Molecular characterization of volume-sensitive SKCachannels in human liver cell lines

2002 ◽  
Vol 282 (1) ◽  
pp. G116-G122 ◽  
Author(s):  
Richard Roman ◽  
Andrew P. Feranchak ◽  
Marlyn Troetsch ◽  
Jeffrey C. Dunkelberg ◽  
Gordon Kilic ◽  
...  
Keyword(s):  
Cell Volume ◽  
Human Liver ◽  
Fluorescent Protein ◽  
Recombinant Adenovirus ◽  
Chinese Hamster Ovary Cells ◽  
Cell Volume Regulation ◽  
Chinese Hamster ◽  
Protein Fusion ◽  
Fusion Construct ◽  
Human Liver Cell

In human liver, Ca2+-dependent changes in membrane K+permeability play a central role in coordinating functional interactions between membrane transport, metabolism, and cell volume. On the basis of the observation that K+conductance is partially sensitive to the bee venom toxin apamin, we aimed to assess whether small-conductance Ca2+-sensitive K+(SKCa) channels are expressed endogenously and contribute to volume-sensitive K+efflux and cell volume regulation. We isolated a full-length 2,140-bp cDNA (hSK2) highly homologous to rat brain rSK2 cDNA, including the putative apamin-sensitive pore domain, from a human liver cDNA library. Identical cDNAs were isolated from primary human hepatocytes, human HuH-7 hepatoma cells, and human Mz-ChA-1 cholangiocarcinoma cells. Transduction of Chinese hamster ovary cells with a recombinant adenovirus encoding the hSK2-green fluorescent protein fusion construct resulted in expression of functional apamin-sensitive K+channels. In Mz-ChA-1 cells, hypotonic (15% less sodium glutamate) exposure increased K+current density (1.9 ± 0.2 to 37.5 ± 7.1 pA/pF; P < 0.001). Apamin (10–100 nM) inhibited K+current activation and cell volume recovery from swelling. Apamin-sensitive SKCachannels are functionally expressed in liver and biliary epithelia and likely contribute to volume-sensitive changes in membrane K+permeability. Accordingly, the hSK2 protein is a potential target for pharmacological modulation of liver transport and metabolism through effects on membrane K+permeability.

Single-cell imaging of graded Ins(1,4,5)P3 production following G-protein-coupled-receptor activation

2001 ◽  
Vol 356 (1) ◽  
pp. 137-142 ◽  
Author(s):  
Mark S. NASH ◽  
Kenneth W. YOUNG ◽  
Gary B. WILLARS ◽  
R. A. John CHALLISS ◽  
Stefan R. NAHORSKI
Keyword(s):  
Chinese Hamster Ovary ◽  
Metabotropic Glutamate Receptor ◽  
Fluorescent Protein ◽  
Single Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Receptor Activation ◽  
Pleckstrin Homology Domain ◽  
Fusion Construct ◽  
Group I

The pleckstrin homology domain of phospholipase Cδ1 (PHPLCδ) binds Ins(1,4,5)P3 and PtdIns(4,5)P2 specifically, and can be used to detect changes in Ins(1,4,5)P3 in single cells. A fusion construct of PHPLCδ and enhanced green fluorescent protein (EGFP–PHPLCδ) associates with the plasma membrane due to its association with PtdIns(4,5)P2. However, PHPLCδ has greater affinity for Ins(1,4,5)P3 than PtdIns(4,5)P2, and translocates to the cytosol as Ins(1,4,5)P3 levels rise. Prolonged activation of group I metabotropic glutamate receptor 1α expressed in Chinese-hamster ovary cells or endogenous M3 muscarinic receptors in SH-SY5Y neuroblastoma cells gave an initial transient peak in translocation, followed by a sustained plateau phase. This closely followed changes in cell population Ins(1,4,5)P3 mass, but not PtdIns(4,5)P2 levels, which decreased monophasically, as determined by radioreceptor assay. Translocation thus provides a real-time method to follow increases in Ins(1,4,5)P3. Graded changes in Ins(1,4,5)P3 in Chinese-hamster ovary-lac-mGlu1α cells could be detected with increasing glutamate concentrations, and dual loading with fura 2 and EGFP–PHPLCδ showed that changes in intracellular Ca2+ concentration closely paralleled Ins(1,4,5)P3 production. Moreover, Ins(1,4,5)P3 accumulation and intracellular Ca2+ mobilization within single cells is graded in nature and dependent on both agonist concentration and receptor density.

A glycosylated antitumor ether lipid kills cells via paraptosis-like cell death

2009 ◽  
Vol 87 (2) ◽  
pp. 401-414 ◽  
Author(s):  
Pranati Samadder ◽  
Robert Bittman ◽  
Hoe-Sup Byun ◽  
Gilbert Arthur
Keyword(s):  
Cell Death ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ether Lipid ◽  
Protein Fusion ◽  
Protein Marker ◽  
Anticancer Properties ◽  
Lc3 Puncta ◽  
Green Fluorescent

Glycosylated antitumor ether lipids (GAELs) have superior anticancer properties relative to the alkyllysophospholipid class, but there have been no studies of the mechanisms of these compounds. The prototype GAEL, 1-O-hexadecyl-2-O-methyl-3-O-(2′-amino-2′-deoxy-β-d-glucopyranosyl)-sn-glycerol (Gln), effectively killed mouse embryonic fibroblasts (MEFs) lacking key molecules involved in caspase-dependent apoptosis, and cell death was not prevented by caspase inhibitors. Gln did not cause a loss of mitochondrial membrane potential, even in rounded-up dying cells. Gln stimulated the appearance and accumulation of LC3-II, a protein marker for autophagy, in a variety of cells, including wild-type MEFs, but not in MEFs lacking ATG5, a key protein required for autophagy. Gln induced LC3 puncta formation in Chinese hamster ovary cells stably expressing a LC3–green fluorescent protein fusion protein. Thus, Gln appears to induce autophagy. Autophagy was mTOR-independent and was not inhibited by 3-methyladenine or wortmannin. Although Gln is toxic, cellular ability to undergo autophagy was not essential for its toxicity. Furthermore, the GAEL analog 2-deoxy-C-Glc induced LC3 puncta formation but did not kill the cells. Gln, but not 2-deoxy-C-Glc, caused the accumulation of cytoplasmic acidic vacuoles in the cells. Our data suggest that GAELs may activate autophagy; however, GAELs do not kill cells by apoptosis or autophagy but rather by a paraptosis-like cell death mechanism.

scholarly journals α4β1 Integrin Regulates Lamellipodia Protrusion via a Focal Complex/Focal Adhesion-independent Mechanism

2002 ◽  
Vol 13 (9) ◽  
pp. 3203-3217 ◽  
Author(s):  
Karen A. Pinco ◽  
Wei He ◽  
Joy T. Yang
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Time Lapse ◽  
Random Motility ◽  
Protein Fusion ◽  
Green Fluorescent

α4β1 integrin plays an important role in cell migration. We show that when ectopically expressed in Chinese hamster ovary cells, α4β1 is sufficient and required for promoting protrusion of broad lamellipodia in response to scratch-wounding, whereas α5β1 does not have this effect. By time-lapse microscopy of cells expressing an α4/green fluorescent protein fusion protein, we show that α4β1 forms transient puncta at the leading edge of cells that begin to protrude lamellipodia in response to scratch-wounding. The cells expressing a mutant α4/green fluorescent protein that binds paxillin at a reduced level had a faster response to scratch-wounding, forming α4-positive puncta and protruding lamellipodia much earlier. While enhancing lamellipodia protrusion, this mutation reduces random motility of the cells in Transwell assays, indicating that lamellipodia protrusion and random motility are distinct types of motile activities that are differentially regulated by interactions between α4β1 and paxillin. Finally, we show that, at the leading edge, α4-positive puncta and paxillin-positive focal complexes/adhesions do not colocalize, but α4β1 and paxillin colocalize partially in ruffles. These findings provide evidence for a specific role of α4β1 in lamellipodia protrusion that is distinct from the motility-promoting functions of α5β1 and other integrins that mediate cell adhesion and signaling events through focal complexes and focal adhesions.

scholarly journals A Sperm-Associated WD Repeat Protein Orthologous to Chlamydomonas PF20 Associates with Spag6, the Mammalian Orthologue of Chlamydomonas PF16

2002 ◽  
Vol 22 (22) ◽  
pp. 7993-8004 ◽  
Author(s):  
Zhibing Zhang ◽  
Rossana Sapiro ◽  
David Kapfhamer ◽  
Maja Bucan ◽  
Jeff Bray ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Chromosome 1 ◽  
Chromosome 2 ◽  
Flagellar Motility ◽  
Protein Fusion ◽  
Ovary Cells ◽  
Central Apparatus ◽  
Green Fluorescent

ABSTRACT cDNAs were cloned for the murine and human orthologues of Chlamydomonas PF20, a component of the alga axoneme central apparatus that is required for flagellar motility. The mammalian genes encode transcripts of 1.4 and 2.5 kb that are highly expressed in testis. The two transcripts appear to arise from alternative transcription start sites. The murine Pf20 gene was mapped to chromosome 1, syntenic with the location of the human gene on chromosome 2. An antibody generated against an N-terminal sequence of mouse Pf20 recognized a 71-kDa protein in sperm and testis extracts. Immunocytochemistry localized Pf20 to the tails of permeabilized sperm; electron microscope immunocytochemistry showed that Pf20 was located in the axoneme central apparatus. A murine Pf20-green fluorescent protein fusion protein expressed in Chinese hamster ovary cells accumulated in the cytoplasm. When coexpressed with Spag6, the mammalian orthologue of Chlamydomonas PF16, Pf20 was colocalized with Spag6 on polymerized microtubules. Yeast two-hybrid assays demonstrated interaction of the Pf20 WD repeats with Spag6. Pf20 was markedly reduced in sperm collected from mice lacking Spag6, which are infertile due to a motility defect. Our observations provide the first evidence for an association between mammalian orthologues of two Chlamydomonas proteins known to be critical for axoneme structure and function.

scholarly journals Hydrophobic Regions Adjacent to Transmembrane Domains 1 and 5 Are Important for the Targeting of the 70-kDa Peroxisomal Membrane Protein

2007 ◽  
Vol 282 (46) ◽  
pp. 33831-33844 ◽  
Author(s):  
Yoshinori Kashiwayama ◽  
Kota Asahina ◽  
Masashi Morita ◽  
Tsuneo Imanaka
Keyword(s):  
Amino Acid ◽  
Membrane Protein ◽  
Protein Targeting ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Intracellular Localization ◽  
Chinese Hamster ◽  
Transmembrane Domains ◽  
Peroxisomal Membrane ◽  
Peroxisomal Membrane Protein

The 70-kDa peroxisomal membrane protein (PMP70) is a major component of peroxisomal membranes. Human PMP70 consists of 659 amino acid residues and has six putative transmembrane domains (TMDs). PMP70 is synthesized on cytoplasmic ribosomes and targeted posttranslationally to peroxisomes by an unidentified peroxisomal membrane protein targeting signal (mPTS). In this study, to examine the mPTS within PMP70 precisely, we expressed various COOH-terminally or NH2-terminally deleted constructs of PMP70 fused with green fluorescent protein (GFP) in Chinese hamster ovary cells and determined their intracellular localization by immunofluorescence. In the COOH-terminally truncated PMP70, PMP70(AA.1-144)-GFP, including TMD1 and TMD2 of PMP70, was still localized to peroxisomes. However, by further removal of TMD2, PMP70(AA.1-124)-GFP lost the targeting ability, and PMP70(TMD2)-GFP did not target to peroxisomes by itself. The substitution of TMD2 in PMP70(AA.1-144)-GFP for TMD4 or TMD6 did not affect the peroxisomal localization, suggesting that PMP70(AA.1-124) contains the mPTS and an additional TMD is required for the insertion into the peroxisomal membrane. In the NH2-terminal 124-amino acid region, PMP70 possesses hydrophobic segments in the region adjacent to TMD1. By the disruption of these hydrophobic motifs by the mutation of L21Q/L22Q/L23Q or I70N/L71Q, PMP70(AA.1-144)-GFP lost targeting efficiency. The NH2-terminally truncated PMP70, GFP-PMP70(AA.263-375), including TMD5 and TMD6, exhibited the peroxisomal localization. PMP70(AA.263-375) also possesses hydrophobic residues (Ile307/Leu308) in the region adjacent to TMD5, which were important for targeting. These results suggest that PMP70 possesses two distinct targeting signals, and hydrophobic regions adjacent to the first TMD of each region are important for targeting.

Impaired cell volume regulation in Na(+)-H+ exchange-deficient mutants

1989 ◽  
Vol 257 (6) ◽  
pp. C1158-C1165 ◽  
Author(s):  
D. Rotin ◽  
S. Grinstein
Keyword(s):  
Chinese Hamster Ovary ◽  
Volume Regulation ◽  
Chinese Hamster Ovary Cells ◽  
Cell Volume Regulation ◽  
Chinese Hamster ◽  
Cell Types ◽  
Influx Rate ◽  
Volume Increase ◽  
Ovary Cells ◽  
Na Uptake

To elucidate the mechanism of regulatory volume increase (RVI) in Chinese hamster ovary cells, Na(+)-H+ exchange-deficient mutants, called AP-1, were derived from WT-5 cells, a wildtype subclone. The absence of functional antiports in AP-1 cells was established through measurements of intracellular pH (pHi) and Na+ uptake. Cells exposed to hypotonic medium initially swelled but regained near-normal volume within minutes. When isotonicity was then restored, WT-5 cells shrank immediately and then carried out RVI, which was inhibited by 0.1 mM amiloride. This amiloride-sensitive RVI was absent in the AP-1 mutants, suggesting involvement of Na(+)-H+ exchange. In some cell types, RVI is mediated by Na(+)-K(+)-2Cl- cotransport. Bumetanide-sensitive 86Rb+ (K+) influx was detectable in both WT-5 and AP-1 cells, suggesting the presence of Na(+)-K(+)-2Cl- cotransport. Bumetanide-sensitive influx was stimulated by osmotic shrinking in WT-5 cells, and only slightly in AP-1 cells. However, Na(+)-K(+)-2Cl- cotransport did not contribute to volume regulation, since bumetanide (50 microM) failed to inhibit RVI in osmotically shrunken WT-5 cells. The inability of cotransport to induce a volume gain in WT-5 cells was attributable to the simultaneous stimulation of Na(+)-K(+)-2Cl- efflux. The rate of efflux was similar in magnitude to the corresponding influx rate so that net Na(+)-K(+)-2Cl- cotransport was negligible. These results show that RVI in osmotically shrunken Chinese hamster ovary cells is mediated by the Na(+)-H+ antiport and that, although stimulated, Na(+)-K(+)-2Cl- cotransport does not contribute to anisosmotic volume regulation.

scholarly journals Visualization of Phosphoinositides That Bind Pleckstrin Homology Domains: Calcium- and Agonist-induced Dynamic Changes and Relationship to Myo-[3H]inositol-labeled Phosphoinositide Pools

1998 ◽  
Vol 143 (2) ◽  
pp. 501-510 ◽  
Author(s):  
Péter Várnai ◽  
Tamás Balla
Keyword(s):  
Fluorescent Probe ◽  
Fluorescent Protein ◽  
Dynamic Imaging ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Protein Fusion ◽  
Fusion Construct ◽  
Cellular Expression ◽  
Ph Domains ◽  
Green Fluorescent

Phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) pools that bind pleckstrin homology (PH) domains were visualized by cellular expression of a phospholipase C (PLC)δ PH domain–green fluorescent protein fusion construct and analysis of confocal images in living cells. Plasma membrane localization of the fluorescent probe required the presence of three basic residues within the PLCδ PH domain known to form critical contacts with PtdIns(4,5)P2. Activation of endogenous PLCs by ionophores or by receptor stimulation produced rapid redistribution of the fluorescent signal from the membrane to cytosol, which was reversed after Ca2+ chelation. In both ionomycin- and agonist-stimulated cells, fluorescent probe distribution closely correlated with changes in absolute mass of PtdIns(4,5)P2. Inhibition of PtdIns(4,5)P2 synthesis by quercetin or phenylarsine oxide prevented the relocalization of the fluorescent probe to the membranes after Ca2+ chelation in ionomycin-treated cells or during agonist stimulation. In contrast, the synthesis of the PtdIns(4,5)P2 imaged by the PH domain was not sensitive to concentrations of wortmannin that had been found inhibitory of the synthesis of myo-[3H]inositol– labeled PtdIns(4,5)P2. Identification and dynamic imaging of phosphoinositides that interact with PH domains will further our understanding of the regulation of such proteins by inositol phospholipids.

scholarly journals Generation of Transduction-Competent Retroviral Vectors by Infection with a Single Hybrid Vaccinia Virus

2003 ◽  
Vol 77 (12) ◽  
pp. 7017-7025 ◽  
Author(s):  
Christian Konetschny ◽  
Georg W. Holzer ◽  
Carsten Urban ◽  
Thomas Hämmerle ◽  
Josef Mayrhofer ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Host Range ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Retroviral Vectors ◽  
Host Cells ◽  
Rabbit Kidney ◽  
Single Infection ◽  
Broad Host Range

ABSTRACT Recombinant vaccinia viruses that express defective retroviral vectors upon a single infection event in normal host cells were constructed. The gag-pol and envelope genes and a retroviral vector unit were inserted as vaccinia virus promoter-controlled transcription units at three separate loci. The triple recombinant virus was used to infect such diverse cell types as monkey and rabbit kidney, human lung, and primary chicken cells, resulting in the production of transduction-competent defective retroviral vectors. Infection of Chinese hamster ovary cells, which are nonpermissive for vaccinia virus replication, also resulted in production of retroviral vectors and secondary permanent transduction of the host cells. Since vaccinia virus supports the expression of cytotoxic proteins, the vesicular stomatitis virus G glycoprotein could be chosen as the envelope allowing a broad host range of transduction. Functionality of particles was monitored by expression of the green fluorescent protein in transduced 3T3 cell clones. This is the first description of a single chimeric virus encoding and releasing functional retroviral vectors, providing proof of principle of the new concept. No replication-competent retrovirus was detectable by sensitive reverse transcriptase assays. Since vaccinia virus has a broad host range, is extremely robust, and can be obtained at high titers and safe nonreplicating vaccinia virus strains are available, the hybrid system may open new perspectives for gene delivery.

scholarly journals New cell line development for antibody-producing Chinese hamster ovary cells using split green fluorescent protein

2012 ◽  
Vol 12 (1) ◽  
pp. 24 ◽  
Author(s):  
Yeon-Gu Kim ◽  
Byoungwoo Park ◽  
Jung Ahn ◽  
Joon-Ki Jung ◽  
Hong Lee ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Line Development ◽  
Ovary Cells ◽  
New Cell Line ◽  
Green Fluorescent
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