Intercisternal substances of the Golgi apparatus

Summary In media of high ionic strength, neutral pH, low temperature, and varying ion composition, plant dictyosomes were disassembled into component cisternae. The effective ions included phosphotungstate and several halides. Constituents of the intercisternal or bonding regions were revealed through electron microscope analysis. These included intercisternal elements and electrontransparent plaques of undetermined composition. The intercisternal plaques were confined to the central platelike regions of cisternae and were distinct from the intercisternal fibers. The findings demonstrate that plant dictyosomes can be dissociated into component cisternae. With monovalent halide salts, the unstacking process was sufficiently mild to reveal constituents of the intercisternal region as well as yield intact single cisternae.     

Comparison of the ultrastructure of conventionally fixed and high pressure frozen/freeze substituted root tips ofNicotiana andArabidopsis

Summary To circumvent the limitations of chemical fixation (CF) and to gain more reliable structural information about higher plant tissues, we have cryofixed root tips ofNicotiana andArabidopsis by high pressure freezing (HPF). Whereas other freezing techniques preserve tissue to a relatively shallow depth, HPF in conjunction with freeze substitution (FS) resulted in excellent preservation of entire root tips. Compared to CF, in tissue prepared by HPF/FS: (1) the plasmalemma and all internal membranes were much smoother and often coated on the cytoplasmic side by a thin layer of stained material, (2) the plasmalemma was appressed to the cell wall, (3) organelle profiles were rounder, (4) the cytoplasmic, mitochondrial, and amyloplast matrices were denser, (5) vacuoles contained electron dense material, (6) microtubules appeared to be more numerous and straighter, with crossbridges observed between them, (7) cisternae of endoplasmic reticulum (ER) were wider and filled with material, (8) Golgi intercisternal elements were more clearly resolved and were observed between both Golgi vesicles and cisternae, and (9) larger vesicles were associated with Golgi stacks. This study demonstrates that HPF/FS can be used to successfully preserve the ultrastructure of relatively large plant tissues without the use of intracellular cryoprotectants.Abbreviations CF chemical fixation - ER endoplasmic reticulum - FF freeze fracture - FS freeze substitution - HPF high pressure freezing Dedicated to the memory of Professor Oswald Kiermayer     

Macromolecular differentiation of Golgi stacks in root tips ofArabidopsis andNicotiana seedlings as visualized in high pressure frozen and freeze-substituted samples

Summary The plant root tip represents a fascinating model system for studying changes in Golgi stack architecture associated with the developmental progression of meristematic cells to gravity sensing columella cells, and finally to young and old, polysaccharideslime secreting peripheral cells. To this end we have used high pressure freezing in conjunction with freeze-substitution techniques to follow developmental changes in the macromolecular organization of Golgi stacks in root tips ofArabidopsis andNicotiana. Due to the much improved structural preservation of all cells under investigation, our electron micrographs reveal both several novel structural features common to all Golgi stacks, as well as characteristic differences in morphology between Golgi stacks of different cell types.Common to all Golgi stacks are clear and discrete differences in staining patterns and width of cis, medial and trans cisternae. Cis cisternae have the widest lumina (30 nm) and are the least stained. Medial cisternae are narrower (20 nm) and filled with more darkly staining products. Most trans cisternae possess a completely collapsed lumen in their central domain, giving rise to a 4–6 nm wide dark line in cross-sectional views. Numerous vesicles associated with the cisternal margins carry a non-clathrin type of coat. A trans Golgi network with clathrin coated vesicles is associated with all Golgi stacks except those of old peripheral cells. It is easily distinguished from trans cisternae by its blebbing morphology and staining pattern. The zone of ribosome exclusion includes both the Golgi stack and the trans Golgi network.Intercisternal elements are located exclusively between trans cisternae of columella and peripheral cells, but not meristematic cells. In older peripheral cells only trans cisternae exhibit slime-related staining. Golgi stacks possessing intercisternal elements also contain parallel rows of freeze-fracture particles in their trans cisternal membranes. We propose that intercisternal elements serve as anchors of enzyme complexes involved in the synthesis of polysaccharide slime molecules to prevent the complexes from being dragged into the forming secretory vesicles by the very large slime molecules. In addition, we draw attention to the similarities in composition and apparent site of synthesis of xyloglucans and slime molecules.Dedicated to the memory of Professor Oswald Kiermayer     

The Symplechosome: a Unique Cell Organelle of some Basidiomycetes*

An unusual cell organelle of some basidiomycetes, the symplechosome, is described and illustrated in detail using Saccoblastia farinacea as an example. Symplechosomes are structurally similar, but not identical to “classical” dictyosomes of green plants and animals. As is typical for dictyosomes, each symplechosome consists of a stack of platelike cisternae. The central portions of the symplechosome-cisternae are flattened, and adjacent cisternae are separated in the mid-region by an intercisternal space of constant width. In contrast to dictyosomes, the intracisternal spaces are completely obliterated in the central area, and hexagonally arranged bars extend between adjacent cisternae. Identical bars often connect the symplechosomes with mitochondria. Symplechosomes are highly complex-structured organelles which differ significantly from the simple individual Golgi cisternae or “Golgi bodies” observed in asco- and basidiomycetes.     

Changes of dictyosome ultrastructure during the cell cycle in onion root meristematic cells. A morphometric and stereological study

Summary Dictyosome ultrastructure changes during the cell cycle in onion root meristematic cells. Changes were mainly related to cisternae and intercisternal spaces morphology. Taking each dictyosome to be composed of three different regions (CIS, medial, and TRANS), several quantitative changes were detected in some of the compartments. Many of the planimetric parameters evaluated showed higher values in medical cisternae, while CIS and TRANS remained nearly constant. We have also found an increased activity of dictyosomes, as indicated by increase in the volume fraction of vesicular attached structures. This reaches maximum values at ana-telophase in coincidence with the onset and progression of cytokinesis.Abbreviations A anaphase - Ac mean area occupied by cisternae per stack section - C CIS - CCS cell cycle stage - D_A mean total dictyosome area - I_SA mean area occupied by intercisternal spaces per stack section - M metaphase - N mean number of cisternae per stack - P prophase - S.E. standard error - T telophase - T TRANS - V_v volume density     

Zur Feinstruktur der schleimsezernierenden Drüsenhaare auf der Ochrea von Rumex und Rheum

Zusammenfassung Die Drüsenhaare auf der Ochrea in Knospen von Rumex und Rheum sezernieren einen Polysaccharid-Schleim, der keine sauren Komponenten enthält (vgl. Kling, 1961). Diese Drüsen wurden licht- und elektronen-mikroskopisch untersucht. Die Befunde sind: Die Ausscheidung des Schleimes erfolgt über den Golgi-Apparat. Die Dictyosomen sind relativ groß; die Sekretvesikel sind mit den Golgi-Zisternen durch die Tubuli der netzartigen Zisternenränder verbunden. In diesen Vesikeln wurde mit der Perjodsäure-Silbermethenamin-Methode (die der PAS-Reaktion vergleichbar ist) Kohlenhydrat nachgewiesen. Zwischen den Golgi-Zisternen werden mit Glutaraldehyd-OsO₄-Fixierungen Fibrillen dargestellt. Die Dictyosomen sind deutlich polar gebaut; die Polarität ist von ihrem Aktivitätszustand unabhängig. Der Schleim hebt häufig die Cuticula ab und tritt durch größere und kleinere Poren aus. Die Außenwände der Stielzellen und die Wände zwischen ihnen sind völlig cutinisiert; der Stiel bildet so eine Sperre im Apoplasten. In den Zellkernen treten gelegentlich spindelförmige Einschlüsse auf.

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Fine structure of mucus-secreting gland hairs on the ochrea of Rumex and Rheum

Summary The gland hairs on the ochrea in buds of Rumex and Rheum secrete a mucopolysaccharide which contains no acidic components (compare also Kling, 1961). These glands were studied by light and electron microscopy. The results are: The mucilage is secreted by the Golgi apparatus. The dictyosomes are relatively large; the secretory vesicles are connected with the Golgi cisternae by a network of tubules. It was proved by the silver methenamine method (comparable to the PAS reaction) that the Golgi vesicles contain a carbohydrate. The glutaraldehyde-OsO₄-fixation preserves intercisternal fibrils between the Golgi cisternae. The dictyosomes show a polarity in the development of the cisternae which is not dependent on the stage of activity of the Golgi apparatus. The extruded mucilage accumulates in spaces between the cell wall and the cuticule. It penetrates the cuticule through pores of different sizes. The outer walls of the cells of the stalk and the walls between these cells are completely cutinized; therefore the stalk functions as a barrier in the apoplast. — Sometimes the nuclei contain spindlelike inclusions.

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Herrn Prof. Dr. Dr. h. c. Dr. h. c. Richard Harder zum 80. Geburtstag gewidmet.     

Nonclathrin Vesicle Coats and Filament Networks in the Transition Zone and trans-Golgi Region of the Golgi Complex of Paramecium

Understanding vesicle trafficking to and through the Golgi stack has been greatly elucidated recently, but the question of what holds the endoplasmic reticulum (ER) and Golgi stack together in many cell types and an explanation of anterograde trafficking in the ER-Golgi transitional zone have not yet been adequately explained. We have studied these problems using both the thin sectioning and the quick-freeze deep-etch (QF-DE) technique on Paramecium cells harvested at different culture ages. Although the Golgi apparatus of Paramecium is made up of many sets of more reduced stacks of cisternae than those of many mammalian cells, the stacks in Paramecium always bear a close relationship to a transitional element of the ER from which non-clathrin-coated transition vesicles arise. In QF-DE replicas two networks of filaments are clearly shown; one is in this ER-Golgi transition zone and the other is on the trans side of the Golgi stack. The network associated with the trans-Golgi region links a number of vesicular elements. The network in the transition zone spans the distance between the ER and the cis-cisterna of the Golgi stack and has branches extending to the coats of the enmeshed nonclathrin-coated transition vesicles. These coats consist of a layer of 11-nm globular elements (the same size as coatomer complexes) which surround the 40-nm-diameter transition vesicles. We conclude that the filamentous network holds the ER and Golgi stack together and prevents the dispersal of the transition vesicles away from this zone. This network may also delineate and stabilize the transitional element within the ER and, finally, help organize anterograde transition vesicle trafficking in this ER-Golgi transition zone.     

Reevaluation of the effects of brefeldin A on plant cells using tobacco Bright Yellow 2 cells expressing Golgi-targeted green fluorescent protein and COPI antisera

Brefeldin A (BFA) causes a block in the secretory system of eukaryotic cells by inhibiting vesicle formation at the Golgi apparatus. Although this toxin has been used in many studies, its effects on plant cells are still shrouded in controversy. We have reinvestigated the early responses of plant cells to BFA with novel tools, namely, tobacco Bright Yellow 2 (BY-2) suspension-cultured cells expressing an in vivo green fluorescent protein-Golgi marker, electron microscopy of high-pressure frozen/freeze-substituted cells, and antisera against Atgamma-COP, a component of COPI coats, and AtArf1, the GTPase necessary for COPI coat assembly. The first effect of 10 microg/mL BFA on BY-2 cells was to induce in <5 min the complete loss of vesicle-forming Atgamma-COP from Golgi cisternae. During the subsequent 15 to 20 min, this block in Golgi-based vesicle formation led to a series of sequential changes in Golgi architecture, the loss of distinct Golgi stacks, and the formation of an endoplasmic reticulum (ER)-Golgi hybrid compartment with stacked domains. These secondary effects appear to depend in part on stabilizing intercisternal filaments and include the continued maturation of cis- and medial cisternae into trans-Golgi cisternae, as predicted by the cisternal progression model, the shedding of trans-Golgi network cisternae, the fusion of individual Golgi cisternae with the ER, and the formation of large ER-Golgi hybrid stacks. Prolonged exposure of the BY-2 cells to BFA led to the transformation of the ER-Golgi hybrid compartment into a sponge-like structure that does not resemble normal ER. Thus, although the initial effects of BFA on plant cells are the same as those described for mammalian cells, the secondary and tertiary effects have drastically different morphological manifestations. These results indicate that, despite a number of similarities in the trafficking machinery with other eukaryotes, there are fundamental differences in the functional architecture and properties of the plant Golgi apparatus that are the cause for the unique responses of the plant secretory pathway to BFA.     

Primary fixation in osmium-potassium ferrocyanide: the staining of glycogen, glycoproteins, elastin, an intranuclear reticular structure, and intercisternal trabeculae

Primary fixation in an osmium-potassium ferrocyanide (K4Fe(CN)6) mixture combines selective fixation, staining, and extraction of various cellular components; membranes, glycogen, glycoproteins, and elastin are preserved and stained. An intranuclear reticular structure that is composed of 3-6 nm fibers and permeates the entire nucleus, except for the nuclear pores, is demonstrated by electron microscopic examination of tissues prepared in an osmium-potassium ferrocyanide fixative. Condensations of the reticulum parallel the distribution of heterochromatin in interphase nuclei. This preparative procedure also reveals a network of trabeculae that are associated with the cisternae of rough endoplasmic reticulum and connect the parallel cisternae in hepatocytes, plasmacytes, neurons, and pancreatic ancinar cells. The intercisternal trabeculae are associated with both free and bound ribosomes.     

Polysomes and intracisternal accumulations in enucleate sieve elements of rice (Oryza sativa L.)

Polysomes in sieve elements of rice (Oryza sativa L.) were studied with the electron microscope. The polysomes were found on the rough endoplasmic reticulum (ER) present in immature sieve elements and also on the cisternae of aggregated ER in the parietal layer of mature, enucleate sieve elements. In the immature sieve elements the ER cisternae existed as narrow profiles while in the mature sieve elements the ER cisternae were considerably dilated and contained a fibrillar material and, occasionally, electron-opaque inclusions. In addition to the aggregated ER, single profiles of ER were found applied to the lateral walls and also the sieve plates. These cisternae also bore ribosomes and were separated from the plasmalemma by a narrow, dense space. In the mature sieve elements much of the surface of the ER membranes was covered with polysomes. The dimensions of the polysomes are described and the possibility that they contribute to the formation of the fibrillar material in the intracisternal space is discussed.Abbreviations ER endoplasmic reticulum     

The Golgi apparatus ofPhytophthora cinnamomi makes three types of secretory or storage vesicles concurrently

Summary The formation of three types of vesicles in the oomycetePhytophthora cinnamomi was investigated using ultrastructural and immunocytochemical techniques. All three vesicles are synthesised at the same time; one type serves a storage role; the others undergo regulated secretion. A monoclonal antibody Lpv-1 that is specific for glycoproteins contained in the storage vesicles labelled the endoplasmic reticulum (ER), elements in the transition region between ER and Golgi stack, and cis, medial and trans Golgi cisternae. Cpa2, a monoclonal antibody specific for glycoproteins contained within secretory dorsal vesicles labelled the transition region, cis cisternae and a trans-Golgi network. Vesicles possessing a structure characteristic of mature secretory ventral vesicles were observed in close association with the trans face of Golgi stacks. The results suggest that all three vesicles are formed by the Golgi apparatus. Double immunogold labelling with Lpv-1 and Cpa-2 showed that these two sets of glycoproteins occurred within the same Golgi cisternae, indicating that both products pass through and are sorted concurrently within a single Golgi stack.     

Secretory traffic triggers the formation of tubular continuities across Golgi sub-compartments

The organization of secretory traffic remains unclear, mainly because of the complex structure and dynamics of the secretory pathway. We have thus studied a simplified system, a single synchronized traffic wave crossing an individual Golgi stack, using electron tomography. Endoplasmic-reticulum-to-Golgi carriers join the stack by fusing with cis cisternae and induce the formation of intercisternal tubules, through which they redistribute their contents throughout the stack. These tubules seem to be pervious to Golgi enzymes, whereas Golgi vesicles are depleted of both enzymes and cargo. Cargo then traverses the stack without leaving the cisternal lumen. When cargo exits the stack, intercisternal connections disappear. These findings provide a new view of secretory traffic that includes dynamic intercompartment continuities as key players.     

Golgi structure in three dimensions: functional insights from the normal rat kidney cell

Three-dimensional reconstructions of portions of the Golgi complex from cryofixed, freeze-substituted normal rat kidney cells have been made by dual-axis, high-voltage EM tomography at approximately 7-nm resolution. The reconstruction shown here ( approximately 1 x 1 x 4 microm3) contains two stacks of seven cisternae separated by a noncompact region across which bridges connect some cisternae at equivalent levels, but none at nonequivalent levels. The rest of the noncompact region is filled with both vesicles and polymorphic membranous elements. All cisternae are fenestrated and display coated buds. They all have about the same surface area, but they differ in volume by as much as 50%. The trans-most cisterna produces exclusively clathrin-coated buds, whereas the others display only nonclathrin coated buds. This finding challenges traditional views of where sorting occurs within the Golgi complex. Tubules with budding profiles extend from the margins of both cis and trans cisternae. They pass beyond neighboring cisternae, suggesting that these tubules contribute to traffic to and/or from the Golgi. Vesicle-filled "wells" open to both the cis and lateral sides of the stacks. The stacks of cisternae are positioned between two types of ER, cis and trans. The cis ER lies adjacent to the ER-Golgi intermediate compartment, which consists of discrete polymorphic membranous elements layered in front of the cis-most Golgi cisterna. The extensive trans ER forms close contacts with the two trans-most cisternae; this apposition may permit direct transfer of lipids between ER and Golgi membranes. Within 0.2 microm of the cisternae studied, there are 394 vesicles (8 clathrin coated, 190 nonclathrin coated, and 196 noncoated), indicating considerable vesicular traffic in this Golgi region. Our data place structural constraints on models of trafficking to, through, and from the Golgi complex.     

THE RESPONSE OF VENTRAL HORN NEURONS TO AXONAL TRANSECTION

The morphological changes induced in the frog ventral horn neurons by axonal transection have been studied with the electron microscope. During the first 2 wk after axotomy the neuronal nucleus becomes more translucent and the nucleolus becomes enlarged and less compact. The cisternae of the granular endoplasmic reticulum vesiculate and ribosomes dissociate from membranes. Free ribosomes and polysomes are dispersed in the cytoplasmic matrix. Neurofilaments and neurotubules are increased in number. These structures appear to be important in the regeneration of the axon. It is proposed that neurotubules, neurofilaments, and axoplasmic matrix are synthesized by the free polyribosomes in the chromatolytic neuron. By the fourth postoperative week, the neurons show evidence of recovery. The cytoplasm is filled with profiles of granular endoplasmic reticulum and many intercisternal polysomes. The substances being manufactured by the newly formed granular endoplasmic reticulum are not clearly defined, but probably include elements essential to electrical and chemical conduction of impulses. The significance of these observations in respect to recent studies of axoplasmic flow is discussed.     

STRUCTURE OF ISOLATED PLANT GOLGI APPARATUS REVEALED BY NEGATIVE STAINING

Sucrose-gradient-purified dictyosomes of plant Golgi apparatus appear, after glutaraldehyde stabilization, as stacks of highly fenestrate and tubate cisternae when negatively stained with phosphotungstic acid, shadowed with heavy metal, or OsO₄-stained in thin section. The tubular proliferations (diameter 200 to 400 A) extend for several microns from the central region and are united at intervals into an anastomosing network. Associated with the tubules are two kinds of vesicles which are distinguishable on the basis of texture, size, shape, and staining characteristics. One vesicle type is rough-surfaced, nearly spherical, and of uniform dimensions (diameter approximately 600 A). Metal shadowing shows that these vesicles remain spherical after drying. The other vesicle type is smooth-surfaced and varies in both size and shape. Intercisternal elements are revealed, by negative staining, on the surface of internal cisternae after fragmentation of the dictyosome. The progressive differentiation of cisternae from the forming face to the maturing face is observed in thin sections of these isolated preparations. The morphological characteristics observed in negatively stained dictyosomes indicate regions of functional specialization within the dictyosome cisternae and reveal a dictyosome structure more extensive than that envisioned from sections.     

Evolution of Tandemly Repeated Sequences: What Happens at the End of an Array?

Tandemly repeated sequences are a major component of the eukaryotic genome. Although the general characteristics of tandem repeats have been well documented, the processes involved in their origin and maintenance remain unknown. In this study, a region on the paternal sex ratio (PSR) chromosome was analyzed to investigate the mechanisms of tandem repeat evolution. The region contains a junction between a tandem array of PSR2 repeats and a copy of the retrotransposon NATE, with other dispersed repeats (putative mobile elements) on the other side of the element. Little similarity was detected between the sequence of PSR2 and the region of NATE flanking the array, indicating that the PSR2 repeat did not originate from the underlying NATE sequence. However, a short region of sequence similarity (11/15 bp) and an inverted region of sequence identity (8 bp) are present on either side of the junction. These short sequences may have facilitated nonhomologous recombination between NATE and PSR2, resulting in the formation of the junction. Adjacent to the junction, the three most terminal repeats in the PSR2 array exhibited a higher sequence divergence relative to internal repeats, which is consistent with a theoretical prediction of the unequal exchange model for tandem repeat evolution. Other NATE insertion sites were characterized which show proximity to both tandem repeats and complex DNAs containing additional dispersed repeats. An ``accretion model' is proposed to account for this association by the accumulation of mobile elements at the ends of tandem arrays and into ``islands' within arrays. Mobile elements inserting into arrays will tend to migrate into islands and to array ends, due to the turnover in the number of intervening repeats. Received: 18 August 1997 / Accepted: 18 September 1998     

Effect of colchicine on the Golgi complex of rat pancreatic acinar cells

Colchicine administered to adult rats at a dosage of 0.5 mg/100 g of body weight effected a disorganization of the Golgi apparatus in pancreatic acinar cells. The results obtained after various periods of treatment (10 min to 6 h) showed (a) changes in all components of the Golgi complex, and (b) occurrence of large vacuoles that predominated in cytoplasmic areas outside the Golgi region. The alterations in Golgi stacks concerned elements of the proximal and distal side: (a) accumulation of transport vesicles, (b) formation of small, polymorphic secretion granules, and (c) alterations in the cytochemical localization of enzymes and reaction product after osmification. Transport vesicles accumulated and accompanied short, dilated cisternae, which lack mostly the reaction products of thiamine pyrophosphatase, inosine diphosphatase, and acid phosphatase, and osmium deposits after prolonged osmification. After 4 to 6 h of treatment, accumulated transport vesicles occupied extensive cellular areas; stacked cisternae were not demonstrable in these regions. The changes on the distal Golgi side included GERL elements: condensing vacuoles were diminished; they were substituted by small, polymorphic zymogen granules, which appeared to be formed by distal Golgi cisternae and by rigid lamellae. Unusually extended coated regions covered condensing vacuoles, rigid lamellae, and polymorphic secretion granules. A cytochemical distinction between Golgi components and GERL was possible neither in controls nor after colchicine treatment. The cytochemical alterations in Golgi components were demonstrable 20-30 min following administration of colchicine; at 45 min, initial morphological changes--augmentation of transport vesicles and formation of polymorphic zymogen granules--became apparent. 20 min after administration of colchicine, conspicuous groups of large vacuoles occurred. They were located mostly in distinct fields between cisternae of the endoplasmic reticulum, and were accompanied by small osmium--reactive vesicles. Stacked cisternae were not demonstrable in these fields. Vacuoles and vesicles were devoid of reaction products of thiamine pyrophosphatase, inosine diphosphatase, and acid phosphatase. The results provide evidence that formation of stacked Golgi cisternae is impaired after colchicine treatment. The colchicine--induced disintegration of the Golgi complex suggests a regulatory function of microtubules in the organization of the Golgi apparatus.     

Group IV Phospholipase A2α Controls the Formation of Inter-Cisternal Continuities Involved in Intra-Golgi Transport

The organization of intra-Golgi trafficking and the nature of the transport intermediates involved (e.g., vesicles, tubules, or tubular continuities) remain incompletely understood. It was recently shown that successive cisternae in the Golgi stack are interconnected by membrane tubules that form during the arrival of transport carriers from the endoplasmic reticulum. Here, we examine the mechanisms of generation and the function of these tubules. In principle, tubule formation might depend on several protein- and/or lipid-based mechanisms. Among the latter, we have studied the phospholipase A₂ (PLA₂)-mediated generation of wedge-shaped lysolipids, with the resulting local positive membrane curvature. We show that the arrival of cargo at the Golgi complex induces the recruitment of Group IVA Ca²⁺-dependent, cytosolic PLA₂ (cPLA₂α) onto the Golgi complex itself, and that this cPLA₂α is required for the formation of the traffic-dependent intercisternal tubules and for intra-Golgi transport. In contrast, silencing of cPLA₂α has no inhibitory effects on peri-Golgi vesicles. These findings identify cPLA₂α as the first component of the machinery that is responsible for the formation of intercisternal tubular continuities and support a role for these continuities in transport through the Golgi complex.     

High -resolution scanning electron-microscopic studies on the three-dimensional structure of mitochondria and sarcoplasmic reticulum in the different twitch muscle fibers of the frog

Summary The three-dimensional structure of the mitochondria and sarcoplasmic reticulum (SR) in the three types of twitch fibers, i.e., the red, white and intermediate skeletal muscle fibers, of the vastus lateralis muscle of the Japanese meadow frog (Rana nigromaculata nigromaculata Hallowell) was examined by high resolution scanning electron microscopy, after removal of the cytoplasmic matrices.The small red fibers have numerous mitochondrial columns of large diameter, while the large white fibers have a small number of mitochondrial columns of small diameter. In the medium-size intermediate fibers, the number and diameter of the mitochondrial columns are intermediate between those of the red and white fibers.In all three types of fibers, the terminal cisternae and transverse tubules form triads at the level of each Z-line. The thick terminal cisternae continue into much thinner flat intermediate cisternae, through a transitional part where a row of tiny indentations can be observed. Numerous slender longitudinal tubules originating from the intermediate cisternae, extend longitudinally or obliquely and form elongated oval networks of various sizes in front of the A-band, then fuse to form the H-band collar (fenestrated collar) around the myofibrils. On the surface of the H-band collar, small fenestrations as well as tiny hollows are seen. The three-dimensional structure of SR is basically the same in all three muscle fiber-types. However, the SR is sparse on the surface of mitochondria, so the mitochondria-rich red fiber has a smaller total volume of SR than the mitochondria-poor white fiber. The volume of SR of the intermediate fiber is intermediate between other the two.     

Immunolocalization of the oligosaccharide trimming enzyme glucosidase II

We used immunoelectron microscopy to localize glucosidase II in pig hepatocytes. The enzyme trims the two inner alpha 1,3-linked glucoses from N-linked oligosaccharide precursor chains of glycoproteins. Immunoreactive enzyme was concentrated in rough (RER) and smooth (SER) endoplasmic reticulum but not detectable in Golgi apparatus cisternae. Transitional elements of RER and smooth membraned structures close to Golgi apparatus cisternae contained labeling for glucosidase II. Specific labeling was also found in autophagosomes. These results indicate strongly that glucosidase II acts on glycoproteins before their transport to, and processing in Golgi apparatus cisternae, and suggest that an important transitional region for glucosidase II exists between RER and Golgi apparatus cisternae. Degradation in autophagolysosomes could form a normal catabolic pathway for glucosidase II.