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.     

Ceramide forms channels in mitochondrial outer membranes at physiologically relevant concentrations

Recent evidence suggests that the ability of ceramides to induce apoptosis is due to a direct action on mitochondria. Mitochondria are known to contain enzymes responsible for ceramide synthesis and hydrolysis and mitochondrial ceramide levels have been shown to be elevated prior to the mitochondrial phase of apoptosis. Ceramides have been reported to induce the release of intermembrane space proteins from mitochondria, which has been linked to their ability to form large channels in membranes. The aim of this study was to determine if the membrane concentration of ceramide required for the formation of protein permeable channels is within the range that is present in mitochondria during the induction phase of apoptosis. Only a very small percentage of the ceramide actually inserts into the mitochondrial membranes. The permeability of the mitochondrial outer membrane correlates directly with the level of ceramide in the membrane. Importantly, the concentration of ceramide at which significant channel formation occurs is consistent with the level of mitochondrial ceramide that occurs during the induction phase of apoptosis (4 pmol ceramide/nanomole phospholipid). Similar results were obtained with short- and long-chain ceramide. Ceramide channel formation is specific to mitochondrial membranes in that no channel formation occurs in the plasma membranes of erythrocytes even at concentrations 20 times higher than those required for channel formation in mitochondrial outer membranes. Thus, ceramide channels are good candidates for the pathway by which proapoptotic proteins are released from mitochondria during the induction phase of apoptosis.     

Protein import into mitochondria: ATP-dependent protein translocation activity in a submitochondrial fraction enriched in membrane contact sites and specific proteins

To identify the membrane regions through which yeast mitochondria import proteins from the cytoplasm, we have tagged these regions with two different partly translocated precursor proteins. One of these was bound to the mitochondrial surface of ATP-depleted mitochondria and could subsequently be chased into mitochondria upon addition of ATP. The other intermediate was irreversibly stuck across both mitochondrial membranes at protein import sites. Upon subfraction of the mitochondria, both intermediates cofractionated with membrane vesicles whose buoyant density was between that of inner and outer membranes. When these vesicles were prepared from mitochondria containing the chaseable intermediate, they internalized it upon addition of ATP. A non-hydrolyzable ATP analogue was inactive. This vesicle fraction contained closed, right-side-out inner membrane vesicles attached to leaky outer membrane vesicles. The vesicles contained the mitochondrial binding sites for cytoplasmic ribosomes and contained several mitochondrial proteins that were enriched relative to markers of inner or outer membranes. By immunoelectron microscopy, two of these proteins were concentrated at sites where mitochondrial inner and outer membranes are closely apposed. We conclude that these vesicles contain contact sites between the two mitochondrial membranes, that these sites are the entry point for proteins into mitochondria, and that the isolated vesicles are still translocation competent.     

The mitochondrial compartment

Mitochondria are vital organelles that perform a variety of fundamental functions ranging from the synthesis of ATP through to being intimately involved in programmed cell death. Comprised of at least six compartments: outer membrane, inner boundary membrane, intermembrane space, cristal membranes, intracristal space, and matrix, mitochondria have a complex, dynamic internal structure. This internal dynamism is reflected in the pleomorphy and motility of mitochondria. Mitochondria contain their own DNA (mtDNA), encoding a small number of vital genes, but this role as a genetic vault is not compatible with the role of mitochondria in bioenergetics since electron transport results in the generation of reactive oxygen species (ROS) that induce lesions in the mtDNA. It is hypothesized that ROS shape the morphological organization of the higher plant cell mitochondrial population into a discontinuous whole, and that ROS are a selective pressure affecting the organization of the mitochondrial genome. This review describes how inter- and intra-mitochondrial compartmentalization underpins the biology of this complex organelle.     

Alteration of cytochrome C oxidase activity during spermatogenesis in Carcinus maenas

During spermatogenesis in the crab, Carcinus maenas, mitochondria in the developing spermatids degenerate. In close association with mitochondrial fragments in late spermatids, an elaborate lattice-like complex of fused membranes is formed and lies in a position adjacent to the acrosome. Mature sperm possess no mitochondria, but a whorl of membranes is present alongside the acrosome in the diffuse nucleus. To ascertain whether or not cytochrome c oxidase activity is lost as the mitochondria disintegrate, differentiating Carcinus maenas spermatids and mature sperm have been studied cytochemically. Cytochfome c oxidase activity was localised by means of the osmophilic compound 3,3'-diaminobenzidine (DAB). Cytochrome c oxidase activity is confined to the cristae of mitochondria in the testis. As spermatids mature most mitochondria are lost. A few mitochondrial fragments may be caught up within the lamellar complex. While they are recognisalbe as mitochondria they retain cytochrome c oxidase activity. The lamellar complex does not show this enzyme activity. These results therefore suggest that the mature sperm of Carcinus maenas do not contain the enzymes normally incorporated in the mitochondrial membrane, capable of oxidative phosphorylation.     

The mitochondrial compartment

Mitochondria are vital organelles that perform a variety of fundamental functions ranging from the synthesis of ATP through to being intimately involved in programmed cell death. Comprised of at least six compartments: outer membrane, inner boundary membrane, intermembrane space, cristal membranes, intracristal space, and matrix, mitochondria have a complex, dynamic internal structure. This internal dynamism is reflected in the pleomorphy and motility of mitochondria. Mitochondria contain their own DNA (mtDNA), encoding a small number of vital genes, but this role as a genetic vault is not compatible with the role of mitochondria in bioenergetics since electron transport results in the generation of reactive oxygen species (ROS) that induce lesions in the mtDNA. It is hypothesized that ROS shape the morphological organization of the higher plant cell mitochondrial population into a discontinuous whole, and that ROS are a selective pressure affecting the organization of the mitochondrial genome. This review describes how inter- and intra-mitochondrial compartmentalization underpins the biology of this complex organelle.     

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.     

Mitochondrial Placement and Function in Insect Ion-transporting Cells

The ion-transporting epithelia of insects possess some unusualmorphological adaptations which promote close juxtapositionof mitochondria and the ion-transporting plasma membranes. Aparticularly striking example of this adaptation is providedby the movement of branches of mitochondria into and out ofthe apical microvilli in the Malpighian tubules. In the hemipteranRhodmus prohxus, the microvilli in the resorptive lower tubuleare small and contain no mitochondria during the non-transportingperiod. When ion transport is stimulated,either in vivo or invitro, there is a concomitant growth in microvillar volume andsurface area. In addition, branches of mitochondria enter thesemicrovilli. It has been shown that these mitochondrial movementsare driven by an actinassociated process involving the microvillarcore microfilaments. The stimulation for this movement in vivois the insect diuretic hormone. In the lepidopteran Calpodesethhus, the rates of fluid transport which the Malpighian tubulescan sustain vary during the insect's life stages. Larvae andadults show rapid transport, while pupal Malpighian tubulesshow none. In the larvae and adults, microvilli in the Malpighiantubules are large and contain mitochondria. In the pupae, reducedtransport is associated with mitochondrial retraction and microvillarshrinkage. These ultrastructural changes appear tobe regulatedby the insect's developmental hormones. In the Malpighian tubulesof adult female mosquitoes of the genus Aedes, intracellularinfection by microfiliarial nematodes has hen shown to causemitochondrial retraction and reduced rates of fluid transport.A model is presented which serves to summarize currently proposedmechanisms of membrane and mitochondrial function in the ion-transportingepithelia of insects.     

Subcellular localization of VDAC in mitochondria and ER in the cerebellum

The voltage-dependent anion channel (VDAC) provides passage for adenine nucleotides, Ca2+ and other metabolites into and from mitochondria. Here, the intracellular localization and oligomeric organization of VDAC in brain mitochondria and ER are demonstrated. Immunohistochemical staining of VDAC in rat cerebellum showed high labeling of the Purkinje neurons. Immunogold labeling and EM analysis of the cerebellar molecular layer showed specific VDAC immunostaining of the mitochondrial outer membrane, highly enhanced in contact sites between mitochondria or between mitochondria and associated ER. Purified ER membranes contain VDAC, but not other mitochondrial proteins. Chemical cross-linking of isolated mitochondria, ER or purified VDAC demonstrated the existence of VDAC in oligomeric form. Based on the enrichment of VDAC in the junctional face of closely associated mitochondrial and ER membranes and the existence of VDAC oligomers, we propose an involvement of VDAC in specialized intermembrane communication between mitochondria or between ER and mitochondria, serving to complement the tight structural and functional coupling observed between these organelles.     

The signal distinguishing between targeting of outer membrane β-barrel protein to plastids and mitochondria in plants

The proteome of the outer membrane of mitochondria and chloroplasts consists of membrane proteins anchored by α-helical or β-sheet elements. While proteins with α-helical transmembrane domains are present in all cellular membranes, proteins with β-barrel structure are specific for these two membranes. The organellar β-barrel proteins are encoded in the nuclear genome and thus, have to be targeted to the outer organellar membrane where they are recognized by surface exposed translocation complexes. In the last years, the signals that ensure proper targeting of these proteins have been investigated as essential base for an understanding of the regulation of cellular protein distribution. However, the organellar β-barrel proteins are unique as most of them do not contain a typical targeting information in form of an N-terminal cleavable targeting signal. Recently, it was discovered that targeting and surface recognition of mitochondrial β-barrel proteins in yeast, humans and plants depends on the hydrophobicity of the last β-hairpin of the β-barrel. However, we demonstrate that the hydrophobicity is not sufficient for the discrimination of targeting to chloroplasts or mitochondria. By domain swapping between mitochondrial and chloroplast targeted β-barrel proteins atVDAC1 and psOEP24 we demonstrate that the presence of a hydrophilic amino acid at the C-terminus of the penultimate β-strand is required for mitochondrial targeting. A mutation of the chloroplast β-barrel protein psOEP24 which mimics such profile is efficiently targeted to mitochondria. Thus, we present the properties of the signal for mitochondrial targeting of β-barrel proteins in plants.     

Electron Microscopic Analysis of a Spherical Mitochondrial Structure

Mitochondria undergo dynamic structural alterations to meet changing needs and to maintain homeostasis. We report here a novel mitochondrial structure. Conventional transmission electron microscopic examination of murine embryonic fibroblasts treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, found that more than half of the mitochondria presented a ring-shaped or C-shaped morphology. Many of these mitochondria seemed to have engulfed various cytosolic components. Serial sections through individual mitochondria indicated that they formed a ball-like structure with an internal lumen surrounded by the membranes and containing cytosolic materials. Notably, the lumen was connected to the external cytoplasm through a small opening. Electron tomographic reconstruction of the mitochondrial spheroids demonstrated the membrane topology and confirmed the vesicular configuration of this mitochondrial structure. The outside periphery and the lumen were defined by the outer membranes, which were lined with the inner membranes. Matrix and cristae were retained but distributed unevenly with less being kept near the luminal opening. Mitochondrial spheroids seem to form in response to oxidative mitochondrial damage independently of mitophagy. The structural features of the mitochondrial spheroids thus represent a novel mitochondrial dynamics.     

Mitochondrial Ceramide and the Induction of Apoptosis

In most cell types, a key event in apoptosis is the release of proapoptotic intermembrane space proteins from mitochondria to the cytoplasm. In general, it is the release of these intermembrane space proteins that is responsible for the activation of caspases and DNases that are responsible for the execution of apoptosis. The mechanism for the increased permeability of the mitochondrial outer membrane during the induction phase of apoptosis is currently unknown and highly debated. This review will focus on one such proposed mechanism, namely, the formation of ceramide channels in the mitochondrial outer membrane. Ceramides are known to play a major regulatory role in apoptosis by inducing the release of proapoptotic proteins from the mitochondria. As mitochondria are known to contain the enzymes responsible for the synthesis and hydrolysis of ceramide, there exists a mechanism for regulating the level of ceramide in mitochondria. In addition, mitochondrial ceramide levels have been shown to be elevated prior to the induction phase of apoptosis. Ceramide has been shown to form large protein permeable channels in planar phospholipid and mitochondrial outer membranes. Thus, ceramide channels are good candidates for the pathway with which proapoptotic proteins are released from mitochondria during the induction phase of apoptosis.     

Transport of glutamate and aspartate across the membranes of rat liver mitochondria

Chromatographic analyses have indicated that aspartate and glutamate constitute from 50–70% of the total free amino acids in freshly isolated mitochondria. Radioactive tracer studies indicate that while the l-isomers of glutamate and aspartate are rapidly accumulated by mitochondria, the d-isomers of these amino acids do not penetrate the mitochondrial membrane. The action of two inhibitory compounds, 1-fluoro-2,4-dinitrobenzene (Sanger's reagent) and tannic acid, on the transport of l-glutamate and l-aspartate has been examined. A marked inhibition of l-glutamate transfer by 1-fluoro-2,4-dinitrobenzene is observed. A corresponding effect on the transport of either l-aspartate or the anionic substrate, succinate has not been found. Tannic acid, an agent previously known to inhibit certain carrier-mediated solute fluxes in mitochondria, is shown also to inhibit the uptake of both l-glutamate and l-aspartate. These findings are consistent with the view that the mitochondrial membranes of rat liver cells contain distinct, stereospecific transport mechanisms for aspartate and glutamate.     

Phylogenetic distribution of superoxide dismutase supports an endosymbiotic origin for chloroplasts and mitochondria

Three isozymes of superoxide dismutase (SOD) have been identified and characterized. The iron and manganese isozymes (Fe-SOD and Mn-SOD, respectively) show extensive primary sequence and structural homology, suggesting a common evolutionary ancestor. In contrast, the copper/zinc isozyme (CuZn-SOD) shows no homology with Fe-SOD or Mn-SOD, suggesting an independent origin for this enzyme. The three isozymes are unequally distributed throughout the biological kingdoms and are located in different subcellular compartments. Obligate anaerobes and aerobic diazotrophs contain Fe-SOD exclusively. Facultative aerobes contain either Fe-SOD or Mn-SOD or both. Fe-SOD is found in the cytosol of cyanobacteria while the thylakoid membranes of these organisms contain a tightly bound Mn-SOD. Similarly, most eukaryotic algae contain Fe-SOD in the chloroplast stroma and Mn-SOD bound to the thylakoids. Most higher plants contain a cytosol-specific and a chloroplast-specific CuZn-SOD, and possibly a thylakoid-bound Mn-SOD as well. Plants also contain Mn-SOD in their mitochondria. Likewise, animals and fungi contain a cytosolic CuZn-SOD and a mitochondrial Mn-SOD. The Mn-SOD found in the mitochondria of eukaryotes shows strong homology to the prokaryotic form of the enzyme. Taken together, the phylogenetic distribution and subcellular localization of the SOD isozymes provide strong support for the hypothesis that the chloroplasts and mitochondria of eukaryotic cells arose from prokaryotic endosymbionts.     

Characterization of CDPdiacylglycerol hydrolase in mitochondrial and microsomal fractions from rat lung

CDPdiacylglycerol pyrophosphatase (E.C. 3.6.1.26) activity has been examined in rat lung mitochondrial and microsomal fractions. While the mitochondrial hydrolase exhibited a broad pH optimum from pH 6-8, the microsomal activity decreased rapidly above pH 6.5. Apparent Km values of 36.2 and 23.6 microM and Vmax values of 311 and 197 pmol.min-1.mg protein-1 were observed for the mitochondrial and microsomal preparations, respectively. Addition of parachloromercuriphenylsulphonic acid led to a marked inhibition of the microsomal fraction but slightly stimulated the mitochondrial activity at low concentrations. Mercuric ions were inhibitory with both fractions. Although biosynthetic reactions utilizing CDPdiacylglycerol require divalent cations, addition of Mg2+, Mn2+, Ca2+, Zn2+, Co2+, and Cu2+ all inhibited the catabolic CDPdiacylglycerol hydrolase activity in both fractions. EDTA and EGTA also produced an inhibitory effect, especially with the mitochondrial fraction. Although addition of either adenine or cytidine nucleotides led to a decrease in activity with both fractions, the marked susceptibility to AMP previously reported for this enzyme in Escherichia coli membranes, guinea pig brain lysosomes, and pig liver mitochondria was not observed. These results indicate that rat lung mitochondria and microsomes contain specific CDPdiacylglycerol hydrolase activities, which could influence the rate of formation of phosphatidylinositol and phosphatidylglycerol for pulmonary surfactant.     

Blue Light-Reducible Cytochromes in Membrane Fractions from Neurospora crassa

We have assayed absorbance changes generated by blue light in plasma membranes, endoplasmic reticulum, and mitochondrial membranes from Neurospora crassa. Light minus dark difference spectra, obtained anaerobically in the presence of ethylenediaminetetraacetate, indicated that b-type cytochromes could be photoreduced in all three membranes. In plasma membranes, a b-type cytochrome with a distinct difference spectrum was photoreducible without addition of exogenous flavin. Addition of riboflavin greatly stimulated the photoreduction of cytochromes in endoplasmic reticulum and mitochondrial membranes. In its spectral characteristics the cytochrome on the endoplasmic reticulum resembled cytochrome b₅ or nitrate reductase, while the cytochrome in mitochondrial membranes had the same spectrum as cytochrome b of the mitochondrial respiratory chain.

Cytochromes in the three membrane fractions reacted differently to blue light in the presence of various inhibitors. Potassium azide inhibited reduction of plasma membrane cytochrome b, with 50% inhibition at 1.0 millimolar. The same concentration of azide stimulated photoreduction of cytochromes in both endoplasmic reticulum and mitochondria. Although photoreduction of cytochromes in all three membranes was inhibited by salicylhydroxamic acid, cytochromes in plasma membranes were more sensitive to this inhibitor than those in endoplasmic reticulum and mitochondria. Cells grown to induce nitrate reductase activity showed an elevated amount of blue light-reducible cytochrome b in the endoplasmic reticulum.

     

Beef-heart submitochondrial particles: a mixture of mitochondrial inner and outer membranes.

1. EPR spectra at 9 GHz and 83 degrees K of NADH-reduced anaerobic beef-heart submitochondrial particles, prepared from mitochondria by sonication and centrifugation, contain a signal (gz equals to 2.01, gy equals to 1.94, gx equals to 1.89) due to an iron-sulphur center of the mitochondrial outer membrane. 2. The ratio of inner and outer membranes in submitochondrial particles is not greatly different from that in beef-heart mitochondria. 3. Beef-heart submitochondrial particles free from outer-membrane contamination have been prepared by free-flow electrophoresis. EPR spectra at 83 degrees K of such particles are presented.     

Electrophysiological characterization of contact sites in brain mitochondria.

From morphological and biochemical studies it has been recognized that the regions where the outer and inner membranes of mitochondria come in close contact (contact sites) can be the route mechanism through which mitochondria interact directly with the cytoplasm. We have studied these regions electrophysiologically with the patch clamp technique, with the aim of understanding if this direct interaction is mediated by high conductance ion channels similar to the channel already detected in the inner membrane of mitochondria (Sorgato M. C., Keller, B. U., and Stühmer, W. (1987) Nature 330, 498-500). Contact sites isolated from rat brain mitochondria were thus incorporated into liposomes subsequently enlarged sufficiently to be patch clamped. This study shows that these particular fractions contain ion channels with conductances ranging from approximately 5 picosiemens to 1 nanosiemens (in symmetrical 150 mM KCl). Most of these channels are not voltage-dependent and can be open at physiological potentials sustained by respiring mitochondria. The lack of voltage sensitivity seems not to be the outcome of methodological artifacts, as voltage-gated channels are detected in giant liposomes containing either the outer mitochondrial membrane or a partially purified fraction of the inner mitochondrial membrane. These data therefore indicate that channels present in mitochondrial contact sites have properties which render them amenable to perform several of the functions hypothesized for these regions, particularly that of translocating macromolecules from the cytoplasm to the matrix of mitochondria.     

A yeast mitochondrial presequence functions as a signal for targeting to plant mitochondria in vivo.

To date, the presequence of the mitochondrial beta-subunit of ATPase from tobacco is the only signal sequence that has been shown to target a foreign protein into plant mitochondria in vivo. Here we report that the presequence of a yeast mitochondrial protein directs bacterial beta-glucuronidase (GUS) specifically into the mitochondrial compartment of transgenic tobacco plants. Fusions between the presequence of the mitochondrial tryptophanyl-tRNA-synthetase gene from yeast and the GUS gene have been introduced into tobacco plants and yeast cells. In both systems, proteins containing the complete yeast mitochondrial presequence are efficiently imported in the mitochondria. Measurements of GUS activity in different subcellular fractions indicate that there is no substantial misrouting of the chimeric proteins in plant cells. In vitro synthesized GUS fusion proteins have a higher molecular weight than those found inside yeast and tobacco mitochondria, suggesting a processing of the precursors during import. Interestingly, fusion proteins translocated across the mitochondrial membranes of tobacco have the same size as those that are imported into yeast mitochondria. We conclude that the processing enzyme in plant mitochondria may recognize a proximate or even the same cleavage site within the mitochondrial tryptophanyl-tRNA-synthetase presequence as the matrix protease from yeast.     

A new method for studying the genetic control of specific mitochondrial proteins in Paramecium aurelia

Summary Mitochondria from one syngen (or sub-species) of Paramecium aurelia have been introducted into a different syngen by preparing erythromycin-resistant mitochondria from syngen 1 and micro-injecting them into erythromycin-sensitive syngen 7 cells. The recipient sensitive cells were then placed in erythromycin to inhibit the replication of the sensitive mitochondria. Such selected clones contain a syngen 7 nucleus but a mitochondrial genome which is derived from syngen 1 erythromycin-resistant mitochondria.Using this method it has been shown that the mitochondrial enzyme fumarase is not coded by the mitochondrial genome, and by implication, is coded by the nuclear genome. The use of this technique as a method for determining if specific mitochondrial proteins are controlled by nuclear or mitochondrial genes is discussed.