hepatocytes.

Some characteristics of the mitochondria of hepatocytes and of three hepatoma cell lines have been compared. By means of stereologic analysis of electron micrographs of cross-sections through cells the volume of mitochondria per unit volume of cell cytoplasm and the surface areas of the mitochondrial envelope and cristae membranes have been measured. The relative mitochondrial volume in the cytoplasm decreases with increasing growth rate but the surface area of outer and cristae membranes per unit volume of mitochondria is not altered. The internal organization of hepatoma mitochondria, however, differs distinctly from that of normal liver mitochondria as evident from electron micrographs; the hepatoma cells contain mitochondria in which parallel cristae appear to cross the whole mitochondrial profile unlike the irregular, short cristae seen in normal liver mitochondria. Furthermore, in the fast-growing hepatoma cells the mitochondrial matrix appears less dense than in the hepatocyte. Hepatoma cells contain less organized rough endoplasmic reticulum than normal liver cells and the spatial relationship of the mitochondria to the rough cisternae, seen in the hepatocyte, is absent in the fast-growing hepatoma cell lines. It is concluded that hepatoma cells have fewer mitochondria than normal liver cells, but that the organelles have a normal content of inner membranes.