Electron Microscopic Detection of Proteins and Protein Complexes in Mammalian Cells Using APEX-tagged, Conditionally Stable Nanobodies

A number of viral agents are recognized as and suspected of causing the clinical condition “gastroenteritis.” In our attempts to establish an animal model for studies of this entity, we have been examining the nonhuman primate to ascertain what viruses may be found in the intestinal tract of “normal” animals as well as animals with diarrhea. Several virus types including coronavirus, adenovirus, herpesvirus, and picornavirus (Table I) were detected in our colony; however, rotavirus, astrovirus, and calicivirus have not yet been observed. Fecal specimens were prepared for electron microscopy by procedures reported previously.

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Use of Uranium-Labeled Antibodies for Electron Microscopic Localization of Various Antigens in Mammalian Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100060696 ◽

1967 ◽

Vol 25 ◽

pp. 136-137

Author(s):

J. P. Petrali ◽

E. J. Donati ◽

L. A. Sternberger

Keyword(s):

Endoplasmic Reticulum ◽

Hela Cells ◽

Mammalian Cells ◽

Specific Antiserum ◽

Electron Microscopic ◽

E Coli ◽

Cytoplasmic Membranes ◽

Viral Progeny ◽

Ultrathin Sections ◽

Electron Microscopic Localization

Specific contrast is conferred to subcellular antigen by applying purified antibodies, exhaustively labeled with uranium under immunospecific protection, to ultrathin sections. Use of Seligman’s principle of bridging osmium to metal via thiocarbohydrazide (TCH) intensifies specific contrast. Ultrathin sections of osmium-fixed materials were stained on the grid by application of 1) thiosemicarbazide (TSC), 2) unlabeled specific antiserum, 3) uranium-labeled anti-antibody and 4) TCH followed by reosmication. Antigens to be localized consisted of vaccinia antigen in infected HeLa cells, lysozyme in monocytes of patients with monocytic or monomyelocytic leukemia, and fibrinogen in the platelets of these leukemic patients. Control sections were stained with non-specific antiserum (E. coli).In the vaccinia-HeLa system, antigen was localized from 1 to 3 hours following infection, and was confined to degrading virus, the inner walls of numerous organelles, and other structures in cytoplasmic foci. Surrounding architecture and cellular mitochondria were unstained. 8 to 14 hours after infection, antigen was localized on the outer walls of the viral progeny, on cytoplasmic membranes, and free in the cytoplasm. Staining of endoplasmic reticulum was intense and focal early, and weak and diffuse late in infection.

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Structure of contact sites between the outer and inner mitochondrial membranes investigated by HVEM tomography

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100167299 ◽

1996 ◽

Vol 54 ◽

pp. 966-967

Author(s):

C.A. Mannella ◽

K.F. Buttle ◽

K.A. O‘Farrell ◽

A. Leith ◽

M. Marko

Keyword(s):

Liver Mitochondrion ◽

Adenine Nucleotide ◽

Protein Complexes ◽

Mitochondrial Membranes ◽

Electron Microscopic ◽

Contact Sites ◽

Transmission Electron ◽

Precursor Proteins ◽

Membrane Contacts ◽

And Function

Early transmission electron microscopy of plastic-embedded, thin-sectioned mitochondria indicated that there are numerous junctions between the outer and inner membranes of this organelle. More recent studies have suggested that the mitochondrial membrane contacts may be the site of protein complexes engaged in specialized functions, e.g., import of mitochondrial precursor proteins, adenine nucleotide channeling, and even intermembrane signalling. It has been suggested that the intermembrane contacts may be sites of membrane fusion involving non-bilayer lipid domains in the two membranes. However, despite growing interest in the nature and function of intramitochondrial contact sites, little is known about their structure.We are using electron microscopic tomography with the Albany HVEM to determine the internal organization of mitochondria. We have reconstructed a 0.6-μm section through an isolated, plasticembedded rat-liver mitochondrion by combining 123 projections collected by tilting (+/- 70°) around two perpendicular tilt axes. The resulting 3-D image has confirmed the basic inner-membrane organization inferred from lower-resolution reconstructions obtained from single-axis tomography.

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Cytological changes induced by platinum-thymine in sarcoma-180 cells: Electron microscopy study

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010015900x ◽

1990 ◽

Vol 48 (3) ◽

pp. 290-291

Author(s):

Gustav Ofosu

Keyword(s):

Mammalian Cells ◽

Antitumor Agent ◽

Microscopy Study ◽

Electron Microscopy Study ◽

Limiting Factor ◽

Sarcoma 180 ◽

Electron Microscopic ◽

Cytological Changes

Platinum-thymine has been found to be a potent antitumor agent, which is quite soluble in water, and lack nephrotoxicity as the dose-limiting factor. The drug has been shown to interact with DNA and inhibits DNA, RNA and protein synthesis in mammalian cells in vitro. This investigation was undertaken to elucidate the cytotoxic effects of piatinum-thymine on sarcoma-180 cells in vitro ultrastructurally, Sarcoma-180 tumor bearing mice were treated with intraperitoneal injection of platinum-thymine 40mg/kg. A concentration of 60μg/ml dose of platinum-thymine was used in in vitro experiments. Treatments were at varying time intervals of 3, 7 and 21 days for in vivo experiments, and 30, 60 and 120 min., 6, 12, and 24th in vitro. Controls were not treated with platinum-thymine.Electron microscopic analyses of the treated cells in vivo and in vitro showed drastic cytotoxic effect.

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Tafazzins from Drosophila and mammalian cells assemble in large protein complexes with a short half-life

Mitochondrion ◽

10.1016/j.mito.2015.01.002 ◽

2015 ◽

Vol 21 ◽

pp. 27-32 ◽

Cited By ~ 8

Author(s):

Yang Xu ◽

Ashim Malhotra ◽

Steven M. Claypool ◽

Mindong Ren ◽

Michael Schlame

Keyword(s):

Half Life ◽

Mammalian Cells ◽

Protein Complexes ◽

Large Protein ◽

Short Half Life

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Immunohistochemical and electron microscopic detection of hepatitis B surface and core antigens

Experimental and Molecular Pathology ◽

10.1016/0014-4800(78)90036-9 ◽

1978 ◽

Vol 29 (2) ◽

pp. 156-169 ◽

Cited By ~ 11

Author(s):

G.A. Cabral ◽

F. Gyorkey ◽

P. Gyorkey ◽

J.L. Melnick ◽

G.R. Dreesman

Keyword(s):

Hepatitis B ◽

Electron Microscopic ◽

Microscopic Detection

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Electron microscopic detection of PAS-positive substances with thiosemicarbazide

Histochemistry and Cell Biology ◽

10.1007/bf00263369 ◽

1971 ◽

Vol 27 (1) ◽

pp. 63-68 ◽

Cited By ~ 11

Author(s):

Jitka Šťastná ◽

Pavel Trávník

Keyword(s):

Electron Microscopic ◽

Microscopic Detection

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Analysis of Antigenicity and Topology of E2 Glycoprotein Present on Recombinant Hepatitis C Virus-Like Particles

Journal of Virology ◽

10.1128/jvi.76.15.7672-7682.2002 ◽

2002 ◽

Vol 76 (15) ◽

pp. 7672-7682 ◽

Cited By ~ 110

Author(s):

Reginald F. Clayton ◽

Ania Owsianka ◽

Jim Aitken ◽

Susan Graham ◽

David Bhella ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Mammalian Cells ◽

Function Analysis ◽

Recombinant Baculovirus ◽

Microscopic Analysis ◽

Surface Topology ◽

Viral Glycoprotein ◽

Electron Microscopic ◽

Virus Like Particles

ABSTRACT Purification of hepatitis C virus (HCV) from sera of infected patients has proven elusive, hampering efforts to perform structure-function analysis of the viral components. Recombinant forms of the viral glycoproteins have been used instead for functional studies, but uncertainty exists as to whether they closely mimic the virion proteins. Here, we used HCV virus-like particles (VLPs) generated in insect cells infected with a recombinant baculovirus expressing viral structural proteins. Electron microscopic analysis revealed a population of pleomorphic VLPs that were at least partially enveloped with bilayer membranes and had viral glycoprotein spikes protruding from the surface. Immunogold labeling using specific monoclonal antibodies (MAbs) demonstrated these protrusions to be the E1 and E2 glycoproteins. A panel of anti-E2 MAbs was used to probe the surface topology of E2 on the VLPs and to compare the antigenicity of the VLPs with that of truncated E2 (E2660) or the full-length (FL) E1E2 complex expressed in mammalian cells. While most MAbs bound to all forms of antigen, a number of others showed striking differences in their abilities to recognize the various E2 forms. All MAbs directed against hypervariable region 1 (HVR-1) recognized both native and denatured E2660 with comparable affinities, but most bound either weakly or not at all to the FL E1E2 complex or to VLPs. HVR-1 on VLPs was accessible to these MAbs only after denaturation. Importantly, a subset of MAbs specific for amino acids 464 to 475 and 524 to 535 recognized E2660 but not VLPs or FL E1E2 complex. The antigenic differences between E2660, FL E1E2, and VLPs strongly point to the existence of structural differences, which may have functional relevance. Trypsin treatment of VLPs removed the N-terminal part of E2, resulting in a 42-kDa fragment. In the presence of detergent, this was further reduced to a trypsin-resistant 25-kDa fragment, which could be useful for structural studies.

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