Synthesis and deposition of zein in protein bodies of maize endosperm

The origin of protein bodies in maize (Zea mays L.) endosperm was investigated to determine whether they are formed as highly differentiated organelles or as protein deposits within the rough endoplasmic reticulum. Electron microscopy of developing maize endosperm cells showed that membranes surrounding protein bodies were continuous with rough endoplasmic reticulum membranes. Membranes of protein bodies and rough endoplasmic reticulum both contained cytochrome c reductase activity indicating a similarity between these membranes. Furthermore, the proportion of alcohol-soluble protein synthesized by polyribosomes isolated from protein body or rough endoplasmic reticulum membranes was similar, and the alcohol-soluble or -insoluble proteins showed identical [¹⁴C]leucine labeling. These results demonstrated that protein bodies form simply as deposits within the rough endoplasmic reticulum.

Messenger RNA that directed synthesis of only the smaller molecular weight zein subunit was separated from mRNA that synthesized both subunits by sucrose gradient centrifugation. This result demonstrated that separate but similar sized mRNAs synthesize the major zein components. In vitro translation products of purified mRNAs or polyribosomes were approximately 2,000 daltons larger than native zein proteins, suggesting that the proteins are synthesized as zein precursors. When intact rough endoplasmic reticulum was placed in the in vitro protein synthesis system, proteins corresponding in molecular weight to the native zein proteins were obtained.

     

Morphology and function of the endoplasmic reticulum

Comparison with the findings in the cells of other plants and animals showed that the endoplasmic reticulum in the root apex ofFagopyrum has the same general character and function as in other biological objects, i.e. in secretory processes and especially in this case in the transport of the substances produced. Detailed studies of the morphology and activity of the endoplasmic reticulum showed some functional differences which are characteristic for this object. The endoplasmic reticulum participates apparently in the transport of the mass of known but functionally and nomenclatorically controversial formations which here are called dense bodies. Dense bodies exist inFagopyrum in a considerable amount as compared to other objects. Frequent contact of the dense bodies with the ends of the endoplasmic reticulum, contact with the endoplasmic retieulum passing through the plasmodesm, accumulation of the dense bodies along the cell wall and in proximal distance of the plasmodesms and intensive staining of some plasmodesms was observed. The dense vacuoles in this object represent dilated spaces of the endoplasmic reticulum which apparently have the function of reservoirs of the dense mass. The endoplasmic reticulum in the calyptra cells appears to participate in the formation of the cell walls. This object differs hereby from others, where the participation of the Golgi apparatus has been observed in this function.     

Ultrastructure of Mitosis and Cytokinesis In the Colorless Flagellate Katablepharis Ovalis Skuja

ABSTRACT. Mitosis and cytokinesis in Katablepharis ovalis , a colorless flagellate, was investigated. Two new flagella are produced prior to prophase, resulting in a motile quadriflagellate cell during mitosis. the inner array of microtubules of the feeding apparatus disappears before prophase begins. the nuclear envelope disperses during prophase, apparently being converted into rough endoplasmic reticulum. the chromatin condenses and the nucleolus disperses with spindle microtubules appearing oriented perpendicular to the longitudinal axis of the cell. At metaphase, the chromatin is condensed as a single disc-shaped mass and rough endoplasmic reticulum flanks the chromatin mass on each side. Groups of spindle microtubules pass through tunnels in the rough endoplasmic reticulum and through electron-translucent areas of the chromatin. the spindle microtubules end at a number of minipoles in the cytoplasm. Vesicles, ribosomes, mitochondria and endoplasmic reticulum migrate among the spindle microtubules. There is no polar body or any electrondense area associated with the spindle poles. the basal bodies of the flagella remain attached to the axonemes and do not participate in mitosis. In anaphase, the chromatin separates and migrates to the poles. During telophase, the nuclear envelope reforms from the rough endoplasmic reticulum and the nucleoli reappear. the spindle microtubules are persistent during telophase. Cytokinesis occurs by longitudinal fission, starting at the anterior end and progressing posteriorly. Cytokinesis may be driven by elongation of the spindle microtubules since there is no visible structure associated with the furrowing.     

Dual Localization of Squalene Epoxidase, Erg1p, in Yeast Reflects a Relationship between the Endoplasmic Reticulum and Lipid Particles

Squalene epoxidase, encoded by the ERG1 gene in yeast, is a key enzyme of sterol biosynthesis. Analysis of subcellular fractions revealed that squalene epoxidase was present in the microsomal fraction (30,000 × g) and also cofractionated with lipid particles. A dual localization of Erg1p was confirmed by immunofluorescence microscopy. On the basis of the distribution of marker proteins, 62% of cellular Erg1p could be assigned to the endoplasmic reticulum and 38% to lipid particles in late logarithmic-phase cells. In contrast, sterol Δ²⁴-methyltransferase (Erg6p), an enzyme catalyzing a late step in sterol biosynthesis, was found mainly in lipid particles cofractionating with triacylglycerols and steryl esters. The relative distribution of Erg1p between the endoplasmic reticulum and lipid particles changes during growth. Squalene epoxidase (Erg1p) was absent in an erg1 disruptant strain and was induced fivefold in lipid particles and in the endoplasmic reticulum when the ERG1 gene was overexpressed from a multicopy plasmid. The amount of squalene epoxidase in both compartments was also induced approximately fivefold by treatment of yeast cells with terbinafine, an inhibitor of the fungal squalene epoxidase. In contrast to the distribution of the protein, enzymatic activity of squalene epoxidase was only detectable in the endoplasmic reticulum but was absent from isolated lipid particles. When lipid particles of the wild-type strain and microsomes of an erg1 disruptant were mixed, squalene epoxidase activity was partially restored. These findings suggest that factor(s) present in the endoplasmic reticulum are required for squalene epoxidase activity. Close contact between lipid particles and endoplasmic reticulum may be necessary for a concerted action of these two compartments in sterol biosynthesis.     

Mutants of the Yeast Yarrowia lipolytica Defective in Protein Exit from the Endoplasmic Reticulum Are Also Defective in Peroxisome Biogenesis

Mutations in the SEC238 and SRP54 genes of the yeast Yarrowia lipolytica not only cause temperature-sensitive defects in the exit of the precursor form of alkaline extracellular protease and of other secretory proteins from the endoplasmic reticulum and in protein secretion but also lead to temperature-sensitive growth in oleic acid-containing medium, the metabolism of which requires the assembly of functionally intact peroxisomes. The sec238A and srp54KO mutations at the restrictive temperature significantly reduce the size and number of peroxisomes, affect the import of peroxisomal matrix and membrane proteins into the organelle, and significantly delay, but do not prevent, the exit of two peroxisomal membrane proteins, Pex2p and Pex16p, from the endoplasmic reticulum en route to the peroxisomal membrane. Mutations in the PEX1 and PEX6 genes, which encode members of the AAA family of N-ethylmaleimide-sensitive fusion protein-like ATPases, not only affect the exit of precursor forms of secretory proteins from the endoplasmic reticulum but also prevent the exit of the peroxisomal membrane proteins Pex2p and Pex16p from the endoplasmic reticulum and cause the accumulation of an extensive network of endoplasmic reticulum membranes. None of the peroxisomal matrix proteins tested associated with the endoplasmic reticulum in sec238A, srp54KO, pex1-1, and pex6KO mutant cells. Our data provide evidence that the endoplasmic reticulum is required for peroxisome biogenesis and suggest that in Y. lipolytica, the trafficking of some membrane proteins, but not matrix proteins, to the peroxisome occurs via the endoplasmic reticulum, results in their glycosylation within the lumen of the endoplasmic reticulum, does not involve transport through the Golgi, and requires the products encoded by the SEC238, SRP54, PEX1, and PEX6 genes.     

Preperoxisomal vesicles can form in the absence of Pex3

We demonstrate that the peroxin Pex3 is not required for the formation of peroxisomal membrane structures in yeast pex3 mutant cells. Notably, pex3 mutant cells already contain reticular and vesicular structures that harbor key proteins of the peroxisomal receptor docking complex—Pex13 and Pex14—as well as the matrix proteins Pex8 and alcohol oxidase. Other peroxisomal membrane proteins in these cells are unstable and transiently localized to the cytosol (Pex10, Pmp47) or endoplasmic reticulum (Pex11). These reticular and vesicular structures are more abundant in cells of a pex3 atg1 double deletion strain, as the absence of Pex3 may render them susceptible to autophagic degradation, which is blocked in this double mutant. Contrary to earlier suggestions, peroxisomes are not formed de novo from the endoplasmic reticulum when the PEX3 gene is reintroduced in pex3 cells. Instead, we find that reintroduced Pex3 sorts to the preperoxisomal structures in pex3 cells, after which these structures mature into normal peroxisomes.     

Heterogeneous distribution of glycosyltransferases in the endoplasmic reticulum of castor bean endosperm

Endoplasmic reticulum membranes stripped of attached ribosomes were isolated from homogenates of germinating castor bean (Ricinus communis L.) endosperm by sucrose density gradient centrifugation. The isolated endoplasmic reticulum fraction was further separated into two major membrane subfractions by centrifugation on a flotation gradient. Both subfractions appeared to be derived from the endoplasmic reticulum inasmuch as they share several enzymic markers including cholinephosphotransferase, NADH-cytochrome c reductase, and glycoprotein fucosyl-transferase and phase separation of membrane polypeptides using Triton X-114 revealed a striking similarity in both their hydrophilic and hydrophobic protein components. The endoplasmic reticulum membrane subfractions contain glycoproteins which were readily labeled by incubating intact endosperm tissue with radioactive sugars prior to fractionation.

Castor bean endosperm endoplasmic reticulum apparently exhibits a degree of enzymic heterogeneity, however, since the enzymes responsible for the synthesis of dolicholpyrophosphate N-acetylglucosamine and dolicholmonophosphate mannose together with their incorporation into the oligosaccharide-lipid precursor of protein N-glycosylation were largely recovered in a single endoplasmic reticulum subfraction.

     

LYSOSOMES AND GERL IN NORMAL AND CHROMATOLYTIC NEURONS OF THE RAT GANGLION NODOSUM

The rat ganglion nodosum was used to study chromatolysis following axon section. After fixation by aldehyde perfusion, frozen sections were incubated for enzyme activities used as markers for cytoplasmic organelles as follows: acid phosphatase for lysosomes and GERL (a Golgi-related region of smooth endoplasmic reticulum from which lysosomes appear to develop) (31–33); inosine diphosphatase for endoplasmic reticulum and Golgi apparatus; thiamine pyrophosphatase for Golgi apparatus; acetycholinesterase for Nissl substance (endoplasmic reticulum); NADH-tetra-Nitro BT reductase for mitochondria. All but the mitochondrial enzyme were studied by electron microscopy as well as light microscopy. In chromatolytic perikarya there occur disruption of the rough endoplasmic reticulum in the center of the cell and segregation of the remainder to the cell periphery. Golgi apparatus, GERL, mitochondria and lysosomes accumulate in the central region of the cell. GERL is prominent in both normal and operated perikarya. Electron microscopic images suggest that its smooth endoplasmic reticulum produces a variety of lysosomes in several ways: (a) coated vesicles that separate from the reticulum; (b) dense bodies that arise from focal areas dilated with granular or membranous material; (c) "multivesicular bodies" in which vesicles and other material are sequestered; (d) autophagic vacuoles containing endoplasmic reticulum and ribosomes, presumably derived from the Nissl material, and mitochondria. The number of autophagic vacuoles increases following operation.     

Ultrastructure of the vitelline follicles of Gorgoderina vitelliloba (Trematoda: Gorgoderidae)

An electron microscope study of the vitelline follicles of Gorgoderina vitelliloba indicates that they contain vitelline cells in various stages of development. Juvenile cells are small and characterised by a little cytoplasm. During differentiation a large amount of granular endoplasmic reticulum develops. In more mature cells, indistinct Golgi complexes give rise to globules of shell protein which migrate to form clusters at the periphery of the cell. Further maturation results in the appearance of large lipid bodies in the vitelline cell cytoplasm.Developing vitelline cells are ensheathed by nurse cell cytoplasm containing numerous small vacuoles which appear to be derived from smooth endoplasmic reticulum. It is suggested that nurse cells may have a role in selection and transport of nutrient material for vitelline cells and that they manufacture precursors of lipid which is subsequently stored as a food reserve in mature vitelline cells. Possible transport sites between parenchymal cells and nurse cells were identified.     

Low-pH-induced apoptosis: role of endoplasmic reticulum stress-induced calcium permeability and mitochondria-dependent signaling

The acidic microenvironment around tumor cells is a major determinant in cancer growth, metabolism, and metastasis. However, its role in cancer physiology is still not clearly understood. In the present investigation, an attempt has been made to explore the effect of acidic environment on physiology of cancer cells. Exposure of Raji cells to extracellular acidic environment was associated with enhanced cytosolic calcium level and endoplasmic reticulum stress response. X-box binding protein 1 (XBP1) splicing, CCAAT/enhancer-binding protein homologous protein (CHOP), and glucose-regulated protein 78 kDa (GRP78) upregulation suggested endoplasmic reticulum stress generation. On the other hand, real-time-based upregulation of Bax gene expression and flow cytometric analysis of cytochrome c release as well as enhanced active caspase-3 further confirmed mitochondrion-mediated events leading to induction of apoptosis. The expression of TP53 and p21 was upregulated. These observations collectively strongly suggest that both endoplasmic reticulum stress-mediated calcium release and Bax targeting might be altering mitochondrion membrane potential which in turn could induce secondary apoptotic signals; subsequently, endoplasmic reticulum stress can also lead to nuclear localization of Nuclear factor-κB (NF-κB) which in turn favors p53 mediated apoptotic signals.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-014-0568-6) contains supplementary material, which is available to authorized users.     

Ultrastructure of the salt glands of the mangrove,Avicennia marina (Forssk.) Vierh., as indicated by the use of selective membrane staining

Each salt-excreting gland of the mangrove Avicennia marina (Forsskål) Vierh. consists of two to four collecting cells, one stalk cell, and eight to twelve excretory cells. Differential membrane staining by zinc iodide-osmium tetroxide (as a post-fixative) or phosphotungstic acid (as a section-stain) was used to characterise the ultrastructure of the glands. A large amount of tubular endoplasmic reticulum was found in the stalk and excretory cells of the gland, but not in the collecting cells. The ultrastructural arrangement of the endoplasmic reticulum indicates that salt is loaded from the apoplasm into the endoplasmic reticulum of the symplasm at the base of the stalk cell, traverses both cell types in the endoplasmic reticulum, and is excreted at the outer edge of the gland by an eccrine-type mechanism. Increasing development of the tubular endoplasmic reticulum accompanied differentiation of the gland cells.Abbreviations ER endoplasmic reticulum - PTA phosphotungstic acid - ZIO zinc iodide-osmium tetroxide     

Development of secretory activity in the long hyaline gland of the male migratory grasshopper,Melanoplus sanguinipes (Fabr.) (Orthoptera : Acrididae)

Changes in the ultrastructure of epithelial cells from long hyaline glands of male Melanoplus sanguinipes (Fabr.) (Orthoptera : Acrididae) have been examined during sexual maturation and after allatectomy. In newly emerged males, the long hyaline gland epithelium is composed of 1–3 cell layers. The cells contain almost no rough endoplasmic reticulum, inconspicuous Golgi complexes, and large numbers of free ribosomes and polysomes. Within 24 hr, the cells undergo considerable reorganization to form a 1-cell-thick layer. Changes in cytostructure include proliferation of the rough endoplasmic reticulum and the development of several elaborate Golgi complexes. The developing lumen contains a coarse fibrous material. By 3 days postemergence, columnar epithelial cells are clearly capable of considerable synthesis and export of secretory protein. Rough endoplasmic reticulum, and large, elaborate Golgi complexes are the major structural features of the cytoplasm. From day 3 to sexual maturity (day 7), no major ultrastructural changes occur, although massive accumulation of secretion in the lumen causes the epithelium to become cuboidal or flattened. Isoelectric focusing of soluble proteins from long hyaline gland secretions shows that maturing glands contain increasing numbers and quantities of secretory proteins.Allatectomy has minor effects on long hyaline gland ultrastructure. A reduction in the density of rough endoplasmic reticulum and ribosomes suggests that glands from operated males are metabolically less active. This is confirmed by qualitative and quantitative changes in the amount of secretion as revealed by isoelectric focusing. The observations are discussed in terms of the juvenile hormone control of long hyaline gland maturation.     

Morphology of the salivary glands of Mastotermes darwiniensis Froggatt (Isoptera : Mastotermitidae)

The salivary glands of Mastotermes darwiniensis (Isoptera : Mastotermitidae) workers consist of acini made up of peripherally and centrally placed cells with storage cells. A network of small efferent ductules is associated with each acinus. Intracellular ductules of the central and peripheral cells are filled with protruding microvilli. Central cells contain an abundance of rough endoplasmic reticulum and vacuoles. Continua of rough endoplasmic reticulum and vacuoles are present. Pinocytosis does not appear to be involved in secretion by the central cells. Peripheral cells are compartmental, having electron-lucent vesicle accumulations away from intracellular ductules and the basal plasmalemma invaginations. Peripheral cell morphology is similar to cells which are associated with fluid movement. Storage cells contain a large vacuole and exhibit micropinocytosis. Two axon profile types are present near and on the acini and salivary ducts, one of which appears associated with neuronal end plates.     

Identification of c-myc-dependent proteins in the medulloblastoma cell line D425Med

High c-myc levels are linked to poor prognosis in medulloblastoma (MB), and it was the aim of the current study to search for c-myc-dependent proteins in the MB cell line D425Med. For this purpose D425Med cells and cells with knocked-down c-myc (by siRNA) were analysed by a gel-based differential proteomics study using mass spectrometry. Heterogeneous nuclear ribonucleoproteins C1/C2, heterogeneous nuclear ribonucleoprotein A/B, stathmin, endoplasmic reticulum protein ERp29 precursor and guanidinoacetate N-methyltransferase were c-myc dependently expressed. Signalling, the protein machinery, metabolism and endoplasmic reticulum function may be affected and these results enable studying tumour tissue for these proteins as potential dignity markers or pharmacological targets.     

滞育和非滞育棉铃虫前胸腺的形态解剖学比较研究

该文用解剖镜和电子显微镜对滞育和非滞育棉铃虫Helicoverpa armigera前胸腺的形态解剖结构进行了比较研究。结果发现滞育棉铃虫的前胸腺细胞及其细胞间隙相对较小,不易着色,细胞核规则,较小,细胞质中几乎见不到光滑内质网和粗面内质网,线粒体极少,这些观察到的现象充分说明滞育棉铃虫的前胸腺活性较低。     

Organelle Membranes from Germinating Castor Bean Endosperm: II. ENZYMES, CYTOCHROMES, AND PERMEABILITY OF THE GLYOXYSOME MEMBRANE

Glyoxysome ghosts were isolated from germinating castor bean endosperms using established methods. Electron microscopic examination showed that some matrix material was retained within the glyoxysomal membrane. Two cytochrome reductases and phosphorylcholine glyceride transferase co-sedimented with the alkaline lipase, a known component of the glyoxysome membrane, in sucrose gradient centrifugation of osmotically shocked glyoxysomes. The activities of these enzymes in the glyoxysome membranes were compared to those in the endoplasmic reticulum relative to phospholipid content. On this basis, the phosphorylcholine glyceride transferase was 10-fold more active in the endoplasmic reticulum, whereas the lipase was 50-fold more active in the glyoxysome membrane. The cytochrome reductases were only 2-fold more active in the endoplasmic reticulum, indicating that they are components of the two membranes. Difference spectroscopy of the glyoxysome membrane suspension revealed the presence of a b5-type cytochrome similar to that found in the endoplasmic reticulum. Since the glyoxysome membrane is apparently derived from the endoplasmic reticulum, components of the endoplasmic reticulum such as these are likely to be incorporated into the glyoxysome membrane during biogenesis.     

A comparative study on the ultrastructure of cultured cells and protoplasts of soybean during cell division

Summary Cultured soybean cells recovered from a marked decrease in cell division 20 hours after removal of their cell walls with enzymes and exhibited sustained mitotic activity. Mitosis was essentially similar in both cultured cells and protoplasts. At prophase microtubules aggregated in a clear zone surrounding the nucleus prior to forming the spindle. During metaphase and anaphase chromosomal microtubules were attached to diffuse kinetochores and extended to broad spindle poles; few interzonal microtubules were evident. Considerable endoplasmic reticulum was present at the spindle poles throughout division and may contribute to the new nuclear envelope at telophase. A typical phragmoplast consisting of vesicles, overlapping microtubules and associated electron-dense material appeared earlier in the protoplasts and developed into a thicker cell plate than found in the cultured cells.Supported by the National Research Council of Canada, Grant A6304.     

Morphology and ultrastructure of the dufour's and venom gland in the ant,Myrmica rubra (L.) (Hymenoptera : Formicidae)

The morphology and fine structure of the Dufour's and venom gland, as well as their entrance into the sting, are described in the myrmicine ant, Myrmica rubra (Hymenoptera : Formicidae). The epithelial cells that constitute the Dufour's gland wall, contain a well-developed smooth endoplasmic reticulum. Older workers, compared with younger ones, show an increasing number of multilamellar inclusions. The venom gland secretory cells are arranged in 2 free filaments that carry the secretion to the reservoir. Their cytoplasm shows an intracellular collecting ductule with surrounding microvillar sheath, and an abundance of free ribosomes. However, a well-organized granular endoplasmic reticulum, which is typical in species with a more powerful sting, does not occur. Both the Dufour's and venom gland ducts are characterized by the insertion of extensive muscle fibres, which act as a precise and mutually independent control mechanism for the discharging activities of the 2 glands.     

The Fine Structure of Blastema Cells and Differentiating Cartilage Cells in Regenerating Limbs of Amblystoma Larvae

Regenerating forelimbs of larval salamanders, Amblystoma punctatum, were fixed in OsO₄ at various intervals after amputation and were sectioned for study with the electron microscope. The dedifferentiated cells comprising the early blastema were found to have a fine structure similar to that of other undifferentiated cells and to have lost all of the identifying morphological features of their tissues of origin. The cytoplasm of such cells is characterized by numerous free ribonucleoprotein granules and a discontinuous vesicular endoplasmic reticulum. The cells have more abundant cytoplasm and are in closer contact with each other than was previously realized. The layer of condensed ground substance investing most differentiated cell types is lacking. After a period of rapid cell division, the morphology of the blastema cell changes. Cytoplasm is now sparse and contains a high concentration of free ribonucleoprotein granules, but little endoplasmic reticulum. The differentiating cartilage cell, however, develops an extensive, highly organized endoplasmic reticulum and the Golgi apparatus also appears to become more highly differentiated and more extensive at this time. Small vesicles appear throughout the cytoplasm at the time the new cisternae originate and may contribute to their formation. These and other changes in the cytoplasmic organelles are discussed.