Association between intermediate-sized filaments and mitochondria in rat Leydig cells

The cytoplasmic filamentous structures of the testis Leydig cells have been studied in the rat after double fixation with glutaraldehyde and osmium-potassium ferrocyanide. A good preservation of the cytoplasmic filaments was obtained and their abundance in the rat Leydig cells become apparent. Judging from their diameter (8–11 nm) and general morphology most of the filaments observed belong to the intermediate-sized type. The close association between filament bundles and mitochondria is described. Some images show cross-bridges between individual filaments and mitochondria and their attachment to the mitochondria outer membrane.     

Attachment of mitochondria to intermediate filaments in adrenal cells: relevance to the regulation of steroid synthesis.

The rate of steroid synthesis is regulated by the rate of transport of cholesterol from lipid droplets to mitochondria. We have previously demonstrated that lipid droplets in adrenal cells are tightly attached to intermediate filaments. Here we now show that mitochondria colocalize with intermediate filaments in modified double indirect immunofluorescence and by electron microscopy of extracted adrenal cells. Direct contact between mitochondria and intermediate filaments was established by examination of stereo pairs of electron micrographs from extracted cells. The attachment of both droplets and mitochondria to intermediate filaments suggests possible mechanisms for this form of intracellular transport of cholesterol to mitochondria and hence for the regulation of steroid synthesis.     

Interrelationships of endoplasmic reticulum, mitochondria, intermediate filaments, and microtubules--a quadruple fluorescence labeling study.

To study the interrelationships of endoplasmic reticulum, mitochondria, intermediate filaments, and microtubules, we have developed a quadruple fluorescence labeling procedure to visualize all four structures in the same cell. We applied this approach to study cellular organization in control cells and in cells treated with the microtubule drugs vinblastine or taxol. Endoplasmic reticulum was visualized by staining glutaraldehyde-fixed cells with the dye 3,3'-dihexyloxacarbocyanine iodide. After detergent permeabilization, triple immunofluorescence was carried out to specifically visualize mitochondria, vimentin intermediate filaments, and microtubules. Mitochondria in human fibroblasts were found to be highly elongated tubular structures (lengths up to greater than 50 microns), which in many cases were apparently fused to each other. Mitochondria were always observed to be associated with endoplasmic reticulum, although endoplasmic reticulum also existed independently. Intermediate filament distribution could not completely account for endoplasmic reticulum or mitochondrial distributions. Microtubules, however, always codistributed with these organelles. Microtubule depolymerization in vinblastine treated cells resulted in coaggregation of endoplasmic reticulum and mitochondria, and in the collapse of intermediate filaments. The spatial distributions of organelles compared with intermediate filaments were not identical, indicating that attachment of organelles to intermediate filaments was not responsible for organelle aggregation. Mitochondrial associations with endoplasmic reticulum, on the other hand, were retained, indicating this association was stable regardless of endoplasmic reticulum form or microtubules. In taxol-treated cells, endoplasmic reticulum, mitochondria, and intermediate filaments were all associated with taxol-stabilized microtubule bundles.     

Microfilaments and microtubules control the shape, motility, and subcellular distribution of cortical mitochondria in characean internodal cells

Summary. The shape, motility, and subcellular distribution of mitochondria in characean internodal cells were studied by visualizing fluorescent dyes with confocal laser scanning microscopy and conducting drug-inhibitor experiments. Shape, size, number, and distribution of mitochondria varied according to the growth status and the metabolic activity within the cell. Vermiform (sausage-shaped), disc-, or amoeba-like mitochondria were present in elongating internodes, whereas very young cells and older cells that had completed growth contained short, rodlike organelles only. Mitochondria were evenly distributed and passively transported in the streaming endoplasm. In the cortex, mitochondria were sandwiched between the plasma membrane and the stationary chloroplast files and distributed in relation to the pattern of pH banding. Highest mitochondrial densities were found at the acid, photosynthetically more active regions, whereas the alkaline sites contained fewer and smaller mitochondria. In the cortex of elongating cells, small mitochondria moved slowly along microtubules or actin filaments. The shape and motility of giant mitochondria depended on the simultaneous interaction with both cytoskeletal systems. There was no microtubule-dependent motility in the cortex of nonelongating mature cells and mitochondria only occasionally travelled along actin filaments. These observations suggest that mitochondria of characean internodes possess motor proteins for microtubules and actin filaments, both of which can be used either as tracks for migration or for immobilization. The cortical cytoskeleton probably controls the spatiotemporal distribution of mitochondria within the cell and promotes their association with chloroplasts, which is necessary for exchange of metabolites during photosynthesis and detoxification.Correspondence and reprints: Arbeitsgruppe Pflanzenphysiologie, Fachbereich Zellbiologie, Universität Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria.     

Association of mitochondria with intermediate filaments and of polyribosomes with cytoplasmic actin

Summary Anti-mitochondrial autoantibody and fluorescent derivatives of insulin stain phase-dense mitochondria in acetone-fixed monolayers of fibroblasts. Double fluorochrome studies show mitochondria in close topographic association with intermediate filaments. In cells treated with vinblastine or colchicine, mitochondria are relocated in sites closely associated with coils of perinuclear intermediate filaments. In contrast, autoantibody to polyribosomes stains granules aligned in the long axis of well spread embryonic cells, in the direction of actin-containing fibrils, an arrangement that is lost in cells pretreated with the actin filament disrupting drug cytochalasin B. In more mature fibroblasts, antiribosomal antibody reacts with phase-dense rough endoplasmic reticulum and this staining pattern is not affected by cytochalasin B. The observations suggest that mitochondria are associated with intermediate filaments and that free polyribosomes, but not polyribosomes attached to rough endoplasmic reticulum, are associated with cytoplasmic actin.Supported by a grant from the Anti-Cancer Council of Victoria. We thank Mrs. I. Burns for technical assistance and Dr. H.A. Ward and staff for preparation of fluorescent conjugates     

Intermediate (10 nm) filaments in human malignant mesothelioma.

Human malignant mesotheliomas were studied by electron microscopy. Three main types of cells were seen--submesothelial epithelioid cells, epithelial lining cells and fibroblast-like cells. In submesothelial epithelioid cells prominent arrays of intermediate (10 nm) filaments were often seen attached to plasma membrane, mitochondria, nuclei and concentric whorls of rough endoplasmic reticulum. The other types of cell found in the tumors, epithelial lining cells and fibroblast-like cells, lacked such distinct filaments. The intermediate filaments were especially abundant in cells with extensive whorling of endoplasmic reticulum. The association of intermediate filaments with such deranged cytoplasmic organization suggests that they play a role in the altered behavior of malignant cells.     

Unusual mitochondrial ultrastructure in the pig adrenal cortex

Unusually large mitochondria with few cristae were observed in the cells of the boundary layer between the zonae fasciculata and reticularis of the pig adrenal. These mitochondria occasionally contained parallel arrays of beaded filaments which appeared to be composed of repetitive electron opaque particles, measuring 10 to 11 nm in diameter. The possibility that these filaments are arranged in closely packed arrays of tubular structures with a central filament is discussed.     

LIGHT AND ELECTRON MICROSCOPE STUDIES OF MORPHOLOGICAL CHANGES OF MITOCHONDRIA DURING SPERMATOGENESIS IN THE GRASSHOPPER

1. Observations on the morphological changes of mitochondria preparatory to the formation of the nebenkern, as well as changes within the nebenkern, are reported. 2. Mitochondria enlarge and divide during the meiotic divisions. 3. The mitochondria of the spermatid elongate, become filamentous, form a crescent, and partially encircle the nucleus. 4. Nodes which develop on either end of the crescent become entwined as they move toward each other. 5. The two nodes coalesce to form a filamentous or early type nebenkern which is described by others as chromophilic. 6. Internal rearrangement and partial dissolution of the filaments result in the development of the late or chromophobic nebenkern which separates into two distinct bodies. 7. The nebenkern moieties send out processes toward the centrosome, and after making contact, elongate and occupy part of the space between the tail filaments and sheath of the spermatozoon.     

Giant mitochondria in the A cells of the pancreas of a lizard: Varanus niloticus

Pancreatic A cells of the lizard Varanus niloticus are characterized by the presence of two types of mitochondria: (a) normal, small mitochondria (about 0.4 X 1 micron), and (b) giant mitochondria, measuring up to 9 micron in length and 1 micron in diameter. Giant mitochondria show various shapes. Their matrix is filled with tubules, filaments, and dense granules. Transverse sections of tubules are polygonal in shape and about 20 nm in diameter. They are grouped in bundles. The filaments, about 9-10 nm in diameter, are arranged in parallel layers crossing each other at a 57 degree angle. In a closely related species, Varanus exanthematicus, pancreatic A cells do not show these peculiar features.     

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.     

Filaments associated with the endoplasmic reticulum in the streaming cytoplasm of Chara corallina

Perfused Chara cells capable of resuming ATP-dependent cytoplasmic streaming in low free Ca++ solutions have been examined by electron microscopy for myosin-like filaments. Filaments 44 nm in diameter and up to 3 micron in length have been found associated with the endoplasmic reticulum that along with mitochondria, microbodies and dictyosomes from the endoplasm becomes immobilised around the sub-cortical actin bundles when ATP is depleted. Such endoplasmic filaments have not been detected in association with mitochondria or microbodies and they have not been found in the stationary cortex. These filaments are extracted from the perfused cell by ATP unless motility-inhibiting levels of cytochalasin B are present. The filaments are not detectable in cells inactivated in solutions containing high (10(-4) M) Ca++ concentrations even when the Ca++ level is subsequently lowered. Consistent with their being required for motility, cytoplasmic streaming cannot be effeiciently reactivated by ATP in such filament-depleted cells. The possibility is discussed that the filaments contain myosin and that the endoplasmic reticulum with which they are associated has a major role in generating and transmitting the motive force for streaming.     

The 2G2 Antibody Recognizes an Acidic 110-kDa Human Mitochondrial Protein

Using fluorescence microscopy, the mouse monoclonal antibody 2G2 was found to label mitochondria in human cells, as assessed by double staining with either Rhodamine 123 or a polyclonal antibody to mitochondrial matrix HSP-60 proteins. No reactivity to the 2G2 antibody was detected in cells from mouse, rat and chicken. Immunoblotting analysis demonstrated that the 2G2 antigen corresponds to a human protein with a relative mobility of 110 kDa and an approximate isoelectric point of 6.5 that co-partitions with HSP-60 proteins during isolation of mitochondria from HeLa cells. Close examination of the 2G2 staining pattern in HeLa and Fanconi's anaemia cells revealed differences in the morphology and organization of mitochondria in these two cell types. In HeLa cells, mitochondria appear as individual tubular compartments of variable length and are closely associated with vimentin filaments, particularly at the periphery of the nucleus. In Fanconi's anaemia cells, mitochondria have a filamentous shape and form an interconnected cytoplasmic reticulum running in parallel with both vimentin filaments and microtubules. After stabilization with aldehyde- or alcohol-based fixation protocols that optimize the preservation of cytoskeletal components, the epitope targeted by the 2G2 antibody may serve as a valuable marker in the investigation of relationships between mitochondria and other cellular structures in human cells.     

Effects of antimitotic and antimitochondrial agents on the cellular distribution of microtubules and mitochondria

Using antibodies to a mitochondrial molecular chaperone class of protein, which is specifically altered in mutants resistant to microtubule (MT) inhibitors, the effect of a number of MT and mitochondrial inhibitors on the cellular distribution of mitochondria and various cytoskeletal filaments was examined. Treatment of Chinese hamster ovary (CHO) or chicken embryo fibroblast (CEF) cells with the MT inhibitors podophyllotoxin, colchicine, nocodazole and vinblastine caused depolymerization of cellular MTs, but had no significant effect on the distribution patterns of mitochondria. This is attributed to the association of mitochondria with intermediate filaments (IFs) which are not destroyed under these conditions. In contrast to MT inhibitors, treatment of CEFs with the potassium ionophores nonactin and valinomycin caused aggregation of mitochondria towards the perinuclear region of the cells, without having any apparent effect on cellular MTs. This observation suggests that mitochondrial membrane potential, which is abolished by these drugs, play a role in the cellular distribution of mitochondria. In cells recovering from the effects of MT inhibitors, mitochondria have been found to surround the MT organizing complexes and upon complete recovery a realignment of MTs with mitochondria takes place. These observations suggest that MT growth in cells does not occur in a completely random manner but that mitochondria may play some role in their directional growth.     

Observations on the ultrastructure of developing myocardium of rat embryos

Timed pregnancies were obtained in Sprague-Dawley rats and early ultrastructural differentiation of myocardium of embryos of 10, 11, 12, 13, and 14 days was investigated and compared with that of newborn. Ten-day myocardium is characterized by loosely packed cells; the cytoplasm is typified by a dearth of organelles. Both thick (myosin) and thin (actin) filaments become identifiable for the first time in the 10-day myocardium where the heart is pulsating but circulation is not established. These filaments are not visible in the embryos of 9-day-old myocardium. The formation of these filaments is observed to continue throughout the period covered in this investigation. Concomitant with the appearance of the myofilaments is the synthesis of Z band material. By the eleventh day of gestation and during the subsequent days there is a rapid proliferation and differentiation of most of the organelles. The myofilaments become organized into fully formed striated fibrils. Intercalated discs appear as. small wavy lines on the eleventh day and become plicated in later stages and serve as cell boundaries and points of attachment for myofilaments and fibrils. There is a perceptible change in the number and morphology of mitochondria from the tenth to eleventh day and later stages of development when the heart becomes functional. Similarly, there is a rapid proliferation and differentiation of granular endoplasmic reticulum and Golgi bodies. Large quantities of free ribosomes are dispersed in the cytoplasm of 10-day myocardium; however, in later stages there is a progressive reduction in the distribution of these particles. An intimate association of ribosomes and polysomes with the developing myofibrils is discernible. The T -system and sarcoplasmic reticulum begin to appear in II-day myocardium. The embryonic myocardium displays intense mitotic activity throughout its development and a unique feature of embryonic myocardial cells is the simultaneous occurrence of myofilament synthesis and mitotic activity within the same cells.     

Protein kinase C regulates mitochondrial motility

Functions of mitochondria depend on their intracellular localization, which often coincides with regions of the highest ATP consumption. Transport of mitochondria along microtubules and actin microfilaments and their anchoring by cytoskeletal structures are regulated by exogenous endogenous factors. Changes in mitochondrial motility induced by protein kinase C activity modulators were studied using video microscopy of live fibroblasts expressing a GFP-tagged mitochondrial marker. Intracellular activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (TPA) significantly enhances mitochondrial mobility, whereas the protein kinase C inhibitor bis-indolylmaleimide fully suppresses it. The activating effect of TPA on mitochondrial movement is not due to rearrangements in the actin cytoskeleton, since it persists in cells treated with latrunculin B disrupting F-actin. No effect of TPA on mitochondrial mobility is observed in MFT-16 cells devoid of intermediate vimentin filaments. The data obtained suggest that protein kinase C regulates the interactions of mitochondria with intermediate filaments.     

A new cytoplasmic structure in the basidiomycete Helicogloea: The microscala

Certain cells of the hyphae of Helicogloea variabilis and H. lagerheimii contain an assemblage of endoplasmic reticulum with or without mitochondria cross-linked by short filaments in a three-dimensional geometric array. Each complex of filaments and membranes is termed a microscala. Developmental and phylogenetic significance of the microscala are considered. The short filaments may represent an unrecognized cytoskeletal element in fungi.     

Mitochondria-associated myosin 19 processively transports mitochondria on actin tracks in living cells

Mitochondria are fundamentally important in cell function, and their malfunction can cause the development of cancer, cardiovascular disease, and neuronal disorders. Myosin 19 (Myo19) shows discrete localization with mitochondria and is thought to play an important role in mitochondrial dynamics and function; however, the function of Myo19 in mitochondrial dynamics at the cellular and molecular levels is poorly understood. Critical missing information is whether Myo19 is a processive motor that is suitable for transportation of mitochondria. Here, we show for the first time that single Myo19 molecules processively move on actin filaments and can transport mitochondria in cells. We demonstrate that Myo19 dimers having a leucine zipper processively moved on cellular actin tracks in demembraned cells with a velocity of 50 to 60 nm/s and a run length of ∼0.4 μm, similar to the movement of isolated mitochondria from Myo19 dimer-transfected cells on actin tracks, suggesting that the Myo19 dimer can transport mitochondria. Furthermore, we show single molecules of Myo19 dimers processively moved on single actin filaments with a large step size of ∼34 nm. Importantly, WT Myo19 single molecules without the leucine zipper processively move in filopodia in living cells similar to Myo19 dimers, whereas deletion of the tail domain abolished such active movement. These results suggest that Myo19 can processively move on actin filaments when two Myo19 monomers form a dimer, presumably as a result of tail–tail association. In conclusion, Myo19 molecules can directly transport mitochondria on actin tracks within living cells.     

DEGENERATION IN THE EFFERENT NERVE ENDINGS IN THE COCHLEA AFTER AXONAL SECTION

Both roots of the olivo-cochlear nerve bundle to one ear were transected in the brain stems of 12 chinchillas. The animals were sacrificed at times ranging from 2 to 35 days after surgery. The normal olivo-cochlear terminals on the external hair cells in the cochleas of the control ears contained many mitochondria and small vesicles of constant size. The earliest evidence for degeneration was the presence of fine 100 A filaments in the proximal parts of the terminals. These were visible at 2 days. Animals sacrificed at later times showed a greater number of filaments and fewer vesicles, but few mitochondrial changes. After 1 week, disintegration of the terminals was more prominent. A few terminals showed mitochondrial swelling and lysis of the plasma membrane but few or no filaments within the first week. These latter terminals were interpreted as representing a more rapid process of disintegration than those terminals characterized by numerous filaments and seemingly unchanged mitochondria.     

Vimentin intermediate filaments protect mitochondria from oxidative stress

The role of intermediate filaments (IFs) in eukaryotic cells is still unclear. The disturbance of mitochondria distribution and function, in particular the enhanced production of reactive oxygen species (ROS) and decreased membrane potential, is observed in cells devoid of IFs. The aim of this work was to study the dependence of mitochondria sensitivity to oxidative stress on the presence of vimentin IFs. It was found that mitochondria are less sensitive to ROS in cells containing vimentin than in cells devoid of vimentin. Besides, mitochondrial membrane potential was demonstrated to increase upon regeneration of vimentin IFs in the cells. Substitution of Pro-57 by Arg in N-terminal part of the vimentin molecule abandoned its protective ability and the effect on mitochondrial membrane potential.     

Ultrastructural features of the columellar muscle and contractile protein analyses in different muscle groups of Megalobulimus abbreviatus (Gastropoda, Pulmonata)

In Megalobulimus abbreviatus, the ultrastructural features and the contractile proteins of columellar, pharyngeal and foot retractor muscles were studied. These muscles are formed from muscular fascicles distributed in different planes that are separated by connective tissue rich in collagen fibrils. These cells contain thick and thin filaments, the latter being attached to dense bodies, lysosomes, sarcoplasmic reticulum, caveolae, mitochondria and glycogen granules. Three types of muscle cells were distinguished: T1 cells displayed the largest amount of glycogen and an intermediate number of mitochondria, suggesting the highest anaerobic metabolism; T2 cells had the largest number of mitochondria and less glycogen, which suggests an aerobic metabolism; T3 cells showed intermediate glycogen volumes, suggesting an intermediate anaerobic metabolism. The myofilaments in the pedal muscle contained paramyosin measuring between 40 and 80 nm in diameter. Western Blot muscle analysis showed a 46-kDa band that corresponds to actin and a 220-kDa band that corresponds to myosin filaments. The thick filament used in the electrophoresis showed a protein band of 100 kDa in the muscles, which may correspond to paramyosin.