Engineering Intracellular Protein Sensors in Mammalian Cells

10.3791/60878 ◽  
2020 ◽  
Author(s):  
Marina Duhkinova ◽  
Claudia Crina ◽  
Ron Weiss ◽  
Velia Siciliano
Keyword(s):  
Mammalian Cells ◽  
Intracellular Protein ◽  
Protein Sensors

scholarly journals The Metabolic Chemical Reporter Ac46AzGal Could Incorporate Intracellular Protein Modification in the Form of UDP-6AzGlc Mediated by OGT and Enzymes in the Leloir Pathway

2021 ◽  
Vol 9 ◽  
Author(s):  
Jiajia Wang ◽  
Biao Dou ◽  
Lu Zheng ◽  
Wei Cao ◽  
Peiyu Dong ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Protein Modification ◽  
Time Dependent ◽  
Dependent Manner ◽  
Intracellular Protein ◽  
Galactose Metabolism ◽  
Naturally Occurring ◽  
Leloir Pathway ◽  
Chemical Reporter ◽  
Complex Glycans

Galactose is a naturally occurring monosaccharide used to build complex glycans that has not been targeted for labeling as a metabolic reporter. Here, we characterize the cellular modification of proteins by using Ac46AzGal in a dose- and time-dependent manner. It is noted that a vast majority of this labeling of Ac46AzGal occurs intracellularly in a range of mammalian cells. We also provided evidence that this labeling is dependent on not only the enzymes of OGT responsible for O-GlcNAcylation but also the enzymes of GALT and GALE in the Leloir pathway. Notably, we discover that Ac46AzGal is not the direct substrate of OGT, and the labeling results may attribute to UDP-6AzGlc after epimerization of UDP-6AzGal via GALE. Together, these discoveries support the conclusion that Ac46AzGal as an analogue of galactose could metabolically label intracellular O-glycosylation modification, raising the possibility of characterization with impaired functions of the galactose metabolism in the Leloir pathway under certain conditions, such as galactosemias.

Intracellular protein degradation in mammalian cells: recent developments

2009 ◽  
Vol 66 (15) ◽  
pp. 2427-2443 ◽  
Author(s):  
Erwin Knecht ◽  
Carmen Aguado ◽  
Jaime Cárcel ◽  
Inmaculada Esteban ◽  
Juan Miguel Esteve ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Mammalian Cells ◽  
Intracellular Protein ◽  
Intracellular Protein Degradation ◽  
Recent Developments

scholarly journals Intracellular protein scaffold-mediated display of random peptide libraries for phenotypic screens in mammalian cells

2001 ◽  
Vol 8 (5) ◽  
pp. 521-534 ◽  
Author(s):  
Beau Peelle ◽  
James Lorens ◽  
Weiqun Li ◽  
Jakob Bogenberger ◽  
Donald G Payan ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Peptide Libraries ◽  
Intracellular Protein ◽  
Protein Scaffold ◽  
Random Peptide ◽  
Random Peptide Libraries

Mammalian Cells Express Two VPS4 Proteins Both of Which are Involved in Intracellular Protein Trafficking

2001 ◽  
Vol 312 (3) ◽  
pp. 469-480 ◽  
Author(s):  
Sibylle Scheuring ◽  
Ralf A. Röhricht ◽  
Britta Schöning-Burkhardt ◽  
Andreas Beyer ◽  
Sibylle Müller ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Mammalian Cells ◽  
Intracellular Protein

Ultrastructural Alterations in Hela Cells Induced by Spider Venom

1967 ◽  
Vol 25 ◽  
pp. 20-21
Author(s):  
Dale E. McClendon ◽  
Paul N. Morgan ◽  
Bernard L. Soloff
Keyword(s):  
Growth Medium ◽  
Mammalian Cells ◽  
Carcinoma Cells ◽  
Venom Protein ◽  
Sterile Saline ◽  
Carbon Coated ◽  
Available Information ◽  
Loxosceles Reclusa ◽  
Essential Growth ◽  
Solution Cultures

It has been observed that minute amounts of venom from the brown recluse spider, Loxosceles reclusa, are capable of producing cytotoxic changes in cultures of certain mammalian cells (Morgan and Felton, 1965). Since there is little available information concerning the effect of venoms on susceptible cells, we have attempted to characterize, at the electron microscope level, the cytotoxic changes produced by the venom of this spider.Cultures of human epithelial carcinoma cells, strain HeLa, were initiated on sterile, carbon coated coverslips contained in Leighton tubes. Each culture was seeded with approximately 1x105 cells contained in 1.5 ml of a modified Eagle's minimum essential growth medium prepared in Hank's balanced salt solution. Cultures were incubated at 36° C. for three days prior to the addition of venom. The venom was collected from female brown recluse spiders and diluted in sterile saline. Protein determinations on the venom-were made according to the spectrophotometric method of Waddell (1956). Approximately 10 μg venom protein per ml of fresh medium was added to each culture after discarding the old growth medium. Control cultures were treated similarly, except that no venom was added. All cultures were reincubated at 36° C.

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

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.

Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

1972 ◽  
Vol 30 ◽  
pp. 350-351
Author(s):  
K. Shankar Narayan ◽  
Kailash C. Gupta ◽  
Tohru Okigaki
Keyword(s):  
Ultraviolet Light ◽  
Mammalian Cells ◽  
Biological Effects ◽  
Survival Rates ◽  
Chinese Hamster ◽  
Cell Types ◽  
Differential Sensitivity ◽  
Ultrastructural Changes ◽  
Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

Androgen-induced plasticity at a “vocal” neuromuscular synapse

1994 ◽  
Vol 52 ◽  
pp. 32-33
Author(s):  
Darcy B. Kelley ◽  
Martha L. Tobias ◽  
Mark Ellisman
Keyword(s):  
Xenopus Laevis ◽  
Motor Neurons ◽  
Sexual Differentiation ◽  
Molecular Basis ◽  
Neuromuscular Synapse ◽  
Sexually Dimorphic ◽  
Intracellular Protein ◽  
Developmental Program ◽  
Androgen Action ◽  
Clawed Frog

Brain and muscle are sexually differentiated tissues in which masculinization is controlled by the secretion of androgens from the testes. Sensitivity to androgen is conferred by the expression of an intracellular protein, the androgen receptor. A central problem of sexual differentiation is thus to understand the cellular and molecular basis of androgen action. We do not understand how hormone occupancy of a receptor translates into an alteration in the developmental program of the target cell. Our studies on sexual differentiation of brain and muscle in Xenopus laevis are designed to explore the molecular basis of androgen induced sexual differentiation by examining how this hormone controls the masculinization of brain and muscle targets.Our approach to this problem has focused on a highly androgen sensitive, sexually dimorphic neuromuscular system: laryngeal muscles and motor neurons of the clawed frog, Xenopus laevis. We have been studying sex differences at a synapse, the laryngeal neuromuscular junction, which mediates sexually dimorphic vocal behavior in Xenopus laevis frogs.

Dissection of the molecular mechanisms of protein targeting to the peroxisome using immunofluorescence and immunocryoelectron microscopies

1990 ◽  
Vol 48 (3) ◽  
pp. 44-45
Author(s):  
G-A. Keller ◽  
S. J. Gould ◽  
S. Subramani ◽  
S. Krisans
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Protein Targeting ◽  
Firefly Luciferase ◽  
Peroxisomal Enzyme ◽  
Transfected Cells ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Series Of Experiments ◽  
Peroxisomal Targeting Signal

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

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