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.     

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.     

Cytoskeletal organization and cell organelle transport in basal epithelial cells of the freshwater sponge Spongilla lacustris

Summary Different antibodies against actin, tubulin and cytokeratin were utilized to demonstrate the spatial organization of the cytoskeleton in basal epithelial cells of the freshwater sponge Spongilla lacustris. Accordingly, actin is localized in a cortical layer beneath the plasma membrane and in distinct fibers within the cytoplasmic matrix. Microtubules exhibit a different distributional pattern by radiating from a perinuclear sheath and terminating at, the cell periphery; in contrast, intermediate filaments are lacking. Cytoplasmic streaming activity was studied by in-vivo staining of mitochondria and endoplasmic reticulum by means of fluorescent dyes. Single-frame analysis of such specimens revealed a regular shuttle movement of mitochondria and other small particles between the cell nucleus and the plasma membrane, which can be stopped in a reversible manner with the use of colcemid or colchicine but not with cytochalasin D. The results point to the microtubular system as a candidate for cell organelle transport, whereas the actomyosin system rather serves for changes in cellular shape and motility.     

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.     

Actin microfilaments facilitate the retrograde transport from the Golgi complex to the endoplasmic reticulum in mammalian cells

The morphology and subcellular positioning of the Golgi complex depend on both microtubule and actin cytoskeletons. In contrast to microtubules, the role of actin cytoskeleton in the secretory pathway in mammalian cells has not been clearly established. Using cytochalasin D, we have previously shown that microfilaments are not involved in the endoplasmic reticulum–Golgi membrane dynamics. However, it has been reported that, unlike botulinum C2 toxin and latrunculins, cytochalasin D does not produce net depolymerization of actin filaments. Therefore, we have reassessed the functional role of actin microfilaments in the early steps of the biosynthetic pathway using C2 toxin and latrunculin B. The anterograde endoplasmic reticulum-to-Golgi transport monitored with the vesicular stomatitis virus-G protein remained unaltered in cells treated with cytochalasin D, latrunculin B or C2 toxin. Conversely, the brefeldin A-induced Golgi membrane fusion into the endoplasmic reticulum, the Golgi-to-endoplasmic reticulum transport of a Shiga toxin mutant form, and the subcellular distribution of the KDEL receptor were all impaired when actin microfilaments were depolymerized by latrunculin B or C2 toxin. These findings, together with the fact that COPI-coated and uncoated vesicles contain β/γ-actin isoforms, indicate that actin microfilaments are involved in the endoplasmic reticulum/Golgi interface, facilitating the retrograde Golgi-to-endoplasmic reticulum membrane transport, which could be mediated by the orchestrated movement of transport intermediates along microtubule and microfilament tracks.     

Organelle distribution in chick neuroepithelial cells: effects of colchicine and cytochalasin B

The intracellular distribution of mitochondria, cytoplasmic inclusions and rough endoplasmic reticulum cisternae of chick neuroepithelial cells was investigated at neurulation stages 6, 8, 10 and 12. These neuroepithelial cells were subdivided into three zones: apical, median and basal and the distribution percentages of distribution of these organelles were obtained. Mitochondrial distribution was related to the energy supply that mitochondria provide for apical microfilament contraction. Cytoplasmic inclusions were distributed preferentially in the apical zone of the neuroepithelial cells during the four stages. Rough endoplasmic reticulum cisternae were homogeneously distributed in the three zones at stages 10 and 12, but at stages 6 and 8 there are more elevated percentages of rough endoplasmic reticulum in the apical zones than in the other zones. Experimental treatments with colchicine and cytochalasin B does not modify the patterns of mitochondria and rough endoplasmic reticulum cisternae but alters the distribution of cytoplasmic inclusions. Finally, there is a correlation in the normal neurulating neuroepithelial cells between the distributions of mitochondria and rough endoplasmic reticulum distribution and between the distributions of mitochondria and cytoplasmic inclusions distribution. This relationship is retained in the treated neuroepithelial cells.     

Identification of mRNA/protein (mRNP) complexes containing Puralpha,mStaufen, fragile X protein,and myosin Va and their association with rough endoplasmic reticulum equipped with a kinesin motor

Puralpha, which is involved in diverse aspects of cellular functions, is strongly expressed in neuronal cytoplasm. Previously, we have reported that this protein controls BC1 RNA expression and its subsequent distribution within dendrites and that Puralpha is associated with polyribosomes. Here, we report that, following treatment with EDTA, Puralpha was released from polyribosomes in mRNA/protein complexes (mRNPs), which also contained mStaufen, Fragile X Mental Retardation Protein (FMRP), myosin Va, and other proteins with unknown functions. As the coimmunoprecipitation of these proteins by an anti-Puralpha antibody was abolished by RNase treatment, Puralpha may assist mRNP assembly in an RNA-dependent manner and be involved in targeting mRNPs to polyribosomes in cooperation with other RNA-binding proteins. The immunoprecipitation of mStaufen- and FMRP-containing mRNPs provided additional evidence that the anti-Puralpha detected structurally or functionally related mRNA subsets, which are distributed in the somatodendritic compartment. Furthermore, mRNPs appear to reside on rough endoplasmic reticulum equipped with a kinesin motor. Based on our present findings, we propose that this rough endoplasmic reticulum structure may form the molecular machinery that mediates and regulates multistep transport of polyribosomes along microtubules and actin filaments, as well as localized translation in the somatodendritic compartment.     

The Sertoli cell in lizards

Sertoli cells of lizards are characterized by variable size, abundant smooth and rough endoplasmic reticulum, multivesicular bodies, lipid vacuoles probably related to the spermatogenic cycle, and mitochondria of normal size. The cytoskeleton contains actin, particularly abundant in the cell periphery, vimentin all around the nucleus and throughout the rest of the cytoplasm. Moreover, microtubules are distributed in the cell periphery. The junctional complexes demonstrate the presence of a very efficient blood-testis barrier, containing tight, gap, tight-gap, septate-like, desmosome-like, and "Sertoli-Sertoli" junctions. In the last, the actin layer interposed between the plasma membrane and the subsurface cistern is absent. The desmosome-like junctions are surrounded by 7-nm filaments and not by intermediate filaments.     

Altered distribution profiles of endoplasmic reticulum subfractions after incubation of Krebs II ascites cells with different concentrations of cytochalasin B

Information on the interaction between endoplasmic reticulum (ER) membranes and components of the skeletal network of the cell was gained by treating cells with the antimicrofilament agent cytochalasin B prior to cell disruption by nitrogen cavitation. Treatment of Krebs II ascites cells with cytochalasin B (5–10 μg ml^?1) resulted in an increased yield of three ER membrane subfractions — heavy rough (HR), light rough (LR) and smooth (S) membranes, as judged by ³H-choline incorporation in gradient fractions following discontinuous sucrose gradient centrifugation. The major increase was observed in the HR fraction. These results indicate that the actual yield of the respective ER membrane subfractions after cell disruption is dependent on the degree of direct and/or indirect interaction between individual ER membranes and actin containing filaments of the cytoskeleton in the intact cell.     

Free and membrane-bound polyribosomes in BHK cells infected with Sindbis virus.

The data presented in the paper demonstrate that in BHK cells infected with Sindbis virus virtually all the 42S mRNA not in nucleocapsid is associated with free polyribosomes, whereas the 26S mRNA is distributed between free and membrane-bound polyribosomes. We suggest that the 26S RNA polyribosomes are bound to the membranes through the nascent chains of the B1 protein and that a large percentage of 26S RNA polyribosomes free in the cytoplasm may be due to the small amount of rough endoplasmic reticulum in BHK cells. In addition, we found that intracellular nucleocapsid is in the nonmembrane fraction of the cytoplasm of infected cells.     

The ultrastructure of developing wings in the giant silkmoth, Hyalophora cecropia. I. Generalized epidermal cells

The ultrastructure of wing epidermis of the giant silkmoth, Hyalophora cecropia, was studied during pupal diapause and the first half of development to the adult. In diapause, the generalized epidermal cells are characterized by many free ribosomes, some vesicles and small lamellae of rough endoplasmic reticulum, some scattered short mitochondria and a few small Golgi complexes. During the early states of post-diapause development, before and after the time of apolysis (separation of the epidermis from the overlying cuticle), there is a marked increase in structures often associated with synthetic functions, such as polyribosomes, lamellate rough endoplasmic reticulum and Golgi complexes. On day five of post-apolysis development, just after the appearance of scale-forming and socket-forming cells, the generalized epidermal cells lay down the cuticulin layer of the adult cuticle. At this stage and later, the polyribosomes and lamellate rough endoplasmic reticulum decrease in abundance. Cell nuclei show three phases of temporary transition from predominantly lobed to predominantly round profile, which correspond to periods of reported DNA synthesis. Throughout this developmental process, therefore, there is good correlation of fine structure with changes in macromolecular synthesis recorded elsewhere.     

Cytochalasin B binding and actin content of endoplasmic reticulum subfractions isolated from L-929 cells

Heavy rough (HR) endoplasmic reticulum (ER) membranes and a dense fraction of light rough (LR) membranes (LR I) of L-929 cells bind H-cytochalasin B extremely poorly in comparison to smooth (S) membranes and a fraction of LR membranes of low density (LR II). The LR and S subfractions of ER are apparently heterogeneous membrane populations with respect to cytochalasin B binding. The separation of proteins in HR and LR subfractions by electrophoresis followed by immunoblotting with monoclonal antibodies against actin showed that actin was not present in the former membranes while there were large amounts in the LR subfraction. It is concluded that membranes in the LR II fraction of ER are associated with actin-containing microfilaments of the cytoskeleton, but that HR membranes are not.     

Carbocyanine dyes stain the sarcoplasmic reticulum of beating heart cells

In search of a fluorescent dye suitable for monitoring membrane potentials of beating heart cells, we noticed that the carbocyanine dyes, CC₅ and CC₆, show a unique pattern of intracellular distribution in vital and glutaraldehyde-fixed cardiomyoblasts. This distribution is clearly different from that observed in fibroblasts. In heart cells, it parallels the localization of actin-myosin containing myofilaments as visualized by fluorescent antibody staining but it does not correspond to the localization of actin filaments or the microtubules. In fibroblasts these dyes stain only fine filaments and granules in the perinuclear space which correspond to the endoplasmic reticulum. This observation is evidence in support of the hypothesis that carbocyanine dyes accumulate selectively in the sarcoplasmic reticulum. It indicates that certain carbocyanine dyes may be useful tools to differentiate between muscle cells and connective tissue cells in cell cultures.     

OBSERVATIONS ON THE ASSOCIATION OF HELICAL POLYRIBOSOMES AND FILAMENTS WITH VINCRISTINE-INDUCED CRYSTALS IN EARLE'S L-CELL FIBROBLASTS

Earle's L-929 fibroblasts from cultures treated with 5–10 µg/ml of vincristine sulfate have a large number of eosinophilic, proteinaceous crystals in their cytoplasm. In electron micrographs, large arrays of helical polyribosomes, stacks of Golgi lamellae, and membranes of granular endoplasmic reticulum are seen in the cytoplasm of these cells. "Stalks" of fine granular material, approximately 300 A wide, are often seen in association with the arrays of the helical polyribosomes. In many sections rows of helical polyribosomes and filaments emerging from individual polyribosomes are seen in intimate contact with the crystals. A gradual reduction in the number of crystals and crystal-bearing cells is seen in cultures removed from the drug-containing medium and reincubated in fresh medium. In electron micrographs, these reincubated cells show large aggregates of filamentous material in the cytoplasm, and in many sections filaments are seen in continuity with the crystals.     

The role of actin filaments in the organization of the endoplasmic reticulum in honeybee photoreceptor cells

Close to the bases of the photoreceptive microvilli, arthropod photoreceptors contain a dense network of endoplasmic reticulum that is involved in the regulation of the intracellular calcium concentration, and in the biogenesis of the photoreceptive membrane. Here, we examine the role of the cytoskeleton in organizing this submicrovillar endoplasmic reticulum in honeybee photoreceptors. Immunofluorescence microscopy of taxol-stabilized specimens, and electron-microscopic examination of high-pressure frozen, freeze-substituted retinae demonstrate that the submicrovillar cytoplasm lacks microtubules. The submicrovillar region contains a conspicuous F-actin system that codistributes with the submicrovillar endoplasmic reticulum. Incubation of retinal tissue with cytochalasin B leads to depolymerization of the submicrovillar F-actin system, and to disorganization and disintegration of the submicrovillar endoplasmic reticulum, indicating that an intact F-actin cytoskeleton is required to maintain the architecture of this domain of the endoplasmic reticulum. We have also developed a permeabilized cell model in order to study the physiological requirements for the interaction of the endoplasmic reticulum with actin filaments. The association of submicrovillar endoplasmic reticulum with actin filaments appears to be independent of ATP, Ca²⁺ and Mg²⁺, suggesting a tight static anchorage.     

Polyribosomal attachment to rat liver and hepatoma endoplasmic reticulum in vitro. A method for its study

A system for study and measurement of the attachment in vitro of exogenous polyribosomes to membranes has been presented. Its main features are use of low temperature, post-microsomal supernatant, pyrophosphate and citric acid to remove ribosomes from the surface of rough endoplasmic reticulum, and a method for quantitative separation of unattached from membrane-associated polyribosomes. The following were found. (1) Rough endoplasmic reticulum, from which ribosomes had been removed by treatment with pyrophosphate and citrate, bound over 50% of added polyribosomes, whereas the untreated (or control) rough and smooth endoplasmic reticulum and the smooth endoplasmic reticulum treated with pyrophosphate-citrate did not bind polyribosomes. (2) The polyribosome-binding capacity of rough endoplasmic reticulum stripped of its ribosomes decayed upon storage of the membranes at 0-4 degrees C. The half-life of this decay was about 6 days whereas that of the polyribosome-binding capacity of hepatoma stripped rough endoplasmic reticulum was about 1.5 days. (3) Preparations of stripped rough endoplasmic reticulum after reassociation with polyribosomes in vitro were quite similar to preparations of native rough endoplasmic reticulum as viewed with the electron microscope. Evidence is presented to support the contention that association of polyribosomes with membranes was the result of polyribosomal reattachment to the membranes rather than trapping of the polyribosomes between vesicles of the membranes.     

Binding of rat liver and hepatoma polyribosomes to stripped rough endoplasmic reticulum in vitro. Biological or an artifact?

Exposed thiol groups do not appear to be related to the binding of (32)P-labelled polyribosomes to stripped rough endoplasmic reticulum in vitro. Treating stripped rough endoplasmic reticulum with GSSG did not diminish binding of polyribosomes, suggesting that binding in vitro has no correlation with the inhibition of protein synthesis in vitro reported by Kosower et al. (1971). Thiol reagents, which are known to dissociate ribosomes, did not significantly decrease binding of (32)P-labelled polyribosomes to stripped rough endoplasmic reticulum. Denaturing the protein of (32)P-labelled polyribosomes or stripped rough endoplasmic reticulum of liver or hepatoma with heat, trichloroacetic acid, or HClO(4) did not alter the binding in vitro. Therefore, the practice of measuring the binding of (32)P-labelled polyribosomes to stripped rough endoplasmic reticulum in vitro (Shires et al., 1971b) is an unsuitable indicator of biological significance in the intact cell.     

Polyribosomes associated with microfilaments in cultured lens cells

Epithelial hamster lens cells, transformed by SV40 can be grown in suspension culture. Triton X-100 extraction of these cells grown under conditions when ribosome run off is blocked releases about 40% of the total amount of polyribosomes, designated as free- and loosely-bound polyribosomes. The Triton ghosts retain the remaining polysomal population which can be released by a combined treatment with deoxycholate and Nonidet P 40. Electron microscopic examination of the ghosts reveals microfilament-associated ribosome clusters next to a fraction of polysomes still attached to membranes. Preincubation of the cells with cytochalasin D prior to polyribosome isolation enables us to discriminate between these two latter polysome populations. The experiments indicate that about 25% of the polyribosomes are attached to microfilaments, while the remaining 35% are tightly bound to the membranes of the endoplasmic reticulum. When the different polyribosome classes were translated in a reticulocyte lysate, no significant differences could be observed in the patterns of the newly synthesized polypeptides. In all cases actin was one of the major products synthesized de novo.     

The mechanism of cytoplasmic streaming in characean algal cells: sliding of endoplasmic reticulum along actin filaments

Electron microscopy of directly frozen giant cells of characean algae shows a continuous, tridimensional network of anastomosing tubes and cisternae of rough endoplasmic reticulum which pervade the streaming region of their cytoplasm. Portions of this endoplasmic reticulum contact the parallel bundles of actin filaments at the interface with the stationary cortical cytoplasm. Mitochondria, glycosomes, and other small cytoplasmic organelles enmeshed in the endoplasmic reticulum network display Brownian motion while streaming. The binding and sliding of endoplasmic reticulum membranes along actin cables can also be directly visualized after the cytoplasm of these cells is dissociated in a buffer containing ATP. The shear forces produced at the interface with the dissociated actin cables move large aggregates of endoplasmic reticulum and other organelles. The combination of fast-freezing electron microscopy and video microscopy of living cells and dissociated cytoplasm demonstrates that the cytoplasmic streaming depends on endoplasmic reticulum membranes sliding along the stationary actin cables. Thus, the continuous network of endoplasmic reticulum provides a means of exerting motive forces on cytoplasm deep inside the cell distant from the cortical actin cables where the motive force is generated.     

Translation and the cytoskeleton: a mechanism for targeted protein synthesis

This review describes the critical evidence that in eukaryotic cells polyribosomes, mRNAs and components of the protein synthetic machinery are associated with the cytoskeleton. The role of microtubules, intermediate filaments and microfilaments are discussed; at present most evidence suggests that polyribosomes interact with the actin filaments. The use of non-ionic detergent/deoxycholate treatment in the isolation of cytoskeletal-bound polysomes is described and the conclusion reached that at low salt concentrations this leads to mixed preparations of polysomes derived from both the cytoskeleton and the endoplasmic reticulum. At present the best approach for isolation of cytoskeletal-bound polysomes appears to involve extraction with salt concentrations greater than 130 mM after an initial non-ionic detergent treatment. Such polysomes appear to be enriched in certain mRNAs and thus it is suggested that they are involved in translation of a unique set of proteins. The evidence for mRNA localisation is presented and the role of the cytoskeleton in transport and localisation of RNA discussed. Recent data on the role of the 3 untranslated region in the targeting of mRNAs both to particular regions of the cell and for translation on cytoskeletal-bound polysomes is described. The hypothesis is developed that the association of polysomes with the cytoskeleton is the basis of a mechanism for the targeting of mRNAs and the compartmentalization of protein synthesis.Abbreviations CBP cytoskeletal-bound polysomes - FP free polysomes - MBP membrane-bound polysomes - ER endoplasmic reticulum