scholarly journals Enigmatic origin of the poxvirus membrane from the endoplasmic reticulum shown by 3D imaging of vaccinia virus assembly mutants

2017 ◽  
Vol 114 (51) ◽  
pp. E11001-E11009 ◽  
Author(s):  
Andrea S. Weisberg ◽  
Liliana Maruri-Avidal ◽  
Himani Bisht ◽  
Bryan T. Hansen ◽  
Cindi L. Schwartz ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Vaccinia Virus ◽  
Virus Assembly ◽  
Transmembrane Protein ◽  
Membrane Dynamics ◽  
Deletion Mutants ◽  
Viral Membrane ◽  
Transmission Electron

The long-standing inability to visualize connections between poxvirus membranes and cellular organelles has led to uncertainty regarding the origin of the viral membrane. Indeed, there has been speculation that viral membranes form de novo in cytoplasmic factories. Another possibility, that the connections are too short-lived to be captured by microscopy during a normal infection, motivated us to identify and characterize virus mutants that are arrested in assembly. Five conserved vaccinia virus proteins, referred to as Viral Membrane Assembly Proteins (VMAPs), that are necessary for formation of immature virions were found. Transmission electron microscopy studies of two VMAP deletion mutants had suggested retention of connections between viral membranes and the endoplasmic reticulum (ER). We now analyzed cells infected with each of the five VMAP deletion mutants by electron tomography, which is necessary to validate membrane continuity, in addition to conventional transmission electron microscopy. In all cases, connections between the ER and viral membranes were demonstrated by 3D reconstructions, supporting a role for the VMAPs in creating and/or stabilizing membrane scissions. Furthermore, coexpression of the viral reticulon-like transmembrane protein A17 and the capsid-like scaffold protein D13 was sufficient to form similar ER-associated viral structures in the absence of other major virion proteins. Determination of the mechanism of ER disruption during a normal VACV infection and the likely participation of both viral and cell proteins in this process may provide important insights into membrane dynamics.

scholarly journals Pollen, Tapetum, and Orbicule Development inColletia paradoxaandDiscaria americana(Rhamnaceae)

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
M. Gotelli ◽  
B. Galati ◽  
D. Medan
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Pollen Grains ◽  
Pollen Grain ◽  
Ultrastructural Changes ◽  
Transmission Electron ◽  
The Family ◽  
Tapetal Cells ◽  
Grain Formation

Tapetum, orbicule, and pollen grain ontogeny inColletia paradoxaandDiscaria americanawere studied with transmission electron microscopy (TEM). The ultrastructural changes observed during the different stages of development in the tapetal cells and related to orbicule and pollen grain formation are described. The proorbicules have the appearance of lipid globule, and their formation is related to the endoplasmic reticulum of rough type (ERr). This is the first report on the presence of orbicules in the family Rhamnaceae. Pollen grains are shed at the bicellular stage.

scholarly journals Effects of ubiquitin C-terminal hydrolase L1 deficiency on mouse ova

Reproduction ◽  
2012 ◽  
Vol 143 (3) ◽  
pp. 271-279 ◽  
Author(s):  
Sayaka Koyanagi ◽  
Hiroko Hamasaki ◽  
Satoshi Sekiguchi ◽  
Kenshiro Hara ◽  
Yoshiyuki Ishii ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Proteomic Analysis ◽  
Ubiquitin Proteasome System ◽  
Underlying Mechanism ◽  
Wild Type ◽  
Transmission Electron ◽  
Ubiquitin Proteasome

Maternal proteins are rapidly degraded by the ubiquitin–proteasome system during oocyte maturation in mice. Ubiquitin C-terminal hydrolase L1 (UCHL1) is highly and specifically expressed in mouse ova and is involved in the polyspermy block. However, the role of UCHL1 in the underlying mechanism of polyspermy block is poorly understood. To address this issue, we performed a comprehensive proteomic analysis to identify maternal proteins that were relevant to the role of UCHL1 in mouse ova using UCHL1-deficientgad. Furthermore, we assessed morphological features ingadmouse ova using transmission electron microscopy. NACHT, LRR, and PYD domain-containing (NALP) family proteins and endoplasmic reticulum (ER) chaperones were identified by proteomic analysis. We also found that the ‘maternal antigen that embryos require’ (NLRP5 (MATER)) protein level increased significantly ingadmouse ova compared with that in wild-type mice. In an ultrastructural study,gadmouse ova contained less ER in the cortex than in wild-type mice. These results provide new insights into the role of UCHL1 in the mechanism of polyspermy block in mouse ova.

Interrelations between the Parasitophorous Vacuole ofToxoplasma gondiiand Host Cell Organelles

2005 ◽  
Vol 11 (2) ◽  
pp. 166-174 ◽  
Author(s):  
Rodrigo Cardoso Magno ◽  
Lorian Cobra Straker ◽  
Wanderley de Souza ◽  
Marcia Attias
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Host Cell ◽  
Fluorescent Probes ◽  
Parasitophorous Vacuole ◽  
Laser Scanning Microscopy ◽  
Cell Organelles ◽  
Apical Side ◽  
Transmission Electron

Toxoplasma gondii, the causative agent of toxoplasmosis, is capable of actively penetrating and multiplying in any nucleated cell of warm-blooded animals. Its survival strategies include escape from fusion of the parasitophorous vacuole with host cell lysosomes and rearrangement of host cell organelles in relation to the parasitophorous vacuole. In this article we report the rearrangement of host cell organelles and elements of the cytoskeleton of LLCMK2 cells, a lineage derived from green monkey kidney epithelial cells, in response to infection byT. gondiitachyzoites. Transmission electron microscopy made on flat embedded monolayers cut horizontally to the apical side of the cells or field emission scanning electron microscopy of monolayers scraped with scotch tape before sputtering showed that association of mitochondria to the vacuole is much less frequent than previously described. On the other hand, all parasitophorous vacuoles were surrounded by elements of the endoplasmic reticulum. These data were complemented by observations by laser scanning microscopy using fluorescent probes from mitochondria and endoplasmic reticulum and reinforced by three-dimensional reconstruction from serial sections observed by transmission electron microscopy and labeling of mitochondria and endoplasmic reticulum by fluorescent probes.

Studies on the ontogeny and ultrastructure of the sclerotium of Botrytis cinerea Pers. ex Nocca & Balbis

1982 ◽  
Vol 28 (12) ◽  
pp. 1347-1354 ◽  
Author(s):  
H. J. Willetts ◽  
Suzanne Bullock
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Botrytis Cinerea ◽  
Transmission Electron ◽  
Polyphosphate Bodies ◽  
Vacuolated Cells

The ontogeny and ultrastructure of sclerotia of the fungus Botrytis cinerea Pers. ex Nocca & Balbis were studied by light, scanning, and transmission electron microscopy. Exudation droplets of various sizes and colour accumulated on sclerotial surfaces during development and eventually disappeared. A surface hyphal weft was present over sclerotia at maturity, forming a dense covering which often obscured the underlying rind. The rind consisted of highly vacuolated cells with thick, pigmented walls that remained intact even in old sclerotia. The cortex was poorly defined and usually consisted of only one layer of cells. The prosenchymatous medulla constituted the main volume of mature sclerotia. The ultrastructure of young sclerotial hyphae was similar to that of actively growing vegetative hyphae. Hyphae of mature sclerotia contained fewer nuclei and profiles of mitochondria and endoplasmic reticulum than young sclerotial hyphae. Electron-dense structures, tentatively identified as protein and polyphosphate bodies, were observed in hyphae of the cortex and medulla. Sclerotia of B. cinerea were structurally similar to those of Sclerotinia spp.

Effect of Endoplasmic Reticulum Stress on Resveratrol-Induced Cardioprotection

2014 ◽  
Vol 651-653 ◽  
pp. 227-230
Author(s):  
Hammayun Ayub ◽  
Shakir Ahmed ◽  
Ayesha Yasin ◽  
Yi Dong Zhang ◽  
Zhuo Wang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Western Blotting ◽  
Mitochondrial Permeability Transition ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Mptp Opening ◽  
Transmission Electron

To investigate if resveratrol prevent the mitochondrial permeability transition pore (mPTP) opening through inhibition of endoplasmic reticulum stress (ERS). Methods: Rat heart tissue-derived cardiac H9c2 myoblast cell line was cultured. Fluorescence images of mitochondrial membrane potential were obtained with confocal microscopy. Western blotting analyzes the ERS marker protein GRP78 expression. Transmission electron microscopy detects the subcellular structure. Results: Exposure of cardiac H9c2 cells to 100 μM 2-DG, the ERS inducer, for 20 min caused a marked decrease in mitochondrial specific tetramethylrhodamine ethyl ester (TMRE) fluorescence. Resveratrol significantly prevented the loss of TMRE fluorescence. Western blotting revealed that resveratrol decreased GRP78 expression. Experiments with transmission electron microscopy revealed that resveratrol prevented 2-DG-induced swelling of endoplasmic reticulum and mitochondrial damages. Conclusions: These data suggest that inhibition of ERS leads to the prevention of mPTP opening. Resveratrol prevents the mPTP opening through inhibition of ERS.

The role of endoplasmic reticulum during gametogenesis in the aquatic fungus Allomyces macrogynus

1991 ◽  
Vol 69 (2) ◽  
pp. 336-341 ◽  
Author(s):  
Tommy C. Sewall ◽  
Jeffrey C. Pommerville
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Osmium Tetroxide ◽  
Cleavage Furrow ◽  
Aquatic Fungus ◽  
Transmission Electron ◽  
Allomyces Macrogynus ◽  
Zinc Iodide

The Chytridiomycete Allomyces macrogynus generates new membranes for cleavage furrow and nuclear-cap formation during gametogenesis and zoosporogenesis. Transmission electron microscopy after impregnation with a mixture of zinc iodide and osmium tetroxide clearly demonstrated changes in the endoplasmic reticulum. Endoplasmic reticulum was intensely stained but did not appear to contribute to the formation of the unstained flagellar membranes or cleavage furrows. However, the relative cytoplasmic volume of endoplasmic reticulum decreased as positively stained nuclear-cap membrane formed. These observations are consistent with the hypothesis that flagellar membranes and cleavage furrows are derived from trans-Golgi equivalents, whereas the nuclear-cap membrane is derived from the endoplasmic reticulum. Key words: Allomyces macrogynus, Chytridiomycetes, endoplasmic reticulum, gametogenesis, zoosporogenesis.

Autophagy is associated with male sterility in pistillate flowers of Maytenus obtusifolia (Celastraceae)

2018 ◽  
Vol 66 (2) ◽  
pp. 108 ◽  
Author(s):  
Isabella Veríssimo Nader Haddad ◽  
Lygia Dolores Ribeiro de Santiago-Fernandes ◽  
Silvia Rodrigues Machado
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Male Sterility ◽  
Reproductive Development ◽  
Vital Role ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Anatomical Analysis ◽  
Transmission Electron

Programmed cell death (PCD) is defined as a sequence of genetically regulated events leading to controlled and organised cellular degradation. It plays a vital role in plant development; however, little is known about the role of PCD in reproductive development. Sterility in pistillate flowers of Maytenus obtusifolia Mart. has been shown to be related to cytoplasmic male sterility (CMS) based on reproductive biology and anatomical analysis. The recurrent PCD led us to investigate changes in the tapetum and sporogenic tissue during the establishment of male sterility using light and transmission electron microscopy combined with the use of TUNEL (terminal deoxynucleotidyl transferase mediated dUDP end-labelling) assay. The interruption of pollen development in pistillate flowers is a result of premature PCD in the tapetum and consequently in the sporogenic cells. Autophagy, via macroautophagy, occurs in the sporogenic cells and involves the formation of autophagosomes, through rough endoplasmic reticulum, and of complex macroautophagic structures. In the final stage of PCD, massive autophagy takes place. Male sterility in female individuals is thus reasonably interpreted as sporophytic CMS associated to autophagy.

scholarly journals Sequence-Independent Targeting of Transmembrane Proteins Synthesized within Vaccinia Virus Factories to Nascent Viral Membranes

2007 ◽  
Vol 81 (6) ◽  
pp. 2646-2655 ◽  
Author(s):  
Matloob Husain ◽  
Andrea S. Weisberg ◽  
Bernard Moss
Keyword(s):  
Endoplasmic Reticulum ◽  
Vaccinia Virus ◽  
Binding Sites ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Transmembrane Proteins ◽  
Structural Features ◽  
Specific Amino Acid ◽  
Viral Membrane ◽  
Virus Factory

ABSTRACT The primary membrane of vaccinia virus, as well as those of other poxviruses, forms within a discrete cytoplasmic factory region. We recently determined the existence of an operative pathway from the endoplasmic reticulum within the virus factory to nascent viral membranes and demonstrated that a viral protein could be diverted from this pathway to Golgi membranes by the addition of COPII-binding sites (M. Husain, A. S. Weisberg, and B. Moss, Proc. Natl. Acad. Sci. USA, 103:19506-19511, 2006). Here we describe an investigation of the structural features that are required for transit of proteins to the viral membrane. Deletion of either the N-terminal domain or the C-terminal cytoplasmic tail from the conserved A9 protein did not prevent its incorporation into viral membranes, whereas deletion of the transmembrane domain resulted in its distribution throughout the cytoplasm. Nevertheless, replacement of the A9 transmembrane domain with the corresponding region of a nonpoxvirus transmembrane protein or of a vaccinia virus extracellular envelope protein allowed viral membrane targeting, indicating no requirement for a specific amino acid sequence. Remarkably, the epitope-tagged A9 transmembrane domain alone, as well as a heterologous transmembrane domain lacking a poxvirus sequence, was sufficient for viral membrane association. The data are consistent with a sequence-independent pathway in which transmembrane proteins that are synthesized within the virus factory and lack COPII or other binding sites that enable conventional endoplasmic reticulum exiting are incorporated into nascent viral membranes.

scholarly journals A Universal Approach to Analyzing Transmission Electron Microscopy with ImageJ

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2177
Author(s):  
Jacob Lam ◽  
Prasanna Katti ◽  
Michelle Biete ◽  
Margaret Mungai ◽  
Salma AshShareef ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Open Source Software ◽  
Imaging Modality ◽  
National Institutes Of Health ◽  
Transmission Electron ◽  
Straightforward Method ◽  
Universal Approach ◽  
Length Width

Transmission electron microscopy (TEM) is widely used as an imaging modality to provide high-resolution details of subcellular components within cells and tissues. Mitochondria and endoplasmic reticulum (ER) are organelles of particular interest to those investigating metabolic disorders. A straightforward method for quantifying and characterizing particular aspects of these organelles would be a useful tool. In this protocol, we outline how to accurately assess the morphology of these important subcellular structures using open source software ImageJ, originally developed by the National Institutes of Health (NIH). Specifically, we detail how to obtain mitochondrial length, width, area, and circularity, in addition to assessing cristae morphology and measuring mito/endoplasmic reticulum (ER) interactions. These procedures provide useful tools for quantifying and characterizing key features of sub-cellular morphology, leading to accurate and reproducible measurements and visualizations of mitochondria and ER.

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