Secretory organelles in ECL cells of the rat stomach: an immunohistochemical and electron-microscopic study

ECL cells are numerous in the rat stomach. They produce and store histamine and chromogranin-A (CGA)-derived peptides such as pancreastatin and respond to gastrin with secretion of these products. Numerous electron-lucent vesicles of varying size and a few small, dense-cored granules are found in the cytoplasm. Using confocal and electron microscopy, we examined these organelles and their metamorphosis as they underwent intracellular transport from the Golgi area to the cell periphery. ECL-cell histamine was found to occur in both cytosol and secretory vesicles. Histidine decarboxylase, the histamine-forming enzyme, was in the cytosol, while pancreastatin (and possibly other peptide products) was confined to the dense cores of granules and secretory vesicles. Dense-cored granules and small, clear microvesicles were more numerous in the Golgi area than in the docking zone, i.e. close to the plasma membrane. Secretory vesicles were numerous in both Golgi area and docking zone, where they were sometimes seen to be attached to the plasma membrane. Upon acute gastrin stimulation, histamine was mobilized and the compartment size (volume density) of secretory vesicles in the docking zone was decreased, while the compartment size of microvesicles was increased. Based on these findings, we propose the following life cycle of secretory organelles in ECL cells: small, electron-lucent microvesicles (pro-granules) bud off the trans Golgi network, carrying proteins and secretory peptide precursors (such as CGA and an anticipated prohormone). They are transformed into dense-cored granules (approximate profile diameter 100 nm) while still in the trans Golgi area. Pro-granules and granules accumulate histamine, which leads to their metamorphosis into dense-cored secretory vesicles. In the Golgi area the secretory vesicles have an approximate profile diameter of 150 nm. By the time they reach their destination in the docking zone, their profile diameter is between 200 and 500 nm. Exocytosis is coupled with endocytosis (membrane retrieval), and microvesicles in the docking zone are likely to represent membrane retrieval vesicles (endocytotic vesicles).     

Pyridoxal phosphatase: cytochemical localization in GERL and other organelles of rat neurons.

A phosphatase, hydrolyzing pyridoxal-5-phosphate (P5P), a physiologically active component of the vitamin B6 complex and an essential co-enzyme in the synthesis of neurotransmitters, has been localized cytochemically in the perikarya of neurons in the peripheral, autonomic and central nervous systems of the rat. Neurons in dorsal root ganglia, sympathetic ganglia and ventral horn of spinal cord were studied by light and electron microscopy, while Purkinje cells, neurons in the dentate nucleus of the cerebellum, thalamus, and hypothalamus were studied by light microscopy only. Optimal conditions for demonstrating this activity in aldehyde-fixed tissue were determined with dorsal root ganglia. At the optimal pH of 5.0, neurons in these ganglia and in all other neurons studied show pyridoxal-5-phosphatase (P5Pase) activity in GERL. Small neurons in dorsal root ganglia also display enzyme activity in the endoplasmic reticulum (ER); activities in GERL and ER are also appreciably high at neutral pH. Small and large neurons in these ganglia, and neurons of sympathetic ganglia, show variable P5Pase activity in the Golgi apparatus. These localizations differ from the usual sites of both acid phosphatase and alkaline phosphatase activities. The P5Pase activity, demonstrated cytochemically, is a new acid hydrolase activity in GERL.     

Secretory organelles of Paramecium cells (trichocysts) are not remarkably acidic compartments.

Acridine orange (AO) trapping in conjunction with fluorescence microscopy was applied to Paramecium cells. Trichocysts were not labeled when analyzed with an image intensification system (as opposed to a lysosomal population). Only with increasing intensity of ultraviolet light (UV) did trichocysts (and to some extent the cytosol) exhibit orange fluorescence, both effects being paralleled by increasing cell damage. Therefore, in comparison with the reported cytosolic pH (6.8), trichocysts cannot be considered as essentially acidic compartments. This is supported by experiments in vitro, using isolated cortex fragments or isolated fractions of membrane-bounded trichocysts (greater than or equal to 90% non-leaky). Again, during UV illumination orange fluorescence was observed even in the absence of ATP and Mg2+. Furthermore, this AO fluorescence and the condensation state of trichocyst contents were not affected by NH3 or by any of the widely differing ion- and H(+)-exchange inhibitors or ionophores tested. Decondensation of trichocyst contents occurred only when Ca2+ ionophore A23187 or X537A was incorporated into trichocyst membranes and when Ca2+ was then added. In this case all trichocysts partially decondensed within their intact membranes. We conclude that AO might be trapped in trichocysts by the abundant acidic secretory components during observation with UV light, rather than by acidic luminal pH.     

The structure of microbodies and their associations with other organelles in zoosporangia ofEntophlyctis variabilis

Summary The ultrastructure of microbodies in developing zoosporangia ofEntophlyctis variabilis was studied by three dimensional reconstructions from serial sections and by cytochemical localization of catalase activity. The morphology of microbodies and the spatial association of microbodies with other organelles varied during fungal development. In incipient zoo-sporangia, granular dilations resembling microbodies arose from rough ER. Young, enlarging zoosporangia contained elongate, contorted microbodies continuous with ER and aligned along bundles of microtubules. Oval, paired microbodies, lying on each side of an ER cisternae, were found in all zoosporangia, but in older zoosporangia this configuration of microbodies predominated. Analysis of serial sections revealed that these oval, paired microbodies were sometimes continuous with each other, with ER, and also apparently with the ER cisterna interposed between them. Other paired, oval microbodies were clearly discrete. Constrictions were found along the length of elongate microbodies and at junctions between oval microbodies. These constrictions may represent stages in fragmentation of microbodies from pre-existing microbodies. These observations suggest that microbodies originated in three ways: 1. as local dilations in tubular ER, 2. as lateral buds from opposite sides of ER cisternae, and 3. as fragments from elongate microbodies.Microbodies were consistently spatially associated with ER, nuclear envelopes, and mitochondria. The cisterna of ER passing between paired microbodies sometimes extended into a branching, tubular system of ER which curved around the side of one microbody and lay between this microbody and the forming face of a dictyosome. The cytochemical localization of thiamine pyrophosphatase activity in this cisterna when it is not associated with dictyosomes suggests a role in metabolic control. These spatial associations indicate that the microbody assemblage with other organelles represents functional units where propinquity to other organelles and intraluminal continuities insure a system for transport of substrates and products.     

Transport-dependent maturation of organelles in neurons

Some organelles show a spatial gradient of maturation along the neuronal process where more mature organelles are found closer to the cell body. This gradient is set up by progressive maturation steps that are aided by differential organelle distribution as well as transport. Autophagosomes and endosomes mature as they acquire lysosomal membrane proteins and decrease their luminal pH as they are retrogradely transported towards the cell body. The acquisition of lysosomal proteins along the neuronal processes likely occurs through fusion or membrane exchange events with Golgi-derived donor transport carriers that are transported anterogradely from the cell body. The mechanisms by which endosomes and autophagosomes mature might be applicable to other organelles that are transported along neuronal processes. Defects in axonal transport may also contribute to the accumulation of immature organelles in neurons. Such accumulations have been seen in neurons of neurodegenerative models.     

Dense-granule organelles of Toxoplasma gondii: their role in the host-parasite relationship

The dense-granule organelles of Toxoplasma gondii are secretory vesicles that play a major role in the structural modifications of the parasitophorous vacuole. In this article, Marie-France Cesbron-Delauw reviews work on a prominent set of molecules recently characterized as components of the dense granules, and discusses various aspects on their post-translational trafficking and delivery within the host cell.     

Galanin-immunoreactive neurons in the guinea-pig small intestine: their projections and relationships to other enteric neurons

Summary Galanin immunoreactivity was observed in nerve cell bodies and nerve fibres, but not in enteroendocrine cells, in the small intestine of the guinea-pig. Nerve terminals were found in the myenteric plexus, in the circular muscle, in submucous ganglia, around submucous arterioles, and in the mucosa. Lesion studies showed that all terminals were intrinsic to the intestine; those in myenteric ganglia arose from cell bodies in more orally placed ganglia. Myenteric nerve cells were also the source of terminals in the circular muscle. Galanin (GAL) was located in a population of submucous nerve cell bodies that also showed immunoreactivity for vasoactive intestinal peptide (VIP) and in a separate population that was immunoreactive for neuropeptide Y (NPY). Processes of the GAL/VIP neurons supplied submucous arterioles and the mucosal epithelium. Processes of GAL/NPY neurons ran to the mucosa. It is concluded that galanin immunoreactivity occurs in several functionally distinct classes of enteric neurons, amongst which are neurons controlling (i) motility, (ii) intestinal blood flow, and (iii) mucosal water and electrolyte transport.     

Redistribution of Golgi stacks and other organelles during mitosis and cytokinesis in plant cells

We have followed the redistribution of Golgi stacks during mitosis and cytokinesis in living tobacco BY-2 suspension culture cells by means of a green fluorescent protein-tagged soybean alpha-1,2 mannosidase, and correlated the findings to cytoskeletal rearrangements and to the redistribution of endoplasmic reticulum, mitochondria, and plastids. In preparation for cell division, when the general streaming of Golgi stacks stops, about one-third of the peripheral Golgi stacks redistributes to the perinuclear cytoplasm, the phragmosome, thereby reversing the ratio of interior to cortical Golgi from 2:3 to 3:2. During metaphase, approximately 20% of all Golgi stacks aggregate in the immediate vicinity of the mitotic spindle and a similar number becomes concentrated in an equatorial region under the plasma membrane. This latter localization, the "Golgi belt," accurately predicts the future site of cell division, and thus forms a novel marker for this region after the disassembly of the preprophase band. During telophase and cytokinesis, many Golgi stacks redistribute around the phragmoplast where the cell plate is formed. At the end of cytokinesis, the daughter cells have very similar Golgi stack densities. The sites of preferential Golgi stack localization are specific for this organelle and largely exclude mitochondria and plastids, although some mitochondria can approach the phragmoplast. This segregation of organelles is first observed in metaphase and persists until completion of cytokinesis. Maintenance of the distinct localizations does not depend on intact actin filaments or microtubules, although the mitotic spindle appears to play a major role in organizing the organelle distribution patterns. The redistribution of Golgi stacks during mitosis and cytokinesis is consistent with the hypothesis that Golgi stacks are repositioned to ensure equal partitioning between daughter cells as well as rapid cell plate assembly.     

Molecular evolution of maize catalases and their relationship to other eukaryotic and prokaryotic catalases

We have compared the nucleotide and protein sequences of the three maize catalase genes with other plant catalases to reconstruct the evolutionary relationship among these catalases. These sequences were also compared with other eukaryotic and prokaryotic catalases. Phylogenies based on distances and parsimony analysis show that all plant catalases derive from a common ancestral catalase gene and can be divided into three distinct groups. The first, and major, group includes maizeCatl, barleyCat1, riceCatB and most of the dicot catalases. The second group is an apparent dicot-specific catalase group encompassing the tobaccoCat2 and tomatoCat. The third is a monocot-specific catalase class including the maize Cat3, barley Cat2, and riceCatA. The maize Cat2 gene is loosely related to the first group. The distinctive features of monocot-specific catalases are their extreme high codon bias at the third position and low degree of sequence similarity to other plant catalases. Similarities in the intron positions for several plant catalase genes support the conclusion of derivation from a common ancestral gene. The similar intron position between bean catalases and human catalase implies that the animal and plant catalases might have derived from a common progenitor gene sequence. Correspondence to: J.G. Scandalios     

Muscle intermediate filaments and their links to membranes and membranous organelles

Intermediate filaments (IFs) play a key role in the integration of structure and function of striated muscle, primarily by mediating mechanochemical links between the contractile apparatus and mitochondria, myonuclei, the sarcolemma and potentially the vesicle trafficking apparatus. Linkage of all these membranous structures to the contractile apparatus, mainly through the Z-disks, supports the integration and coordination of growth and energy demands of the working myocyte, not only with force transmission, but also with de novo gene expression, energy production and efficient protein and lipid trafficking and targeting. Desmin, the most abundant and intensively studied muscle intermediate filament protein, is linked to proper costamere organization, myoblast and stem cell fusion and differentiation, nuclear shape and positioning, as well as mitochondrial shape, structure, positioning and function. Similar links have been established for lysosomes and lysosome-related organelles, consistent with the presence of widespread links between IFs and membranous structures and the regulation of their fusion, morphology and stabilization necessary for cell survival.     

A comparative chromosome banding analysis of the Ursidae and their relationship to other carnivores

Trypsin G-banded karyotypes of eight species of Ursidae were prepared from retrovirus-transformed skin fibroblast cultures. The banding patterns of all bears are highly conserved, even though their diploid numbers range from 42 to 72. A comprehensive analysis of the homologous banding patterns within the Ursidae and with a hypothesized ancestral carnivore karyotype permitted the reconstruction of three significant chromosomal reorganization events that occurred during the evolution of the modern ursids. The first was a multichromosomal fissioning away from the biarmed (2n = 44) primitive carnivore karyotype, leading to six species of the Ursinae subfamily (2n = 78). The second was a comprehensive chromosome fusion in the lineage that led to the Ailuropodinae (giant panda) subfamily (2n = 44). The third event was a second, independent, but less extensive, centromeric fusion occurring in the line that led to the Tremarctinae (spectacled bear) subfamily (2n = 52). Ursidae karyotypes are not only highly conserved within the family but also exhibit extensive chromosome banding homology with other carnivore families.     

Phytohormone contents in Corylus avellana and their relationship to age and other developmental processes

Endogenous levels of indole-3-acetic acid, abscisic acid, and cytokinins (Z-type: dihydrozeatin, dihydrozeatin riboside, zeatin, and zeatin riboside; iP-type: N ⁶-isopentenyl adenine and N ⁶-isopentenyl adenosine), were determined in leaves of hazelnut (Corylus avellana L.) (adult material from spring, autumn and forced outgrowth, and juvenile material). Our results showed high levels of indole-3-acetic acid, abscisic acid and total cytokinins in spring samples and low levels of the same hormones in autumn and forced outgrowth materials. The ratios of iP-type/Z-type cytokinins were low in autumn and spring leaves, while they were high in the juvenile and forced outgrowth samples. Both juvenile and forced-outgrowth hazel tissues also showed a high morphogenetic potential, suggesting that the ratio of iP-type/Z-type cytokinins may be a good index of in vitro potential of hazelnut materials.     

Peptide-based targeting of fluorophores to organelles in living cells

Peptides carrying organelle-specific import or retention sequences can target the fluorophore BODIPY(581/591) to the nucleus, peroxisomes, endoplasmic reticulum (ER), or the trans-Golgi network (TGN). The peroxisomal peptide contains the PTS1 sequence AKL. For targeting to the ER or TGN, the peptides carry the retention sequences KDEL and SDYQRL, respectively. A peptide carrying the nuclear leader sequence of the simian virus SV40 large tumor antigen, KKKRK, was used to direct the fluorophore to the nucleus. The fluorescent peptides for peroxisomes, ER, and the TGN spontaneously incorporate into living fibroblasts at 37 degrees C and accumulate in their target organelles within minutes. The uptake is still significant at 4 degrees C, indicating that endocytosis is not required for internalization. The highly charged nuclear peptide (net charge +4) does not spontaneously internalize. However, by transient permeabilization of the plasma membrane, this fluorescent peptide was found to rapidly accumulate in the nucleus. These fluorescent peptides open new opportunities to follow various aspects of specific organelles such as their morphology, biogenesis, dynamics, degradation, and their internal parameters (pH, redox).     

Ultrastructural relationship of the phagophore with surrounding organelles

Phagophore nucleates from a subdomain of the endoplasmic reticulum (ER) termed the omegasome and also makes contact with other organelles such as mitochondria, Golgi complex, plasma membrane and recycling endosomes during its formation. We have used serial block face scanning electron microscopy (SB-EM) and electron tomography (ET) to image phagophore biogenesis in 3 dimensions and to determine the relationship between the phagophore and surrounding organelles at high resolution. ET was performed to confirm whether membrane contact sites (MCSs) are evident between the phagophore and those surrounding organelles. In addition to the known contacts with the ER, we identified MCSs between the phagophore and membranes from putative ER exit sites, late endosomes or lysosomes, the Golgi complex and mitochondria. We also show that one phagophore can have simultaneous MCSs with more than one organelle. Future membrane flux experiments are needed to determine whether membrane contacts also signify lipid translocation.