Interaction of intravenously injected liposomes with mouse liver mitochondria. A fluorescence and electron microscopy study

Megamitochondria, resulting from cuprizone feeding of Swiss ICR mice, were fluorescent in hepatocytes after the intravenous injection to mice of a liposome-encapsulated acridine orange-DNA complex (AO-DNA). Flow cytofluorimetric analysis of isolated megamitochondria showed that the proportion of liposome-encapsulated AO-DNA which localized in megamitochondria increased from 0.02% of the dose injected per liver cell at 3 min after injection to an average of 0.34% at 1 h after injection. Megamitochondria showed negligible fluorescence by fluorescence activated cell sorting (FACS) analysis when free AO-DNA was intravenously injected. Transmission electron micrographs of mouse liver tissue after intravenous injection of liposomes encapsulating iron dextran showed an association of the liposomes with megamitochondria which appeared identical to liposome association with normal mitochondria. These results support and extend our earlier observation that a fraction of the liposomes injected intravenously into mice associate with mitochondria in the liver, and possibly deliver their aqueous contents there.     

Structural changes of mitochondria related to apoptosis: swelling and megamitochondria formation.

Recently we have found that the formation of megamitochondria in culture cells of various sources, induced by chemicals capable of generating free radicals, is followed by apoptotic changes of the cell. Detailed analysis on functional and morphological aspects of megamitochondria has enabled us to speculate that the formation of megamitochondria may be a prerequisite for free radical-mediated apoptosis: free radicals modify the mitochondrial membranes resulting in the fusion of adjacent mitochondria (megamitochondria formation). If the intracellular level of free radicals is continuously kept high, the permeability transition pores of the megamitochondria membranes are opened and megamitochondria become swollen. Oxygen consumption and the ability to synthesise ATP by swollen megamitochondria decrease distinctly. At the same time, cytochrome c is released from swollen megamitochondria into the cytoplasm. If lowered rates of the generation of reactive oxygen species from swollen megamitochondria, possibly due to decrease in their oxygen consumption, are effective enough to lower the intracellular level of free radicals, megamitochondria may return to normal. If not, decrease in the membrane potential of megamitochondria membranes causes the release of apoptosis-inducing factor into the cytoplasm. Cytochrome c and apoptosis-inducing factor thus released into the cytoplasm may cause cytoplasmic and nuclear apoptotic changes. Experimental data to support this hypothesis are presented.     

Channels in the mitochondrial outer membrane: Evidence from patch clamp studies

Patch clamp techniques were applied to outer mitochondrial membranes of giant mitochondria from mice kept on a cuprizone diet or to vesicles produced by fusing membranes derived from the outer membrane ofNeurospora mitochondria. In the negative range of potentials the conductances decreased with increases in the magnitude of voltage, suggesting the closing of channels. Experiments in which mitochondria were treated with the polyanion polymethacrylate maleate styrene (1:2:3) or succinic anhydride suggest that the channels correspond to VDAC. Although sometimes conductance also decreased with increasing potential over a narrow range of positive potentials, more commonly the conductances increased. Although this phenomenon may represent a detachment of the patch, the changes in conductance are reversible, suggesting that they correspond to the formation or the opening of channels.     

Supported double membranes

Planar model membranes, like supported lipid bilayers and surface-tethered vesicles, have been proven to be useful tools for the investigation of complex biological functions in a significantly less complex membrane environment. In this study, we introduce a supported double membrane system that should be useful for studies that target biological processes in the proximity of two lipid bilayers such as the periplasm of bacteria and mitochondria or the small cleft between pre- and postsynaptic neuronal membranes. Large unilamellar vesicles (LUV) were tethered to a preformed supported bilayer by a biotin–streptavidin tether. We show from single particle tracking (SPT) experiments that these vesicle are mobile above the plane of the supported membrane. At higher concentrations, the tethered vesicles fuse to form a second continuous bilayer on top of the supported bilayer. The distance between the two bilayers was determined by fluorescence interference contrast (FLIC) microscopy to be between 16 and 24 nm. The lateral diffusion of labeled lipids in the second bilayer was very similar to that in supported membranes. SPT experiments with reconstituted syntaxin-1A show that the mobility of transmembrane proteins was not improved when compared with solid supported membranes.     

Giant mitochondria in the human myocardium — Morphogenesis and fate

Electron-microscopical examination of myocardial biopsy material obtained from a 58-year-old man revealed giant mitochondria having a length of 30 micron. Such giant mitochondria (also called megamitochondria) evolve by fusion of the membranes of numerous large individual organelles. Initially they are polymorphous and of diverse shapes, but later they are seen to be arranged among and parallel with the filaments of the myocardial fibres, where they present a smooth, cigar-like appearance. Deposits of glycogen in the giant mitochondria result from the accidental inclusion of glycogen granules during fusion. The abundance of cristae, which often form dense stacks in the megamitochondria, is evidence for the genuine synthesis of new cristal material. The aetiological and exact pathogenetic mechanisms of the evolution of giant mitochondria in the myocardium, as also their function, remain unclear. Particularly large specimens are obviously inefficient and disturbing to the cell. They are degraded by autophagy.     

A characterization of cuprizone-induced giant mouse liver mitochondria

Cuprizone affects the liver of treated mice in a random manner, causing no appreciable change in some cases and inducing the formation of megamito-chondria with altered properties in others. Lack of a full appreciation of this variability may be at the origin of some discrepancies in published work dealing with the properties of cuprizone mouse liver mitochondria (CMLM). CMLM from fully affected livers were remarkably labile and difficult to isolate in a coupled state by homogenization and centrifugation techniques. The integral respiratory chain proteins of CMLM were functionally normal, with the exception of succinic dehydrogenase which showed considerable inhibition. Coupled morphological and functional analysis provided evidence that these properties were independent of CMLM size, a matter which had remained doubtful thus far and bears on the validity of literature reports.Abbreviations MLM mouse liver mitochondria - CMLM cuprizone mouse liver mitochondria - PMS phenazine methosulfate - Mops 4-morphoinopropanesulfonic acid - TMPD tetramethyl-p-phenylenediamine - AA Antimycin A - RCR respiratory control ratio - BSA bovine serum albumin     

ATPase complex and oxidative phosphorylation in chloramphenicol-induced megamitochondria from mouse liver.

1. Functional properties of the ATPase complex are investigated in megamitochondria isolated from livers of weanling mice fed a diet containing 2% chloramphenicol, as an inhibitor of mitochondrial protein synthesis. 2. Whereas the specific activity of ATPase remains unchanged in chloramphenicol-induced megamitochondria, about 40% of the enyzme activity is resistant to inhibition by oligomycin, triethyltin or venturicidin. It is concluded that the ATPase complex lacks one or more components whose synthesis or accumulation is dependent on mitochondrial translation. The inhibitor-resistant ATPase portion appears tightly bound to the mitochondrial membrane. 3. Respiratory chain phosphorylation is tightly coupled in isolated megamitochondria. ATP synthesis and ATP-Pi exchange are diminished by 40%, as compared to control mitochondria, but both processes are sensitive to oligomycin, triethyltin or venturicidin. 4. The decrease in ATP synthesis and ATP-Pi exchange in megamitochondria correlates quite well with the emergence of inhibitor-resistant ATPase. 5. The following electron transport activities in the megmitochondria are reduced: NADH-cytochrome c reductase, by 60%, cytochrome oxidase, by 80%; the amount of antimycin required to gain complete inhibition of the bc1-segment is diminished by more than 50%. On the other hand succinate dehydrogenase activity is increased by 50%. 6. Chloramphenicol-induced megamitochondria appear to be a useful system for studying the role of mitochondrial translation in the assembly of mammalian mitochondria.     

Ultrastructural investigation of spermiogenesis in Peripatopsis capensis (Onychophora)

Early spermatids of the onychophoran Peripatopsis capensis are spherical cells with a centrally located nucleus, numerous mitochondria, Golgi complexes, microtubules and two centrioles. During spermiogenesis, Golgi vesicles migrate to one side of the cell where they form a tight aggregate, which is later shed. The mature spermatozoon has no acrosome. Several mitochondria fuse to form a middle piece containing three large mitochondria. Nucleus and middle-piece elongate, presumably under the influence of helically twisted microtubules. Outside this set of microtubules a continuous layer of endoplasmic reticulum cisternae is formed which separates the interior portion of the cell from an external cytoplasmic rim, which is later shed. Outside the 9 + 2 complex, the tail presents nine accessory microtubules, and a peripheral layer of microtubules beneath the plasma membrane. The enforcement of the tail structure may be related to the fertilization biology of this animal, which is by “hypodermal” impregnation.     

Penetration of spermatozoon into the ovum and transformation of the sperm nucleus into the male pronucleus in the domestic fowl,Gallus gallus

Summary The apex of the sperm head which has undergone the acrosome reaction comes in contact with the plasma membrane of the ovum. After the entire surface of the inner acrosomal membrane has come into close contact with the plasma membrane of the ovum, the two membranes fuse to form a continuous membrane. All parts of the spermatozoon that are devoid of plasma membrane penetrate into the ooplasm. As the head of the spermatozoon moves deeper into the ooplasm, the chromatin begins to disperse, and the head of spermatozoon is transformed into a large spherical nucleus with low electron density. At a later stage of the transformation, many small vesicles appear around the nucleus and subsequently fuse to form two continuous membranes. These membranes represent the male pronuclear envelope. The condensation of the chromatin occurs in places in the nucleus, so that the male pronucleus is formed. During the course of the formation of the male pronucleus, the subacrosomal rod and tail become detached from the head and disintegrate.The authors are greatly indebted to assoc. Prof. Dr. Osamu Koga for his valuable advices. The authors also wish to thank Mr. Takayuki Mori for his helpful suggestions and technical advices. This investigation was supported by a grant from the Ministry of Education of Japan (156185)     

Ultrastructural aspects and amino acid composition of the purified inner and outer membranes of human liver mitochondria as compared to rat liver mitochondria.

1. The mitochondria isolated from human or rat liver were fractionated into submitochondrial particles and purified inner and outer membrane. According to different marker enzymes the inner membranes were enriched about 5-6-fold and the outer membranes about 12-14-fold. The electron microscopical appearance of the membranes was that expected on the basis of enzymic characterization. 2. A comparison of the average amino acid composition of the membrane proteins from the two types of mitochondria has been made. In the case of submitochondrial particles there were statistically significant differences between the human and rat hydrolysates for only five amino acids. Analysing the purified mitochondrial membranes there were significant differences between the two species for nine amino acids in the case of outer membranes and for 12 amino acids in the case of inner membranes. 3. With one exception all amino acids that were increased or decreased in the outer membrane exhibited a similar trend in the inner membrane of human compared with rat liver mitochondria. It appears that liver mitochondrial membranes have a species-dependent pattern of amino acid composition of their proteins.     

The adherence of human neutrophils and eosinophils to schistosomula: evidence for membrane fusion between cells and parasites

Human neutrophils and eosinophils adhere to the surface of schistosomula of Schistosoma mansoni that have been preincubated with antischistosomular sera with or without complement. Neutrophils are seen to form small (< 0.5 micrometer), heptalaminar and large (5-8 micrometer), pentalaminar fusions with the normal pentalaminar parasite surface membrane. By freeze-fracture techniques, attachment areas 5-8 micrometer in diameter are seen to form between neutrophils and schistosomula. These areas have three zones--an edge and two centrally located areas, one of which is rich and one of which is poor in intramembrane particles (IMPs). The edge zone is continuous around the attachment areas and is usually composed of a skip-fracture that passes out of the schistosomular outer membrane into the inner membrane. In some cases, the edge zone is made up of a string of IMPs. The IMP-rich central areas have an IMP concentration similar to that of unattached neutrophil membranes, are raised off of the surface of the schistosomulum, and have two normal schistosomular membranes underneath indicating that they are indeed unattached. the IMP-poor central areas are composed of a fused or hybrid membrane that is continuous with the neutrophil plasma membrane but that bears the same spatial relationship to the schistosomular inner membrane that the normal outer membrane does. Similar changes are seen in samples prepared with glycerination. Eosinophils generally do not fuse with the schistosomular outer membrane but, instead, discharge their granular contents onto the surface of the schistosomula and appear to adhere to the parasite through this discharged material. It is suggested that schistosomula have a capability to fuse with mammalian cells and that this fusion proceeds from a fusion of the outer leaflets to a fusion of the bilayers, as appears also to be the case in other systems.     

Kinetics of Lucifer yellow CH efflux in giant mitochondria

The fluorescent dye Lucifer yellow CH was microinjected electrophoretically into giant mitochondria isolated from mice maintained on a diet containing cuprizone. The dye was retained by the mitochondria, indicating that it was contained in a space bounded by a selectively permeable membrane. The labelling was reversible by reversing the polarity of the current. A study of the disappearance of the fluorescence indicates that the permeability of the mitochondrial membrane to the dye (probably the lithium and/or the potassium salts) ranges from 10(-7) to 10(-8) cm/s.     

CHANGES IN THE SPERMATOZOON DURING FERTILIZATION IN HYDROIDES HEXAGONUS (ANNELIDA) : II. Incorporation with the Egg

This, the last of a series of three papers, deals with the final events which lead to the incorporation of the spermatozoon with the egg. The material used consisted of moderately polyspermic eggs of Hydroides hexagonus, osmium-fixed at various times up to five minutes after insemination. The first direct contact of sperm head with egg proper is by means of the acrosomal tubules. These deeply indent the egg plasma membrane, and consequently at the apex of the sperm head the surfaces of the two gametes become interdigitated. But at first the sperm and egg plasma membranes maintain their identity and a cross-section through the region of interdigitation shows these two membranes as a number of sets of two closely concentric rings. The egg plasma membrane rises to form a cone which starts to project into the hole which the spermatozoon earlier had produced in the vitelline membrane by means of lysis. But the cone does not literally engulf the sperm head. Instead, where they come into contact, sperm plasma membrane and egg plasma membrane fuse to form one continuous membranous sheet. At this juncture the two gametes have in effect become mutually incorporated and have formed a single fertilized cell with one continuous bounding membrane. At this time, at least, the membrane is a mosaic of mostly egg plasma membrane and a patch of sperm plasma membrane. The evidence indicates that the fusion of the two membranes results from vesiculation of the sperm and egg plasma membranes in the region at which they come to adjoin. Once this fusion of membranes is accomplished, the egg cytoplasm intrudes between the now common membrane and the internal sperm structures, such as the nucleus, and even extends into the flagellum; finally these sperm structures come to lie in the main body of the egg. The vesiculation suggested above appears possibly to resemble pinocytosis, with the difference that the vesicles are formed from the plasma membranes of two cells. At no time, however, is the sperm as a whole engulfed and brought to the interior of the egg within a large vesicle.     

ATPase complex and oxidative phosphorylation in chloramphenicol-induced megamitochondria from mouse liver

1. 1. Functional properties of the ATPase complex are investigated in megamitochondria isolated from livers of weanling mice fed a diet containing 2% chloramphenicol, as an inhibitor of mitochondrial protein synthesis.

2. 2. Whereas the specific activity of ATPase remains unchanged in chloramphenicol-induced megamitochondria, about 40% of the enzyme activity is resistant to inhibition by oligomycin, triethyltin or venturicidin. It is concluded that the ATPase complex lacks one or more components whose synthesis or accumulation is dependent on mitochondrial translation. The inhibitor-resistant ATPase portion appears tightly bound to the mitochondrial membrane.

3. 3. Respiratory chain phosphorylation is tightly coupled in isolated megamitochondria. ATP synthesis and ATP-P_i exchange are diminished by 40%, as compared to control mitochondria, but both processes are sensitive to oligomycin, triethyltin or venturicidin.

4. 4. The decrease in ATP synthesis and ATP-P_i exchange in megamitochondria correlates quite well with the emergence of inhibitor-resistant ATPase.

5. 5. The following electron transport activities in the megamitochondria are reduced: NADH-cytochrome c reductase, by 60%, cytochrome oxidase, by 80%; the amount of antimycin required to gain complete inhibition of the bc₁-segment is diminished by more than 50%. On the other hand succinate dehydrogenase activity is increased by 50%.

6. 6. Chloramphenicol-induced megamitochondria appear to be a useful system for studying the role of mitochondrial translation in the assembly of mammalian mitochondria.

The effect of choline deficiency on the outer membranes of rat liver mitochondria

The outer membranes of mitochondria prepared from the liver of rats kept 12 days on a choline-deficient diet were analyzed for changes in phospholipid and protein content. The total amount of phospholipid in the outer membranes was not affected by the deficiency. There was, however, a significant decrease in the amount of phosphatidylcholine and an increase in phosphatidylethanolamine. The alterations in the membrane phospholipids were reflected in a reduction in the fluorescence of the membrane probe, 8-anilino-1-naphthalene sulfonate. Choline deficiency also affected the protein composition of the outer membranes as judged by electrophoretic analysis; however, the activity of several enzymes which serve as markers for the outer membrane was not affected by the deficiency.     

Reconstitution of membrane proteins: sequential incorporation of integral membrane proteins into preformed lipid bilayers

Several integral membrane proteins can be inserted sequentially into preformed unilamellar vesicles (ULV's) composed of dimyristoylphosphatidylcholine (DMPC) and cholesterol in a gel phase. Thus, proteoliposomes of DMPC, cholesterol, and bacteriorhodopsin from Halobacterium halobium rapidly incorporate UDPglucuronosyltransferase (EC 2.4.1.17) from pig liver microsomes, cytochrome oxidase from beef heart mitochondria, and additional bacteriorhodopsin, added sequentially. This process of spontaneous incorporation can be regulated to produce complex artificial membranes that contain phospholipids and proteins at ratios (mol/mol) equivalent to what is found in biological membranes. The ability of the lipid-protein bilayers to incorporate additional integral membrane proteins is not affected by annealing of the proteoliposomes at 37 degrees C nor by the order of addition of the proteins. Bacteriorhodopsin-containing vesicles formed by the sequential addition of integral membrane proteins demonstrate light-driven proton pumping. Therefore, they have retained a vesicular structure. Vesicles containing one or two different proteins will fuse with each other at 21 degrees C or with ULV's devoid of proteins. Incorporation of bacteriorhodopsin or UDPglucuronosyltransferase into proteoliposomes containing DMPC, with or without cholesterol as impurity, also occurs above the phase transition for DMPC. The presence of a protein in a liquid-crystalline bilayer provides the necessary condition for promoting the spontaneous incorporation of other membrane proteins into preformed bilayers.     

Effects of coenzyme Q10 on changes in the membrane potential and rate of generation of reactive oxygen species in hydrazine- and chloramphenicol-treated rat liver mitochondria.

Effects of CoQ10 and cycloheximide (CHX) on hydrazine- and chloramphenicol (CP)-induced morphological and some functional changes of mitochondria using cultured rat hepatocytes and effects on the process of recovery from CP intoxication using mouse liver were examined. Results obtained are summarized as follows: (1) The formation of megamitochondria induced in the hepatocytes cultured for 22 h in the presence of 2 mM hydrazine or CP (300 microgram/ml) was suppressed by pretreatment of hepatocytes with CoQ10 (1 microM) or CHX (0.5 microgram/ml). This was proved by electron microscopic analysis of mitochondria. (2) Treatment of hepatocytes with hydrazine for 48 h or longer caused decreases in the membrane potential of mitochondria, which were suppressed by CoQ10. (3) Treatment of hepatocytes with hydrazine for 22 h or longer caused remarkable increases in intracellular levels of reactive oxygen species in hepatocytes, which were suppressed by CoQ10. (4) The process of recovery from the CP-induced changes of mitochondria in mouse liver was accelerated by CoQ10 and CHX.     

Enrichment and biochemical characterization of boundary membrane contact sites from rat-liver mitochondria

A subfraction of mitochondrial membranes was prepared from osmotically lysed rat liver mitochondria by density gradient centrifugation which contained the inner boundary membrane and the contact sites between this membrane and the outer membrane. The fraction was composed of inner and outer limiting membrane components as shown by the presence of specific marker enzymes, monoamine oxidase and glycerolphosphate oxidase. Surface proteolysis analysis, studies of cytochrome c permeability, and electron microscopy revealed the localization of the inner membrane component within a right-side-out outer membrane vesicle. Moreover, the outer membrane component in this fraction exhibited a higher capacity to bind hexokinase and had a higher specific activity of glutathione transferase than the pure outer membrane. In freeze-fracture analyses the fraction showed fracture plane deflections which may be specific for hydrophobic interactions between the two membranes.     

Biosynthesis of rat liver transhydrogenase in vivo and in vitro

The biosynthesis of pyridine dinucleotide transhydrogenase, a homodimeric inner mitochondrial membrane redox-linked proton pump, has been studied in isolated rat hepatocytes. Newly synthesized transhydrogenase, having an apparent molecular weight identical to the enzyme of isolated liver mitochondria, was selectively immunoprecipitated from detergent extracts of isolated hepatocytes which were labeled with [35S]methionine. That the enzyme is a nuclear gene product is indicated since 1) synthesis was inhibited by cycloheximide, but not by chloramphenicol and 2) no synthesis could be demonstrated in hepatocyte ghosts which are competent only in mitochondrial translation. In addition to the mature form of the enzyme, a species about 2000 daltons larger was also immunoprecipitated from pulse-labeled cells. The half-life of the larger form during a subsequent chase at 37 degrees C was about 2 min, whereas the mature form was not degraded. The relationship between the two forms of the enzyme was established by in vitro studies. A protein approximately 2000 daltons larger than mature transhydrogenase was immunoisolated from a rabbit reticulocyte lysate system programmed with sucrose gradient fractionated rat liver mRNA. This protein was converted to a species having the same size as mature enzyme after incubation with either intact rat liver mitochondria or a soluble matrix fraction derived from mitoplasts. These studies indicate that transhydrogenase is synthesized in the cytoplasm as a higher molecular weight precursor which is post-translationally processed to the mature protein by a soluble matrix protease during or after membrane insertion.     

LIVER PARENCHYMAL CELL INJURY : III. The Nature of Calcium-Associated Electron-Opaque Masses in Rat Liver Mitochondria Following Poisoning with Carbon Tetrachloride

Accumulation of calcium in the mitochondria of rat liver parenchymal cells at 16 and 24 hours after poisoning with carbon tetrachloride is associated with an increase in amount of liver inorganic phosphate, the persistence of mitochondrial adenosine triphosphatase activity, and the formation of electron-opaque intramitochondrial masses in cells with increased calcium contents. These masses, which form within the mitochondrial matrix adjacent to internal mitochondrial membranes, resemble those observed in isolated mitochondria which accumulate calcium and inorganic phosphate; are present in a locus similar to that of electron opacities which result from electron-histochemical determination of mitochondrial ATPase activity; and differ in both appearance and position from matrix granules of normal mitochondria. After poisoning, normal matrix granules disappear from mitochondria prior to their accumulation of calcium. As calcium-associated electron-opaque intramitochondrial masses increase in size, mitochondria degenerate in appearance. At the same time, cytoplasmic membrane systems of mid-zonal and centrilobular cells are disrupted by degranulation of the rough endoplasmic reticulum and the formation of labyrinthine tubular aggregates. The increase in amount of inorganic phosphate in rat liver following poisoning is balanced by a decreased amount of phosphoprotein. These chemical events do not appear to be related, however, as the inorganic phosphate accumulated is derived from serum inorganic phosphate.