The classic Golgi apparatus and vacuoles

The dense vacuoles, considered to be the classic Golgi apparatus in the root meristem ofFagopyrum, were studied by the following methods: 1. Impregnation methods for the demonstration of the Golgi apparatus, 2. cytochemical methods, 3. electron microscopic methods in the light microscope and 4. the electron microscope. A comparison was made with the classic Golgi apparatus in animal cells in the light and electron microscope. Dense vacuoles inFagopyrum and also evidently in other plants, were taken for the classic Golgi apparatus on account of their morphological similarity to the Golgi apparatus in animal cells on impregnation with silver and osmium and their staining preperties with lipoid methods. Dense vacuoles differ from the classic Golgi apparatus in other chemical properties, such as content of phenol substances, etc. No formations were found in animal cells which were similar to dense vacuoles on investigating by electron microscopy. In the electron microscope dense vacuoles have the appearance of derivatives of the normal light vacuoles known in plant cells. They therefore belong to vacuome of plant cell and cannot be analogous to the classic Golgi apparatus in animal cells. Thus the use of the term Golgi apparatus for dense vacuoles is not well founded. A comparison was made of fixation and impregnation used in the light microscope with fixation in the electron microscope. After fixation with permanganate, dense vacuoles have the same shape as after impregnation. After fixation with permanganate, they stain an intense black in the same way as after impregnation with silver and osmium. The form of the vacuoles is dependent on the fixation used. The comparison was made in the light microscope.     

The electron microscopic and classic Golgi apparatus

The relationship of the membrane structure, designated in electron microscopy as the Golgi apparatus, to the classic Golgi apparatus in the light microscope were studied withFagopyrum. Comparison of these structures in plant cells with the same or similar structures in animal cells led to the following conclusions: there exist two groups of formations, impregnable with osmium or silver, considered as the classic Golgi apparatus. The first group contains the active membrane structures. These are the dictyosomes and the anastomosing form of the electron microscopic Golgi apparatus. To this group belongs also the endoplasmatic reticulum, which in plant cells forms dense vacuoles, having the appearance of the classic Golgi apparatus, and in animal cells occasionally has a similar arrangement as the anastomosing form of the Golgi apparatus. The second group comprises formation containing reserve and secretion material, i.e. predominantly products of the activity of the electron microscopic Golgi apparatus and of the endoplasmic reticulum (matter of the dense vacuoles, lipochondria, secretory granula etc.). In the plant cells, especially ofFygopyrum, the dictyosomes contained in the structures of the first group are separated from the formations of a reserve character in the second group, formed in the lumen of the endoplasmic reticulum (dense vacuoles). The identity of the dictyosomes with the osmiophilic platelets, considered by some authors in the light microscope as the classic Golgi apparatus, has not been proved up to present, because of the one-sidedness of the methods used nowadays. WithFagopyrum no foundation has been observed for the assumed formation of net-form structures by grouping of the dictyosomes. Structures similar to the net-form of the classic Golgi apparatus in the animal cell form only dense vacuoles. On the basis of the differentiation of both types of formations in the plant cell, the foundations were laid for the characterization of the classic Golgi apparatus in the animal cell. The net-form of the classic Golgi apparatus in the animal cell is obviously not artificial, but reflects the ultrastructural arrangement of the electron microscopic Golgi apparatus or of the endoplasmic reticulum. The problem of the suitability and specification of the name Golgi apparatus in the animal and plant cell was also discussed. In contrast to the opinion of some authors, it does not appear useful to remove the name golgi apparatus, designating the dictyosomes and the anastomosing forms of the smooth membranes.     

Golgi apparatus in parasitic protists (review of the literature)

This review summarizes modem data on Golgi apparatus of parasitic protists and demonstrates how the parasitic lifestyle determines functional and structural peculiarities of secretory systems in unrelated groups of unicellular parasites, in comparison to ones of "model systems", mammalian and yeast cells. The review covers the most well-studied protists, predominantly of high medical importance, belonging to following taxons: Parabasalia (Trichomonas), Diplomonada (Giardia), Entamoebidae (Entamoeba), parasitic Alveolata of the phyllum Apicomplexa (Toxoplasma and Plasmodium), and Kinetoplastida (Trypanosoma and Leishmania). Numerous recent publications demonstrated that studies on intracellular traffic in the mentioned above parasites essentially advanced our knowledge of Golgi function, traditionally based on research of cultured mammalian and yeast cells. Morphology of Golgi organelle in eukaryotes from various taxonomic groups has been compared. Within three of total six the highest taxons of Eukatyota (Adl et al., 2005) there exist at minimum eight groups represented by species lacking Golgi dictiosomes. However, biochemical and (or) molecular (genomic) evidences indicate that the organelle with functions of Golgi was present in every studied so far lineage of eukaryotes. Loss of Golgi organelle is a secondary event, which has been proven by identification of Golgi genes in the genomes of Golgi-lacking lineages. This loss might have occurred independently several times in the course of evolution. Neither the number of stacks, nor the size of the organelle correlates with intensity of secretion, or the position of the species on the evolutionary tree (in terms of presumably early/lately diverged lineages).     

The yeast Golgi apparatus: Insights and mysteries

The Golgi apparatus is known to modify and sort newly synthesized secretory proteins. However, fundamental mysteries remain about the structure, operation, and dynamics of this organelle. Important insights have emerged from studying the Golgi in yeasts. For example, yeasts have provided direct evidence for Golgi cisternal maturation, a mechanism that is likely to be broadly conserved. Here, we highlight features of the yeast Golgi as well as challenges that lie ahead.     

The Golgi apparatus in epidermal mucoid and ellipsoid-vacuolate cells ofAeolidia papillosa andCoryphella rufibranchialis (Nudibranchia)

Summary The epidermal cell layer of the apical end of the ceras was investigated in two species of aeolid nudibranchs. Based on cellular inclusions, mostly two cell types were found: mucoid and ellipsoid-vacuolate cells. Mucoid cells ofCoryphella rufibranchialis have large heterogeneous and fibrillar secretory granules whereas inAeolidia papillosa, the granules are homogeneous, but vary in electron density from one cell to another. Ellipsoid-vacuolate cells contained large quantities of small vacuoles with an included ellipsoidal structure. Both species contained very numerous ellipsoid-vacuolate cells. Secretory granules and ellipsoid-vacuoles appear to arise from the Golgi apparatus and these contents stain with PAS, suggesting a polysaccharide composition. Mucoid cells contained both secretory granules and ellipsoid-vacuoles which may arise from the same Golgi apparatus.     

Differentiation and continuity of the Golgi apparatus during carposporogenesis inPolysiphonia (Rhodophyta)

Summary Changes in the morphology of the Golgi apparatus during carposporogenesis occur gradually and are observed first in proximal cisternae. As successive cisternae mature, the appearance of the Golgi apparatus is gradually altered. Morphological changes are observed in the contents of differentiating cisternae and detached vesicles, and in the mode of vesicle detachment. The reprogramming of the Golgi apparatus between consecutive developmental stages does not appear to occur abruptly. Cisternae transitional between successive stages are observed to have features characteristic of both. Several unusual morphological features of the Golgi apparatus and detached vesicles are discussed.     

The actin-binding protein comitin (p24) is a component of the Golgi apparatus

Comitin (p24) was first identified in Dictyostelium discoideum as a membrane-associated protein which binds in gel overlay assays to G and F actin. To analyze its actin-binding properties we used purified, bacterially expressed comitin and found that it binds to F actin in spin down experiments and increases the viscosity of F actin solutions even under high-salt conditions. Immunofluorescence studies, cell fractionation experiments and EM studies of vesicles precipitated with comitin-specific monoclonal antibodies showed that comitin was present in D. discoideum on: (a) a perinuclear structure with tubular or fibrillary extensions; and (b) on vesicles distributed throughout the cell. In immunofluorescence experiments using comitin antibodies NIH 3T3 fibroblasts showed a similar staining pattern as D. discoideum cells. Using bona fide Golgi markers the perinuclear structure was identified as the Golgi apparatus. The results were supported by an electron microscopic study using cryosections. Based on these data we propose that also in Dictyostelium the stained perinuclear structure is the Golgi apparatus. In vivo the perinuclear structure was found to be attached to the actin and the microtubule network. Alteration of the actin network or depolymerization of the microtubules led to its dispersal into vesicles distributed throughout the cell. These results suggest that the Golgi apparatus in D. discoideum is connected to the actin network by comitin. This protein seems also to be present in mammalian cells.     

The lipids of the Golgi apparatus subfractions from rat liver.

Golgi apparatus were isolated from untreated rat liver and separated into three fractions. One consisted mainly of vesicles, a second of tubular particles (dictyosomes) and the third was a mixed fraction. Large differences between these fractions could be seen in the electron microscope and by enzyme analysis. The total lipid content of the vesicles was 3.5-times greater than that of the dictyosomes and the neutral lipid value was 7-times greater. The ratio of phospholipids to protein was approximately the same in the three fractions. However, the phospholipid patterns differed between the vesicle and dictyosome fractions.     

CALNUC (nucleobindin) is localized in the Golgi apparatus in insect cells

A mouse monoclonal antibody 12B1 was raised against Golgi fractions from Sf21 insect cells and selected as Golgi-specific by immunostaining of the cells. The antigen was purified from the cells by immunoaffinity chromatography with the monoclonal antibody, and its N-terminal and internal amino acid sequences were determined. Based on the partial amino acid sequences, cDNA encoding the antigen protein was cloned and sequenced. The amino acid sequence deduced from the cDNA nucleotide sequence showed a homology to those of CALNUC family proteins, CALNUC (or nucleobindin, a calcium-binding Golgi protein with DNA-binding activity) and protein NEFA (a cell surface protein with DNA-binding, EF-hand, and acidic domains). The insect protein had two EF-hand loops at the same sites as the mammalian CALNUC family proteins, but had no leucine zipper which the mammalian homologues commonly have. An electron microscopic immunoperoxidase study demonstrated that the insect protein was localized in the cis-Golgi cisternae and cis-Golgi networks. Since this localization is identical to that of mammalian CALNUC, the insect protein was considered to be a homologue of CALNUC rather than that of NEFA. Assays involving proteinase K digestion, sodium carbonate extraction and Triton X-114 extraction revealed that the insect CALNUC-like protein was a soluble protein tightly associated with the luminal surface of Golgi membranes as reported for mammalian CALNUC. The insect protein was also shown to have calcium-binding activity as does mammalian CALNUC. These data verify that the insect protein is CALNUC. The existence of CALNUC in insect cells suggests that CALNUC is an essential calcium-binding Golgi protein in a wide range of the animal kingdom. A phylogenetic tree analysis, however, suggested that NEFA was derived from CALNUC long after the segregation of a mammalian ancestor from an insect ancestor.     

The Golgi apparatus and cell polarity: Roles of the cytoskeleton,the Golgi matrix,and Golgi membranes

Membrane trafficking plays a crucial role in cell polarity by directing lipids and proteins to specific subcellular locations in the cell and sustaining a polarized state. The Golgi apparatus, the master organizer of membrane trafficking, can be subdivided into three layers that play different mechanical roles: a cytoskeletal layer, the so-called Golgi matrix, and the Golgi membranes. First, the outer regions of the Golgi apparatus interact with cytoskeletal elements, mainly actin and microtubules, which shape, position, and orient the organelle. Closer to the Golgi membranes, a matrix of long coiled–coiled proteins not only selectively captures transport intermediates but also participates in signaling events during polarization of membrane trafficking. Finally, the Golgi membranes themselves serve as active signaling platforms during cell polarity events. We review here the recent findings that link the Golgi apparatus to cell polarity, focusing on the roles of the cytoskeleton, the Golgi matrix, and the Golgi membranes.     

GRP94 (endoplasmin) co-purifies with and is phosphorylated by Golgi apparatus casein kinase

Certain picornaviruses encode proteinases which cleave the translation initiation factor eIF4G, a member of the eIF4F complex which recruits mRNA to the 40S ribosomal subunit during initiation of protein synthesis in eukaryotes. We have compared the efficiency of eIF4G cleavage in rabbit reticulocyte lysates during translation of mRNAs encoding the foot-and-mouth disease virus leader proteinase (L^pro) or the human rhinovirus 2A^pro. Under standard translation conditions, L^pro cleaved 50% of eIF4G within 4 min after initiation of protein synthesis, whereas 2A^pro required 15 min. At these times, the molar ratios of proteinase to eIF4G were 1:130 for L^pro and 1:12 for 2A^pro, indicating a much more efficient in vitro cleavage than previously observed. The molar ratios are similar to those observed during viral infection in vivo.     

The Golgi apparatus in developing endosperm of wheat (Triticum aestivum L.)

The ultrastructure and distribution of the Golgi apparatus in developing wheat endosperm was investigated using a zinc iodide-osmium tetroxide staining complex in conjunction with low and high voltage electron microscopy. Dictyosomes were numerous in starchy endosperm and aleurone at 15 days after anthesis, and during the period of rapid storage protein deposition 25 d after anthesis. Fewer dictyosomes were seen in maturing endosperm. Two types of vesicles were associated with the dictyosomes; small, heavily-stained vesicles were sited at the ends of fine tubules which extend from the cisternae, and larger less-stained vesicles were associated with the periphery of the cisternae. Stereo-pairs of micrographs up to 1 m thick were taken to demonstrate the interconnections between cisternal and tubular endoplasmic reticulum. Elements of tubular ER were closely associated with dictyosomes, but connections were not observed. These results are discussed in relation to the transport of endosperm storage proteins from their site of synthesis on the cisternal ER to their site of storage, the protein bodies.     

The blood concentration of intercellular adhesion molecule-1 (sICAM-1) and vascular cell adhesion molecule-1 (sVCAM-1) in patients with active thyroid-associated orbitopathy before and after methylprednisolone treatment

BACKGROUND: The soluble forms of vascular cell adhesion molecule-1 (sVCAM-1) and intercellular adhesion molecule-1 (sICAM-1) have been found to be increased in the blood of patients with Graves disease. The aim of this study is evaluation of the serum concentrations of soluble forms of adhesion molecules ICAM-1 and VCAM-1 in patients with thyroid-associated orbitopathy (TAO) before and after methylprednisolone treatment. MATERIAL AND METHODS: The study was performed in 40 Graves disease, hyperthyroid and euthyroid patients with a clinically active form of TAO. Serum concentrations of sVCAM-1 and sICAM-1 in TAO patients were determined by enzymelinked immunoabsorbent assay (ELISA) before and after intensive pulse methylprednisolone treatment. RESULTS: We did not find any significant changes in the studied parameters between TAO patients with hyperthyroidism and those with euthyroidism. The serum concentrations of sICAM-1 and sVCAM-1 were significantly increased in patients with TAO before methylprednisolone therapy when compared with the control group. After treatment serum concentrations of sICAM-1 and sVCAM-1 decreased significantly but were still significantly higher than for the control group. CONCLUSION: From the results obtained we can conclude that Graves orbitopathy itself but not thyroid function is probably responsible for the elevated level of the adhesion molecules studied.