Backscattered electron image of osmium‐impregnated/macerated tissues as a novel technique for identifying the cis‐face of the Golgi apparatus by high‐resolution scanning electron microscopy

The osmium maceration method with scanning electron microscopy (SEM) enabled to demonstrate directly the three‐dimensional (3D) structure of membranous cell organelles. However, the polarity of the Golgi apparatus (that is, the cis–trans axis) can hardly be determined by SEM alone, because there is no appropriate immunocytochemical method for specific labelling of its cis‐ or trans‐faces. In the present study, we used the osmium impregnation method, which forms deposits of reduced osmium exclusively in the cis‐Golgi elements, for preparation of specimens for SEM. The newly developed procedure combining osmium impregnation with subsequent osmium maceration specifically visualised the cis‐elements of the Golgi apparatus, with osmium deposits that were clearly detected by backscattered electron‐mode SEM. Prolonged osmication by osmium impregnation (2% OsO₄ solution at 40°C for 40 h) and osmium maceration (0.1% OsO₄ solution at 20°C for 24 h) did not significantly impair the 3D ultrastructure of the membranous cell organelles, including the Golgi apparatus. This novel preparation method enabled us to determine the polarity of the Golgi apparatus with enough information about the surrounding 3D ultrastructure by SEM, and will contribute to our understanding of the global organisation of the entire Golgi apparatus in various differentiated cells.     

Perspectives on golgi apparatus form and function

In 1898, Camillio Golgi reported a new cellular constituent with the form of an extensive intracellular network (the apparato reticolare interno), which now bears his name. However, the history of Golgi's apparatus is replete with controversy regarding its reality, what components of the cell should be included under its aegis, and what terminology should be used when referring to it. Electron microscopy has resolved many of these controversies and it is appropriate that this volume emphasize that aspect of Golgi apparatus discovery. The principal structural component of the Golgi apparatus is the stack of cisternae, or dictyosome. As determined both biochemically and at the level of electron microscopy, the dictyosome is a highly ordered and polarized structure. The maintenance of order within the stack is thought to result from either intercisternal bonding constituents, or filamentous structures (or both) that bridge the space between adjacent cisternae. Mechanisms proposed for movement of membrane and product into and out of the dictyosome (i.e., the Golgi apparatus stack) include a serial mode which functions exclusively by the formation, displacement, and loss of cisternae from the stack, and a parallel mode which functions exclusively by the movement of membrane, product, or precursor molecules directly into the peripheral edges of the cisternae. In the parallel mode, all cisternae can be accessed either singly or simultaneously, at least in theory, at any position within the stack. It is probable that both the serial and the parallel modes function concomitantly and need not be mutually exclusive. Finally, the peripheral tubules of the cisternae represent a major membranous constituent of the cell with potentially unique functions. These tubules interconnect cisternae of adjacent stacks and may represent the major site of receptors for the shuttle (i.e., parallel) type of transfer among cisternae. Peripheral tubules as extensions of the cisternal lumina into the cytoplasm presumably have other functions, but these, like the tubules themselves, have only rarely been accommodated into functional models of Golgi apparatus dynamics in secretion or membrane flow.     

Plant Golgi ultrastructure

The plant Golgi apparatus (sensu lato: Golgi stack + Trans Golgi Network, TGN) is a highly polar and mobile key organelle lying at the junction of the secretory and endocytic pathways. Unlike its counterpart in animal cells it does not disassemble during mitosis. It modifies glycoproteins sent to it from the endoplasmic reticulum (ER), it recycles ER resident proteins, it sorts proteins destined for the vacuole from secretory proteins, it receives proteins internalised from the plasma membrane and either recycles them to the plasma membrane or retargets them to the vacuole for degradation. In functional terms the Golgi apparatus can be likened to a car factory, with incoming (COPII traffic) and returning (COPI traffic) railway lines at the entry gate, and a distribution centre (the TGN) at the exit gate of the assembly hall. In the assembly hall we have a conveyor belt system where the incoming car parts are initially assembled (in the cis-area) then gradually modified into different models (processing of secretory cargo) as the cars pass along the production line (cisternal maturation). After being released the trans-area, the cars (secretory cargos) are moved out of the assembly hall and passed on to the distribution centre (TGN), where the various models are placed onto different trains (cargo sorting into carrier vesicles) for transport to the car dealers. Cars with motor problems are returned to the factory for repairs (endocytosis to the TGN). This simple analogy also incorporates features of quality control at the COPII entry gate with defective parts being returned to the manufacturing center (the ER) via the COPI trains (vesicles). In recent years, numerous studies have contributed to our knowledge on Golgi function and structure in both animals, yeast and plants. This review, rather than giving a balanced account of the structure as well as of the function of the Golgi apparatus has purposely a marked slant towards plant Golgi ultrastructure integrating findings from the mammalian/animal field.     

ERES (ER exit sites) and the "secretory unit concept"

The higher plant Golgi apparatus consists of hundreds of individual Golgi stacks which move along the cortical ER, propelled by the actomysin system. Anterograde and retrograde transport between the endoplasmic reticulum (ER) and the plant Golgi occurs over a narrow interface (around 500 nm) and is generally considered to be mediated by COP-coated vesicles. Previously, ER exit sites (ERES) have been identified on the basis of to localization of transiently expressed COPII-coat proteins. As a consequence it has been held that ERES in higher plants are intimately associated with Golgi stacks, and that both move together as an integrated structure: the "secretory unit". Using a new COPII marker, as well as YFP-SEC24 (a bona fide COPII coat protein), we have made observations on tobacco leaf epidermis at high resolution in the CLSM. Our data clearly shows that COPII fluorescence is associated with the Golgi stacks rather than the surface of the ER and probably represents the temporary accumulation of COPII vesicles in the Golgi matrix prior to fusion with the cis-Golgi cisternae. We have calculated the numbers of COPII vesicles which would be required to provide a typical Golgi-associated COPII-fluorescent signal as being much less than 20. We have discussed the consequences of this and question the continued usage of the term "secretory unit".     

Three-dimensional reconstruction of a plant dictyosome from series of ultrathin sections using computer image processing

A single dictyosome from an actively secreting ovary gland cell of Aptenia cordifolia has been reconstructed in 3-D from a series of twenty-nine electron micrographs by computer image processing. The reconstruction is presented under different viewing angles in the form of shaded perspective displays. From these displays the entire dictyosome, surrounded by numerous vesicles, appears to be more a spherical than a flat body. The plate-like region of the dictyósome is demonstrated when only a portion of the electron micrographs is used for the image processing, leading to ‘cut-off’ displays. Since some upper planes were removed, such ‘cut-off’ displays revealed both tubular connections between cisternae of the dictyosome and the neighbouring endoplasmic reticulum as well as tubular continuities between adjacent Golgi cisternae within the same stack. Possible consequences of both types of interconnections on transport and processing of proteins and glycoproteins are discussed.     

Ultrastructural changes of the olfactory bulb in manganesetreated mice

The effect of manganese toxicity on the ultrastructure of the olfactory bulb was evaluated. Male albino mice were injected intraperitoneally with MnCl₂ (5 mg/Kg/day) five days per week during nine weeks. The control group received NaCl (0.9%). The olfactory bulbs of five mice from each group were processed for transmission electron microscopy after 2, 4, 6 and 9 weeks of manganese treatment. On week 2, some disorganization of the myelin sheaths was observed. After 4 weeks, degenerated neurons with dilated cisternae of rough endoplasmic reticulum and swollen mitochondria appeared. A certain degree of gliosis with a predominance of astrocytes with swollen mitochondria, disorganization of the endomembrane system, dilation of the perinuclear cisternae and irregularly shaped nuclei with abnormal chromatin distribution were observed after 6 weeks. Some glial cells showed disorganization of the Golgi apparatus. On week 9, an increase in the number of astrocytes, whose mitochondrial cristae were partially or totally erased, and a dilation of the rough endoplasmic reticulum were found. Neurons appear degenerated, with swollen mitochondria and a vacuolated, electron dense cytoplasm. These changes seem to indicate that the olfactory bulb is sensitive to the toxic effects of manganese.     

Comparisons of golgi structure and dynamics in plant and animal cells

The Golgi apparatus of both higher plant and animal cells sorts and packages macromolecules which are in transit to and from the cell surface and to the lysosome (vacuole). It is also the site of oligosaccharide and polysaccharide synthesis and modification. The underlying similarity of function of plant and animal Golgi is reflected in similar morphological features, such as cisternal stacking. There are, however, several fundamental differences between the Golgi of plant and animal cells, reflecting, in large part, the fact that the extracellular matrices and lysosomal systems differ between these kingdoms. These include (1) the form and replication of the Golgi during cell division; (2) the disposition of the Golgi in the interphase cell; (3) the nature of “anchoring” the Golgi in the cytoplasm; (4) the genesis, extent, and nature of membranes at the trans side of the stack; (5) targeting signals to the lysosome (vacuole); and (6) physiological regulation of secretion events (constitutive vs. regulated secretion). The degree of participation of the Golgi in endocytosis and membrane recycling is becoming clear for animal cells, but has yet to be explored in detail for plant cells.     

Functional organisation of the endomembrane network in the digestive gland of the Venus flytrap: revisiting an old story with a new microscopy toolbox

Up-to-date imaging approaches were used to address the spatiotemporal organisation of the endomembrane system in secretory cells of Dionaea muscipula. Different ‘slice and view’ methodologies were performed on resin-embedded samples to finally achieve a 3D reconstruction of the cell architecture, using ultrastructural tomography, array tomography, serial block face-scanning electron microscopy (SBF-SEM), correlation, and volume rendering at the light microscopy level. Observations of cryo-fixed samples by high-pressure freezing revealed changes of the endomembrane system that occur after trap activation and prey digestion. They provide evidence for an original strategy that adapts the secretory machinery to a specific and unique case of stimulated exocytosis in plant cells. A first secretion peak is part of a rapid response to deliver digestive fluids to the cell surface, which delivers the needed stock of digestive materials ‘on site’. The second peak of activity could then be associated with the reconstruction of the Golgi apparatus (GA), endoplasmic reticulum (ER) and vacuolar machinery, in order to prepare for a subsequent round of prey capture. Tubular continuum between ER and Golgi stacks observed on ZIO-impregnated tissues may correspond to an efficient transfer mechanism for lipids and/or proteins, especially for use in rapidly resetting the molecular GA machinery. The occurrence of one vacuolar continuum may permit continuous adjustment of cell homeostasy. The subcellular features of the secretory cells of Dionaea muscipula outline key innovations in the organisation of plant cell compartmentalisation that are used to cope with specific cell needs such as the full use of the GA as a protein factory, and the ability to create protein reservoirs in the periplasmic space. Shape-derived forces of the pleiomorphic vacuole may act as signals to accompany the sorting and entering flows of the cell.     

Extended depth from focus reconstruction using NIH ImageJ plugins: Quality and resolution of elevation maps

In this work, NIH ImageJ plugins for extended depth‐from‐focus reconstructions (EDFR) based on spatial domain operations were compared and tested for usage optimization. Also, some preprocessing solutions for light microscopy image stacks were evaluated, suggesting a general routine for the ImageJ user to get reliable elevation maps from grayscale image stacks. Two reflected light microscope image stacks were used to test the EDFR plugins: one bright‐field image stack for the fracture of carbon‐epoxy composite and its darkfield corresponding stack at same (x,y,z) spatial coordinates. Image quality analysis consisted of the comparison of signal‐to‐noise ratio and resolution parameters with the consistence of elevation maps, based on roughness and fractal measurements. Darkfield illumination contributed to enhance the homogeneity of images in stack and resulting height maps, reducing the influence of digital image processing choices on the dispersion of topographic measurements. The subtract background filter, as a preprocessing tool, contributed to produce sharper focused images. In general, the increasing of kernel size for EDFR spatial domain‐based solutions will produce smooth height maps. Finally, this work has the main objective to establish suitable guidelines to generate elevation maps by light microscopy. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Ultrastructure and distribution of superficial neuromasts of blind cavefish,Phreatichthys andruzzii,juveniles

Transmission and scanning electron microscopy (TEM, SEM) were used to study the ultrastructure of superficial neuromasts in 15 six-month old blind cavefish juveniles, Phreatichthys andruzzii (Cyprinidae). In five specimens examined with SEM, the number of superficial neuromasts over the fish body (480–538) was recorded. They were localized mainly on the head (362–410), including the dorsal surface, the mentomandibular region, and laterally from the mouth to the posterior edge of the operculum. Neuromasts were also present laterally on the trunk and near the caudal fin (116–140). A significantly higher number of neuromasts were present on the head compared to the trunk (t-test, P < 0.05). Superficial neuromasts of the head and those along the trunk were similar in ultrastructure. Each neuromast comprised sensory hair cells surrounded by nonsensory support cells (mantle cells and supporting basal cells) with the whole covered by a cupula. Each hair cell was pear-shaped, 15–21 μm high and 4–6 μm in diameter, with a single long kinocilium and several short stereocilia. Most support cells were elongated, with nuclei occupying a large portion of the cytoplasm. In the margin of the neuromast, mantle cells were particularly narrow. Both types of support cells had well-developed Golgi apparatus and rough endoplasmic reticulum. The number of hair cells and nonsensory support cells of the anterior lateral line (head) did not differ significantly from those of the posterior lateral line (trunk) (t-test, P > 0.05). Microsc. Res. Tech. 2009. © 2009 Wiley-Liss, Inc.     

Tales of tethers and tentacles: golgins in plants

As plant Golgi bodies move through the cell along the actin cytoskeleton, they face the need to maintain their polarized stack structure whilst receiving, processing and distributing protein cargo destined for secretion. Structural proteins, or Golgi matrix proteins, help to hold cisternae together and tethering factors direct cargo carriers to the correct target membranes. This review focuses on golgins, a protein family containing long coiled-coil regions, summarizes their known functions in animal cells and highlights recent findings about plant golgins and their putative roles in the plant secretory pathway.     

Scheduling algorithms for a two-machine flexible manufacturing system

The flexible manufacturing system (FMS) considered in this paper is composed of two CNC machines working in series—a punching machine and a bending machine connected through rollers acting as a buffer system of finite capacity. The main difference between the present problem and the standard two-machine flow shop problem with finite intermediate capacity is precisely the buffer system, which in our problem consists of two stacks of parts supported by rollers: the first stack contains the output of the punching machine, while the second stack contains the input for the bending machine. When the second stack is empty, the first stack may be moved over. Furthermore, the capacity of each stack depends on the particular part type being processed.The FMS can manufacture a wide range of parts of different types. Processing times on the two machines are usually different so that an unbalance results in their total workload. Furthermore, whenever there is a change of the part type in production, the machines must be properly reset—that is, some tools need to be changed or repositioned.A second important difference between the present problem and the usual two-machine flow shop problem is the objective. Given a list ofp part types to be produced in known quantities, the problem considered here is how to sequence or alternate the production of the required part types so as to achieve various hierarchical targets: minimize the makespan (the total time needed to complete production) and, for instance, compress the idle periods of the machine with less workload into a few long enough intervals that could be utilized for maintenance or other reasons.Although Johnson's rule is optimal in some particular cases, the problem addressed in the paper isNP-hard in general: heuristic procedures are therefore provided.     

Fine structure of the male accessory glands of Triatoma rubrofasciata (De Geer, 1773) (Hemiptera, Triatominae)

Male of Triatoma rubrofasciata has four elongated sac-like reproductive mesodermic accessory glands, lined by an inner single layer of secretory cells, with basal plasma membrane infolds and short apical microvilli, and externally enveloped by a thin visceral muscle layer. The secretory cells have a well-developed rough endoplasmic reticulum, Golgi complex, mitochondria, and secretory granules. In one day old adult the gland cells are poorly developed, presenting small, electron-transparent secretory granules scattered among the rough endoplasmatic reticulum, whereas in three days old adult these cells have the cisternae of the rough endoplasmatic reticulum varing size degree, filled with granular electrondense content. In five days old males the secretory granules increase in diameter, being released to the gland lumen. Therefore, there is an increase of the secretory activity according to male maturation.     

Traffic through the golgi apparatus as studied by radioautography

The ability to radiolabel biological molecules, in conjunction with radioautographic or cell fractionation techniques, has brought about a revolution in our knowledge of dynamic cellular processes. This has been particularly true since the 1940's, when isotopes such as ³⁵S and ¹⁴C became available, since these isotopes could be incorporated into a great variety of biologically important compounds. The first dynamic evidence for Golgi apparatus involvement in biosynthesis came from light microscope radioautographic studies by Jennings and Florey in the 1950's, in which label was localized to the supranuclear Golgi region of goblet cells soon after injection of ³⁵S-sulfate. When the low energy isotope tritium became available, and when radioautography could be extended to the electron microscope level, a great improvement in spatial resolution was achieved. Studies using ³H-amino acids revealed that proteins were synthesized in the rough endoplasmic reticulum, migrated to the Golgi apparatus, and thence to secretion granules, lysosomes, or the plasma membrane. The work of Neutra and Leblond in the 1960's using ³H-glucose provided dramatic evidence that the Golgi apparatus was involved in glycosylation. Work with ³H-mannose (a core sugar in N-linked side chains), showed that this sugar was incorporated into glycoproteins in the rough endoplasmic reticulum, providing the first radioautographic evidence that glycosylation of proteins did not occur solely in the Golgi apparatus. Studies with the tritiated precursors of fucose, galactose, and sialic acid, on the other hand, showed that these terminal sugars are mainly added in the Golgi apparatus. With its limited spatial resolution, radioautography cannot discriminate between label in adjacent Golgi saccules. Nonetheless, in some cell types, radioautographic evidence (along with cytochemical and cell fractionation data) has indicated that the Golgi is subcompartmentalized in terms of glycosylation, with galactose and sialic acid being added to glycoproteins only within the trans-Golgi compartment. In the last ten years, radioautographic tracing of radioiodinated plasma membrane molecules has indicated a substantial recycling of such molecules to the Golgi apparatus.     

Mineralization patterns in elasmobranch fish

This article reviews current findings on the organic matrix and the mineralization patterns in elasmobranchs, including an analysis of the role of the dental epithelial cells and the odontoblasts during odontogenesis. Our electron micrographs demonstrated that tubular vesicles limited by a unit membrane occupied the bulk of the elasmobranch enameloid matrix during the stage of enameloid matrix formation. It is likely that the tubular vesicles originated from the odontoblast processes. Two types of electron-dense fibrils, with cross-striations at intervals of approximately either 17 nm or 55 nm, respectively, were detected in the enameloid matrix. These data suggest that odontoblasts were strongly involved in enameloid matrix formation and in initial enameloid mineralization. Two types of odontoblasts, dark and light cells, were recognized during the stage of dentinogenesis. The light cells contained numerous mitochondria, intermediate filaments, and microtubules that extended their processes into the dentin. The dark cells possessed a well-developed Golgi apparatus and many cisternae in the rough endoplasmic reticulum, which suggests that the dark cells are involved in the formation of dentin. The inner dental epithelial (IDE) cells exhibited a well-developed Golgi apparatus, many mitochondria, cisternae of smooth endoplasmic reticulum, vesicles, vacuoles, and granules during the mineralization and maturation stages. During the stages of mineralization and early maturation, ACPase-positive granules were visible in the IDE cells and ALPase and Ca-ATPase activities were found at the lateral and proximal cell membrane of the IDE cells, suggesting that the IDE cells are involved in the removal of enameloid organic matrix and in the process of mineralization during later stages of enameloid formation. Our data indicate that elasmobranch enameloid is distinct from teleost enameloid, based on its organic content, on the mechanisms of its mineralization, and on the role of IDE cells concerning enameloid formation.     

A unique mechanism of nuclear division in <i>Giardia lamblia</i> involves components of the ventral disk and the nuclear envelope

The fine structure of the binucleate, parasitic protist Giardia lamblia during interphase and divisional stages was studied by serial thin sectioning and three-dimensional reconstructions. The earlier sign of nuclear division is the development of a few peripheral areas of densely packed chromatin directly attached to the inner nuclear envelope. An intracytoplasmic sheet of ventral disk components grows from the cell periphery towards one of the nuclei, apparently constricting this nucleus, which becomes located at a ventral bulge. After the basal bodies become duplicated, a full nuclear division occurs in trophozoites, giving two pairs of parent-daughter nuclei. This full division occurs in a dorsal-ventral direction, with the resulting nuclear pairs located at the sides of the two sets of basal bodies. A new ventral disk is formed from the diskderived sheets in the cell harboring the four nuclei. Cytokinesis is polymorphic, but at early stages is dorsalto-dorsal. Encysting trophozoites show the development of Golgi cisternae stacks and dense, specific secretory granules. 3-D reconstructions show that cysts contain a single pair of incompletely strangled nuclei. The dividing Giardia lacks a typical, microtubular spindle either inside or outside the nuclei. The nuclear envelope seems to be the only structure involved in the final division of the parent-daughter nuclei.     

Scale formation in algae

Scale biogenesis in algae represents a unique model system to study the transport of secretory macromolecules through the Golgi apparatus (GA) and their exocytosis. The larger scales can be visualized in the light microscope, and thus the kinetics of scale assembly, transport, and secretion can be studied in vivo. In addition, scales are osmiophilic and readily visible in conventional transmission electron microscopy; thus, details of scale assembly and sorting can be studied without invoking immunolabeling techniques. The following are distinctive features of scale biogenesis in algae: (1) transport of scales through the GA-stack occurs by cisternal progression; (2) scale secretion may be very rapid (in some cases a single GA-cisterna leaves the stack every 15–20 s); (3) sorting of different scale types does not occur in the GA, but in a post-GA-compartment. Recent progress in the analysis of scale formation in the green flagellates Tetraselmis and Scherffelia is reviewed.     

Fine structural analysis of the epithelial cells in the hepatopancreas of Palaemonetes argentinus (Crustacea, Decapoda, Caridea) in intermoult.

The aim of this study is to describe the ultrastructure of the hepatopancreas of P. argentinus in intermoult. P. argentinus hepatopancreas was studied using standard TEM techniques. Each tubule consists of four cellular types: E (embryonic), F (fibrillar), R (resorptive) and B (blister like). E-cells have embryonic features and some of them were found in mitosis. F, R and B cells possess an apical brush border. F-cells have a central or basal nucleus, a conspicuous RER, and dilated Golgi cisternae. R cells show a polar organization of organelles in three areas: apical, with numerous mitochondria and sER tubules, a central area with the nucleus and RER, and a basal area containing a sER-like tubule system and mitochondria. B-cells were observed at different stages of their life cycle. In an early differentiation stage they comprise an apical endocytotic complex and Golgi vesicles. The fusion of endocytotic and Golgi vesicles originates subapical vacuoles. During maturation, a big central vacuole is formed by coalescence of subapical vacuoles. The central vacuole is eliminated by holocrine secretion. The ultrastructure suggests that F-cells synthesize proteins, R-cells storage nutrients and B-cells have a secretory or excretory function, and confirms the independent origin of F, B and R cells from the embryonic cells.     

A fast method to study the secretory activity of neuroendocrine cells at the ultrastructural level

Cryo field emission scanning electron microscopy (cryo-FE-SEM) is a versatile technique that allows the investigation of the three-dimensional organization of cells at the ultrastructural level over a wide range of magnifications. Unfortunately, cryopreparation of the specimens for this technique remains cumbersome, in particular because ice crystal formation must be prevented during freezing. Here we report that a light prefixation with glutaraldehyde and incubation in glycerol as cryoprotectant or a high-pressure freezing approach are both excellent procedures for cryopreparation of animal cells to be used in combination with cryo-FE-SEM. Using the proopiomelanocortin-producing intermediate pituitary melanotrope cells of Xenopus laevis as a physiologically inducible neuroendocrine system, we compared the ultrastructural characteristics of inactive and hyperactive neuroendocrine cells. The overall quality of the ultrastructural images was comparable for the two cryopreparation procedures, although some fine structures were better conserved using high-pressure freezing. Melanotrope cells in a secretory inactive state contained numerous storage granules and a poorly developed endoplasmic reticulum (ER), while large amounts of rough ER were present in hyperactive cells. Thus, the cryo-FE-SEM approach described here allows a fast ultrastructural study on the secretory activity of neuroendocrine cells.