scholarly journals Export from Pericentriolar Endocytic Recycling Compartment to Cell Surface Depends on Stable, Detyrosinated (Glu) Microtubules and Kinesin

2002 ◽  
Vol 13 (1) ◽  
pp. 96-109 ◽  
Author(s):  
Sharron X. Lin ◽  
Gregg G. Gundersen ◽  
Frederick R. Maxfield
Keyword(s):  
Elevated Temperature ◽  
Cell Surface ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Temperature Sensitive ◽  
Microtubule Organizing Center ◽  
Endocytic Recycling ◽  
In Cells

A significant fraction of internalized transferrin (Tf) concentrates in the endocytic recycling compartment (ERC), which is near the microtubule-organizing center in many cell types. Tf then recycles back to the cell surface. The mechanisms controlling the localization, morphology, and function of the ERC are not fully understood. We examined the relationship of Tf trafficking with microtubules (MTs), specifically the subset of stable, detyrosinated Glu MTs. We found some correlation between the level of stable Glu MTs and the distribution of the ERC; in cells with low levels of Glu MTs concentrated near to the centriole, the ERC was often tightly clustered, whereas in cells with higher levels of Glu MTs throughout the cell, the ERC was more dispersed. The clustered ERC in Chinese hamster ovary cells became dispersed when the level of Glu MTs was increased with taxol treatment. Furthermore, in a temperature-sensitive Chinese hamster ovary cell line (B104-5), the cells had more Glu MTs when the ERC became dispersed at elevated temperature. Microinjecting purified anti-Glu tubulin antibody into B104-5 cells at elevated temperature induced the redistribution of the ERC to a tight cluster. Microinjection of anti-Glu tubulin antibody slowed recycling of Tf to the cell surface without affecting Tf internalization or delivery to the ERC. Similar inhibition of Tf recycling was caused by microinjecting anti-kinesin antibody. These results suggest that stable Glu MTs and kinesin play a role in the organization of the ERC and in facilitating movement of vesicles from the ERC to the cell surface.

scholarly journals Functional expression of the human transferrin receptor cDNA in Chinese hamster ovary cells deficient in endogenous transferrin receptor.

1987 ◽  
Vol 105 (1) ◽  
pp. 207-214 ◽  
Author(s):  
T E McGraw ◽  
L Greenfield ◽  
F R Maxfield
Keyword(s):  
Cell Surface ◽  
Chinese Hamster Ovary ◽  
Transferrin Receptor ◽  
Diphtheria Toxin ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Functional Expression ◽  
Genetically Engineered ◽  
Ovary Cell ◽  
Ovary Cells

Transferrin (Tf) receptor-variant Chinese hamster ovary cells have been isolated by selection for resistance to two Tf-toxin conjugates. The hybrid toxins contain Tf covalently linked to ricin A chain or a genetically engineered diphtheria toxin fragment. The Tf-receptor-variant (TRV) cells do not have detectable cell-surface Tf receptor; they do not bind fluorescein-Tf or 125I-Tf. TRV cells are at least 100-fold more resistant to the Tf-diphtheria toxin conjugate than are the parent cells. The TRV cells have retained sensitivity to native diphtheria toxin, indicating that the increased resistance to the conjugate is correlated with the loss of Tf binding. The endocytosis of fluorescein-labeled alpha 2-macroglobulin is normal in TRV cells, demonstrating that the defect does not pleiotropically affect endocytosis. Since these cells lack endogenous Tf receptor activity, they are ideally suited for studies of the functional expression of normal or altered Tf receptors introduced into the cells by cDNA transfection. One advantage of this system is that Tf binding and uptake can be used to monitor the behavior of the transfected receptor. A cDNA clone of the human Tf receptor has been transfected into TRV cells. In the stably expressing transfectants, the behavior of the human receptor is very similar to that of the endogenous Chinese hamster ovary cell Tf receptor. Tf binds to cell surface receptors, and is internalized into the para-Golgi region of the cell. Iron is released from Tf, and the apo-Tf and its receptor are recycled back to the cell surface. Thus, the TRV cells can be used to study the behavior of genetically altered Tf receptors in the absence of interfering effects from endogenous receptors.

Surface polypeptides of the Chinese hamster ovary cell; an immunochemical study

1976 ◽  
Vol 54 (2) ◽  
pp. 185-191 ◽  
Author(s):  
M. Behar-Bannelier ◽  
R. L. Juliano
Keyword(s):  
Cell Surface ◽  
Chinese Hamster Ovary Cell ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Immunochemical Study ◽  
Ovary Cells ◽  
Molecular Weights ◽  
Label Technique

Antibodies elicited by the injection of live Chinese hamster ovary cells (CHO) into rabbits precipitated four major components from detergent extracts of CHO membranes. The four components, of molecular weights 200 000, 125 000, 95 000 and 41 000 daltons, corresponded to cell surface components identified by the lactoperoxidase surface label technique.

scholarly journals Isolation of Chinese hamster ovary cell lines temperature conditional for the cell-surface expression of integral membrane glycoproteins.

1989 ◽  
Vol 108 (2) ◽  
pp. 339-353 ◽  
Author(s):  
J Hearing ◽  
E Hunter ◽  
L Rodgers ◽  
M J Gething ◽  
J Sambrook
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Ovary Cell ◽  
Cell Surface Expression ◽  
Membrane Glycoproteins

A procedure is described to select mutants of Chinese hamster ovary cells that are conditionally defective for the cell-surface expression of integral membrane glycoproteins, including the hemagglutinin (HA) of influenza virus. Using a combination of cell sorting and biochemical screening, seven cell lines were obtained that express more cell-surface HA at 32 degrees C than at 39 degrees C. The production of infectious vesicular stomatitis virus, whose growth requires insertion of an integral membrane protein into the plasma membrane, was also temperature conditional in the majority of these mutant cell lines. Five of the lines synthesized apparently normally core-glycosylated HA at the elevated temperature but the protein was neither displayed on the cell surface nor accumulated intracellularly. In these cell lines, little or no terminally glycosylated HA molecules were observed after synthesis at 39 degrees C. By contrast, the core glycosylation of HA and several other integral membrane proteins was abnormal in the remaining two cell lines at both permissive and restrictive temperatures, due to a lesion in a cellular gene(s) that affects the formation of and/or the addition of mannose-rich oligosaccharide chains to newly synthesized polypeptides. Although HA was transported to the plasma membrane at both 32 and 39 degrees C, it did not accumulate on the cell surface at the higher temperature, apparently because of an increased rate of degradation.

Complementation of a methotrexate uptake defect in Chinese hamster ovary cells by DNA-mediated gene transfer

1989 ◽  
Vol 9 (4) ◽  
pp. 1754-1758
Author(s):  
T M Underhill ◽  
W F Flintoff
Keyword(s):  
Gene Transfer ◽  
Dna Sequences ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Wild Type ◽  
Ovary Cells ◽  
Ovary Cell Line ◽  
Human Specific

A methotrexate-resistant Chinese hamster ovary cell line deficient in methotrexate uptake has been complemented to methotrexate sensitivity by transfection with DNA isolated from either wild-type Chinese hamster ovary or human G2 cells. Primary and secondary transfectants regained the ability to take up methotrexate in a manner similar to that of wild-type cells, and in the case of those transfected with human DNA, to contain human-specific DNA sequences. The complementation by DNA-mediated gene transfer of this methotrexate-resistant phenotype provides a basis for the cloning of a gene involved in methotrexate uptake.

Temperature-sensitive DNA mutant of Chinese hamster ovary cells with a thermolabile ribonucleotide reductase activity

1990 ◽  
Vol 10 (11) ◽  
pp. 5688-5699
Author(s):  
B E Wojcik ◽  
J J Dermody ◽  
H L Ozer ◽  
B Mun ◽  
C K Mathews
Keyword(s):  
Dna Synthesis ◽  
Ribonucleotide Reductase ◽  
Chinese Hamster Ovary ◽  
Reductase Activity ◽  
Chinese Hamster ◽  
Temperature Shift ◽  
Ovary Cell ◽  
Temperature Sensitive ◽  
Dntp Pool ◽  
Ribonucleotide Reductase Activity

JB3-B is a Chinese hamster ovary cell mutant previously shown to be temperature sensitive for DNA replication (J. J. Dermody, B. E. Wojcik, H. Du, and H. L. Ozer, Mol. Cell. Biol. 6:4594-4601, 1986). It was chosen for detailed study because of its novel property of inhibiting both polyomavirus and adenovirus DNA synthesis in a temperature-dependent manner. Pulse-labeling studies demonstrated a defect in the rate of adenovirus DNA synthesis. Measurement of deoxyribonucleoside triphosphate (dNTP) pools as a function of time after shift of uninfected cultures from 33 to 39 degrees C revealed that all four dNTP pools declined at similar rates in extracts prepared either from whole cells or from rapidly isolated nuclei. Ribonucleoside triphosphate pools were unaffected by a temperature shift, ruling out the possibility that the mutation affects nucleoside diphosphokinase. However, ribonucleotide reductase activity, as measured in extracts, declined after cell cultures underwent a temperature shift, in parallel with the decline in dNTP pool sizes. Moreover, the activity of cell extracts was thermolabile in vitro, consistent with the model that the JB3-B mutation affects the structural gene for one of the ribonucleotide reductase subunits. The kinetics of dNTP pool size changes after temperature shift are quite distinct from those reported after inhibition of ribonucleotide reductase with hydroxyurea. An indirect effect on ribonucleotide reductase activity in JB3-B has not been excluded since human sequences other than those encoding the enzyme subunits can correct the temperature-sensitive growth defect in the mutant.

Chinese hamster ovary cell mutants defective in the internalization of ricin

1982 ◽  
Vol 2 (5) ◽  
pp. 535-544
Author(s):  
B Ray ◽  
H C Wu
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Electron Microscopic ◽  
Ovary Cells ◽  
Internalization Process ◽  
In The Wild ◽  
Mutant Cells

Chinese hamster ovary mutants simultaneously resistant to ricin and Pseudomonas toxin have been isolated. Two mutant cell lines (4-10 and 11-2) were found to retain normal levels of binding of both ricin and Pseudomonas toxin. They were defective in the internalization of [125I]ricin into the mutant cells, as measured by both a biochemical assay for ricin internalization and electron microscopic autoradiographic studies. Although pretreatment of Chinese hamster ovary cells with a Na+/K+ ionophore, nigericin, resulted in an enhancement of the cytotoxicities of ricin and Pseudomonas toxin in the wild-type Chinese hamster ovary cells, preculture of the mutant cells did not alter the susceptibility of the mutant cells to either toxin. These results provide further evidence that there is a common step in the internalization process for ricin and Pseudomonas toxin.

Moderate-level gene amplification in methotrexate-resistant Chinese hamster ovary cells is accompanied by chromosomal translocations at or near the site of the amplified DHFR gene

1984 ◽  
Vol 4 (1) ◽  
pp. 69-76
Author(s):  
W F Flintoff ◽  
E Livingston ◽  
C Duff ◽  
R G Worton
Keyword(s):  
Gene Amplification ◽  
Structural Gene ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Chromosome 2 ◽  
Dhfr Gene ◽  
Cell Biol ◽  
A Site

In previous studies, we have described several classes of methotrexate-resistant Chinese hamster ovary cell lines. Although the RI class is resistant because of an altered target enzyme, dihydrofolate reductase, the RIII class derived from RI cells is somewhat more resistant because of a moderate amplification of the altered dhfr structural gene (Flintoff et al., Mol. Cell. Biol. 2:275-285, 1982). In one RIII line, a translocation between the short arm (p) of chromosome 2 and the long arm (q) of chromosome 5 was observed, and the amplified RIII gene complex was mapped to the p arm of the 2p-marker chromosome derived from the translocation (Worton et al., Mol. Cell. Biol. 1:330-335, 1981). We tested the hypothesis that chromosomal translocation is a general feature of RIII cells and that such translocation involves a site at or near the dhfr structural gene. Thus, we examined four independently derived RIII-type mutants and found that each had a moderate amplification of the dhfr gene sequences, and karyotype analysis revealed that each carried a translocation involving the 2p arm at or near band 2p25. That this chromosomal rearrangement involves a site near the dhfr locus was demonstrated by mapping the altered but unamplified structural gene coding for the RI phenotype to the short arm of an unaltered chromosome 2. This suggests that a highly specific rearrangement involving an exchange at or near the site of the unamplified gene is a necessary prerequisite for the amplification process. A model for gene amplification involving chromosomal rearrangements and sister chromatid exchange is described.

scholarly journals Isolation of Chinese hamster ovary cells that overproduce asparaginyl-tRNA synthetase.

1984 ◽  
Vol 4 (9) ◽  
pp. 1939-1941
Author(s):  
R E Cirullo ◽  
J J Wasmuth
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Trna Synthetase ◽  
Temperature Sensitive ◽  
Ovary Cells ◽  
Resistant Phenotype ◽  
Lines Of Evidence

Temperature-resistant revertants, derived from the temperature-sensitive CHO asparaginyl-tRNA synthetase mutant, Asn-5, were isolated and characterized. Several lines of evidence indicate that the temperature-resistant phenotype of the revertants is due to their overproducing the same altered enzyme present in the Asn-5 parent.

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