scholarly journals A Chinese hamster ovary cell mutant defective in the non-endocytic uptake of fluorescent analogs of phosphatidylserine: isolation using a cytosol acidification protocol.

1995 ◽  
Vol 128 (5) ◽  
pp. 793-804 ◽  
Author(s):  
K Hanada ◽  
R E Pagano
Keyword(s):  
Plasma Membrane ◽  
Cho Cells ◽  
Screening Method ◽  
Endocytic Pathway ◽  
Ovary Cell ◽  
Acidic Ph ◽  
Intact Cells ◽  
Energy Dependent ◽  
Mutant Cells ◽  
Cytosolic Acidification

Transmembrane movement of phosphatidylserine (PS) and various PS analogs at the plasma membrane is thought to occur by an ATP-dependent, protein-mediated process. To isolate mutant CHO cells defective in this activity, we first obtained conditions which inhibited the endocytic, but not the non-endocytic pathway of lipid internalization since PS may enter cells by a combination of these two pathways. We found that acidic treatment of cells, which blocks clathrin-dependent endocytosis, enhanced the energy-dependent uptake of 1-palmitoyl-2-(6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl -sn- glycero-3-phosphoserine (C6-NBD-PS) in CHO cells from donor vesicles (liposomes) by about twofold. Control experiments demonstrated that the enhanced uptake of C6-NBD-PS at acidic pH was not due to: (a) an increase in the capacity of the plasma membrane to incorporate C6-NBD-PS from the donor vesicles; (b) a decrease in the rate of loss of C6-NBD-PS from the cells; or (c) fusion or engulfment of the donor vesicles. When cytosolic acidification (to pH 6.3) was imposed without acidification of the extracellular medium, C6-NBD-PS uptake by intact cells was increased by about 50% compared to control values determined in the absence of acidification. These results suggested that a protein and energy dependent system(s) for transbilayer movement of the fluorescent PS was stimulated by cytosolic acidification. A screening method for mutant cells defective in the non-endocytic uptake of fluorescent PS analogs with replica cell colonies at acidic pH was then devised. After selection of mutagenized CHO-K1 cells by in situ screening, we obtained a mutant cell line in which uptake of fluorescent PS analogs was reduced to about 25% of the wild type level at either pH 6.0 or 7.4. Control experiments demonstrated that the reduced uptake of fluorescent PS analogs in the mutant cells was unrelated to multidrug resistance, and that endocytosis of another plasma membrane lipid marker occurred normally in the mutant cells. These results suggested that a non-endocytic pathway responsible for uptake of fluorescent PS analogs was specifically affected in the mutant cells.

Effect of altered oligosaccharide structure on the cell surface number, distribution and turnover of the high molecular weight acidic glycoproteins of CHO cells

1984 ◽  
Vol 67 (1) ◽  
pp. 1-23
Author(s):  
L.A. Fitzgerald ◽  
J.B. Denny ◽  
G.A. Baumbach ◽  
C.M. Ketcham ◽  
R.M. Roberts
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
High Molecular Weight ◽  
Cho Cells ◽  
Carbohydrate Structure ◽  
Membrane Glycoproteins ◽  
Number Distribution ◽  
Mutant Lines ◽  
Mutant Cells ◽  
Surface Glycoproteins

The influence of altered carbohydrate structure on the surface number, distribution and turnover of plasma membrane glycoproteins has been studied in Chinese hamster ovary (CHO) cells by comparing three lines that are resistant to the cytotoxic effects of wheat germ agglutinin (WGA) with parental CHO cells. The glycoproteins investigated were members of a group of high molecular weight acidic glycoproteins (HMWAG). On parental cells these represent the major surface components that become labelled by lactoperoxidase-catalysed iodination. They are the only plasma membrane glycoproteins that bind WGA. The mutant lines also possess iodinatable surface polypeptides of high molecular weight, but these were less acidic and electrophoretically less diffuse than those from parental cells. These polypeptides in general did not bind [125I]WGA when two-dimensional polyacrylamide gels were overlaid with iodinated lectin. Mutant cells treated with fluorescein-conjugated WGA showed low surface fluorescence. However, the nuclear envelope and a small region in the perinuclear zone fluoresced strongly. Together, these results confirm that the surface glycoproteins of mutant cells had altered carbohydrate structure. Mouse antiserum prepared against the HMWAG, however, bound equally effectively to the mutant lines as to the parental lines. Indirect immunofluorescence experiments showed that the HMWAG had a fairly uniform distribution over the surface, and that internalization induced by second antibody occurred at a similar rate and in a similar manner in all lines, including the mutants. Electron microscopic observations using immunoperoxidase procedures confirmed the similarities in glycoprotein distribution on mutant and parental cells. Two mouse monoclonal antibodies raised against the HMWAG also revealed no difference in the number or topography of surface glycoproteins. Finally, the half-lives of several HMWAG in a parental and a mutant line (15B) maintained on low-serum medium were compared by means of a 125I/131I double-label technique. Half-lives of HMWAG from the former averaged 12 h and from the latter 11 h. It is concluded that the lack of complex termini on oligosaccharides of this particular group of CHO plasma membrane glycoproteins has no effect on their number, distribution or turnover.

End13p/Vps4p is required for efficient transport from early to late endosomes in Saccharomyces cerevisiae

2001 ◽  
Vol 114 (10) ◽  
pp. 1935-1947 ◽  
Author(s):  
R. Zahn ◽  
B.J. Stevenson ◽  
S. Schroder-Kohne ◽  
B. Zanolari ◽  
H. Riezman ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Dyes ◽  
Membrane Receptors ◽  
Endocytic Pathway ◽  
Aaa Atpase ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Plasma Membrane Receptors ◽  
Vacuolar Protein ◽  
Mutant Cells

end13-1 was isolated in a screen for endocytosis mutants and has been shown to have a post-internalisation defect in endocytic transport as well as a defect in vacuolar protein sorting (Vps(-) phenotype), leading to secretion of newly synthesised vacuolar proteins. Here we demonstrate that END13 is identical to VPS4, encoding an AAA (ATPase associated with a variety of cellular activities)-family ATPase. We also report that the end13-1 mutation is a serine 335 to phenylalanine substitution in the AAA-ATPase domain of End13p/Vps4p. It has been reported that mutant cells lacking End13p/Vps4p (end13(vps4)((Dgr;)) accumulate endocytosed marker dyes, plasma membrane receptors and newly synthesised vacuolar hydrolase precursors in an endosomal compartment adjacent to the vacuole (prevacuolar compartment, or PVC). We find, however, that the end13 mutants have defects in transport of endocytosed fluorescent dyes, plasma membrane receptors and ligands from small peripherally located early endosomes to larger late endosomes, which are often located adjacent to the vacuole. Our results indicate that End13p/Vps4p may play an important role in multiple steps of membrane traffic through the endocytic pathway.

SFV infection in CHO cells: cell-type specific restrictions to productive virus entry at the cell surface

1997 ◽  
Vol 110 (1) ◽  
pp. 95-103 ◽  
Author(s):  
M. Marsh ◽  
R. Bron
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Semliki Forest Virus ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Types ◽  
Endocytic Pathway ◽  
Baby Hamster Kidney

Alphaviruses, such as Semliki Forest virus, normally enter cells by penetration from acidic organelles of the endocytic pathway. The virions are internalised intact from the cell surface before undergoing acid-induced fusion in endosomes. To investigate the possibility that endocytosis might play a role in delivering virions to specific sites for replication, we compared SFV infection of baby hamster kidney (BHK) cells and Chinese hamster ovary (CHO) cells following either normal virus fusion in endosomes or experimentally-induced fusion at the cell surface. Whereas baby hamster kidney cells were infected efficiently following fusion in endosomes or at the plasma membrane, Chinese hamster ovary cells were only infected following fusion from endocytic organelles. Virions fused at the plasma membrane of CHO cells failed to initiate viral RNA and protein synthesis. Similar results were observed when CHO cells were challenged with a rhabdovirus, vesicular stomatitis virus. These data suggest that in certain cell types a barrier, other than the plasma membrane, can prevent infection by alpha- and rhabdoviruses fused at the cell surface. Moreover, they suggest the endocytic pathway provides a mechanism for bringing viral particles to a site, or sites, in the cell where replication can proceed.

Over-Expression of Prolylcarboxypeptidase Enhances Plasma Prekallikrein Activation on Chinese Hamster Ovary Cell Membranes.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3698-3698
Author(s):  
Zia Shariat-Madar ◽  
Ehsan Rahimy ◽  
Fakhri Mahdi ◽  
Alvin H. Schmaier
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Cellular Localization ◽  
Cell Membranes ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Intact Cells ◽  
Transfected Cells ◽  
Over Expression

Abstract Recent investigations indicate that there is a physiologic plasma prekallikrein (PK) activator on the membrane of endothelial cells (HUVEC). PK complexes with high molecular weight kininogen (HK) and HK is PK’s receptor on HUVEC. When assembled on HUVEC, PK is activated to plasma kallikrein independent of FXIIa by the serine protease prolylcarboxypeptidase (PRCP) (Km=9 nM). PRCP has been shown to be a PK activator when isolated from HUVEC (JBC277:17962, 2002) and produced as a recombinant protein (Blood103:4554, 2004). To additionally confirm that human PRCP is a physiologic PK activator, PRCP was over-expressed in Chinese hamster ovary cells (CHO). CHO were transfected with full-length PRCP under the control of a CMV promoter and CHO rPRCP was expressed as a fusion protein with C-terminal enhanced green fluorescent protein (EGFP). The presence of rPRCP in transfected CHO was detected by real time RT-PCR, immunoblot and immunoprecipitation. In CHO cells, PRCP mRNA and PK activation are 2–3-fold higher than controls. The increase in PRCP-induced PK activation on the transfected CHO cells parallels the increase in PRCP antigen expression on the transfected cells, as determined by anti-PRCP and anti-GFP antibodies. PK activation of the transfected cells is blocked by siRNA to the translation initiation site of PRCP, anti-PRCP antibody, and Z-Pro-Pro-aldehyde dimethyl acetate, with IC50 of the last 2 inhibitors of 0.01 and 7.0 mM respectively. Cellular localization of PRCP in intact cells using confocal microscopy and flow cytometry indicate that its over-expression results in its placement on the CHO cell plasma membrane. These investigations independently confirm that prolylcarboxypeptidase is expressed on cell membranes and its expression increases PK activation. The endothelial cell membrane expression of PRCP resulting in PK activation contributes to the constitutive level of bradykinin in the intravascular compartment to regulate blood pressure homeostasis.

scholarly journals Morphology of the Yeast Endocytic Pathway

1998 ◽  
Vol 9 (1) ◽  
pp. 173-189 ◽  
Author(s):  
Cristina Prescianotto-Baschong ◽  
Howard Riezman
Keyword(s):  
Time Course ◽  
Strong Support ◽  
Multivesicular Body ◽  
Endocytic Pathway ◽  
Dependent Manner ◽  
Uniform Size ◽  
Endocytic Vesicles ◽  
Energy Dependent ◽  
Mutant Cells ◽  
Positively Charged

Positively charged Nanogold (Nanoprobes, Stony Brook, NY) has been developed as a new marker to follow the endocytic pathway in yeast. Positively charged Nanogold binds extensively to the surface of yeast spheroplasts and is internalized in an energy-dependent manner. Internalization of gold is blocked in the end3 mutant. During a time course of incubation of yeast spheroplasts with positively charged Nanogold at 15°C, the gold was detected sequentially in small vesicles, a peripheral, vesicular/tubular compartment that we designate as an early endosome, a multivesicular body corresponding to the late endosome near the vacuole, and in the vacuole. Experiments examining endocytosis in the sec18mutant showed an accumulation of positively charged Nanogold in approximately 30–50 nm diameter vesicles. These vesicles most likely represent the primary endocytic vesicles as no other intermediates were detected in the mutant cells, and they correspond in size to the first vesicles detected in wild-type spheroplasts at 15°C. These data lend strong support to the idea that the internalization step of endocytosis in yeast involves formation of small vesicles of uniform size from the plasma membrane.

scholarly journals Singlet oxygen damages the function of Photosystem II in isolated thylakoids and in the green alga Chlorella sorokiniana

2021 ◽  
Author(s):  
Faiza Bashir ◽  
Ateeq Ur Rehman ◽  
Milán Szabó ◽  
Imre Vass
Keyword(s):  
Photosystem Ii ◽  
Singlet Oxygen ◽  
Screening Method ◽  
Physiological Role ◽  
Thylakoid Membranes ◽  
Chlorella Sorokiniana ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Intact Cells ◽  
Green Alga Chlorella

AbstractSinglet oxygen (1O2) is an important damaging agent, which is produced during illumination by the interaction of the triplet excited state pigment molecules with molecular oxygen. In cells of photosynthetic organisms 1O2 is formed primarily in chlorophyll containing complexes, and damages pigments, lipids, proteins and other cellular constituents in their environment. A useful approach to study the physiological role of 1O2 is the utilization of external photosensitizers. In the present study, we employed a multiwell plate-based screening method in combination with chlorophyll fluorescence imaging to characterize the effect of externally produced 1O2 on the photosynthetic activity of isolated thylakoid membranes and intact Chlorella sorokiniana cells. The results show that the external 1O2 produced by the photosensitization reactions of Rose Bengal damages Photosystem II both in isolated thylakoid membranes and in intact cells in a concentration dependent manner indicating that 1O2 plays a significant role in photodamage of Photosystem II.

scholarly journals Analysis of a Chinese hamster ovary cell mutant with defective mobilization of cholesterol from the plasma membrane to the endoplasmic reticulum

1997 ◽  
Vol 38 (10) ◽  
pp. 1973-1987
Author(s):  
N L Jacobs ◽  
B Andemariam ◽  
K W Underwood ◽  
K Panchalingam ◽  
D Sternberg ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Chinese Hamster Ovary Cell ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Cell Mutant

scholarly journals Glycosylation Modulates Plasma Membrane Trafficking of CD24 in Breast Cancer Cells

2021 ◽  
Vol 22 (15) ◽  
pp. 8165
Author(s):  
Amanda Chantziou ◽  
Kostas Theodorakis ◽  
Hara Polioudaki ◽  
Eelco de Bree ◽  
Marilena Kampa ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Plasma Membrane ◽  
Subcellular Localization ◽  
Cancer Cells ◽  
Membrane Trafficking ◽  
Breast Cancer Cells ◽  
Endocytic Pathway ◽  
Cell Periphery ◽  
Wild Type ◽  
Mcf 7

In breast cancer, expression of Cluster of Differentiation 24 (CD24), a small GPI-anchored glycoprotein at the cell periphery, is associated with metastasis and immune escape, while its absence is associated with tumor-initiating capacity. Since the mechanism of CD24 sorting is unknown, we investigated the role of glycosylation in the subcellular localization of CD24. Expression and localization of wild type N36- and/or N52-mutated CD24 were analyzed using immunofluorescence in luminal (MCF-7) and basal B (MDA-MB-231 and Hs578T) breast cancer cells lines, as well as HEK293T cells. Endogenous and exogenously expressed wild type and mutated CD24 were found localized at the plasma membrane and the cytoplasm, but not the nucleoplasm. The cell lines showed different kinetics for the sorting of CD24 through the secretory/endocytic pathway. N-glycosylation, especially at N52, and its processing in the Golgi were critical for the sorting and expression of CD24 at the plasma membrane of HEK293T and basal B type cells, but not of MCF-7 cells. In conclusion, our study highlights the contribution of N-glycosylation for the subcellular localization of CD24. Aberrant N-glycosylation at N52 of CD24 could account for the lack of CD24 expression at the cell surface of basal B breast cancer cells.

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