scholarly journals Kinetochore structure, duplication, and distribution in mammalian cells: analysis by human autoantibodies from scleroderma patients.

1981 ◽  
Vol 91 (1) ◽  
pp. 95-102 ◽  
Author(s):  
S Brenner ◽  
D Pepper ◽  
M W Berns ◽  
E Tan ◽  
B R Brinkley
Keyword(s):  
Immunoelectron Microscopy ◽  
Mammalian Cells ◽  
Specific Antigen ◽  
Cell Types ◽  
Electron Microscopic Level ◽  
Immunoperoxidase Technique ◽  
Mitotic Chromosomes ◽  
Electron Microscopic ◽  
Interphase Nuclei ◽  
Kinetochore Region

The specificity of the staining of CREST scleroderma patient serum was investigated by immunofluorescence and immunoelectron microscopy. The serum was found to stain the centromere region of mitotic chromosomes in many mammalian cell types by immunofluorescence. It also localized discrete spots in interphase nuclei which we have termed "presumptive kinetochores." The number of presumptive kinetochores per cell corresponds to the chromosome number in the cell lines observed. Use of the immunoperoxidase technique to localize the antisera on PtK2 cells at the electron microscopic level revealed the specificity of the sera for the trilaminar kinetochore disks on metaphase and anaphase chromosomes. Presumptive kinetochores in the interphase nuclei were also visible in the electron microscope as randomly arranged, darkly stained spheres averaging 0.22 micrometers in diameter. Preabsorption of the antisera was attended using microtubule protein, purified tubulin, actin, and microtubule-associated proteins. None of these proteins diminished the immunofluorescence staining of the sera, indicating that the antibody-specific antigen(s) is a previously unrecognized component of the kinetochore region. In some interphase cells observed by both immunofluorescence and immunoelectron microscopy, the presumptive kinetochores appeared as double rather than single spots. Analysis of results obtained using a microspectrophotometer to quantify DNA in individual cells double stained with scleroderma serum and the DNA fluorescent dye, propidium iodide, led to the conclusion that the presumptive kinetochores duplicate in G2 of the cell cycle.

scholarly journals Murine endodermal cytokeratins Endo A and Endo B are localized in the same intermediate filament.

1986 ◽  
Vol 34 (6) ◽  
pp. 785-793 ◽  
Author(s):  
W E Howe ◽  
F G Klier ◽  
R G Oshima
Keyword(s):  
Immunoelectron Microscopy ◽  
Intermediate Filament ◽  
Microscopic Level ◽  
Cytoskeletal Proteins ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Double Label ◽  
Secondary Antibodies ◽  
Endodermal Cells ◽  
20 Nm

The intracellular distribution of extra-embryonic endodermal, cytoskeletal proteins A (Endo A) and B (Endo B) was investigated by double-label immunofluorescent microscopy and double-label immunoelectron microscopy. In parietal endodermal cells, the immunofluorescent distribution of Endo B was always coincident with that of Endo A and could be distinguished from vimentin, particularly at the periphery of the cell. At the electron microscopic level, antibodies against both Endo A and Endo B recognized both bundles and individual intermediate filaments. Double-label immunoelectron microscopy was achieved by use of two sizes of colloidal gold particles (5 nm and 20 nm) that were stabilized with secondary antibodies. These results show that Endo A and B are found in the same intermediate filament and probably co-polymerize to form such structures.

scholarly journals A New Method to Enhance Contrast of Ultrathin Cryosections for Immunoelectron Microscopy

2003 ◽  
Vol 51 (1) ◽  
pp. 31-39 ◽  
Author(s):  
Toshihiro Takizawa ◽  
Clark L. Anderson ◽  
John M. Robinson
Keyword(s):  
Immunoelectron Microscopy ◽  
Staining Method ◽  
Microscopic Level ◽  
New Method ◽  
Electron Microscopic Level ◽  
Positive Staining ◽  
Immunocytochemical Localization ◽  
Electron Microscopic ◽  
Morphological Detail ◽  
Ultrathin Cryosections

Adequate contrast of ultrathin cryosections is crucial for evaluating morphological detail to assess immunocytochemical localization at the electron microscopic level. We have developed a positive staining method for achieving contrast in ultrathin cryosections, from tissue fixed only in paraformaldehyde, that provides excellent contrast at the electron microscopic level.

scholarly journals Baculovirus Infection of Nondividing Mammalian Cells: Mechanisms of Entry and Nuclear Transport of Capsids

2001 ◽  
Vol 75 (2) ◽  
pp. 961-970 ◽  
Author(s):  
Nico-Dirk van Loo ◽  
Elisabetta Fortunati ◽  
Erich Ehlert ◽  
Martijn Rabelink ◽  
Frank Grosveld ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Nuclear Transport ◽  
Microscopic Analysis ◽  
Cell Types ◽  
Nuclear Pore ◽  
Fusion Event ◽  
Electron Microscopic ◽  
Cytoplasmic Transport ◽  
Baculovirus Infection

ABSTRACT We have studied the infection pathway of Autographa californica multinuclear polyhedrosis virus (baculovirus) in mammalian cells. By titration with a baculovirus containing a green fluorescent protein cassette, we found that several, but not all, mammalian cell types can be infected efficiently. In contrast to previous suggestions, our data show that the asialoglycoprotein receptor is not required for efficient infection. We demonstrate for the first time that this baculovirus can infect nondividing mammalian cells, which implies that the baculovirus is able to transport its genome across the nuclear membrane of mammalian cells. Our data further show that the virus enters via endocytosis, followed by an acid-induced fusion event, which releases the nucleocapsid into the cytoplasm. Cytochalasin D strongly reduces the infection efficiency but not the delivery of nucleocapsids to the cytoplasm, suggesting involvement of actin filaments in cytoplasmic transport of the capsids. Electron microscopic analysis shows the cigar-shaped nucleocapsids located at nuclear pores of nondividing cells. Under these conditions, we observed the viral genome, major capsid protein, and electron-dense capsids inside the nucleus. This suggests that the nucleocapsid is transported through the nuclear pore. This mode of transport seems different from viruses with large spherical capsids, such as herpes simplex virus and adenovirus, which are disassembled before nuclear transport of the genome. The implications for the application of baculovirus or its capsid proteins in gene therapy are discussed.

scholarly journals High- and Low-mobility Populations of HP1 in Heterochromatin of Mammalian Cells

2004 ◽  
Vol 15 (6) ◽  
pp. 2819-2833 ◽  
Author(s):  
Lars Schmiedeberg ◽  
Klaus Weisshart ◽  
Stephan Diekmann ◽  
Gabriele Meyer zu Hoerste ◽  
Peter Hemmerich
Keyword(s):  
Mammalian Cells ◽  
Constitutive Heterochromatin ◽  
Chromatin Condensation ◽  
Cell Types ◽  
Correlation Spectroscopy ◽  
Chromosomal Protein ◽  
Mitotic Chromosomes ◽  
Heterochromatin Protein 1 ◽  
Stable Conformation ◽  
Fluorescence Correlation

Heterochromatin protein 1 (HP1) is a conserved nonhistone chromosomal protein with functions in euchromatin and heterochromatin. Here we investigated the diffusional behaviors of HP1 isoforms in mammalian cells. Using fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) we found that in interphase cells most HP1 molecules (50–80%) are highly mobile (recovery halftime: t1/2 ≈ 0.9 s; diffusion coefficient: D ≈ 0.6–0.7 μm2 s-1). Twenty to 40% of HP1 molecules appear to be incorporated into stable, slow-moving oligomeric complexes (t1/2 ≈ 10 s), and constitutive heterochromatin of all mammalian cell types analyzed contain 5–7% of very slow HP1 molecules. The amount of very slow HP1 molecules correlated with the chromatin condensation state, mounting to more than 44% in condensed chromatin of transcriptionally silent cells. During mitosis 8–14% of GFP-HP1α, but not the other isoforms, are very slow within pericentromeric heterochromatin, indicating an isoform-specific function of HP1α in heterochromatin of mitotic chromosomes. These data suggest that mobile as well as very slow populations of HP1 may function in concert to maintain a stable conformation of constitutive heterochromatin throughout the cell cycle.

Nuclear organization of pre-mRNA splicing factors and their substrates

1994 ◽  
Vol 52 ◽  
pp. 18-19
Author(s):  
S. Huang ◽  
T. J. Deerinck ◽  
M. H. Ellisman ◽  
D. L. Spector
Keyword(s):  
Mammalian Cells ◽  
Large Body ◽  
Mrna Splicing ◽  
Electron Microscopic Level ◽  
Splicing Factors ◽  
Electron Microscopic ◽  
Speckled Pattern ◽  
Interchromatin Granule Clusters ◽  
Interchromatin Granule ◽  
Perichromatin Fibrils

Previous studies from our laboratory as well as other laboratories have shown that a variety of pre-mRNA splicing factors are localized to a subnuclear speckled domain when mammalian cells are immunolabeled with antibodies against these pre-mRNA splicing factors. At the electron microscopic level the speckled pattern is composed of both interchromatin granule clusters and perichromatin fibrils. A large body of evidence has accumulated from both our laboratory and other laboratories which has suggested that the perichromatin fibrils represent nascent transcripts and the interchromatin granule clusters represent storage and/or assembly sites for pre-mRNA splicing factors. The majority of substrates for these splicing factors are pre-mRNAs which contain a poly(A) tail of approximately 200-300 nucleotides. During the past year we have studied the distribution of poly(A)+ RNA in the mammalian cell nucleus and its transport through nuclear pores by fluorescence and electron microscopic in situ hybridization. Poly(A)+ RNA was detected in the nucleus as a speckled pattern which we have found to totally colocalize with pre-mRNA splicing factors at interchromatin granule clusters and perichromatin fibrils.

scholarly journals Molecular trapping of a fluorescent ceramide analogue at the Golgi apparatus of fixed cells: interaction with endogenous lipids provides a trans-Golgi marker for both light and electron microscopy.

1989 ◽  
Vol 109 (5) ◽  
pp. 2067-2079 ◽  
Author(s):  
R E Pagano ◽  
M A Sepanski ◽  
O C Martin
Keyword(s):  
Golgi Apparatus ◽  
Light And Electron Microscopy ◽  
Cell Types ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Fluorescent Marker ◽  
Cellular Lipids ◽  
Endogenous Lipids ◽  
Fluorescent Lipid

We have previously shown that a fluorescent derivative of ceramide, N-(epsilon-7-nitrobenz-2-oxa-1,3-diazol-4-yl-aminocaproyl)-D-eryth ro-sphingosin e (C6-NBD-Cer), vitally stains the Golgi apparatus of cells (Lipsky, N. G., and R. E. Pagano. 1985. Science (Wash. DC). 228:745-747). In the present paper we demonstrate that C6-NBD-Cer also accumulates at the Golgi apparatus of fixed cells and we explore the mechanism by which this occurs. When human skin fibroblasts were fixed with glutaraldehyde and then incubated with C6-NBD-Cer at 2 degrees C, the fluorescent lipid spontaneously transferred into the cells, labeling the Golgi apparatus as well as other intracellular membranes. Subsequent incubations with defatted BSA at 24 degrees C removed excess C6-NBD-Cer from the cells such that fluorescence was then detected only at the Golgi apparatus. Similar results were obtained using other cell types. A method for visualizing the fluorescent lipid at the electron microscopic level, based on the photoconversion of a fluorescent marker to a diaminobenzidine product (Sandell, J. H., and R. H. Masland, 1988. J. Histochem. Cytochem. 36:555-559), is described and evidence is presented that C6-NBD-Cer was localized to the trans cisternae of the Golgi apparatus. While accumulation occurred in cells fixed in various ways, it was inhibited when fixation protocols that extract or modify cellular lipids were used. In addition, Filipin, which forms complexes with cellular cholesterol, labeled the Golgi apparatus of fixed cells and inhibited accumulation of C6-NBD-Cer at the Golgi apparatus. These results are discussed in terms of a simple model based on the physical properties of C6-NBD-Cer and its interactions with endogenous lipids of the Golgi apparatus. Possible implications of these findings for metabolism and transport of (fluorescent) sphingolipids in vivo are also presented.

scholarly journals Immunohistochemical localization of acetylcholine receptors at human endplates using a monoclonal antibody.

1987 ◽  
Vol 35 (5) ◽  
pp. 613-617 ◽  
Author(s):  
L M Smit ◽  
H Veldman ◽  
F G Jennekens
Keyword(s):  
Monoclonal Antibody ◽  
Acetylcholine Receptors ◽  
Immunohistochemical Localization ◽  
Immunohistochemical Method ◽  
Electron Microscopic Level ◽  
Immunoperoxidase Technique ◽  
Electron Microscopic ◽  
Fixed Tissue ◽  
Alpha Bungarotoxin ◽  
Diagnostic Investigations

We describe a simple indirect immunohistochemical method for localization of acetylcholine receptors (AChR) in motor endplates at the light and electron microscopic level. This method involves the use of a monoclonal antibody directed against the main immunogenic region (MIR) of AChRs and is applicable to periodate-lysine-paraformaldehyde (PLP)-fixed tissue. We discuss the advantages of this method, as compared with the alpha-bungarotoxin-immunoperoxidase technique, and stress its value for diagnostic investigations of motor point biopsies from patients with neuromuscular transmission disorders.

scholarly journals Trypanosoma cruzi: mechanism of entry and intracellular fate in mammalian cells.

1976 ◽  
Vol 143 (6) ◽  
pp. 1402-1420 ◽  
Author(s):  
N Nogueira ◽  
Z Cohn
Keyword(s):  
Plasma Membrane ◽  
Trypanosoma Cruzi ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Cell Types ◽  
Peritoneal Macrophages ◽  
Electron Microscopic ◽  
Cytoplasmic Matrix ◽  
Mode Of Entry ◽  
Intracellular Fate

The mode of entry and intracellular fate of epimastigotes and trypomastigotes of Trypanosoma cruzi in cultured cells was studied. Electron microscopic observations indicated the uptake by phagocytosis of both forms into mouse peritoneal macrophages and of trypomastigotes and transition forms into other cultured cell types. In each instance the organisms were initially surrounded by a plasma membrane-derived phagosome. Trypsin and chymotrypsin treatment of the macrophages completely abolished attachment and ingestion of both forms, indicating that protease-sensitive structures on the macrophage plasma membrane mediate ingestion. The macrophage Fc or C3b receptors were not essential for uptake of T. cruzi in the conditions used. Cytochalasin B inhibited ingestion but not the attachment of both forms by macrophages. Epimastigotes were not taken up by HeLa, L cells, and calf embryo fibroblasts. In macrophages, epimastigotes were killed and digested within phagolysosomes. In contrast, trypomastigotes and transition forms escaped from the phagocytic vacuole and then multiplied in the cytoplasmic matrix. Amastigotes released from infected cells exhibited properties similar to those of trypomastigotes and were able to enter all cell types studied and multiply intracellularly.

scholarly journals Cubic membranes: a structure-based design for DNA uptake

2008 ◽  
Vol 5 (26) ◽  
pp. 1023-1029 ◽  
Author(s):  
Zakaria Almsherqi ◽  
Stephen Hyde ◽  
Malarmathy Ramachandran ◽  
Yuru Deng
Keyword(s):  
Mammalian Cells ◽  
Gene Transfection ◽  
Three Dimensional ◽  
Microscopic Analysis ◽  
Cell Types ◽  
Dna Uptake ◽  
Cell Organelles ◽  
Electron Microscopic ◽  
Culture Cells ◽  
Cubic Membrane

Cubic membranes are soft three-dimensional crystals found within cell organelles in a variety of living systems, despite the aphorism of Fedorov: ‘crystallization is death’. They consist of multi-bilayer lipid–protein stacks, folded onto anticlastic surfaces that resemble triply periodic minimal surfaces, forming highly swollen crystalline sponges. Although cubic membranes have been observed in numerous cell types and under different pathophysiological conditions, knowledge about the formation and potential function(s) of non-lamellar, cubic structures in biological systems is scarce. We report that mitochondria with this cubic membrane organization isolated from starved amoeba Chaos carolinense interact sufficiently with short segments of phosphorothioate oligonucleotides (PS-ODNs) to give significant ODNs uptake. ODNs condensed within the convoluted channels of cubic membrane by an unknown passive targeting mechanism. Moreover, the interaction between ODNs and cubic membrane is sufficient to retard electrophoretic mobility of the ODN component in the gel matrix. These ODN–cubic membrane complexes are readily internalized within the cytoplasm of cultured mammalian cells. Transmission electron microscopic analysis confirms ODNs uptake by cubic membranes and internalization of ODN–cubic membrane complexes into the culture cells. Cubic membranes thus may offer a new, potentially benign medium for gene transfection.

Identification of a 40 × 10(3) Mr centromere-associated protein in cultured mammalian cells

1990 ◽  
Vol 97 (4) ◽  
pp. 705-713
Author(s):  
R. Balczon ◽  
M.A. Accavitti ◽  
B.R. Brinkley
Keyword(s):  
Mammalian Cells ◽  
Cell Types ◽  
Immunoblot Analysis ◽  
Structure And Function ◽  
Interphase Nuclei ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Cytoplasmic Microtubules ◽  
And Function

Monoclonal antibodies were raised against a complex of proteins that was purified following the crosslinking of tubulin to the centromeres of CHO chromosomes using Lomant's reagent. One of the clones, hybridoma 32–9, produced antibodies that reacted with a 40 × 10(3) Mr protein present in the crosslinked complex. Furthermore, immunoblot analysis demonstrated that the 40 × 10(3) Mr antigen was present in various mammalian cell types from several different species. Indirect immunofluorescence using the antibody produced by clone 32–9 demonstrated that the 40 × 10(3) Mr antigen was associated with both spindle and cytoplasmic microtubules. In addition, centromere/kinetochore staining was detected in metaphase-arrested cells, while staining of prekinetochores in interphase nuclei was not observed. Unlike microtubule-associated proteins and microtubule-dependent ATPases, the 40 × 10(3) Mr protein did not copurify with microtubules when tubules were assembled from cellular homogenates using taxol and either GTP or GTP and AMP-PNP. Instead, the 40 × 10(3) Mr protein remained associated with the insoluble cellular material. The 40 × 10(3) Mr antigen could be released from the insoluble pelleted material by extraction with 1 M NaCl. Once solubilized, the 40 × 10(3) Mr protein was able to copurify with microtubules in assembly assays in vitro. This monoclonal antibody should serve as a valuable probe for studies of centromere/kinetochore structure and function.

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