Prolonged osmification of stomach epithelium

Summary Osimium impregnation was used upon the mice stomach epithelium to show possible differences in staining during differentiation. In the cells of the stratified gastric epithelium of 14-day-old mice embryos Os black was completely lacking in the Golgi complex. In some but not all cells the staining appears in the perinuclear space and in the endoplasmic reticulum. In the mucoid cells 1 and 8 days after the birth the osmiophility is not uniformly distributed throughout the endomembrane segments, except the cis face of the Golgi complex which is heavily stained. Our results indicate on the variability of the reduction capacity of particular endomembrane segments during differentiation and among the cells at the definite developmental stage.     

Reduction capacity of untreated and of repeatedly isoproterenol treated parotid gland of the mouse

Osmium impregnation was used to show possible differences of reduction capacity of perinuclear space, rough endoplasmic reticulum and the Golgi apparatus of unstimulated mouse parotid gland and in the gland after repeated pharmacological doses of isoproterenol. There were some significant differences between the staining of acinar and duct cells. In all intercalated and striated duct cells the staining is dense in the perinuclear space and in the rough endoplasmic reticulum. Osmiophility was not detected in the Golgi complex of intercalated duct cells. The staining was also lacking in the perinuclear space and endoplasmic reticulum of the acinar cells. The cis face of the Golgi complex and numerous transitional vesicles in the acinar cells showed variability of the reduction capacity of their membrane segments. In chronically treated acinar cells Os black was lacking in the Golgi cisternae, except that the numerous transitional vesicles were heavily stained. These results reveal characteristic differences of reduction capacity of endomembrane compartments in different parotid glandular cells, as well as between untreated and treated acinar cells.     

Reduction capacity of untreated and of repeatedly isoproterenol treated parotid gland of the mouse

Summary Osmium impregnation was used to show possible differences of reduction capacity of perinuclear space, rough endoplasmic reticulum and the Golgi apparatus of unstimulated mouse parotid gland and in the gland after repeated pharmacological doses of isoproterenol. There were some significant differences between the staining of acinar and duct cells. In all intercalated and striated duct cells the staining is dense in the perinuclear space and in the rough endoplasmic reticulum. Osmiophility was not detected in the Golgi complex of intercalated duct cells. The staining was also lacking in the perinuclear space and endoplasmic reticulum of the acinar cells. The cis face of the Golgi complex and numerous transitional vesicles in the acinar cells showed variability of the reduction capacity of their membrane segments. In chronically treated acinar cells Os black was lacking in the Golgi cisternae, except that the numerous transitional vesicles were heavily stained.These results reveal characteristic differences of reduction capacity of endomembrane compartments in different parotid glandular cells, as well as between untreated und treated acinar cells.     

Prolonged osmification as an indicator of the differentiation process in the endomembrane system.

The technique of prolonged osmification was used in the analysis of reducing capacity of perinuclear space, endoplasmic reticulum and cis-Golgi cisternae in different epithelial cells during embryonic differentiation and immediately after the birth. Cells of the mouse gastric and intestinal epithelium and of the exocrine pancreas and mammary gland were analyzed. It was shown that endomembrane compartments exhibit high variability in their capacity to reduce OsO4 into lower valency oxides. Typical staining of cis-Golgi cisternae by osmium black does not occur before the cells achieve the developmental state in which production of specific products starts. The changes in stainability occurring from the perinuclear space and endoplasmic reticulum towards the cis-Golgi cisternae indicate a maturation pathway with no direct correlation to the chemical characteristic of the substances produced in different cell types. In the mammary gland the reduction capacity of endoplasmic reticulum disappeared with the intensive synthesis of lipids. Considering our previous results and those of other authors, the possible reasons for the observed dynamics in reducibility in particular segments of endomembraneous space are discussed.     

Regulation of acrosome formation in mice expressing green fluorescent protein as a marker

Transgenic mice expressing enhanced green fluorescent protein under acrosin promoter were used to study the role of the Golgi complex and of the cytoskeleton during early development of the acrosomic system in exactly defined stages of the seminiferous epithelial cycle during in vitro differentiation. First acrosin expression was found uniformly in the cytoplasm of stage IV pachytene spermatocytes. The steady-state level increased up to stage X pachytene spermatocytes, and in diakinetic primary spermatocytes, acrosin started to accumulate into the Golgi complex. During step 2 of spermiogenesis, several small fluorescent proacrosomic granules were seen in various parts of the Golgi complex, and they fused to a solid acrosomic system at step 3. In cultured stage I-III seminiferous tubule segments, nocodazole slowed down acrosin incorporation and increased the distance of the acrosomic system from the nucleus. Follicle stimulating hormone had an opposite effect by increasing density of the acrosomic system together with activation of the surrounding microtubule network. The observations suggest that microtubules have an important function during the early differentiation of the acrosomic system.     

The Basal Level Ethylene Response is Important to the Wall and Endomembrane Structure in the Hypocotyl Cells of Etiolated Arabidopsis Seedlings

The sub-cellular events that occur during the ethylene-modulated cell elongation were characterized by examining the ultra-structure of etiolated Arabidopsis seedling hypocotyl cells. Preventing the basal level ethylene response facilitated cell elongation, and the cells exhibited wall loosening and separation phenotype. Nearby the wall separation sites were frequently associated with an increase in the cortical rough endoplasmic reticulum (rER) membranes, the presence of paramural bodies, and the circular Golgi formation. The cortical rER proliferation and circular Golgi phenotype were reverted by the protein biosynthesis inhibitor cycloheximide. The cortical rER membranes were longer when the ethylene response was prevented and shortened with elevated ethylene responses. Proteomic changes between wild type and the ethylene-insensitive mutant ethylene insensitive2 (ein2) seedling hypocotyls indicated that distinct subsets of proteins involving endomembrane trafficking, remodeling, and wall modifications were differentially expressed. FM4-64 staining supported the proteomic changes, which indicated reduced endocytosis activity with alleviation of the ethylene response. The basal level ethylene response has an important role in endomembrane trafficking, biological materials transport and maintenance of the endomembrane organization. It is possible that endomembrane alterations may partly associate with the wall modifications, though the biological significance of the alterations should be addressed in future studies.     

Study of the differentiation of secretory cells in the golden hamster oviductal epithelium by use of a monoclonal antibody

The ontogeny of an oviductal zona pellucida glycoprotein, designated ZP-0, in the golden hamster oviductal epithelium was investigated by means of light and electron microscopic immunolabeling studies using a monoclonal antibody. Light microscopic immunohistochemistry showed that ZP-0 appeared in the oviductal epithelium from 9.5 to 10.5 days after birth. At first, ZP-0 appeared in the Golgi region of the epithelial cells, and then increased in amount as development progressed to fill the supranuclear cytoplasm. Electron microscopic observations showed that rough endoplasmic reticulum and Golgi apparatus were well developed in non-ciliated cells of the ampullar and isthmic segments at 10.5 days after birth. Secretory granules appeared near the Golgi apparatus and markedly increased in number until 15.5 days after birth. Ultrastructural immunocytochemistry showed that ZP-0 appeared in the well-developed Golgi apparatus and then was stored within the secretory granules of developing secretory cells. ZP-0 was never present in the ciliated cells. These results indicate a coincidence between the beginning of ZP-0 biosynthesis and the morphological cytodifferentiation of secretory cells in the golden hamster oviduct during postnatal development. This leads us to the conclusion that ZP-0 is a possible differentiation marker of the golden hamster oviductal secretory cell.     

Light dependence of osmium reactivity in mouse photoreceptor cells.

Mouse photoreceptor cells exhibit local accumulations of osmium deposits after prolonged osmic staining at slightly elevated temperatures. Deposits were evident along the membranes of outer segment lamellae, Golgi cisternae and vesicles, nuclear envelopes, and synaptic vesicles. Other membranes within the photoreceptor cells were unreactive. No osmium reactivity was seen in other cells of the retina except for osmiophilic outer segment material which had been phagocytized by the pigment epithelium. In the outer segments, inner segments, and synaptic regions of the photoreceptor cells, the amount of osmium reactivity was increased by light stimulation and decreased following extended dark adaptation. The possible significance of the localized osmium reactivity is discussed.     

Rab-H_1b is essential for trafficking of cellulose synthase and for hypocotyl growth in Arabidopsis thaliana

Cell-wall deposition of cellulose microfibrils is essential for plant growth and development. In plant cells,cellulose synthesis is accomplished by cellulose synthase complexes located in the plasma membrane. Trafficking of the complex between endomembrane compartments and the plasma membrane is vital for cellulose biosynthesis;however, the mechanism for this process is not well understood. We here report that, in Arabidopsis thaliana,Rab-H_1b, a Golgi-localized small GTPase, participates in the trafficking of CELLULOSE SYNTHASE 6(CESA6) to the plasma membrane. Loss of Rab-H_1b function resulted in altered distribution and motility of CESA6 in the plasma membrane and reduced cellulose content. Seedlings with this defect exhibited short, fragile etiolated hypocotyls.Exocytosis of CESA6 was impaired in rab-h1 b cells, and endocytosis in mutant cells was significantly reduced as well. We further observed accumulation of vesicles around an abnormal Golgi apparatus having an increased number of cisternae in rab-h1 b cells, suggesting a defect in cisternal homeostasis caused by Rab-H_1b loss function. Our findings link Rab GTPases to cellulose biosynthesis, during hypocotyl growth, and suggest Rab-H_1b is crucial for modulating the trafficking of cellulose synthase complexes between endomembrane compartments and the plasma membrane and for maintaining Golgi organization and morphology.e     

Improved preservation of the form and contents of wall vesicles and the golgi apparatus in freeze substituted hyphae ofSaprolegnia

Summary Secretory vesicles involved in cell wall synthesis (wall vesicles) and the Golgi apparatus have been compared in conventionally fixed and freeze substituted hyphae of the oomycete fungusSaprolegnia ferax. Wall vesicles freeze substituted in various fluids range from spherical to tubular and contain an intensely staining, phosphorous rich matrix. In contrast diverse conventional fixations cause artefactual constrictions in most tubular vesicles and loss of their intensely staining contents. These data are interpreted to show the existence of an intravesicular skeletal system, with cellular regulation, to determine vesicle morphology and intravesicular synthesis of a hypothetical phosphorylated glycolipid cell wall precursor. Whilst freeze substitution gives superior preservation of wall vesicle morphology, it does not demonstrate any preferential association between wall vesicles and microtubules thus suggesting that microtubules are only indirectly involved in wall vesicle transport. Freeze substitution is superior to conventional fixation for analysis of the Golgi apparatus because it uniquely reveals both differentiation of a specific single cisterna in each Golgi body and greater differences in membrane thicknesses throughout the endomembrane system.     

Arabidopsis vacuolar H-ATPase subunit E isoform 1 is required for Golgi organization and vacuole function in embryogenesis

Vacuolar H(+)-ATPases play an important role in maintaining the pH of endomembrane compartments in eukaryotic cells. The functional relevance of this homeostasis for multicellular development has not been studied in plants. Here, we analyze the biological consequences resulting from the lack of subunit E isoform 1 (VHA-E1) encoded by the Arabidopsis TUFF gene. tuff mutant embryos are lethal, displaying variably enlarged cells with multiple nuclei, large vacuoles containing inclusions, abnormal organization of Golgi stacks, and cell wall defects. Rescue of embryo lethality by cell cycle-regulated expression of VHA-E1 results in abnormal seedlings with non-functional meristems and defective cell differentiation. VHA-E1 is the predominant isoform in embryogenesis whereas VHA-E3 is expressed mainly in the endosperm and surrounding maternal tissues during seed development, and VHA-E2 is pollen-specific. VHA-E1 protein accumulates at endomembrane compartments including vacuoles and endosomes, but appears absent from the plasma membrane. Our results suggest an essential role for VHA-E1 in maintaining a functional secretory system during somatic development but not in the haploid gametophytes.     

Glycosylated polypeptides of soybean root endomembranes

Summary Endoplasmic reticulum, Golgi apparatus, plasma membrane and mitochondria vesicles were isolated from the roots of four-day-old dark-grown soybean [Glycine max (L.) Merr. cv. Wells II] seedlings and characterized by marker enzyme analyses. Glycoproteins of enriched membrane fractions were identified by concanavalin A (con A)-peroxidase staining of polypeptides separated by two-dimensional IEF-SDS-PAGE and transferred to nitrocellulose.Con A bound to many polypeptides in each endomembrane-enriched fraction with several glycopolypeptides common to all fractions. The mitochondria-enriched fraction possessed few glycopolypeptides and those appeared to be highly glycosylated contaminants of endomembrane origin. Comparison of the endomembrane con A-binding patterns revealed changes in relative stain intensity, molecular weight and isoelectric point of several membrane glycopolypeptides suggestive of processing reactions of the endomembrane complex.Abbreviations con A concanavalin A - PM plasma membrane - GA Golgi apparatus - ER endoplasmic reticulum     

Changes of Golgi complex during the induced differentiation of human gastric adenocarcinoma cells in vitro

Golgi complex is a central link related to each kind of organella in cellular metabolic process, its morphological changes are often concerned with the cell differentiation and functional state. The Golgi complexes in tumour cells have the characteristics of poor development and non-typical structure, and closely related to their pathological differential degree. For this reason, having based on appraising the differential effect of human gastric adenocarcinoma cell line MGc 80-3 induced by dBcAMP in vitro, we had made a systematic observation on the changes of Golgi complex during the malignant phenotypical reversion of MGc 80-3 cells, in order to inquire into the relationship between the structural and functional changes of Golgi complex and the malignant phenotypic reversion of cancer cells. It was revealed by ultrathin sectioning and freeze-etching electron microscopy that, in MGc 80-3 cells, there were a few Golgi complexes, their volume was small, the amounts of saccule were few, they arranged irregularly, expanded and inflated, and the intramembranous particles on saccule were rare and not well-distributed. It displayed a non-developmental and non-typical structural state of Golgi complex. But after induced treatment with dBcAMP, the Golgi complexes had grown in number and distributed concentrated, , their volume enlarged, the saccules increased and arranged regularly, and the intermembranous particles on saccules were plentiful and well-distributed. They had restored a well-developed and typical structure of Golgi complex similar to that of the primary culture cell of human normal gastric mucous membrane. It showed that Golgi complex had further changed into a quite developed state during the induced differentiation. This alteration not only inhibited the malignant secretory activity of gastric carcinoma cells but also played a certain regulative role in the changes of the surface components of cancer cells. The structural and functional changes of Golgi complex were considered to be an important expression in the malignant phenotypical reversion of cancer cells, it had an important influence on the differentiation of cancer cells from malignant to normal direction.     

Fluorescence microscopy of the endomembrane system of living plant cells

Abstract The fluorochrome Auramine O has been evaluated as a fluorescent probe for components of the endomembrane system of living plant cells. At 0.001% w/v the compound did not inhibit seedling growth or cytoplasmic streaming but stained the nuclear envelope, endoplasmic reticulum and Golgi apparatus. The three-dimensional, structural interrelationships of these organelles in living tissues could be resolved after minimal tissue preparation. The method is also a valuable control treatment for use in conjunction with electron microscope fixation procedures. It provides a rapid means of examining dynamic changes in the endomembrane system associated with cell development and differentiation and could have application in monitoring the effects of applied physiological or chemical stress.     

Immunohistochemical localization of galactosyltransferase in the normal human ovary and fallopian tube

Galactosyltransferase (UDP-galactose: 2-acetamido-2-deoxy beta-D-glucopyranose beta-(1-4) transferase) in human tissue specimens from ovaries and the corresponding fallopian tubes was localized immunohistochemically for light microscopy. An affinity-purified rabbit anti-human milk galactosyltransferase antibody was used. Intracellular galactosyltransferase was found to be localized to the juxtanuclear (Golgi) region of the secretory cells of the fallopian-tube epithelium and to the ovarian stromal cells involved in steroid-hormone production. Cell-surface galactosyltransferase was localized to ciliated cells of the fallopian-tube epithelium. During the follicular phase of the menstrual cycle, galactosyltransferase was found only in the Golgi regions of theca interna cells of the ovarian graafian follicle, and in the fallopian tube was found predominantly on the cilia of epithelial cells. During the luteal phase, galactosyltransferase was abundant in the Golgi regions of granulosa lutein cells of the corpus luteum, and was predominant in the secretory cells of the tubal epithelium. Galactosyltransferase was not detected on the mesothelial ovarian surface. The results demonstrate that the cellular distribution and location of galactosyltransferase correlates with phenotypic differentiation and varies during the human female hormonal cycle.     

Three-dimensional reconstruction of rabbit-derived Pneumocystis carinii from serial-thin sections. II: Intermediate precyst.

Three-dimensional reconstruction of a binucleate intermediate precyst of Pneumocystis carinii was performed from serial-thin sections using the CATIA (Conception Assistée Tridimensionnelle Inter Active) Dassault system program. The presence of a mitochondrion, complex well-developed endoplasmic structures, and numerous Golgi vesicles was established. A better understanding of the ultrastructure of rabbit-derived P. carinii stages made it possible to formulate hypotheses on the evolution and physiology of the endomembrane system. Thus, the presence of the well-developed endoplasmic saccular structure and more than 230 Golgi vesicles in its vicinity might be implicated in the differentiation of the parasite surface structures and might also be related to nuclear division and individualization of intracystic bodies.     

Masking of sperm maturation antigen by sialic acid in the epididymis of the mouse. An immunohistochemical study

Sperm antigen expression during epididymal transit was examined in 4- to 16-week-old intact and castrated ICR mice, using the avidin-biotin complex (ABC) immunohistochemical method with monoclonal antibody T21 against a flagellar surface antigen. On untreated sections, the antigen was first expressed weakly on sperm in the proximal part of the corpus epididymis, and intraluminal components were stained in 4-week-old mice. Epididymal epithelial cells and their stereocilia, and cells in other reproductive organs were not stained. In contrast, on sections treated with neuraminidase, (1) the initial site of antigen appearance is a more proximal position in treated than in untreated sections, (2) stereocilia stained strongly, (3) the staining intensity of sperm and intraluminal components increased, and (4) some clear cells in the epithelium from the distal position of the caput to the corpus epididymis were stained. These results indicate that the antigen is produced by clear cells of the epididymal epithelium, that the antigenic determinant is masked initially by sialic acid residues, and that expression of the antigenic determinant on the sperm surface during epididymal maturation apparently involves desialylation.