scholarly journals THE USE OF THIOL-SUBSTITUTED CARBOXYLIC ACIDS AS HISTOCHEMICAL SUBSTRATES

1965 ◽  
Vol 13 (8) ◽  
pp. 611-628 ◽  
Author(s):  
MARY BELL ◽  
RUSSELL J. BARRNETT
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Lipid Metabolism ◽  
Light Microscopy ◽  
Nuclear Envelope ◽  
Carboxylic Acids ◽  
Structural Level ◽  
Low Concentrations ◽  
Hydrolysis Of ◽  
Thiolacetic Acid

Thiobutyric, thiocaproic and thiocaprylic acids were synthesized, and enzyme histochemical methods were developed using these thiol-substituted carboxylic acids, as well as thiolacetic acid, as substrates with Pb++ as the capture reagent. The localization of final product of these histochemical reactions, PbS, was studied and compared in a variety of tissues with light microscopy. The enzymatic activities demonstrated were sensitive to low concentrations of E600. The localization of these reactions in several intensely reactive tissues, liver, testis and intestine, were also studied with electron microscopy. At a fine structural level, the final product was deposited primarily in relation to the membranous elements of the smooth and rough varieties of the endoplasmic reticulum, including the nuclear envelope, and of mitochondria. The results of these experiments are discussed, including the possible identity of the enzymes concerned with the hydrolysis of the thiol-substituted substrates. It was suggested that at least three activities were demonstrated in these experiments, one of which was the B type esterase of microsomes, and all of which functioned in lipid metabolism.

scholarly journals Supramolecular Structures of the Dictyostelium Lamin NE81

Cells ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 162
Author(s):  
Marianne Grafe ◽  
Petros Batsios ◽  
Irene Meyer ◽  
Daria Lisin ◽  
Otto Baumann ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Nuclear Envelope ◽  
Model Organism ◽  
Super Resolution ◽  
Nuclear Lamina ◽  
Stimulated Emission ◽  
Structural Level ◽  
Animal Cells ◽  
Nuclear Lamins

Nuclear lamins are nucleus-specific intermediate filaments (IF) found at the inner nuclear membrane (INM) of the nuclear envelope (NE). Together with nuclear envelope transmembrane proteins, they form the nuclear lamina and are crucial for gene regulation and mechanical robustness of the nucleus and the whole cell. Recently, we characterized Dictyostelium NE81 as an evolutionarily conserved lamin-like protein, both on the sequence and functional level. Here, we show on the structural level that the Dictyostelium NE81 is also capable of assembling into filaments, just as metazoan lamin filament assemblies. Using field-emission scanning electron microscopy, we show that NE81 expressed in Xenopous oocytes forms filamentous structures with an overall appearance highly reminiscent of Xenopus lamin B2. The in vitro assembly properties of recombinant His-tagged NE81 purified from Dictyostelium extracts are very similar to those of metazoan lamins. Super-resolution stimulated emission depletion (STED) and expansion microscopy (ExM), as well as transmission electron microscopy of negatively stained purified NE81, demonstrated its capability of forming filamentous structures under low-ionic-strength conditions. These results recommend Dictyostelium as a non-mammalian model organism with a well-characterized nuclear envelope involving all relevant protein components known in animal cells.

scholarly journals A conserved family of proteins facilitates nascent lipid droplet budding from the ER

2015 ◽  
Vol 211 (2) ◽  
pp. 261-271 ◽  
Author(s):  
Vineet Choudhary ◽  
Namrata Ojha ◽  
Andy Golden ◽  
William A. Prinz
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Lipid Metabolism ◽  
Caenorhabditis Elegans ◽  
Lipid Droplet ◽  
Lipid Droplets ◽  
De Novo ◽  
Fat Storage ◽  
Higher Eukaryotes ◽  
Er Membrane

Lipid droplets (LDs) are found in all cells and play critical roles in lipid metabolism. De novo LD biogenesis occurs in the endoplasmic reticulum (ER) but is not well understood. We imaged early stages of LD biogenesis using electron microscopy and found that nascent LDs form lens-like structures that are in the ER membrane, raising the question of how these nascent LDs bud from the ER as they grow. We found that a conserved family of proteins, fat storage-inducing transmembrane (FIT) proteins, is required for proper budding of LDs from the ER. Elimination or reduction of FIT proteins in yeast and higher eukaryotes causes LDs to remain in the ER membrane. Deletion of the single FIT protein in Caenorhabditis elegans is lethal, suggesting that LD budding is an essential process in this organism. Our findings indicated that FIT proteins are necessary to promote budding of nascent LDs from the ER.

scholarly journals Endoplasmic reticulum-to-Golgi transitions upon herpes virus infection

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1804 ◽  
Author(s):  
Peter Wild ◽  
Andres Kaech ◽  
Elisabeth M. Schraner ◽  
Ladina Walser ◽  
Mathias Ackermann
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Golgi Complex ◽  
Progeny Virus ◽  
Perinuclear Space ◽  
Exit Route ◽  
Scanning Electron ◽  
Hsv 1

Background: Herpesvirus capsids are assembled in the nucleus before they are translocated to the perinuclear space by budding, acquiring tegument and envelope, or releasing to the cytoplasm in a “naked” state via impaired nuclear envelope. One model proposes that envelopment, “de-envelopment” and “re-envelopment” are essential steps for production of infectious virus. Glycoproteins gB/gH were reported to be essential for de-envelopment, by fusion of the “primary” envelope with the outer nuclear membrane. Yet, a high proportion of enveloped virions generated from genomes with deleted gB/gH were found in the cytoplasm and extracellular space, suggesting the existence of an alternative exit route.Methods: We investigated the relatedness between the nuclear envelope and membranes of the endoplasmic reticulum and Golgi complex, in cells infected with either herpes simplex virus 1 (HSV-1) or a Us3 deletion mutant thereof, or with bovine herpesvirus 1 (BoHV-1) by transmission and scanning electron microscopy, employing freezing technique protocols that lead to improved spatial and temporal resolution.Results: Scanning electron microscopy showed the Golgi complex as a compact entity in a juxtanuclear position covered by a membrane on thecisface. Transmission electron microscopy revealed that Golgi membranes merge with membranes of the endoplasmic reticulum forming an entity with the perinuclear space. All compartments contained enveloped virions. After treatment with brefeldin A, HSV-1 virions aggregated in the perinuclear space and endoplasmic reticulum, while infectious progeny virus was still produced.Conclusions: The data strongly suggest that virions are intraluminally transported from the perinuclear space via Golgi complex-endoplasmic reticulum transitions into Golgi cisternae for packaging into transport vacuoles. Furthermore, virions derived by budding at nuclear membranes are infective as has been shown for HSV-1 Us3 deletion mutants, which almost entirely accumulate in the perinuclear space. Therefore, de-envelopment followed by re-envelopment is not essential for production of infective progeny virus.

scholarly journals FINE STRUCTURE AND ORGANELLE ASSOCIATIONS IN BROWN ALGAE

1965 ◽  
Vol 26 (2) ◽  
pp. 523-537 ◽  
Author(s):  
G. Benjamin Bouck
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Fine Structure ◽  
Golgi Apparatus ◽  
Nuclear Envelope ◽  
Brown Algae ◽  
Algal Cell ◽  
Plant Cells ◽  
Membrane Systems ◽  
Two Envelopes

The structural interrelationships among several membrane systems in the cells of brown algae have been examined by electron microscopy. In the brown algae the chloroplasts are surrounded by two envelopes, the outer of which in some cases is continuous with the nuclear envelope. The pyrenoid, when present, protrudes from the chloroplast, is also surrounded by the two chloroplast envelopes, and, in addition, is capped by a third dilated envelope or "pyrenoid sac." The regular apposition of the membranes around the pyrenoid contrasts with their looser appearance over the remainder of the chloroplast. The Golgi apparatus is closely associated with the nuclear envelope in all brown algae examined, but in the Fucales this association may extend to portions of the cytoplasmic endoplasmic reticulum as well. Evidence is presented for the derivation of vesicles, characteristic of those found in the formative region of the Golgi apparatus, from portions of the underlying nuclear envelope. The possibility that a structural channeling system for carbohydrate reserves and secretory precursors may be present in brown algae is considered. Other features of the brown algal cell, such as crystal-containing bodies, the variety of darkly staining vacuoles, centrioles, and mitochondria, are examined briefly, and compared with similar structures in other plant cells.

Mitosis in the Fission Yeast Schizosaccharomyces Pombe: A Comparative Study with Light and Electron Microscopy

1971 ◽  
Vol 9 (2) ◽  
pp. 475-507 ◽  
Author(s):  
E. KATHLEEN McCULLY ◽  
C. F. ROBINOW
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Nuclear Envelope ◽  
Schizosaccharomyces Pombe ◽  
Light And Electron Microscopy ◽  
Metaphase Plate ◽  
Middle Part ◽  
Mitotic Chromosomes ◽  
Nucleolar Material ◽  
Differential Expansion

Mitosis in Schizosaccharomyces pombe has been followed in living cells by phase-contrast microscopy and studied in fixed and suitably stained preparations by light microscopy. Successful preservation of nuclear fine structure in this yeast, not previously achieved, has allowed us to confirm and extend the observations made with light microscopy. Without first arranging themselves on a metaphase plate, mitotic chromosomes become grouped in 2 clusters radiating, finger-like, from 2 points of attachment at opposite poles of an elongating nucleus. At these 2 sites electron microscopy reveals the presence of disk-shaped electron-dense organelles which we have called kinetochore equivalents (KCE). At mitosis the KCEs are connected across the nucleus by a narrow bundle of parallel microtubules which we refer to as the spindle. Integration of our observations has led us to propose that at mitosis the separation of the KCEs and their attached chromosomes is initiated by a differential expansion of the nuclear envelope restricted to the region between recently divided KCEs and that expansion of the nuclear envelope later becomes general, resulting in a marked elongation of the nucleus. Displacement of the nuclear contents to the ends of the elongated nucleus gives it the shape of a dumbbell. The elongation of the microtubule bundle keeps in step with the elongation of the nucleus but does not appear to be the cause of it. It may have the function of keeping the separated KCEs rigidly apart. During mitosis the nucleolus persists and stretches out within the unbroken envelope of the nucleus as it elongates. Towards the end of division equal amounts of nucleolar material are found in the rounded ends of the dumbbell-shaped nucleus. The break up of the dumbbell shape into daughter nuclei seems to involve the breaking of its tenuous middle part and a pivoting of its 2 ends in opposite directions. In the course of our work on mitosis we have become aware of features in the cytoplasm of growing S. pombe cells which are described here for the first time. The cells invariably contain several prominent vacuoles containing an extremely electron-dense material which stains metachromatically with toluidine blue and may be polyphosphate. The mitochondria are of special interest for 2 reasons. First, because they have unique mesosome-like membrane invaginations and secondly, because a mitochondrion is regularly associated with the single KCE by the side of the interphase nucleus, as well as with each one of the 2 KCEs that occupy opposite ends of the intranuclear spindle during mitosis.

scholarly journals Enzymatic Activity in the M Band

1959 ◽  
Vol 6 (2) ◽  
pp. 163-170 ◽  
Author(s):  
Russell J. Barrnett ◽  
George E. Palade
Keyword(s):  
Electron Microscopy ◽  
Enzymatic Hydrolysis ◽  
Enzymatic Activity ◽  
Striated Muscles ◽  
Histochemical Reaction ◽  
Esterase Inhibitors ◽  
Enzymatic Nature ◽  
Hydrolysis Of ◽  
Thiolacetic Acid

Experiments which combined histochemistry and electron microscopy were performed in studying the sites of enzymatic hydrolysis of thiolacetic acid in the presence of lead ions in diaphragmatic and cardiac muscle. It was found that in these striated muscles the electron opaque, final product of the histochemical reaction (PbS) was discretely deposited on the swelling of the thick elemental filaments that occurs at the M band. Additional sites of enzymatic activity occurred in mitrochondria and in round sarcoplasmic bodies. A reaction, probably non-enzymatic, also occurred in contraction bands in the area of the Z bands and in the sarcoplasmic reticulum. To ascertain the enzymatic nature of the reaction and to define the enzyme involved, control experiments were carried out and the effect of various esterase inhibitors was assayed. It is suggested that the M band enzyme is a cholinesterase, but the enzymes in the mitochondria and the sarcoplasmic bodies that hydrolyze the substrate appear to be different. A possible role of the M band enzyme is discussed.

The Role of Membrane Proteins and Phospholipids in the Interaction of Ribosomes with Endoplasmic Reticulum Membranes

1975 ◽  
Vol 53 (9) ◽  
pp. 1039-1045 ◽  
Author(s):  
Serge Jothy ◽  
Jean-Louis Bilodeau ◽  
Henry Simpkins
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Binding Sites ◽  
Molecular Weights ◽  
Low Concentrations ◽  
Phospholipid Headgroups ◽  
Hydrolysis Of

Hydrolysis of the membrane proteins and phospholipid headgroups of rat liver rough endoplasmic reticulum membranes showed that the ribosomal binding sites involve membrane proteins susceptible to low concentrations of trypsin, chymotrypsin, and papain. Three membrane proteins having molecular weights of 120 000, 93 000 and 36 000 are found to be altered by trypsin and chymotrypsin treatment. Also the polar headgroup of phosphatidylinositol appears to play a role in the binding process.

scholarly journals AN ELECTRON MICROSCOPE STUDY OF THE ENDOPLASMIC RETICULUM IN NEWT NOTOCHORD CELLS AFTER DISTURBANCE WITH ULTRASONIC TREATMENT AND SUBSEQUENT REGENERATION

1964 ◽  
Vol 20 (1) ◽  
pp. 175-183 ◽  
Author(s):  
G. G. Selman ◽  
A. Jurand
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Light Microscopy ◽  
Ultrasonic Treatment ◽  
Electron Microscope Study ◽  
Triturus Alpestris ◽  
Notochord Cells ◽  
Parallel Arrays ◽  
Subsequent Regeneration

Ultrasonic treatment of the tails of Triturus alpestris tadpoles, at intensities of 8 to 15 watts/cm2, at 1 megacycle/sec., for 5 minutes, disrupted the epidermis and caused pycnosis in individual cells of the muscle and neural tube, but caused no damage to the notochord that could be detected by light microscopy. Electron microscopy showed that this ultrasonic treatment disordered nearly all the endoplasmic reticulum (ER) of the notochord cells into irregularly rounded vesicles, but within 3 hours after treatment some parallel arrays of normal endoplasmic reticulum were seen near, and continuous with, the outer nuclear membrane. In addition, a re-ordering of the previously disordered ER took place throughout the cytoplasm, in some cases. A classification was made of the state of the ER as shown in electron micrographs of material fixed immediately, 3, and 24 hours after treatment. This showed that more than half the total endoplasmic reticulum in notochord cells was normal again by 24 hours after treatment.

XII.—Electron Microscopy of the Epithelial Cells of the Mid-gut and Hepatic Cæca of Blatta Orientalis

1961 ◽  
Vol 68 (2) ◽  
pp. 162-170
Author(s):  
L. T. Threadgold ◽  
R. A. R. Gresson
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Epithelial Cells ◽  
Light Microscopy ◽  
Electron Micrographs ◽  
Anterior Part ◽  
Blatta Orientalis ◽  
Lateral Margins

SynopsisElectron micrographs of the anterior part of the mid-gut and hepatic cæca of Blatta orientalis Linn, show epithelial cells in both secretory and absorptive cycles. The epithelial cells possess microvilli and their lateral margins are provided with irregularly shaped projections that interdigitate with neighbouring cells. Vacuole-like structures were observed in association with the mitochondria of absorptive and degenerating cells. These vacuoles, it is suggested, represent degeneration products. Bundles of saccules and associated groups of vacuoles and vesicles represent the Golgi rods and granules of light microscopy. Epithelial cells engaged in absorption differ from those in which the elaboration of secretion is taking place, in the arrangement of the Golgi saccules and vacuoles, in the size of the Golgi vacuoles, in the distribution of the mitochondria, and in the possession of a less extensive endoplasmic reticulum.

scholarly journals Vaccinia Virus DNA Replication Occurs in Endoplasmic Reticulum-enclosed Cytoplasmic Mini-Nuclei

2001 ◽  
Vol 12 (7) ◽  
pp. 2031-2046 ◽  
Author(s):  
Nina Tolonen ◽  
Laura Doglio ◽  
Sibylle Schleich ◽  
Jacomine Krijnse Locker
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Dna Synthesis ◽  
Vaccinia Virus ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Nuclear Envelope Assembly ◽  
Green Fluorescent Protein Tagging ◽  
Green Fluorescent

Vaccinia virus (vv), a member of the poxvirus family, is unique among most DNA viruses in that its replication occurs in the cytoplasm of the infected host cell. Although this viral process is known to occur in distinct cytoplasmic sites, little is known about its organization and in particular its relation with cellular membranes. The present study shows by electron microscopy (EM) that soon after initial vv DNA synthesis at 2 h postinfection, the sites become entirely surrounded by membranes of the endoplasmic reticulum (ER). Complete wrapping requires ∼45 min and persists until virion assembly is initiated at 6 h postinfection, and the ER dissociates from the replication sites. [3H]Thymidine incorporation at different infection times shows that efficient vv DNA synthesis coincides with complete ER wrapping, suggesting that the ER facilitates viral replication. Proteins known to be associated with the nuclear envelope in interphase cells are not targeted to these DNA-surrounding ER membranes, ruling out a role for these molecules in the wrapping process. By random green fluorescent protein-tagging of vv early genes of unknown function with a putative transmembrane domain, a novel vv protein, the gene product of E8R, was identified that is targeted to the ER around the DNA sites. Antibodies raised against this vv early membrane protein showed, by immunofluorescence microscopy, a characteristic ring-like pattern around the replication site. By electron microscopy quantitation the protein concentrated in the ER surrounding the DNA site and was preferentially targeted to membrane facing the inside of this site. These combined data are discussed in relation to nuclear envelope assembly/disassembly as it occurs during the cell cycle.

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