Cytochemical and electron microscopic demonstration of organelle translocation and the inhibition of such translocation by colchicine during the mitosis of rat hepatocytes

Hepatocyte lysosomes, mitochondria, and peroxisomes show a dramatic translocation during mitosis induced by partial hepatectomy. During prophase, all three organelles move to the perinuclear cytoplasm. In metaphase, they become concentrated in the polar regions. During telophase, these organelles form clusters in the juxtanuclear regions. This organelle translocation is inhibited by the administration of a low concentration of colchicine, suggesting an involvement of microtubules in their movement.     

The mitochondrial tricarboxylate carrier of silver eel: dimeric structure and cytosolic exposure of both N- and C-termini

The mitochondrial tricarboxylate carrier plays a fundamental role in the hepatic fatty acid synthesis. In this study, we investigated the transmembrane organization of this protein in the inner membrane of eel liver mitochondria using anti–N-terminal and anti–C-terminal antibodies. These antibodies recognized the N- and C-termini of the tricarboxylate carrier in intact mitoplasts, thus suggesting a cytosolic exposure of these regions in the membrane-bound protein. This structural arrangement of the tricarboxylate carrier was further confirmed by protease treatment of intact mitoplasts. Moreover, the oligomeric state of the native tricarboxylate carrier was investigated by blue native electrophoresis. A dimeric form of the carrier protein was found when eel liver mitochondria were solubilized with the mild detergent digitonin. These findings suggest an arrangement of the dimeric tricarboxylate carrier into an even number of membrane-spanning domains, with the N-terminal and C-terminal regions oriented toward the intermembrane space of fish mitochondria.     

Scanning electron-microscopic studies on the three-dimensional structure of sarcoplasmic reticulum in the mammalian red,white and intermediate muscle fibers

Summary The three-dimensional structure of the sarcoplasmic reticulum (SR) in the red, white and intermediate striated muscle fibers of the extensor digitorum longus muscle of the rat was examined under a field-emission type scanning electron microscope after removal of cytoplasmic matrices by the osmium-DMSO-osmium procedure.In all three types of fibers, the terminal cisternae and transverse tubules form triads at the level of the A-I junction. Numerous slender sarcotubules, originating from the A-band side terminal cisternae, extend obliquely or longitudinally and form oval or irregular shaped networks of various sizes in front of the A-band, then become continuous with the tiny mesh (fenestrated collar) in front of the H-band. The A-and H-band SR appears as a single sheet of anastomotic tubules. Numerous sarcotubules, originating from the I-band side terminal cisternae, extend in threedimensional directions and form a multilayered network over the I-band and Z-line regions. At the I-band level, paired transversely oriented mitochondria partly embrace the myofibril. The I-band SR network is poorly developed in the narrow space between the paired mitochondria, but is well developed in places devoid of these mitochondria.The three-dimensional structure of the SR is basically the same in all three muscle fiber-types. However, the SR is sparse on the surface of mitochondria, so the mitochondria-rich red fiber has a much smaller total volume of SR than the mitochondria-poor white fiber. Moreover, the volume of SR of the intermediate fiber is intermediate between the two.     

HELICAL FILAMENTS IN ASTROCYTIC MITOCHONDRIA OF THE CORPUS STRIATUM IN THE RAT

Astrocytic mitochondria have been studied in serial electron micrographs of the corpus striatum. Special filaments have been found inside several of these mitochondria located in both the cell body and the processes. They were particularly frequently observed in the pericapillary end-feet. The filaments occur within dilated intracristal spaces provided with several communications with the outer chamber, and form helices which are oriented approximately in the same direction. Each filament is about 30 A thick, the diameter of the helix is 140 A, and the pitch is 120 A. Their possible nature and significance are briefly discussed. There is no clear relationship between these mitochondria and other unusual forms of mitochondria previously described in astrocytes from other regions.     

Ultrastructure of the accessory glands of the Mediterranean flour moth

Five regions are recognized in the accessory glands of the Mediterranean flour moth, Anagasta kuehniella (Zeller), on the basis of cellular morphology and aggregates of secretory material in the lumen. Some variation is found in each of the posterior four regions, especially the third one. In the most anterior region (region 1) the epithelium is composed of a single type of cell, while in each of the other regions there are two classes of cells. The cells of region 1 and one class in each of the other four regions are fairly typical exocrine cells with extensive rough endoplasmic reticula. Secretion is primarily via Golgi-derived vesicles. Apocrine secretion in the form of sloughing off of the apical cytoplasm probably also occurs in all regions but is most prominent in the posterior two regions. One class of cells is very similar in morphology in each of the posterior four regions though their secretory products form characteristic aggregates in the lumen. The second class of cells (foliate cells) occurring in the posterior four segments is most notably characterized by elongate apical projections that extend out into the lumen. The apical projections contain large quantities of glycogen, some microtubules, and, in some cases, many minute mitochondria. The membrane content of the projections is also very high. In the anterior regions, the membranes are mostly fused in pairs and typically form multilayered whorls. Fusion and whorl formation decrease in the posterior regions. The cytoplasm of the foliate cells has a high organelle content including many lysosomes and mitochondria. The latter exhibit considerable polymorphism, with particular forms occurring in the different regions of the glands. The apical projections of the foliate cells are detached during copulation, presumably as the result of nervous stimulation, and become a part of the ejaculate. Replenishment of all secretory material, including the apical projections, occurs after copulation.     

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.     

Differences in the Activation of the Mitochondrial Permeability Transition Among Brain Regions in the Rat Correlate with Selective Vulnerability

Mitochondria from different regions of the brain were prepared, and the activation of the mitochondrial permeability transition (MPT) by calcium was investigated by monitoring the associated mitochondrial swelling. In general, the properties of the MPT in brain mitochondria were found to be qualitatively similar to those observed in liver and heart mitochondria. Thus, swelling was inhibited by adenine nucleotides (AdNs) and low pH (<7.0), whereas thiol reagents and alkalosis facilitated swelling. Cyclosporin A and its nonimmunosuppressive analogue N-methyl-Val-4-cyclosporin A (PKF 220-384) both inhibited swelling and prevented the translocation of cyclophilin D from the matrix to the membranes of cortical mitochondria. However, the calcium sensitivity of the MPT differed in mitochondria from three brain regions (hippocampus > cortex > cerebellum) and is correlated with the susceptibility of these regions to ischemic damage. Depleting mitochondria of AdNs by treatment with pyrophosphate ions sensitized the MPT to [Ca2+] and abolished regional differences, implying regional differences in mitochondrial AdN content. This was confirmed by measurements showing significant differences in AdN content among regions (cerebellum > cortex > hippocampus). Our data add to recent evidence that the MPT may be involved in neuronal death.     

Expression and processing of the TMEM70 protein

TMEM70 protein represents a novel ancillary factor of mammalian ATP synthase. We have investigated import and processing of this factor in human cells using GFP- and FLAG-tagged forms of TMEM70 and specific antibodies. TMEM70 is synthesized as a 29kDa precursor protein that is processed to a 21kDa mature form. Immunocytochemical detection of TMEM70 showed mitochondrial colocalization with MitoTracker Red and ATP synthase. Western blot of subcellular fractions revealed the highest signal of TMEM70 in isolated mitochondria and mitochondrial location was confirmed by mass spectrometry analysis. Based on analysis of submitochondrial fractions, TMEM70 appears to be located in the inner mitochondrial membrane, in accordance with predicated transmembrane regions in the central part of the TMEM70 sequence. Two-dimensional electrophoretic analysis did not show direct interaction of TMEM70 with assembled ATP synthase but indicated the presence of dimeric form of TMEM70. No TMEM70 protein could be found in cells and isolated mitochondria from patients with ATP synthase deficiency due to TMEM70 c.317-2A>G mutation thus confirming that TMEM70 biosynthesis is prevented in these patients.     

Regional and subcellular distribution of choline acetyltransferase in the brain of rats

—Homogenates of corpus striatum, cerebral cortex and hypothalamus excised from rat brain were fractionated on discontinuous Ficoll and sucrose density gradients, and the distribution of choline acetyltransferase (ChAc) in the mitochondrial and synaptosomal fractions was determined. In the hypothalamic and cortical regions the fractions enriched in synaptosomes showed much higher activity of ChAc than those containing mainly mitochondria. On the other hand, the corpus striatum showed an equal distribution of ChAc activity in those two fractions. The localization of ChAc was also studied in the postnuclear supernatants obtained from three brain regions, using continuous sucrose density gradients. The distribution of ChAc was compared to that of monoamine oxidase (MAO), potassium and protein. When the pellets obtained from the fractions collected from the gradient were suspended in sucrose, the peak of ChAc activity was close to that of MAO in all three brain regions. When 0.1 mm EDTA +1% butanol was used in order to liberate the occluded form of ChAc, the maximum liberation occurred in lighter fractions, resulting in a shift of the activity peak toward the top of the gradient. This was found with fractions from hypothalamic and cortical regions. In the striatum, the liberated ChAc remained in the same fractions as the occluded enzyme. The results indicate that ChAc is liberated only in those fractions where it is present in synaptosomes. In agreement with the results on the discontinuous gradients this occurs in particles of lower density than mitochondria in cortex and hypo-thalamus, but in particles of similar density to mitochondria in the corpus striatum, indicating regional differences in the distribution of ChAc in the brain. K+ containing particles centrifuged in less dense fractions than those containing ChAc, indicating that synaptosomes are heterogeneous with respect to these two marker substances.     

Immunocytochemical identification of cytoskeletal linkages to smooth muscle cell nuclei and mitochondria

In avian smooth muscle cells, desmin-containing intermediate filaments (IFs) are a prominent component of the cytoskeleton and are readily seen in several domains, including the axial intermediate filament bundle (IFB). Both the nucleus and some of the mitochondria are partly surrounded by elements of the IFB. By using anti-desmin and protein-A-colloidal gold labeling, we have identified intermediate filaments that form linkages with the nuclear envelope and with mitochondria. These linkage regions seem to occupy a proportionately greater part of the mitochondrial surface than of the nuclear envelope. The existence of these linkages in smooth muscle cells is consistent with results that support similar linkages to mitochondria and other cellular structures in various cells that contain either vimentin or keratin IFs. These linkages could functionally restrain or assist in homeostatically restoring organelles to their normal position after the rearrangement that accompanies the substantial shortening of smooth muscle cells.     

Population dynamics of mitochondria. I. A model for the role to ACTH in the degradation of adrenocortical mitochondria

An allosteric substance has been supposed to be present in the adrenocortical cell and to be involved in the degradation of the adrenocortical mitochondria only when it is present in the cytoplasm as a free form. An allosteric effector has also been assumed to be synthesized in the adrenal cortex strongly depending on the ACTH supply. The allosteric effector combines hypothetically with the allosteric substance to form an association product. In its bound form, the allosteric substance is assumed to be inactive in the degradation reaction of mitochondria. With these assumptions a differential equation has been obtained to describe the decay process of those mitochondria. An algorithm has been developed to compute the dynamical fate of the mitochondria in a simple, iterative way. Experimental results on the mitochondrial decay in the rat adrenal cortex after hypophysectomy have been fitted to the differential equation in a satisfactory manner. It has been stressed that the present hypothesis constitutes in its essence a new working hypothesis on the maintenance of adrenocortical mitochondria under normal physiological conditions.     

Assembly of the iron-sulfur protein into the cytochrome b-c1, complex of yeast mitochondria.

The assembly of the iron-sulfur protein into the cytochrome bc1 complex after import and processing of the precursor form into mitochondria in vitro was investigated by immunoprecipitation of the radiolabeled iron-sulfur protein from detergent-solubilized mitochondria with specific antisera. After import in vitro, the labeled mature form of the iron-sulfur protein was immunoprecipitated by antisera against both the iron-sulfur protein and the entire bc1 complex from mitochondria solubilized with either Triton X-100 or dodecyl maltoside. After sodium dodecyl sulfate solubilization of mitochondria, however, the antiserum against the iron-sulfur protein, but not that against the bc1 complex, immunoprecipitated the radiolabeled iron-sulfur protein. These results suggest that in mitochondria the mature form of the iron-sulfur protein is assembled with other subunits of the bc1 complex that are recognized by the antiserum against the bc1 complex. By contrast, the intermediate and precursor forms of the iron-sulfur protein that accumulated in the matrix when proteolytic processing was blocked with EDTA and o-phenanthroline were not efficiently assembled into the bc1 complex. The import and processing of the iron-sulfur protein also occurred in mitochondria lacking either cytochrome b (W-267) or the iron-sulfur protein (JPJ1). The mature form of the iron-sulfur protein was immunoprecipitated by antisera against the bc1 complex or core protein I after import in vitro into these mitochondria, suggesting that the mature form is associated with other subunits of the bc1 complex in these strains.     

Nerve growth factor signaling regulates motility and docking of axonal mitochondria

Axonal transport is thought to distribute mitochondria to regions of the neuron where their functions are required. In cultured neurons, mitochondrial transport responds to growth cone activity, and this involves both a transition between motile and stationary states of mitochondria and modulation of their anterograde transport activity. Although the exact cellular signals responsible for this regulation remain unknown, we recently showed that mitochondria accumulate in sensory neurons at regions of focal stimulation with NGF and suggested that this involves downstream kinase signaling. Here, we demonstrate that NGF regulation of axonal organelle transport is specific to mitochondria. Quantitative analyses of motility show that the accumulation of axonal mitochondria near a focus of NGF stimulation is due to increased movement into bead regions followed by inhibition of movement out of these regions and that anterograde and retrograde movement are differentially affected. In axons made devoid of F-actin by latrunculin B treatment, bidirectional transport of mitochondria continues, but they can no longer accumulate in the region of NGF stimulation. These results indicate that intracellular signaling can specifically regulate mitochondrial transport in neurons, and they suggest that axonal mitochondria can respond to signals by locally altering their transport behavior and by undergoing docking interactions with the actin cytoskeleton.     

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.     

Discriminatory processing of the precursor forms of cytochrome P-450scc and adrenodoxin by adrenocortical and heart mitochondria

The mitochondrial proteins involved in adrenocortical steroidogenesis are synthesized as higher molecular weight precursors which require processing by the mitochondria to their mature sizes. The post-translational maturation of two of these proteins has been examined: the cholesterol side chain cleavage cytochrome P-450 (P-450scc) and the iron-sulfur protein, adrenodoxin. Total translation products synthesized in a cell-free system programmed by bovine adrenocortical poly(A+) RNA were incubated with isolated bovine adrenocortical or heart mitochondria followed by immunoisolation of radiolabeled P-450scc or adrenodoxin. In the presence of adrenocortical mitochondria, the precursor form of P-450scc was converted into a trypsin-resistant form that had the same molecular weight as mature P-450scc. Unlike adrenocortical mitochondria, heart mitochondria were unable to process the P-450scc precursor which remained unaltered and trypsin-sensitive. In addition, a matrix fraction of heart mitochondria did not cleave the P-450scc precursor. In contrast, the adrenodoxin precursor did not exhibit similar specificity as it was processed to the mature form by both adrenocortical and heart mitochondria. Also, the adrenocortical mitochondria were not restricted to processing endogenous proteins as they imported and cleaved the precursor to ornithine transcarbamylase. The results indicate that some mitochondrial precursor proteins have tertiary structures which allow them to be recognized by all mitochondria while other mitochondrial precursor proteins have structures recognizable by only specialized mitochondria.     

In silico analyses of Fsf1 sequences, a new group of fungal proteins orthologous to the metazoan sideroblastic anemia-related sideroflexin family

Sideroblastic anemias are pathologies observed in metazoan species characterized by accumulation of iron in the mitochondria (sideroblasts), defective erythropoiesis, and iron overload. Some genes have been associated with sideroblastic anemia, e.g. delta-aminolevulinic acid synthase gene (e-ALAS2). Recently, a new sideroblastic-associated protein family was discovered in metazoans and termed sideroflexin (Sfxn). The metazoan Sfxn family comprises five groups of paralogous proteins, present in mitochondria and whose functions are unknown. Using an in silico approach, we have identified and characterized new sideroflexin sequences from the genomes of different fungal species. An in-depth phylogenetic analysis of these new fungal Sfxn sequences (termed Fsf1p) showed that they form a distinct clade within the metazoan Sfxn family. Hydrophobic cluster analysis and transmembrane topological mapping allowed us to compare conserved regions among Fsf1 and Sfxn proteins. The results indicate that Fsf1 probably belongs to an ancient, mitochondrial group of proteins, necessary to maintain the homeostasis of iron within this organelle.     

ER-mitochondria signaling regulates autophagy

The endoplasmic reticulum (ER) and mitochondria form tight functional contacts that regulate several key cellular processes. The formation of these contacts involves “tethering proteins” that function to recruit regions of ER to mitochondria. The integral ER protein VAPB (VAMP associated protein B and C) binds to the outer mitochondrial membrane protein, RMDN3/PTPIP51 (regulator of microtubule dynamics 3) to form one such set of tethers. Recently, we showed that the VAPB-RMDN3 tethers regulate macroautophagy/autophagy. Small interfering RNA (siRNA) knockdown of VAPB or RMDN3 to loosen ER-mitochondria contacts stimulates autophagosome formation, whereas overexpression of VAPB or RMDN3 to tighten contacts inhibit their formation. Artificial tethering of ER and mitochondria via expression of a synthetic linker protein also reduces autophagy and this artificial tether rescues the effects of VAPB- or RMDN3-targeted siRNA loss on autophagosome formation. Finally, our studies revealed that the modulatory effects of ER-mitochondria contacts on autophagy involve their role in mediating ITPR (inositol 1,4,5-trisphosphate receptor) delivery of Ca²⁺ from ER stores to mitochondria.     

Stable Golgi-mitochondria complexes and formation of Golgi Ca(2+) gradients in pancreatic acinar cells

We have determined the localization of the Golgi with respect to other organelles in living pancreatic acinar cells and the importance of this localization to the establishment of Ca(2+) gradients over the Golgi. Using confocal microscopy and the Golgi-specific fluorescent probe 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosine, we found Golgi structures localizing to the outer edge of the secretory granular region of individual acinar cells. We also assessed Golgi positioning in acinar cells located within intact pancreatic tissue using two-photon microscopy and found a similar localization. The mitochondria segregate the Golgi from lateral regions of the plasma membrane, the nucleus, and the basal part of the cytoplasm. The Golgi is therefore placed between the principal Ca(2+) release sites in the apical region of the cell and the important Ca(2+) sink formed by the peri-granular mitochondria. During acetylcholine-induced cytosolic Ca(2+) signals in the apical region, large Ca(2+) gradients form over the Golgi (decreasing from trans- to cis-Golgi). We further describe a novel, close interaction of the peri-granular mitochondria and the Golgi apparatus. The mitochondria and the Golgi structures form very close contacts, and these contacts remain stable over time. When the cell is forced to swell, the Golgi and mitochondria remain juxtaposed up to the point of cell lysis. The strategic position of the Golgi (closer to release sites than the bulk of the mitochondrial belt) makes this organelle receptive to local apical Ca(2+) transients. In addition the Golgi is ideally placed to be preferentially supplied by ATP from adjacent mitochondria.     

Sequencing of the nuclear gene for the yeast cytochrome c1 precursor reveals an unusually complex amino-terminal presequence.

Cytochrome c1 is a component of the mitochondrial respiratory chain in most eukaryotes. The protein is coded by nuclear DNA, synthesized as a larger precursor outside the mitochondria and then cleaved to the mature form in two successive steps during its import into the mitochondria. We have cloned the structural gene for yeast cytochrome c1 by functional complementation of a cytochrome c1-deficient yeast mutant with a yeast genomic library in the yeast-Escherichia coli 'shuttle' vector YEp 13. The complete nucleotide sequence of the gene and of its 5'- and 3'-flanking regions was determined. The deduced amino acid sequence of the yeast cytochrome c1 precursor reveals an unusually long transient amino-terminal presequence of 61 amino acids. This presequence consists of a strongly basic amino-terminal region of 35 amino acids, a central region of 19 uncharged amino acids and an acidic carboxy-terminal region of seven amino acids. This tripartite structure of the presequence resembles that of the precursor of cytochrome c peroxidase and supports a previous suggestion on the import pathways of these two precursors.     

The Neurospora crassa TOB complex: analysis of the topology and function of Tob38 and Tob37

The TOB or SAM complex is responsible for assembling several proteins into the mitochondrial outer membrane, including all β-barrel proteins. We have identified several forms of the complex in Neurospora crassa. One form contains Tob55, Tob38, and Tob37; another contains these three subunits plus the Mdm10 protein; while additional complexes contain only Tob55. As previously shown for Tob55, both Tob37 and Tob38 are essential for viability of the organism. Mitochondria deficient in Tob37 or Tob38 have reduced ability to assemble β-barrel proteins. The function of two hydrophobic domains in the C-terminal region of the Tob37 protein was investigated. Mutant Tob37 proteins lacking either or both of these regions are able to restore viability to cells lacking the protein. One of the domains was found to anchor the protein to the outer mitochondrial membrane but was not necessary for targeting or association of the protein with mitochondria. Examination of the import properties of mitochondria containing Tob37 with deletions of the hydrophobic domains reveals that the topology of Tob37 may be important for interactions between specific classes of β-barrel precursors and the TOB complex.