Experimental changes in the width of the chromatin fibers form chicken erythrocytes

Widths of chromatin fibers prepared by spreading erythrocyte chromatin on water have been measured in different experimental conditions. Chromatin fibers from hemoglobin-free, EDTA-pretreated isolated nuclei, prepared by direct negative staining with uranyl acetate show an average width of 37 Å with a standard deviation of 13 Å. The same chromatin fibers, when previously treated with ethanol show a change in the average width from 37 to 138 Å. The same chromatin, when floated on a hemoglobin solution and then treated with ethanol shows a further enlargement of its average width, from 37 to 313 Å. These changes were compared with the measurements of chromatin fibers from whole erythrocytes spread on water. The average width of these fibers after ethanol treatment is 244 Å. These results show that the 240–250 Å chromatin fibers are the result of conformational changes of a thinner elementary fibril 30–40 Å wide, which are mainly dependent on the action of ethanol on this fibril and on the presence of additional proteins like hemoglobin.     

LYSOSOMES IN THE RAT SCIATIC NERVE FOLLOWING CRUSH

Peripheral nerves undergoing degeneration are favorable material for studying the types, origins, and functions of lysosomes. The following lysosomes are described: (a) Autophagic vacuoles in altered Schwann cells. Within these vacuoles the myelin and much of the axoplasm which it encloses in the normal nerve are degraded (Wallerian degeneration). The delimiting membranes of the vacuoles apparently form from myelin lamellae. Considered as possible sources of their acid phosphatase are Golgi vesicles (primary lysosomes), lysosomes of the dense body type, and the endoplasmic reticulum which lies close to the vacuoles. (b) Membranous bodies that accumulate focally in myelinated fibers in a zone extending 2 to 3 mm distal to the crush. These appear to arise from the endoplasmic reticulum in which demonstrable acid phosphatase activity increases markedly within 2 hours after the nerve is crushed. (c) Autophagic vacuoles in the axoplasm of fibers proximal to the crush. The breakdown of organelles within these vacuoles may have significance for the reorganization of the axoplasm preparatory to regeneration. (d) Phagocytic vacuoles of altered Schwann cells. As myelin degeneration begins, some axoplasm is exposed. This is apparently engulfed by the filopodia of the Schwann cells, and degraded within the phagocytic vacuoles thus formed. (e) Multivesicular bodies in the axoplasm of myelinated fibers. These are generally seen near the nodes of Ranvier.     

Fine structure of the fertilization membranes of sea urchin embryos

The fine structure of the fertilization membranes from S. purpuratus embryos has been studied with the electron microscope. Isolated membranes before and after their full development and membranes formed under the influence of 10⁻³% cystine have been observed. The membrane structure was found to be trilamella: a middle layer about 200 Å thick, which originally was the vitelline membrane, and about 175 Å thick peripheral layers organized by the “crystalline material” from the cortical granules. These surface layers were again found to be trilaminated structure composed of a monolayer of parallel, closely packed flat fibrils, about 160 Å wide and 75 Å thick, adhering on both sides to parallel, 40–50 Å thick filaments separated from each other by about 100 Å and intersecting with the fibrils by an angle of about 75 °.     

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.     

Ultrastructure of posterior silk gland cells and liquid silk in Indian tasar silkworm,Antheraea mylitta drury (Lepidoptera : Saturniidae)

The ultrastructural characteristics of the posterior silk glands of the mature Antheraea mylitta (Lepidoptera : Saturniidae) larvae were clarified. Fibroin globules containing a small dense mass of fibroin fibers are produced in Golgi vacuoles, and released from the apical surface into the lumen by exocytosis. Bundles of microfilaments, which serve as a dynamic skeleton, are well developed. Numerous autophagosomes originating; from mitochondria accumulate in both basal and apical ends of the gland cell; the degenerated materials are released in the basement membrane and into the gland lumen, respectively. These materials invade the central fibroin column, leaving digested vacuoles in the fibroin cocoon filament. Ours may be the first finding of this rare phenomenon in which the degenerated lysosomes originated from mitochondria in both liquid silk in the lumen and cocoon filament.     

The fiber components of insect connective tissue

Connective tissue around the nerve cord and heart have been studied in Calpodes ethlius. Four components at, distinguishable by selective staining and electron microscopy: matrix, collagen fine fibrils less than 60 Å in diameter and broad fibers about 400 Å in diameter after glutaraldehyde only the broad fibers react selectively for peroxidase and stain with phosphotungstic acid. These fibers are most abundant in connective tissue which is elastic. The fine fibrils are arranged parallel to and between the peroxidase reaciive fibers. It is suggested that the peroxidase activity of the fibers may be related to their stabilization. The collagen fibers have the narrow fibrillar form characteristic of Lepidoptera and Coleoptera and have a macroperiod of about 660 Å and a banding pattern matching that found in other insects.     

An actin-like protein from amoebae of dictyostelium discoideum

An actin-like protein has been isolated and purified from amoebae of Dictyostelium discoideum. The 3.7S protein polymerizes upon addition of 0.1 m KCl to a polymer of 26S. An increase in viscosity accompanies this polymerization and electron micrographs have revealed beaded, helical filaments with a diameter of 60–75 Å and an axial periodicity of 350 Å. These F-actin-like filaments produced a 5-fold activation of muscle myosin Mg-ATPase at low ionic strength. When incubated with rabbit muscle heavy meromyosin (HMM) the amoeba F-actin-like protein formed typical “arrowhead” structures with polarized binding of HMM and arrowhead spacings of 350 Å. In SDS polyacrylamide disc gel electrophoresis the purified amoeba protein migrates as a single band corresponding to a molecular weight of 48,000 daltons. The amino acid composition is very similar to that of muscle actin and includes the unusual amino acid 3-methylhistidine.     

ON THE SITE OF SULFATION IN THE CHONDROCYTE

As observed autoradiographically in the cartilage of embryonic rats, radiosulfate is bound and concentrated only in vesicles of the juxtanuclear Golgi apparatus of secreting chondrocytes within 3 minutes of its presentation. From this area, vacuoles migrate peripherally and lodge in the subcortex; their sulfated contents are thence discharged via stomata to the extracellular matrix. The label, apparently often associated with microvesicles at 10 and 20 minutes, is subsequently localized in the dense contents of the larger vacuoles. Bound radiosulfate is not detectable in other organelles. It is concluded that the vesicular component of the Golgi apparatus is the actual site of sulfation. Intracellular hyaluronidase-sensitive metachromatic granules are found chiefly at the cell periphery or mantle, rarely juxtanuclear in the main Golgi zone.     

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.     

Immunocytochemical Localization of the Vacuolar H-ATPase in Maize Root Tip Cells

The vacuolar H⁺-ATPase of maize (Zea mays L.) root tip cells has been localized at the EM level using rabbit polyclonal antibodies to the 69 kilodalton subunit and protein A-colloidal gold. Intracellular gold particles were detected mainly on the tonoplast and Golgi membranes. Only about 27% of the vacuoles were labeled above background. The absence of gold particles on the majority of vacuoles suggests either that the tonoplast H⁺-ATPase is degraded during tissue preparation or that the small vacuoles of root tip cells are specialized with respect to H⁺-ATP ase activity. The pattern of gold particles on the labeled vacuoles ranged from uniform to patchy. Virtually all of the Golgi bodies were labeled by the antibody, but the particle densities were too low to determine whether the H⁺-ATPase was associated with specific regions, such as the trans-face. Cell wall-labeling was also observed which could be partially prevented by the inclusion of gelatin as a blocking agent. The immunocytochemical results confirm previous biochemical studies with isolated membrane fractions (A Chanson, L Taiz 1985 Plant Physiol 78: 232-240).     

THE FINE STRUCTURE OF SYMPATHETIC NEURONS IN X-IRRADIATED FROGS

The effects of whole body x-irradiation on the fine structure of sympathetic neurons were studied in 15 unanesthetized adult frogs (Rana pipiens), as seen at intervals ranging from 1 hour to 2 weeks after single exposures to 1000 r and 2000 r. Using standard procedures, the lumbar sympathetic ganglia of experimental and 20 control animals were prepared for electron microscope examination. Radiation produced conspicuous but irregular and variable deterioration, swelling, and clearing of neuronal lysosomes. These changes may have been due to an increased permeability of lysosomal membranes, causing the entry of fluid into lysosomes and their swelling and deterioration, but a pronounced escape of lysosomal enzymes into the cytoplasm was questionable. Less frequent were the dilatation and the parallel layering or complete fusion and tight packing of the rough-edged endoplasmic reticulum. The number of vacuoles, probably derived from Golgi cisternae, was somewhat increased. These vacuoles were conjectured to serve the "sequestration" of damaged cytoplasmic areas. Abnormal amounts of presumptive glycogen granules occupied some axons of myelinated and unmyelinated fibers, especially of presynaptic nerve fibers. This was assumed to be due to a decreased breakdown of glycogen and probably caused the interruption of the transmission of nerve impulses in presynaptic fibers. The maximal incidence of these alterations seemingly occurred 8 days after exposure to 1000 r, and 1 hour after x-irradiation with 2000 r. Signs of recovery appeared 2 weeks after exposure to 2000 r.     

ELECTRON MICROSCOPE STUDIES ON THE DICTYOSOMES AND ACROBLASTS IN THE MALE GERM CELLS OF THE CRICKET

The dictyosome (Golgi body) in the secondary spermatocyte of the cricket appears in electron micrographs as a duplex structure composed of (a) a group of parallel double-membraned lamellae and (b) a group of associated vacuoles arranged along the compact lamellae in a chain-like fashion. This arrangement of ultramicroscopic structure for the dictyosomes is strikingly comparable to that described for the Golgi apparatus of vertebrates. Accordingly, the two are considered homologous structures. Associated with the duplex structure of the dictyosomes is a differentiated region composed of small vacuoles. This is thought to represent the pro-acrosome region described in light microscope preparations. In the spermatid the dictyosomes fuse, giving rise to the acroblast. Like the dictyosomes, the acroblasts are made up of double-membraned lamellae and associated vacuoles. In addition, a differentiated acrosome region is present which, in some preparations, may display the acrosome vacuole and granule. Both the dictyosomes and acroblasts are distinct from mitochondria.     

Serine-ubiquitination regulates Golgi morphology and the secretory pathway upon Legionella infection

SidE family of Legionella effectors catalyze non-canonical phosphoribosyl-linked ubiquitination (PR-ubiquitination) of host proteins during bacterial infection. SdeA localizes predominantly to ER and partially to the Golgi apparatus, and mediates serine ubiquitination of multiple ER and Golgi proteins. Here we show that SdeA causes disruption of Golgi integrity due to its ubiquitin ligase activity. The Golgi linking proteins GRASP55 and GRASP65 are PR-ubiquitinated on multiple serine residues, thus preventing their ability to cluster and form oligomeric structures. In addition, we found that the functional consequence of Golgi disruption is not linked to the recruitment of Golgi membranes to the growing Legionella-containing vacuoles. Instead, it affects the host secretory pathway. Taken together, our study sheds light on the Golgi manipulation strategy by which Legionella hijacks the secretory pathway and promotes bacterial infection.Subject terms: Biochemistry, Cell biology     

CYTOCHEMISTRY OF GOLGI FRACTIONS PREPARED FROM RAT LIVER

Cytochemical tests for several marker enzymes were applied to liver tissue and to the three Golgi fractions (GF₁, GF₂, GF₃) separated by the procedure of Ehrenreich et al. from liver homogenates of alcohol-treated rats. 5'-Nucleotidase (AMPase) reaction product was found in all three fractions but in different locations: It occurred along the inside of the membrane of VLDL-filled vacuoles in GF₁ and GF₂, and along the outside of the cisternal membranes in GF₃. In the latter it was restricted to the dilated cisternal rims and was absent from the cisternal centers. The AMPase activity found in the fractions by biochemical assay is therefore indigenous to Golgi components and is not due to contamination by plasma membrane. Acid phosphatase (AcPase) reaction product was detected within lysosomal contaminants in GF₁ and within many VLDL-filled vacuoles in GF₁ and GF₂, indicating that AcPase activity is due not only to contaminating lysosomes, but also to enzyme indigenous to Golgi secretory vacuoles. G-6-Pase reaction product was present in GF₃ and within contaminating endoplasmic reticulum fragments, but not in other fractions. Thiamine pyrophosphatase (TPPase) was localized to some of the VLDL-filled vacuoles and cisternae in GF₁ and GF₂, and was not found in the cisternae in GF₃. The results demonstrate the usefulness of cytochemical methods in monitoring the fractionation procedure: They have (a) allowed a reliable identification of contaminants, (b) made possible a distinction between indigenous and contaminating activities, and (c) shown, primarily by the results of the TPPase test, that the procedure achieves a meaningful subfractionation of Golgi elements, with GF₁ and GF₃, representing primarily trans-Golgi elements from the secretory Golgi face, and GF₃ consisting largely of cis-Golgi components from the opposite face.     

Ultrastructure and cytochemistry of glycogen-containing vacuoles in gastrodermal cells in developing hydranths of a hydromedusan coelenterate

Hydranth buds from the colonial hydroid Sertularia pumila (Hydromedusae) were observed by electron microscopy during their development. Before hydranth expansion, the gastrodermal columnar digestive cells had large numbers of vacuoles. These vacuoles contained many membranous components as well as α-glycogen and dense ring- and crescent-shaped bodies. The rings and crescents were not osmiophilic, but did react to periodic acid oxidation in the PA-TSC-SP test for carbohydrate. These structures were digestible by α-amylase and pullulanase. The chemical analyses and the close association of the rings and crescents to α-glycogen particles showed that they may be a highly condensed form of glycogen. Golgi bodies in association with the gastrodermal vacuoles had acid phosphatase activity. This enzyme was only slightly active in the vacuoles. It is suggested that the vacuoles arc primarily storage organelles with a potential for digestion.     

The Endoplasmic Reticulum-Resident Chaperone Heat Shock Protein 47 Protects the Golgi Apparatus from the Effects of O-Glycosylation Inhibition

The Golgi apparatus is important for the transport of secretory cargo. Glycosylation is a major post-translational event. Recognition of O-glycans on proteins is necessary for glycoprotein trafficking. In this study, specific inhibition of O-glycosylation (Golgi stress) induced the expression of endoplasmic reticulum (ER)-resident heat shock protein (HSP) 47 in NIH3T3 cells, although cell death was not induced by Golgi stress alone. When HSP47 expression was downregulated by siRNA, inhibition of O-glycosylation caused cell death. Three days after the induction of Golgi stress, the Golgi apparatus was disassembled, many vacuoles appeared near the Golgi apparatus and extended into the cytoplasm, the nuclei had split, and cell death assay-positive cells appeared. Six hours after the induction of Golgi stress, HSP47-knockdown cells exhibited increased cleavage of Golgi-resident caspase-2. Furthermore, activation of mitochondrial caspase-9 and ER-resident unfolded protein response (UPR)-related molecules and efflux of cytochrome c from the mitochondria to the cytoplasm was observed in HSP47-knockdown cells 24 h after the induction of Golgi stress. These findings indicate that (i) the ER-resident chaperon HSP47 protected cells from Golgi stress, and (ii) Golgi stress-induced cell death caused by the inhibition of HSP47 expression resulted from caspase-2 activation in the Golgi apparatus, extending to the ER and mitochondria.     

Structure, biosynthesis, and function of asparagine-linked glycans on plant glycoproteins

In plants, glycoproteins with asparagine-linked glycans (oligosaccharides) are found in vacuoles, in the extracellular space or matrix, and associated with the endo-membrane system (endoplasmic reticulum, Golgi apparatus, plasma membrane, tonoplast). These glycans are of the high-mannose type, with a structure identical to that found in other organisms (mammals, yeast), or of the complex type with a β1–2 linked xylosyl residue not found in mammalian complex glycans. Asparagine-linked glycans play multiple roles by modifying the physicochemical properties of the polypeptides to which they are attached.     

Ultrastructure of Type 2A Extrafusal Muscle Fibers and Muscle Spindle Intrafusal Fibers of Adult Rats after Prolonged Swimming

We compared the ultrastructure of type 2A extrafusal muscle fibers, the nuclear chain, and other intrafusal fibers of muscle spindle (muscle stretch mechanoreceptor) in adult rats after prolonged swimming (5–10 h/day, 10 days). The Golgi apparatus was expressed moderately in type 2A extrafusal fibers and hypertrophied in the motor B zone of nuclear chain intrafusal fibers. Intense development of the Golgi apparatus in the nuclear chain intrafusal fiber appears to be related to glycogenolysis in the autophagous vacuoles, involvement in the lysosome activity, and plasma membrane renewal. Recapitulation of the mechanism of glycogen autophagy, which is observed in newborn rats during mobilization of glycogen from the liver and muscle, was demonstrated in adult rats under the influence of physical load in the nuclear chain and nuclear bag₂ intrafusal fibers and in type 2A extrafusal fibers. This is accounted for by a weak differentiation of the intrafusal muscle fibers: structurally, they are similar to myotubes and have specific features of blood supply and innervation. Individual features of experimental adult rats may also play a certain role.     

OSMIUM STAINING OF ENDOPLASMIC RETICULUM AND MITOCHONDRIA IN THE RAT ADRENAL CORTEX

The zona fasciculata of the rat adrenal cortex synthesizes and secretes glucocorticoids. As observed after aldehyde fixation, the cells in this zone contain an extensive endoplasmic reticulum (ER), a small Golgi apparatus, a moderate number of lipid droplets, and abundant mitochondria with tubulovesicular cristae. Numerous areas within the endoplasmic reticulum and mitochondrial cristae appear clear. In addition, a small percentage of mitochondria encompasses large, clear areas. After immersion of finely minced adrenal cortex in unbuffered 2% OsO₄ (40–48 hr at 40°C), deposits of osmium are seen within the Golgi apparatus, the entirety of the ER, and occasionally within mitochondria. In some mitochondria, the deposits are within cristae; in others, within vacuoles; in still others, in both cristae and vacuoles. These localizations correspond best to the clear areas found in aldehyde-fixed tissue. Osmium is not deposited in lipid droplets, in bar-containing inclusions, in mitochondrial matrix inclusions, or in the peripheral, outer mitochondrial spaces. Addition of zinc-iodide to OsO₄ increases the amount of Golgi apparatus and mitochondrial staining. Adrenocorticotropin (ACTH) does not affect the localization of deposits; hypophysectomy decreases mitochondrial staining. This study (a) emphasizes the necessity for electron microscopic confirmation of osmium localization when this technique is used as a Golgi apparatus stain; and (b) suggests that the ER-staining pattern may be consistent in cells actively synthesizing steroids or steroid-like compounds.     

Targeting of a Short Peptide Derived from the Cytoplasmic Tail of the G1 Membrane Glycoprotein of Uukuniemi Virus (Bunyaviridae) to the Golgi Complex

Members of the Bunyaviridae family acquire an envelope by budding through the lipid bilayer of the Golgi complex. The budding compartment is thought to be determined by the accumulation of the two heterodimeric membrane glycoproteins G1 and G2 in the Golgi. We recently mapped the retention signal for Golgi localization in one Bunyaviridae member (Uukuniemi virus) to the cytoplasmic tail of G1. We now show that a myc-tagged 81-residue G1 tail peptide expressed in BHK21 cells is efficiently targeted to the Golgi complex and retained there during a 3-h chase. Green-fluorescence protein tagged at either end with this peptide or with a C-terminally truncated 60-residue G1 tail peptide was also efficiently targeted to the Golgi. The 81-residue peptide colocalized with mannosidase II (a medial Golgi marker) and partially with p58 (an intermediate compartment marker) and TGN38 (a trans-Golgi marker). In addition, the 81-residue tail peptide induced the formation of brefeldin A-resistant vacuoles that did not costain with markers for other membrane compartments. Removal of the first 10 N-terminal residues had no effect on the Golgi localization but abolished the vacuolar staining. The shortest peptide still able to become targeted to the Golgi encompassed residues 10 to 40. Subcellular fractionation showed that the 81-residue tail peptide was associated with microsomal membranes. Removal of the two palmitylation sites from the tail peptide did not affect Golgi localization and had only a minor effect on the association with microsomal membranes. Taken together, the results provide strong evidence that Golgi retention of the heterodimeric G1-G2 spike protein complex of Uukuniemi virus is mediated by a short region in the cytoplasmic tail of the G1 glycoprotein.