scholarly journals Steric and not structure-specific factors dictate the endocytic mechanism of glycosylphosphatidylinositol-anchored proteins

2009 ◽  
Vol 186 (4) ◽  
pp. 615-628 ◽  
Author(s):  
Pinkesh Bhagatji ◽  
Rania Leventis ◽  
Jonathan Comeau ◽  
Mohammad Refaei ◽  
John R. Silvius
Keyword(s):  
Mammalian Cells ◽  
Protein Targeting ◽  
Folate Receptor ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fundamental Property ◽  
Membrane Lipid ◽  
Endocytic Pathway ◽  
Ovary Cells ◽  
Lipid Markers

Diverse glycosylphosphatidylinositol (GPI)-anchored proteins enter mammalian cells via the clathrin- and dynamin-independent, Arf1-regulated GPI-enriched early endosomal compartment/clathrin-independent carrier endocytic pathway. To characterize the determinants of GPI protein targeting to this pathway, we have used fluorescence microscopic analyses to compare the internalization of artificial lipid-anchored proteins, endogenous membrane proteins, and membrane lipid markers in Chinese hamster ovary cells. Soluble proteins, anchored to cell-inserted saturated or unsaturated phosphatidylethanolamine (PE)-polyethyleneglycols (PEGs), closely resemble the GPI-anchored folate receptor but differ markedly from the transferrin receptor, membrane lipid markers, and even protein-free PE-PEGs, both in their distribution in peripheral endocytic vesicles and in the manner in which their endocytic uptake responds to manipulations of cellular Arf1 or dynamin activity. These findings suggest that the distinctive endocytic targeting of GPI proteins requires neither biospecific recognition of their GPI anchors nor affinity for ordered-lipid microdomains but is determined by a more fundamental property, the steric bulk of the lipid-anchored protein.

Enhancement of the Cytotoxicity of Liposomal Ricin by the Carboxylic Ionophore Monensin and the Lysosomotropic Amine NH4Cl in Chinese Hamster Ovary Cells

2006 ◽  
Vol 25 (5) ◽  
pp. 349-359 ◽  
Author(s):  
Seemha Bharadwaj ◽  
Shailendra Singh Rathore ◽  
Prahlad C. Ghosh
Keyword(s):  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Brefeldin A ◽  
Endocytic Pathway ◽  
Ovary Cells ◽  
Selective Elimination ◽  
Monensin A ◽  
A Chain ◽  
Ionophore Monensin

Ricin was encapsulated in negatively charged liposomes and its effect on the cytotoxicity was compared with native ricin in Chinese hamster ovarian (CHO) cells. The cytotoxicity of ricin, as measured by a marker protein synthesis (incorporation of 3H-leucine), was reduced markedly (300-fold) following encapsulation in liposomes. Lactose, a potent inhibitor of ricin cytotoxicity, had no effect on the binding, internalization, and cytotoxicity of liposomal ricin, indicating that liposomal ricin enter into mammalian cells by an alternative route, bypassing galactose-mediated endocytic pathway. Both monensin (a carboxylic ionophore) and NH4Cl (a lysosomotropic amine) markedly enhances the cytotoxicity of liposomal ricin, indicating endocytotic uptake of liposomal ricin. The degree of potentiation of the cytotoxicity of liposomal ricin by both monensin and NH4Cl was significantly higher (441- and 51-fold) as compared to native ricin (62.5- and 12.5-fold). The extent of exocytosis of free ricin was found to be much higher as compared to liposomal ricin; on the other hand, the extent of degradation of free and liposomal ricin was identical. Consequently, the intracellular level of liposomal ricin was increased to 3.5-fold. This higher level of intracellular liposomal ricin may allow more efficient ricin A-chain release into the cytosol under the influence of NH4Cl and monensin. Monensin-induced potentiation of liposomal ricin was prevented by brefeldin A, indicating that in the presence of monensin, the liposomal ricin was efficiently routed through the Golgi apparatus en route to the cytosol. Thus, liposomal ricin in combination with monensin may have potential application for selective elimination of malignant cells.

scholarly journals Modulation ofN-Ethylmaleimide-sensitive Factor Activity upon Amino Acid Deprivation

2005 ◽  
Vol 280 (16) ◽  
pp. 16219-16226 ◽  
Author(s):  
Hagai Shorer ◽  
Nira Amar ◽  
Ari Meerson ◽  
Zvulun Elazar
Keyword(s):  
Amino Acid ◽  
Mammalian Cells ◽  
Protein Targeting ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Yellow Fluorescent Protein ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Sensitive Factor ◽  
Vesicular Stomatitis

Adaptation of eukaryotic cells to changing environmental conditions entails rapid regulation of protein targeting and transport to specific organelles. Such adaptation is well exemplified in mammalian cells exposed to nitrogen starvation that are triggered to form and transport autophagosomes to lysosomes, thus constituting an inducible intracellular trafficking pathway. Here we investigated the relationship between the general secretory machinery and the autophagic pathway in Chinese hamster ovary cells grown in the absence of amino acid. Utilizing VSVG-YFP (vesicular stomatitis virus G protein fused to yellow fluorescent protein) and norepinephrine as markers for constitutive and regulated exocytosis, respectively, we found that secretion is attenuated in cells grown in media lacking amino acid. Such decrease in exocytosis stems from partial inhibition ofN-ethylmaleimide-sensitive factor ATPase activity, which in turn causes an accumulation of SNARE complexes at both the Golgi apparatus and the plasma membrane of the starved cells. These findings expose a novel cellular strategy to attenuate secretion of proteins under conditions of limited amino acid supply.

scholarly journals Cell cycle-dependent effects on deoxyribonucleotide and DNA labeling by nucleoside precursors in mammalian cells.

1987 ◽  
Vol 7 (1) ◽  
pp. 532-534 ◽  
Author(s):  
J M Leeds ◽  
C K Mathews
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
G1 Phase ◽  
Ovary Cells ◽  
Dna Labeling ◽  
Metabolic Pool ◽  
Specific Activities ◽  
Cycle Dependent

dCTP pools equilibrated to equivalent specific activities in Chinese hamster ovary cells or in nuclei after incubation of cells with radiolabeled nucleosides, indicating that dCTP in nuclei does not constitute a distinct metabolic pool. In the G1 phase, [5-3H]deoxycytidine labeled dCTP to unexpectedly high specific activities. This may explain reports of replication-excluded DNA precursor pools.

scholarly journals A fluorescent lipid analogue can be used to monitor secretory activity and for isolation of mammalian secretion mutants.

1995 ◽  
Vol 6 (2) ◽  
pp. 135-150 ◽  
Author(s):  
N T Ktistakis ◽  
C Y Kao ◽  
R H Wang ◽  
M G Roth
Keyword(s):  
Chinese Hamster Ovary ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Brefeldin A ◽  
Secretory Activity ◽  
Specific Marker ◽  
Ovary Cells ◽  
Fluorescent Lipid

The use of reporter proteins to study the regulation of secretion has often been complicated by posttranslational processing events that influence the secretion of certain proteins, but are not part of the cellular mechanisms that specifically regulate secretion. This has been a particular limitation for the isolation of mammalian secretion mutants, which has typically been a slow process. To provide a reporter of secretory activity independent of protein processing events, cells were labeled with the fluorescent lipid analogue C5-DMB-ceramide (ceramide coupled to the fluorophore boron dipyrromethene difluoride) and its secretion was followed by fluorescence microscopy and fluorescence-activated cell sorting. Brefeldin A, which severely inhibits secretion in Chinese hamster ovary cells, blocked secretion of C5-DMB-ceramide. At high temperature, export of C5-DMB-ceramide was inhibited in HRP-1 cells, which have a conditional defect in secretion. Using C5-DMB-ceramide as a reporter of secretory activity, several different pulse-chase protocols were designed that selected mutant Chinese hamster ovary cells that were resistant to the drug brefeldin A and others that were defective in the transport of glycoproteins to the cell surface. Mutant cells of either type were identified in a mutagenized population at a frequency of 10(-6). Thus, the fluorescent lipid C5-DMB-ceramide can be used as a specific marker of secretory activity, providing an efficient, general approach for isolating mammalian cells with defects in the secretory pathway.

Further Evidence That the Majority of Primary Nuclear RNA Transcripts in Mammalian Cells Do Not Contribute to mRNA

1982 ◽  
Vol 2 (6) ◽  
pp. 701-707
Author(s):  
M. Salditt-Georgieff ◽  
J. E. Darnell
Keyword(s):  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cdna Clones ◽  
Ovary Cells ◽  
Rna Molecules ◽  
Rna Transcripts ◽  
Polyadenylic Acid ◽  
Nuclear Molecules ◽  
Nuclear Rna

Nuclear RNA from Chinese hamster ovary cells was effectively separated into polyadenylic acid [poly(A)]-containing [poly (A) + ] and non-poly(A)-containing [poly(A) − ] fractions so that ∼90% of the poly(A) was present in the (A) + fraction. Only 25% of the 5′-terminal caps of the large nuclear molecules were present in the (A) + class, but about 70% of the specific mRNA sequences (assayed with cDNA clones) were in the (A) + class. It appears that many long capped heterogeneous nuclear RNA molecules are of a different sequence category from those molecules that are successfully processed into mRNA.

scholarly journals Nicotinic acetylcholine receptor is internalized via a Rac-dependent, dynamin-independent endocytic pathway

2008 ◽  
Vol 181 (7) ◽  
pp. 1179-1193 ◽  
Author(s):  
Sudha Kumari ◽  
Virginia Borroni ◽  
Ashutosh Chaudhry ◽  
Baron Chanda ◽  
Ramiro Massol ◽  
...  
Keyword(s):  
Acetylcholine Receptor ◽  
Nicotinic Acetylcholine Receptor ◽  
Actin Polymerization ◽  
Neuromuscular Junctions ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Endocytic Pathway ◽  
Nicotinic Acetylcholine ◽  
Ovary Cells ◽  
Major Mechanism

Endocytosis of the nicotinic acetylcholine receptor (AChR) is a proposed major mechanism of neuromodulation at neuromuscular junctions and in the pathology of synapses in the central nervous system. We show that binding of the competitive antagonist α-bungarotoxin (αBTX) or antibody-mediated cross-linking induces the internalization of cell surface AChR to late endosomes when expressed heterologously in Chinese hamster ovary cells or endogenously in C2C12 myocytes. Internalization occurs via sequestration of AChR–αBTX complexes in narrow, tubular, surface-connected compartments, which are indicated by differential surface accessibility of fluorescently tagged αBTX–AChR complexes to small and large molecules and real-time total internal reflection fluorescence imaging. Internalization occurs in the absence of clathrin, caveolin, or dynamin but requires actin polymerization. αBTX binding triggers c-Src phosphorylation and subsequently activates the Rho guanosine triphosphatase Rac1. Consequently, inhibition of c-Src kinase activity, Rac1 activity, or actin polymerization inhibits internalization via this unusual endocytic mechanism. This pathway may regulate AChR levels at ligand-gated synapses and in pathological conditions such as the autoimmune disease myasthenia gravis.

Choline Chloride Improves the Desiccation Tolerance of Chinese Hamster Ovary Cells

2010 ◽  
Author(s):  
Nilay Chakraborty ◽  
Michael A. Menze ◽  
Heidi Elmoazzen ◽  
Steve C. Hand ◽  
Mehmet Toner
Keyword(s):  
Chinese Hamster Ovary ◽  
Desiccation Tolerance ◽  
Mammalian Cells ◽  
Cell Injury ◽  
Chinese Hamster Ovary Cells ◽  
Choline Chloride ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Sodium Toxicity ◽  
Ionic Imbalance

Recently there has been much interest in using sugars such as trehalose to preserve mammalian cells in a dry state as an alternative to cryopreservation (1–5). However, some studies indicate that sugars alone may not be sufficient to prevent cell injury during drying. Other factors like sodium toxicity, ionic imbalance and pH excursions during dehydration are a few of the mechanisms that have been hypothesized to decrease the viability of mammalian cells. In the present study, we investigated whether or not substituting sodium chloride with choline chloride (2-hydroxy-N, N,N-trimethylethanaminium chloride) in the preservation medium improves desiccation tolerance of Chinese Hamster Ovary (CHO) cells.

scholarly journals Further Evidence That the Majority of Primary Nuclear RNA Transcripts in Mammalian Cells Do Not Contribute to mRNA

1982 ◽  
Vol 2 (6) ◽  
pp. 701-707 ◽  
Author(s):  
M. Salditt-Georgieff ◽  
J. E. Darnell
Keyword(s):  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cdna Clones ◽  
Ovary Cells ◽  
Rna Molecules ◽  
Rna Transcripts ◽  
Polyadenylic Acid ◽  
Nuclear Molecules ◽  
Nuclear Rna

Nuclear RNA from Chinese hamster ovary cells was effectively separated into polyadenylic acid [poly(A)]-containing [poly (A)+] and non-poly(A)-containing [poly(A)−] fractions so that ∼90% of the poly(A) was present in the (A)+fraction. Only 25% of the 5′-terminal caps of the large nuclear molecules were present in the (A)+class, but about 70% of the specific mRNA sequences (assayed with cDNA clones) were in the (A)+class. It appears that many long capped heterogeneous nuclear RNA molecules are of a different sequence category from those molecules that are successfully processed into mRNA.

scholarly journals Alterations of Gene Structure in Ethyl Methane Sulfonate-Induced Mutants of Mammalian Cells

1982 ◽  
Vol 2 (11) ◽  
pp. 1459-1462 ◽  
Author(s):  
Mark Meuth ◽  
Janet E. Arrand
Keyword(s):  
Gene Structure ◽  
Restriction Enzyme ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Alkylating Agents ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Induced Mutants ◽  
Enzyme Cleavage ◽  
Internal Gene

To determine the types of alterations in gene structure induced by DNA-alkylating agents, we analyzed the restriction enzyme cleavage patterns of adenine phosphoribosyltransferase gene sequences in mutant strains of Chinese hamster ovary cells deficient in this enzyme. Base pair changes as detected by loss of restriction enzyme sites were found, but no major internal gene rearrangements could be detected.

α1C (CaV1.2) L-type calcium channel mediates mechanosensitive calcium regulation

2002 ◽  
Vol 283 (3) ◽  
pp. C1001-C1008 ◽  
Author(s):  
Greg L. Lyford ◽  
Peter R. Strege ◽  
Allan Shepard ◽  
Yijun Ou ◽  
Leonid Ermilov ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Calcium Channel ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Calcium Entry ◽  
Intestinal Smooth Muscle ◽  
Ovary Cells ◽  
Hek 293 ◽  
Rich Domain

Smooth muscle exhibits mechanosensitivity independent of neural input, suggesting that mechanosensitive pathways reside within smooth muscle cells. The native L-type calcium current recorded from human intestinal smooth muscle is modulated by stretch. To define mechanosensitive mechanisms involved in the regulation of smooth muscle calcium entry, we cloned the α1C L-type calcium channel subunit (CaV1.2) from human intestinal smooth muscle and expressed the channel in a heterologous system. This channel subunit retained mechanosensitivity when expressed alone or coexpressed with a β2 calcium channel subunit in HEK-293 or Chinese hamster ovary cells. The heterologously expressed human cardiac α1C splice form also demonstrated mechanosensitivity. Inhibition of kinase signaling did not affect mechanosensitivity of the native channel. Truncation of the α1C COOH terminus, which contains an inhibitory domain and a proline-rich domain thought to mediate mechanosensitive signaling from integrins, did not disrupt mechanosensitivity of the expressed channel. These data demonstrate mechanical regulation of calcium entry through molecularly identified L-type calcium channels in mammalian cells and suggest that the mechanosensitivity resides within the pore forming α1C-subunit.

Sign in / Sign up

Export Citation Format

Share Document