Stacks of flattened smooth endoplasmic reticulum highly enriched in inositol 1,4,5-trisphosphate (InsP3) receptor in mouse cerebellar Purkinje cells.

By immunogold electron microscopy we have shown that in mouse cerebellar Purkinje cells fixed by perfusion with formaldehyde-glutaraldehyde solution, the InsP3 receptor are numerously detected on the stacks of flattened cisterns (OTSU et al, (1990) Cell Struct. Funct., 15: 163-173). In the present experiment we investigated distribution, structure and properties of the stacks by conventional electronmicroscopy, lectin cytochemistry and immunoelectron microscopy. The size and number of stacks were variable depending on their intracellular localization; short stacks with 2-4 parallel cisterns predominate in the perikaryon, long stacks with 4-15 cisterns in the proximal dendrite, and long stacks with 3-4 cisterns in the distal dendrites. The flattened cisterns bind with concanavalin A but not with wheat-germ agglutinin and may contain KDEL proteins loaded with Lys-Asp-Glu-Leu at their C-terminin in their lumens, indicating that the cisterns are derived from ER membranes. The electron dense materials sandwiched between the cisternal membranes are composed of small particles, short cylindrical in shape and approximately 20 nm in diameter, and markedly labeled with anti InsP3R antibody. We suggest that they correspond to the tetramer of the InsP3R or their related molecules. It is not clear whether the stacks of flattened cisterns exist per se in the Purkinje cells or smooth ER existing in singlet in vivo in the Purkinje cells forms stacks during fixation. It is strongly suggested, however, that the smooth ER membranes covered by the InsP3R or their related molecules can easily interact and stack each other in the Purkinje cells.     

Immunogold localization of inositol 1, 4, 5-trisphosphate (InsP3) receptor in mouse cerebellar Purkinje cells using three monoclonal antibodies

Ultrastructural localization of InsP3 receptor in mouse cerebellar Purkinje cells was investigated by immunogold technique using three monoclonal antibodies (mab 10A6, 4C11 and 18A10). The epitopes of the three antibodies were numerously detected on the smooth endoplasmic reticulum (ER) (especially, on the stacks of flattened smooth ER, subsurface cisterns and spine apparatus), scantily on the rough ER and on the outer nuclear membrane, but were not detectable on either the plasmalemma, synaptic densities, mitochondria or Golgi apparatus. Not only mab 4C11 and 10A6 which bind to the N-terminal region of the receptor but also 18A10 which binds to the C-terminal region were localized on the cytoplasmic surface of the ER membranes. This indicates that the C terminus of InsP3 receptor is localized on the cytoplasmic surface of the ER. We noticed that gold particles are usually localized on the fuzzy structure of the cytoplasmic surface of smooth ER, which is suggested to correspond to the feet structure of the ryanodine receptor. In the Nissl body, gold particles were found not only on the ER membranes but also in the cytoplasmic matrix between the rough ER cisterns. We suggest that the peculiar structure of Nissl body, which is composed of parallel cisterns of rough ER, sandwiching a number of free polyribosomes between the cisternal elements, is due to the fact that the major proteins like InsP3 receptor are synthesized mostly on the free polyribosomes and become membrane bound only at the later stage of the biosynthesis.     

The inositol 1,4,5,-trisphosphate receptor in cerebellar Purkinje cells: quantitative immunogold labeling reveals concentration in an ER subcompartment

The Ca2+ mobilization effect of inositol 1,4,5-trisphosphate, the second messenger generated via receptor-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate, is mediated by binding to intracellular receptors, which are expressed in high concentration in cerebellar Purkinje cells. Partially conflicting previous reports localized the receptor to various subcellular structures: elements of ER, both rough and smooth-surfaced, the nuclear envelope, and even the plasma membrane. We have now reinvestigated the problem quantitatively by using cryosections of rat cerebellar tissue immunolabeled with polyclonal monospecific antibodies against the inositol 1,4,5-trisphosphate receptor. By immunofluorescence the receptor was detected only in Purkinje cells, whereas the other cells of the cerebellar cortex remained negative. In immunogold-decorated ultrathin cryosections of the Purkinje cell body, the receptor was concentrated in cisternal stacks (piles of up to 12 parallel cisternae separated by regularly spaced bridges, located both in the deep cytoplasm and beneath the plasma membrane; average density, greater than 5 particles/micron of membrane profile); in cisternal singlets and doublets adjacent to the plasma membrane (average density, approximately 2.5 particles/micron); and in other apparently smooth-surfaced vesicular and tubular profiles. Additional smooth-surfaced elements were unlabeled. Perinuclear and rough-surfaced ER cisternae were labeled much less by themselves (approximately 0.5 particles/micron, two- to threefold the background), but were often in direct membrane continuity with heavily labeled, smooth-surfaced tubules and cisternal stacks. Finally, mitochondria, Golgi cisternae, multivesicular bodies, and the plasma membrane were unlabeled. In dendrites, approximately half of the nonmitochondrial, membrane-bound structures (cisternae, tubules, and vesicles), as well as small cisternal stacks, were labeled. Dendritic spines always contained immunolabeled cisternae and vesicles. The dendritic plasma membrane, of both shaft and spines, was consistently unlabeled. These results identify a large, smooth-surfaced ER subcompartment that appears equipped to play a key role in the control of Ca2+ homeostasis: in particular, in the generation of [Ca2+]i transients triggered by activation of specific receptors, such as the quisqualate-preferring trans(+/-)-1-amino-1,3-cyclopentamedicarboxylic acid glutamatergic receptors, which are largely expressed by Purkinje cells.     

THE VISUALIZATION OF CONCANAVALIN A BINDING SITES IN PURKINJE CELL SOMATA AND DENDRITES OF RAT CEREBELLUM

The localization of concanavalin A (Con A) binding sites in Purkinje cell somata and dendrites has been studied using a peroxidase labeling technique. In the somata, the nuclear, Golgi, and endoplasmic reticulum (ER) membranes are rich in Con A binding sites. The hypolemmal cisternae, which are continuous with the ER from the soma and throughout the dendritic tree of Purkinje cells, are also rich in Con A binding sites. Other cisternae seen in these dendrites do not bind detectable amounts of Con A. The results suggest that a cisternal system, rich in carbohydrate, may be continuous from the nuclear envelope to distal dendritic segments of Purkinje cells. Such a system could play a role in the movement of materials from Purkinje somata to dendrites.     

ER-annulate lamellar associations of mucosal epithelial cells of the rat jejunum

Summary Mucosal cells of the rat jejunum vary greatly in the relative abundance and kind of endoplasmic reticulum (ER). Many cells have few rough surfaced cisternal ER elements; these cells frequently are located in basal villous regions and generally are less differentiated than those near apices. Profiles of rough ER cisternae were often encountered in stacks of 3–4 parallel units in apical cells. A number of differentiating basal cells were found to contain ER-annulate lamellar associations arranged similarly to the ER stacks of apical cells. The annulate lamellae of this complex resemble those described for other rapidly differentiating or embryonic cells and may be derived from the nuclear envelope. They could be involved in the formation of the ER.Supported by Grants (GB-19111 and GU-3161) from the National Science Foundation     

Light and electron microscopic studies in the cerebellum of "trembler" mutant of chickens

Light and electron microscopic analyses of the cerebellar cortex were carried out in inbred trembler mutants of Barred Plymouth Rock (BPR), crossbred trembler (CBT) and crossbred normal (CBN) chickens 6 and 34 days after hatching. The size of the cerebellum of BPR was markedly reduced, but the pattern of fissures appeared essentially normal. Both the molecular and granular layer in the BPR were much thinner than in the CBT and CBN. Ectopic Purkinje cells were observed in the molecular and granular layers, and even in the medulla. The Purkinje cells were more intensely stained in Nissl preparations than those of CBT and CBN. By 34 days, most of the Purkinje cells, which were reduced in size, contained spherical and swollen mitochondria, an undeveloped Golgi apparatus, and many stacks of rough endoplasmic reticulum (ER). The basket cells were reduced in size and exhibited a decrease in stacks of rough ER and an increase in the area of the Golgi apparatus. They contained the swollen mitochondria that were observed in the Purkinje cells. The granule cells were reduced in size, and showed a decrease in the number of cell organelles. The disorder appeared to be intrinsic to Purkinje cells since many types of degeneration were observed in the BPR Purkinje cells.     

Morphological aspects of the galactomannan formation in the endosperm ofTrigonella foenum-graecum L. (Leguminosae)

The mode of deposition (secretion) of galactomannan in the cells of the seed endosperm ofTrigonella foenum-graecum has been studied by electron microscopy. In cells which are just beginning to secrete galactomannan there are stacks of rough endoplasmic reticulum (ER). The intracisternal space (containing the enchylema) of the rough ER then swells, becomes vacuolated and forms a voluminous network, with pockets of cytoplasm entrapped within poculiform rough ER. The enchylema contains material which reacts with periodate-thiocarbohydrazidesilver proteinate in a very similar manner to the galactomannan already deposited in the cell wall. It appears that the galactomannan is formed in the intracisternal space of the rough endoplasmic reticulum and then expelled outside the plasmalemma. This mode of deposition contrasts with that of other plant cell wall polysaccharides whose secretion is mediated by Golgi vesicles.Abbreviation ER endoplasmic reticulum This is part six in a series of papers dealing with galactomannan metabolism. Part five: Planta133, 219–222 (1977)     

Endoplasmic reticulum in developing seeds of Vicia faba

The changes in endoplasmic reticulum (ER) morphology during seed development have been followed using a thick section electron microscope technique. The tissues were stained with a zinc iodineosmium tetroxide complex which preferentially accumulated in the lumen between double membranes. Sections up to 2 m in thickness were examined in a high voltage electron microscope (HVEM) with tilt facility to produce stereo pairs. The micrographs from HVEM showed an increase in the extent of interconnecting tubular and cisternal ER during the protein deposition phase of seed maturation with subsequent degeneration of the cisternae to a reticular form during the final seed maturation phase. No evidence of cisternal ER vesicles was found, instead our work suggests that such structures are artefacts of thin sectioning with the so-called vesicles representing the interconnection of cisternal and tubular ER. The results are discussed with reference to the transport of storage protein from its site of synthesis, the rough cisternal ER, to that of accumulation, the vacuolar protein bodies.Abbreviations ER endoplasmic reticulum - HVEM high voltage electron microscopy     

Retention of membrane proteins by the endoplasmic reticulum

We have used a monoclonal antibody specific for a hydrocarbon-induced cytochrome P450 to localize, by electron microscopy, the epitope- specific cytochrome P450. The cytochrome was found in the rough and smooth endoplasmic reticulum (ER) and the nuclear envelope of hepatocytes. Significant quantities of cytochrome P450 were not found in Golgi stacks. We also could not find any evidence of Golgi- associated processing of the Asn-linked oligosaccharide chains of two well-characterized ER membrane glycoprotein enzymes (glucosidase II and hexose-6-phosphate dehydrogenase), or of the oligosaccharides attached to the bulk of the glycoproteins of the ER membrane. We conclude that these ER membrane proteins are efficiently retained during a process of highly selective export from this organelle.     

Protein accumulation in developing endosperm of a high-protein line of Triticum dicoccoides

Abstract. Endosperm tissue from developing grains of a line of wheat ( Triticum dicoccoides ) which accumulates up to 30% protein in the mature grain, was examined by electron microscopy to establish the ontogeny of the storage protein bodies. Ultrastructural evidence suggests that storage proteins of wheat may be transported from their site of synthesis on the rough endoplasmic reticulum (ER) to protein bodies by two different routes within the endomembrane system. The first route, which probably functions throughout protein deposition, involves the transport of protein from the cisternal rough ER to the protein vacuoles via the Golgi apparatus. The second route, observed 20 d after anthesis, appears to lead directly from dilated regions of the rough ER to protein vacuoles, bypassing the dictyosomes. Phytin inclusions are found in protein vacuoles of starchy endosperm cells adjacent to the aleurone layer of developing grain.     

Ethylene-induced Fine Structure Alterations in Cotton and Sugarbeet Radicle Cells

Electron microscopic examination of cotton (Gossypium hirsutum L.) and sugarbeet (Beta vulgaris L.) radicles subjected to ethylene at 1, 10, 100 microliters per liter and a saturated ethylene atmosphere showed distinct ultrastructural differences compared to aerobic, control tissue and to anaerobic, nitrogen-treated radicles. Short term ethylene treatments of 1 hour induced the formation of cisternal stacks of rough endoplasmic reticulum in undifferentiated radicle cells. Similar profiles of rough endoplasmic reticulum were not observed in control radicles, but were found in nitrogen-treated tissues. Additionally, ribosomes in all ethylene-treated radicles were larger than ribosomes in control and nitrogen-treated cells. By ribosome density determinations it was found that the ethylene-treated ribosomes were swollen and less dense. The cell walls of the ethylene-treated tissue were also expanded and less dense than those of control tissue.     

Ultrastructural, histochemical and cytochemical characterization of intestinal epithelial cells in Aplysia depilans (Gastropoda, Opisthobranchia)

To improve the current knowledge about the digestive system in opisthobranchs, light and electron microscopy methods were used to characterize the epithelial cells in the mid‐intestine of Aplysia depilans. This epithelium is mainly formed by columnar cells intermingled with two types of secretory cells, named mucous cells and granular cells. Columnar cells bear microvilli on their apical surface and most of them are ciliated. Mitochondria, multivesicular bodies, lysosomes and lipid droplets are the main components of the cytoplasm in the region above the nucleus of these cells. Peroxisomes are mainly found in middle and basal regions, usually close to mitochondria. Mucous cells are filled with large secretory vesicles containing thin electron‐dense filaments surrounded by electron‐lucent material in which acidic mucopolysaccharides were detected. The basal region includes the nucleus, several Golgi stacks and many dilated rough endoplasmic reticulum cisternae containing tubular structures. The granular cells are characterized by very high amounts of flat rough endoplasmic reticulum cisternae and electron‐dense spherical secretory granules containing glycoproteins. Enteroendocrine cells containing small electron‐dense granules are occasionally present in the basal region of the epithelium. Intraepithelial nerve fibres are abundant and seem to establish contacts with secretory and enteroendocrine cells.     

Histochemical and ultrastructural changes in the haustorium of date (Phoenix dactylifera L.)

Summary During imbibition ofPhoenix dactylifera embryos, all cotyledon cells show the same changes: protein and lipid bodies degrade, smooth endoplasmic reticulum (ER) increases in amount, and dictyosomes appear. At germination, the distal portion of the cotyledon expands to form the haustorium. At this time, epithelial cells have a dense cytoplasm with many extremely small vacuoles. Many ribosomes are present along with ER, dictyosomes, and mitochondria. The parenchyma cells have large vacuoles and a small amount of peripheral cytoplasm. Between 2 and 6 weeks after germination, epithelial cells still retain the dense cytoplasm and many organelles appear: glyoxysomes, large lipid bodies, amyloplasts, large osmiophilic bodies, and abundant rough and smooth ER which appear to merge into the plasmalemma. A thin electron-transparent inner wall layer with many small internal projections is added to the cell walls. Starch grains appear first in the subsurface and internal parenchyma and subsequently in the epithelium. Lipid bodies, glyoxysomes, protein, and osmiophilic bodies occur in the epithelial and subepithelial cell layers but not in the internal parenchyma. At 8 weeks after germination, the cytoplasm becomes electron transparent, vacuolation occurs, lipid bodies and osmiophilic bodies degrade, and the endomembranes disassemble. After 10 weeks, the cells are empty. These data support the hypothesis that the major functions of the haustorium are absorption and storage.     

Oogenesis in Acerentomon gallicum Jonescu (Protura)

Summary Late stages of oogenesis in Acerentomon gallicum Jonescu have been studied by means of light and electron microscopy. Each of the two ovaries of this species consists of a single panoistic ovariole. Late previtellogenic and early vitellogenic oocytes are enclosed in an electron opaque layer, the so-called primary sheath. The precursors for this sheath are most likely synthesized by follicle cells. The yolk develops through autosynthesis, with free ribosomes, dictyosomes and lamellar bodies being involved in the process. Mature yolk spheres contain proteins and polysaccharides. Besides the organelles that take part in vitellogenesis, mitochondria and cisternal stacks of the rough endoplasmic reticulum occur in the ooplasm.This work was supported by Government Problem Grant ii-1.3.13     

Isolation and functional characterization of an inositol trisphosphate receptor from brain

We have identified an IP3 receptor protein in brain membranes through the binding of radiolabelled IP3. Autoradiographic studies localize the receptor to various areas of the brain with highest densities in Purkinje cells of the cerebellum. IP3 binding is inhibited by physiologic intracellular concentrations of calcium. Purification of the IP3 receptor to homogeneity reveals it to be comprised of four identical subunits of 260 kD each. Antisera to the purified receptor protein have been employed for immunohistochemical studies which, at the electron microscopic level, localize the IP3 receptor to a subdivision of the rough endoplasmic reticulum occurring in synaptic areas and in close association with the nuclear membrane. The IP3 receptor protein is selectively phosphorylated by cyclic AMP (cAMP) dependent protein kinase. This phosphorylation decreases 10-fold the potency of IP3 in releasing calcium from brain membranes.     

Ultrastructure of the chromatophores of Palaemon affinis Heilprin (crustacea: decapoda). The structural basis of pigment migration

The relationships between pigment granules and the prominent smooth endoplasmic reticulum in the chromatophores of the shrimp, Palaemon affinis Heilprin, were investigated by transmission electron microscopy. Different types of pigment granules within the chromatophores were found to exhibit a close structural continuity with the cisternal membranes. The membranes of membrane-bound pigment granules were seen to be continuous with those of the ER cisternae, while pigment granules lacking membranes appear to adhere to the external cisternal surfaces. The reticulum, which seems to form a network enmeshing the pigment granules, is proposed to be part of a continuum linking these granules with their translocating force.     

Three-dimensional ultrastructure of feeding tubes and interconnected endoplasmic reticulum in root-knot nematode-induced giant cells in rose balsam

We investigated the three-dimensional ultrastructure of feeding tubes and the surrounding region in giant cells induced in rose balsam (Impatiens balsamina L.) roots by the root-knot nematode Meloidogyne incognita, using osmium maceration coupled with field emission scanning electron microscopy (FE-SEM). In the roots of 35-day-old galled rose balsam plants, adult nematodes induced the formation of giant cells containing feeding tubes and numerous organelles, including tubular endoplasmic reticulum (ER), cisternal ER, and mitochondria. The feeding tubes were surrounded by fine tubular structures (20–50 nm in diameter), which were in turn surrounded by tubular ER (approximately 120 nm in diameter). The termini of the fine tubular structures appeared to be connected to the surface of the feeding tubes, suggesting that the fine tubular structures were continuous with narrow channels in the feeding tubes. The tubular ER arose from cisternal ER. Large bundles of tubular ER were present near the feeding tube, in the centers of the giant cells, and in the peripheral regions of the giant cells, such as cell wall ingrowths, while smaller bundles of tubular ER formed networks in the giant cells. These observations suggest that tubular ER functions as vascular bundles in giant cells, facilitating the transport of nutrients. We identified capsule-shaped structures (30 μm in diameter) in the giant cells that consisted of smooth, repeatedly branched ER tubules wrapped in several layers of cisternal ER. We propose that lipids and steroids are synthesized at the smooth branched ER and stored in these capsules until needed by the nematode.     

The ependymal secretion of the fetal and adult rat subcommissural organ. morphological aspects linked to the synthesis,storage and release of the secretory products

Different localizations of secretory material are noted in adult and fetal subcommissural organ (SCO) in light microscopy. At the electron microscope level, the secretory ependymocytes reveal frequent associations among mitochondria and ribosomes of the endoplasmic reticulum (ER). In the SCO ependymocytes of the adult rat, the relationship between mitochondria and ribosomes of the ER is observed in the subgolgian zone, the ER cisternal profiles are smooth except where they face the mitochondria. Here, a constant interval of 40-45 nm separates the ribosome-coated ER membrane from the external membrane of the mitochondria. This association evidences a functional cooperation between mitochondria and ER, at least in some phases of the synthesis of the organ's gliosecretory material. By contrast, in the fetus (17-21 fetal day), the synthetic apparatus displays an entirely granular ER. The secretory products are stored as flocculent material which fills the ER cisternae. In the apical zone of the ependymocytes, as the membrane of the dense secretory granules fuses with the apical plasmalemma, the granules release their contents into the ventricular cavity. A possible link between the releasing process and the coated vesicles is discussed.     

The ependymal secretion of the fetal and adult rat subcommissural organ. morphological aspects linked to the synthesis, storage and release of the secretory products

Different localizations of secretory material are noted in adult and fetal subcommissural organ (SCO) in light microscopy. At the electron microscope level, the secretory ependymocytes reveal frequent associations among mitochondria and ribosomes of the endoplasmic reticulum (ER). In the SCO ependymocytes of the adult rat, the relationship between mitochondria and ribosomes of the ER is observed in the subgolgian zone, the ER cisternal profiles are smooth except where they face the mitochondria. Here, a constant interval of 40-45 nm separates the ribosome-coated ER membrane from the external membrane of the mitochondria. This association evidences a functional cooperation between mitochondria and ER, at least in some phases of the synthesis of the organ's gliosecretory material. By contrast, in the fetus (17-21 fetal day), the synthetic apparatus displays an entirely granular ER. The secretory products are stored as flocculent material which fills the ER cisternae. In the apical zone of the ependymocytes, as the membrane of the dense secretory granules fuses with the apical plasmalemma, the granules release their contents into the ventricular cavity. A possible link between the releasing process and the coated vesicles is discussed.     

The Gp78 ubiquitin ligase: probing endoplasmic reticulum complexity

The endoplasmic reticulum (ER) has been classically divided, based on electron microscopy analysis, into parallel ribosome-studded rough ER sheets and a tubular smooth ER network. Recent studies have identified molecular constituents of the ER, the reticulons and DP1, that drive ER tubule formation and whose expression determines expression of ER sheets and tubules and thereby rough and smooth ER. However, segregation of the ER into only two domains remains simplistic and multiple functionally distinct ER domains necessarily exist. In this review, we will discuss the sub-organization of the ER in different domains focusing on the localization and role of the gp78 ubiquitin ligase in the mitochondria-associated smooth ER and on the evidence for a quality control ERAD domain.