Immunoelectron microscopic study of the distribution of porin on outer membranes of rat heart mitochondria

The distribution of porin on the outer membranes of rat heart mitochondria has been studied by means of immunogold labelling with antibodies to the N-terminal part of the human protein. It was found that only a minority of isolated, unfixed mitochondria are labelled by these antibodies, with the gold particles frequently organized in threads or bands. Extensive immunogold labelling is frequently observed on regions of outer membranes stripped away from mitochondria and on regions separating two mitochondrial compartments whose cristae display different configurations (possibly representing two mitoplasts covered by a common outer membrane). Also, pairs of connected mitochondria are sometimes heavily labelled in the neck regions, which may represent the junctions involved in electrical communication between mitochondria in cardiac tissue.     

MEMBRANE JUNCTIONS IN THE INTERMEMBRANE SPACE OF MITOCHONDRIA FROM MAMMALIAN TISSUES

There have been several reports describing paracrystalline arrays in the intermembrane space of mitochondria. On closer inspection these structures appear to be junctions of two adjoining membranes. There are two types. They can be formed between the outer and inner mitochondrial membranes (designated outer-inner membrane junctions) or between two cristal membranes (intercristal membrane junctions). In rat heart, adjoining membranes appeared associated via a central dense midline approximately 30 Å wide. In rat kidney, the junction had a ladder-like appearance with electron-dense "bridges" approximately 80 Å wide, spaced 130 Å apart, connecting the adjoining membranes. We have investigated the conditions which favor the visualization of such structures in mitochondria. Heart mitochondria isolated rapidly from fresh tissue (within 30 min of death) contain membrane junctions in approximately 10–15% of the cross sections. This would indicate that the percentage of membrane junctions in the entire mitochondrion is far greater. Mitochondria isolated from heart tissue which was stored for 1 h at 0°–4°C showed an increased number of membrane junctions, so that 80% of the mitochondrial cross sections show membrane junctions. No membrane junctions are observed in mitochondria in rapidly fixed fresh tissue or in mitochondria isolated from tissue disrupted in fixative. Thus, the visualization of junctions in the intermembrane space of mitochondria appears to be dependent upon the storage of tissue after death. Membrane junctions can also be observed in mitochondria from other stored tissues such as skeletal muscle, kidney, and interstitial cells from large and small intestine. In each case, no such junctions are observed in these tissues when they are fixed immediately after removal from the animal. It would appear that most studies in the literature in which isolated mitochondria from tissues such as heart or kidney were used were carried out on mitochondria which contained membrane junctions. The presence of such structures does not significantly affect normal mitochondrial function in terms of respiratory control and oxidative phosphorylation.     

Corn Mitochondrial Swelling and Contraction-an Alternate Interpretation

Mitochondria isolated from 3-day-old etiolated corn shoots (Zea mays L.) can be categorized into three separate groups, each group characteristic of the cell type from which the mitochondria were isolated. Phloem sieve tubes and some adjacent parenchyma cells contain mitochondria that have few cristae and little amorphous matrix. Mitochondria from meristematic and undifferentiated cells have more cristae and matrix. Vaculate and differentiated cells have mitochondria with well-developed cristae and abundant matrix. Each mitochondrial type exhibits typical in vitro spontaneous swelling and substrate-induced contraction responses. characterized by change or lack of change in cristae size and in density of amorphous material. For the second and third types of mitochondria, swelling and contraction are characterized by a change in degree of cristae size and in matrix density. The first type undergoes few changes upon swelling or contraction. Radical changes of the inner membrane, withdrawal and infolding, are associated with cell differentiation and not with swelling and contraction of isolated corn shoot mitochondria.     

Subcellular distribution of rat liver porin

The subcellular distribution of rat liver porin was investigated using the immunoblotting technique and monospecific antisera against the protein isolated from the outer membrane of rat liver mitochondria. Subfractionation of mitochondria into inner membranes, outer membranes and matrix fractions revealed the presence of porin only in the outer membranes. Porin was also not detected in highly purified subcellular fractions, including plasma membranes, nuclear membranes, Golgi I and Golgi II, microsomes and lysosomes. Thus, liver porin is located exclusively in the outer mitochondrial membrane.     

Cytological differentiation of the interrenal tissue of the Japanese quail,Coturnix coturnix

Summary As reported for several other avian species there are clearly distinguishable subcapsular (SCZ) and inner (IZ) zones of interrenal tissue in the Japanese quail. The SCZ contains large columnar cells (type I) with rounded nuclei, polymorphic mitochondria with shelf-like cristae, and relatively small numbers of lipid droplets. The IZ contains two and possibly three types of cells. Type II consists of large columnar cells with moderately dense cytoplasm containing large numbers of lipid droplets and many rounded mitochondria with tubular cristae. Smooth endoplasmic reticulum (SER) and Golgi apparatus are well developed; coated vesicles occur in the Golgi area and at the cell surface. Type-III cells occur in IZ and especially in its more peripheral areas. They are columnar cells with strikingly clear cytoplasm (in comparison with type II) containing mitochondria with plate-like cristae and tubular SER. Type-IV cells are sparsely distributed in IZ and occur rarely in SCZ. Type IV may be a degenerating phase of type III.After adenohypophysectomy or section of portal vessels type-I cells atrophy somewhat with a decrease in lipid droplets; type-II cells, also atrophy with conspicuous increase in size and number of lipid droplets, enlargement of mitochondria, and gradual disappearance of SER; type-III cells decrease in number whereas type-IV cells increase.After injection of ACTH, type-I cells enlarge and their mitochondria, SER and Golgi apparatus become more conspicuous; there is a decrease in lipid droplets in type-II cells and a development of SER, polysomes and Golgi apparatus; there is also a decrease in lipid droplets and a development of SER in type-III cells after injection of 2IU ACTH and an almost complete disappearance of lipid droplets after 4IU ACTH; type-IV cells increase in number.The investigation reported herein was supported by Scientific Research Grants from the Ministry of Education of Japan to Professor Mikami; and by grants from the Japan Society for the Promotion of Science, the National Science Foundation (USA), and the Graduate School Fund of the University of Washington to Professor Farner     

Water movement from intracristal spaces in isolated liver mitochondria

When analyzing mitochondria isolated in a sucrose medium that had been embedded for thin sectioning according to one low denaturation embedding technique, large intracristal spaces were present in close to 90% of the mitochondria. The two crista membranes were closely apposed in only 40% of all cristae. When the mitochondria were transferred to an incubation medium, the percentage of mitochondria with intracristal spaces was reduced to 40%. About 90% of all cristae were lacking any space separating the two crista membranes. The presence of inorganic phosphate in the medium was required for the closing of the intracristal spaces. The percentage of cristae lacking an intracristal space remained the same after addition of substrate for respiration (state 4) and of ADP (state 3). Inhibition or uncoupling of respiration led to an increase in the percentage of intracristal spaces, showing that oxidative phosphorylation is required to maintain the crista membranes closely apposed. The appearance and disappearance of the intracristal spaces was an indication of water movements across the crista membranes. The mean volume of the mitochondria increased 33% when they were transferred from the sucrose medium to the incubation medium, showing that the removal of water from the cristae was not caused by a passive osmotic effect. Addition of substrate made the volume decrease by 28%. After further addition of ADP, the volume decreased another 23%. No change in volume was associated with inhibition or uncoupling of respiration. The observations revealed that water can move into or out of the cristae independently of water movement out from the entire mitochondrion. Therefore, the water moving out from or into the cristae is translocated across the cristae membrane. The observations are interpreted to reveal the presence of a mechanism that actively prevents water from accumulating in the crista membrane. This mechanism allows for a low water activity to be maintained within the membrane. The variations in the frequency of intracristal spaces occurred without any simultaneous changes in the width of the space appearing between the two surface membranes after isolation of the mitochondria. The observations, therefore, do not agree with the concept that there is an outer compartment that communicates freely with intracristal spaces.     

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.     

Fine structure of the corpus Luteum of the raccoon during pregnancy

Summary Ultrastructure of the granulosa lutein cells of the raccoon from throughout pregnancy has been described. The lutein cells often from epithelial cords which are separated by the connective tissues, capillaries and lymphatics. Based on the arrangements and modifications of the cytoplasmic organelles and inclusions, three types of lutein cells have been recognized. The type I lutein cells predominantly contain tubular, agranular endoplasmic reticulum, juxtanuclear Golgi complexes, a few round to rod-shaped mitochondria, some free ribosomes, and occasional lipid droplets. Occasionally the tubular cristae of mitochondria and tubular smooth endoplasmic reticulum appear contiguous. The type II cells contain abundant lace-like and/or stacked fenestrated endoplasmic reticulum cisternae that frequently form membranous whorls, some tubular, agranular endoplasmic reticulum, mitochondria, and lipid droplets. Mitochondria are usually small, but unusual large ones also occur. The small, rod-to round-shaped mitochondria usually have tubular cristae; but the large, oval, elongate, and cup shaped mitochondria possess tubular, lamellar, plate like, and whorl-like cristae. The plasma membranes of the cells are complexly elaborated and folded, especially when apposing each other. In favorable sections, strands of fenestrated cisternae appose the folds of the plasma membranes. In general, the amount of cytoplasmic organelles and inclusions vary greatly in the cells. The type III cells predominantly contain lipid droplets and sparse cytoplasmic organelles. The type I and II cells are found throughout pregnancy, but the type III cells are observed from mid gestation to term. The cytological features of type I and II cells suggest that they probably secrete most of the steroids, whereas the type III cells primarily store lipids.This research was supported by UPSHS grant AM-11376 and NIH contract 69-2136.     

Fine Structure of Ascosporogenesis in Nematospora coryli Peglion, a Pathogenic Yeast

Observations with the electron microscope of the early stages of ascospore formation in Nematospora coryli Peglion reveal the following sequence of events. Partial dissociation of the nuclear membrane is followed by the appearance of unit membranes. Each unit membrane gives rise to two pairs of double membranes delimiting the ascospores from the epiplasm of the ascus. Enlarged mitochondria which have a granular matrix and limited cristae development are also regularly seen.     

ATP-stimulated protease activity in brown fat mitochondria: response to a 24-h fast in mice.

Brown fat mitochondria have [3H]casein-hydrolyzing activity at pH 8.0 associated with both membrane and soluble fractions. An ATP-stimulated proteolytic activity inhibited by vanadate and N-ethylmaleimide was found in the soluble fraction. Membrane-associated proteolytic activity was inhibited by phenylmethylsulfonyl fluoride and trypsin inhibitor, suggesting that it is a serine protease. A 24-h fast in mice caused a significant loss of mitochondrial proteins from the tissue, but had no effect on protease activity of isolated mitochondria with or without ATP. The ATP-stimulated release of amino acids or peptides from isolated mitochondria, as measured with fluorescamine, was not influenced by food deprivation. Thus, brown fat mitochondria possess an ATP-stimulated proteolytic pathway that does not appear to be involved in the bulk removal of mitochondrial proteins from brown fat of fasting mice.     

Localization of the ATP/ADP translocator in the inner membrane and regulation of contact sites between mitochondrial envelope membranes by ADP. A study on freeze-fractured isolated liver mitochondria.

The frequency of contacts between the mitochondrial envelope membranes was determined in freeze-fractured samples of isolated mitochondria by means of quantifying the frequency of fracture plane deflections between the two membranes. It was observed that the formation of contacts correlated with the concentration of free ADP despite of inhibition of electron transport by antimycin A. The activity of ATPase partially inhibited by oligomycin or depletion of membrane potential by K+ and valinomycin had no effect on the induction of the contacts by ADP. ATP was ineffective in creating contacts irrespective of the presence or absence of a membrane potential, whereas carboxyatractyloside induced the contacts under all conditions in a manner similar to ADP. These results suggest the involvement of the ATP/ADP translocator in regulation of contact sites. As a consequence, we analyzed its distribution in the inner membrane of kidney and liver mitochondria by binding of [3H]atractyloside to subfractions of this membrane. The experiments demonstrated that the translocator was located in the peripheral part of the inner membrane as well as in the portion which formed the cristae.     

Similarities in the polypeptide composition of glyoxysomal and endoplasmic-reticulum membranes from castor-bean endosperm.

Microsomal fractions, glyoxysomes and mitochondria were isolated from homogenates of germinating castor-bean (Ricinus communis) endosperm by sucrose-density-gradient centrifugation. Washed membrane preparations from these cellular fractions were examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. At corresponding developmental stages the endoplasmic-reticulum and glyoxysomal membranes were strikingly similar in polypeptide composition, at least 16 polypeptides being present in membranes isolated from 3-day-old tissue. Supplying [35S]methionine to intact endosperm tissue resulted in the labelling of all membrane polypeptides, the specific radioactivity in the endoplasmic reticulum being greater than for equivalent polypeptides of the glyoxysomal membrane. Washing these membranes with sodium deoxycholate solution extensively solubilized protein components, with the exception of a predominant polypeptide of mol.wt. 55000. Mitochondrial membrane preparations differed from those of the endoplasmic reticulum and glyoxysomes in polypeptide molecular-weight distribution and the [35S]methionine-labelling pattern. The similarity in polypeptide composition between endoplasmic-reticulum and glyoxysomal membranes is discussed in relation to glyoxysome biogenesis.     

Dual Electron Microscopic Localization of Mitochondrial Creatine Kinase in Brain Mitochondria

Mitochondrial creatine kinase in brain mitochondria appears to be located at two different intramitochondrial sites. By using immunogold-labeling techniques, a peripheral immunoreactivity was localized between the two boundary membranes, while an additional, central immunoreactivity was found at the crista surface. The peripheral enzyme was accessible to the antibodies after treatment of the brain mitochondria with 100-300 μg digitonin/mg mitochondrial protein, which left 75% of the activity bound to the membranes. Electron microscopic analyses revealed that 43% of the labeled, peripheral creatine kinase was bound at those places where outer membrane vesicles remained attached to the inner envelope membrane, suggesting that the enzyme is in involved in contact formation between outer and inner mitochondrial membranes. Postembedding staining of mitochondria on thin sections of brain tissue or in the isolated state led to the observation of a second location of creatine kinase inside the mitochondria, along the cristae, which was not accessible to the antibodies in isolated, digitonin-treated mitochondria.     

STUDIES OF THE MITOCHONDRIAL ENERGY-TRANSFER SYSTEM OF BROWN ADIPOSE TISSUE

An investigation of the mechanisms of norepinephrine action and heat production in brown adipose tissue from newborn rabbits has been carried out. Data obtained with the use of biochemical techniques has been correlated with morphological data from electron microscopy. Norepinephrine was found to stimulate the respiration of brown fat in vitro. Inhibitors of glycolysis abolish this effect, whereas inhibitors of oxidative phosphorylation do not, at least not to the same extent. Brown fat is readily permeable to added Krebs cycle intermediates. Substrate level phosphorylation, but no electron transport-coupled phosphorylation, could be demonstrated in isolated mitochondria. It is suggested that the rate of fatty acid oxidation is limited by the availability of phosphate acceptor systems which break down ATP formed at the substrate level and thus provide ADP for further substrate level phosphorylation. The theory of respiratory control by the action of reesterification of fatty acids is discussed in the light of these findings. Under the electron microscope, brown fat mitochondria are characterized by their large size, tightly packed cristae, and by the different types of granules in the matrix. No elementary particles are seen when the mitochondria are examined by the negative-staining technique. The absence of electron transport-coupled phosphorylation together with the apparent absence of elementary particles seems to be of particular significance.     

Immunocytochemical localization of class I H-2 histocompatibility antigens of mouse liver

The subcellular location of class I H-2 histocompatibility antigens was determined for mouse liver using immunocytochemical techniques and correlated with information determined by cell fractionation and analysis in situ. Surface antigens first were localized by standard procedures involving surface labeling with ferritin-labeled antibody. This approach could not be used for internal membranes either in situ or in fractions since the antigens are not expressed at the cytoplasmic surface. For this purpose, thin sections of tissues embedded in Lowicryl were analyzed and quantitated. The in situ analysis confirmed the presence of H-2 antigens on internal membrane compartments as well as on the cell surface and helped rule out the possibility that distributions based on analyses by immunoprecipitation of fractions of internal membranes were influenced greatly by plasma membrane contamination. Quantitation was provided by immunoprecipitation of H-2 antigens from radioiodinated or metabolically labeled isolated and highly purified cell fractions. The findings establish the presence of class I H-2 histocompatibility antigens in endoplasmic reticulum, Golgi apparatus and plasma membrane in the approximate ratios of 1:3:7. No class I H-2 histocompatibility antigens could be detected in mitochondria, salt extracts of isolated membranes or NP-40-insoluble membrane material.     

Studies on ultrastructure and purification of isolated plant mitochondria

Sweetpotato mitochondria, that showed respiratory control, were studied with respect to ultrastructure. If fixed in media containing sucrose at 0.4 M, the cristae were dilated and the matrix was highly condensed. A more orthodox ultrastructural form was observed when the mitochondria were fixed in a medium containing sucrose at 0.25 M, i.e., the matrix was more expanded, the cristae were less dilated, and peripherally, the inner membrane element lay adjacent to the outer membrane element. These results are discussed in terms of a sucrose-accessible space (space between outer and inner membrane elements including intracristal space), and a space relatively inaccessible to sucrose (matrix). Ultrastructural shifts were not observed with change in metabolic steady state of the mitochondria. High resolution electron micrographs showed that the ultrastructure of sweetpotato mitochondria is very similar to that of animal mitochondria.

Purity and homogeneity of mitochondrial fractions were followed both by phase-contrast and electron microscopy. Preparations from sweetpotato, using older methods, were relatively homogeneous with respect to particle type and size, whereas avocado preparations contained a high proportion of chloroplasts and cellular debris. A method of purification involving sucrose-density-gradient centrifugation was developed. Purified mitochondria exhibited respiratory control and appeared similar to unpurified mitochondria under the electron microscope.

     

The significance of promitochondrial structures in rat liver for mitochondrial biogenesis

1. The heavy, light and fluffy mitochondrial fractions obtained by differential centrifugation were further characterized with respect to their protein synthesizing ability in vitro, their nucleic acid content, buoyant density of their DNA and ultrastructure. 2. The light mitochondrial fraction synthesized proteins in vitro at a rate 4-5 times as high as heavy and fluffy mitochondria. The incorporation ability of this fraction was also maximally affected by the thyroid status of the animal. The radioactivity in leucyl-tRNA of the light mitochondrial fraction was about 3-4 times as high as that of the other two fractions. 3. The heavy, light and fluffy mitochondrial fractions contained small but consistent amounts of RNA and DNA. Although the DNA content was the same in all mitochondria fractions, the light mitochondria contained relatively more RNA. The buoyant density of DNA from all the fractions was 1.701g/cm(3). 4. Electron microscopy revealed that the heavy mitochondria have a typical mitochondrial architecture, with densely packed cristae and a well developed double membrane. Light mitochondria were also surrounded by double membranes, but were smaller in size and contained less cristae. The fluffy fraction consisted of a mixture of well formed mitochondria and those in the process of degradation. 5. The significance of these findings in relation to mammalian mitochondrial genesis is discussed.     

The induction of energized configurational changes in plant mitochondria,in vivo

Configurational changes in the mitochondrial membranes of the salt gland ofTamarix aphylla, which are dependent on the biochemical state of the mitochondria, are demonstrated. In the energized state the cristae expand and become closely associated. There is also an increase in the density of the matrix and a formation of strands of material in the matrix and between the closely associated cristae membranes. The energized condition can be discharged by incubation in a medium containing 2,4-dinitrophenol and the mitochondria are comparable to those observed in glands fixed by typical methods for electron microscopy.These studies were supported in part by Grant GB-8199 (W.W.T.) from the National Science Foundation.     

Purification of liver membranes highly enriched in phosphatidylinositol kinase.

Membranes highly enriched in phosphatidylinositol (PtdIns) kinase were purified from rat liver by sucrose density gradient centrifugation of a plasma membrane-depleted microsomal fraction. PtdIns kinase-containing membranes had a lower density than membranes containing Golgi and plasma membrane markers, both in sucrose and Nycodenz gradients, without being completely resolved from these other membranes. They also had a lower density than an endosomal marker. Furthermore, lectin affinity partitioning showed that PtdIns kinase did not reside in plasma membranes. PtdIns kinase in different membrane fractions was of type II and had similar kinetic properties. We suggest that the isolated membranes are the major site for phosphatidylinositol 4-phosphate formation in the liver cell, and that these membranes are part of the exocytic pathway. Thus, PtdIns kinase might be a convenient marker for the exocytic process.