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 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

scholarly journals Expression of TRPC3 in Chinese Hamster Ovary Cells Results in Calcium-activated Cation Currents Not Related to Store Depletion

1997 ◽  
Vol 138 (6) ◽  
pp. 1333-1341 ◽  
Author(s):  
Christof Zitt ◽  
Alexander G. Obukhov ◽  
Carsten Strübing ◽  
Andrea Zobel ◽  
Frank Kalkbrenner ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Membrane Receptors ◽  
Calcium Stores ◽  
Channel Activity ◽  
Ovary Cells ◽  
Cytosolic Side ◽  
Green Fluorescent ◽  
Inside Out

TRPC3 (or Htrp3) is a human member of the trp family of Ca2+-permeable cation channels. Since expression of TRPC3 cDNA results in markedly enhanced Ca2+ influx in response to stimulation of membrane receptors linked to phospholipase C (Zhu, X., J. Meisheng, M. Peyton, G. Bouley, R. Hurst, E. Stefani, and L. Birnbaumer. 1996. Cell. 85:661–671), we tested whether TRPC3 might represent a Ca2+ entry pathway activated as a consequence of depletion of intracellular calcium stores. CHO cells expressing TRPC3 after intranuclear injection of cDNA coding for TRPC3 were identified by fluorescence from green fluorescent protein. Expression of TRPC3 produced cation currents with little selectivity for Ca2+ over Na+. These currents were constitutively active, not enhanced by depletion of calcium stores with inositol-1,4,5-trisphosphate or thapsigargin, and attenuated by strong intracellular Ca2+ buffering. Ionomycin led to profound increases of currents, but this effect was strictly dependent on the presence of extracellular Ca2+. Likewise, infusion of Ca2+ into cell through the patch pipette increased TRPC3 currents. Therefore, TRPC3 is stimulated by a Ca2+-dependent mechanism. Studies on TRPC3 in inside-out patches showed cation-selective channels with 60-pS conductance and short (<2 ms) mean open times. Application of ionomycin to cells increased channel activity in cell-attached patches. Increasing the Ca2+ concentration on the cytosolic side of inside-out patches (from 0 to 1 and 30 μM), however, failed to stimulate channel activity, even in the presence of calmodulin (0.2 μM). We conclude that TRPC3 codes for a Ca2+-permeable channel that supports Ca2+-induced Ca2+-entry but should not be considered store operated.

scholarly journals A single-wavelength flow cytometric approach using redox-sensitive green fluorescent protein probes for measuring redox stress in live cells

BioTechniques ◽  
2021 ◽  
Author(s):  
Elizabeth R Denn ◽  
Joseph M Schober
Keyword(s):  
Oxidative Stress ◽  
Green Fluorescent Protein ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Recovery Phase ◽  
Live Cells ◽  
Redox Stress ◽  
Green Fluorescent

Cellular redox changes are common in apoptosis, immune function, signaling pathways and cancer. The authors aimed to develop a single-wavelength method using the superior fluorescence sensitivity of a flow cytometer for measuring redox-sensitive green fluorescent protein signal during oxidative stress in cell lines. The single-wavelength method was able to discern small differences in oxidative stress between cell lines and between the cytoplasmic and mitochondrial compartments within the same cell line. In Chinese hamster ovary cells, the mitochondrial matrix compartment was more sensitive to oxidative stress compared with MDA-MB-231 cells, and the rapid changes in redox state were followed by a slow recovery phase. The authors conclude that this simplified method is useful and preferred for studies where alterations in overall redox-sensitive green fluorescent protein expression are controlled.

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.

Cholesterol accumulation inhibits ER to Golgi transport and protein secretion: studies of apolipoprotein E and VSVGt

2012 ◽  
Vol 447 (1) ◽  
pp. 51-60 ◽  
Author(s):  
Maaike Kockx ◽  
Donna L. Dinnes ◽  
Kuan-Yen Huang ◽  
Laura J. Sharpe ◽  
Wendy Jessup ◽  
...  
Keyword(s):  
Apolipoprotein E ◽  
Protein Trafficking ◽  
Protein Transport ◽  
Fluorescent Protein ◽  
Regulatory Element ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cholesterol Accumulation ◽  
Golgi Transport ◽  
Green Fluorescent

Cholesterol excess is typical of various diseases including atherosclerosis. We have investigated whether cholesterol accumulation in the ER (endoplasmic reticulum) can inhibit exit of vesicular cargo and secretion of proteins by studying apoE (apolipoprotein E), a significant glycoprotein in human health and disease. CHO (Chinese hamster ovary) cells expressing human apoE under a cholesterol-independent promoter incubated with cholesterol–cyclodextrin complexes showed increased levels of cellular free and esterified cholesterol, inhibition of SREBP-2 (sterol-regulatory-element-binding protein 2) processing, and a mild induction of ER stress, indicating significant accumulation of cholesterol in the ER. Secretion of apoE was markedly inhibited by cholesterol accumulation, and similar effects were observed in cells enriched with lipoprotein-derived cholesterol and in primary human macrophages. Removal of excess cholesterol by a cyclodextrin vehicle restored apoE secretion, indicating that the transport defect was reversible. That cholesterol impaired protein trafficking was supported by the cellular accumulation of less sialylated apoE glycoforms, and by direct visualization of altered ER to Golgi transport of thermo-reversible VSVG (vesicular stomatitis virus glycoprotein) linked to GFP (green fluorescent protein). We conclude that intracellular accumulation of cholesterol in the ER reversibly inhibits protein transport and secretion. Strategies to correct ER cholesterol may restore homoeostatic processes and intracellular protein transport in conditions characterized by cholesterol excess.

Hands-on experiments on glycemia regulation and type 1 diabetes

2015 ◽  
Vol 39 (3) ◽  
pp. 232-239 ◽  
Author(s):  
M. Mingueneau ◽  
A. Chaix ◽  
N. Scotti ◽  
J. Chaix ◽  
A. Reynders ◽  
...  
Keyword(s):  
Type 1 Diabetes ◽  
Green Fluorescent Protein ◽  
Chinese Hamster Ovary ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Pancreatic Cells ◽  
Green Fluorescent

In the present article, we describe a 3-day experimental workshop on glycemia regulation and type 1 diabetes that engages students in open-ended investigations and guided experiments leading to results that are not already known to them. After an initial questioning phase during which students observe PowerPoint slides depicting the glycemia (blood glucose levels) of individuals in various situations, students design, execute, and interpret experiments to address one of the following questions: 1) Which criteria must an animal model of diabetes fulfill? 2) How do pancreatic cells maintain glycemia constant? and 3) Is there a way to produce an insulin protein similar to the one released by human pancreatic cells? Students then 1) measure glycemia and glycosuria in control mice and in a mouse model of type 1 diabetes (Alloxan-treated mice), 2) measure the release of insulin by pancreatic β-cells (INS-1 cell line) in response to different concentrations of glucose in the extracellular medium, and 3) transfect Chinese hamster ovary cells with a plasmid coding for green fluorescent protein, observe green fluorescent protein fluorescence of some of the transfected Chinese hamster ovary cells under the microscope, and observe the characteristics of human insulin protein and its three-dimensional conformation using RASMOL software. At the end of the experimental session, students make posters and present their work to researchers. Back at school, they may also present their work to their colleagues.

scholarly journals Diffusion of a soluble protein, photoactivatable GFP, through a sensory cilium

2010 ◽  
Vol 135 (3) ◽  
pp. 173-196 ◽  
Author(s):  
Peter D. Calvert ◽  
William E. Schiesser ◽  
Edward N. Pugh
Keyword(s):  
Cell Function ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Multiphoton Microscopy ◽  
Chinese Hamster ◽  
Optical Resolution ◽  
Protein Diffusion ◽  
Major Barrier ◽  
Axial Diffusion ◽  
Green Fluorescent

Transport of proteins to and from cilia is crucial for normal cell function and survival, and interruption of transport has been implicated in degenerative and neoplastic diseases. It has been hypothesized that the ciliary axoneme and structures adjacent to and including the basal bodies of cilia impose selective barriers to the movement of proteins into and out of the cilium. To examine this hypothesis, using confocal and multiphoton microscopy we determined the mobility of the highly soluble photoactivatable green fluorescent protein (PAGFP) in the connecting cilium (CC) of live Xenopus retinal rod photoreceptors, and in the contiguous subcellular compartments bridged by the CC, the inner segment (IS) and the outer segment (OS). The estimated axial diffusion coefficients are DCC = 2.8 ± 0.3, DIS = 5.2 ± 0.6, and DOS = 0.079 ± 0.009 µm2 s−1. The results establish that the CC does not pose a major barrier to protein diffusion within the rod cell. However, the results also reveal that axial diffusion in each of the rod’s compartments is substantially retarded relative to aqueous solution: the axial diffusion of PAGFP was retarded ∼18-, 32- and 1,000-fold in the IS, CC, and OS, respectively, with ∼20-fold of the reduction in the OS attributable to tortuosity imposed by the lamellar disc membranes. Previous investigation of PAGFP diffusion in passed, spherical Chinese hamster ovary cells yielded DCHO = 20 µm2 s−1, and estimating cytoplasmic viscosity as Daq/DCHO = 4.5, the residual 3- to 10-fold reduction in PAGFP diffusion is ascribed to sub-optical resolution structures in the IS, CC, and OS compartments.

scholarly journals Asc Modulates the Function of NLRC4 in Response to Infection of Macrophages by Legionella pneumophila

mBio ◽  
2011 ◽  
Vol 2 (4) ◽  
Author(s):  
Christopher L. Case ◽  
Craig R. Roy
Keyword(s):  
Cell Death ◽  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Microbial Infection ◽  
Content Type ◽  
Caspase 1 ◽  
Cell Death Pathway ◽  
Dependent Cell ◽  
Green Fluorescent ◽  
In Cells

ABSTRACTNucleotide-binding domain, leucine-rich repeat containing proteins (NLRs) activate caspase-1 in response to a variety of bacterium-derived signals in macrophages. NLR-mediated activation of caspase-1 byLegionella pneumophilaoccurs through both an NLRC4/NAIP5-dependent pathway and a pathway requiring the adapter protein Asc. Both pathways are needed for maximal activation of caspase-1 and for the release of the cytokines interleukin-1β (IL-1β) and IL-18. Asc is not required for caspase-1-dependent pore formation and cell death induced upon infection of macrophages byL. pneumophila. Here, temporal and spatial localization of caspase-1-dependent processes was examined to better define the roles of Asc and NLRC4 during infection. Imaging studies revealed that caspase-1 localized to a single punctate structure in infected cells containing Asc but not in cells lacking this adapter. Both endogenous Asc and ectopically produced NLRC4 tagged with green fluorescent protein (GFP) were found to localize to caspase-1 puncta followingL. pneumophilainfection, suggesting that NLRC4 and Asc coordinate signaling through this complex during caspase-1 activation. Formation of caspase-1-containing puncta correlated with caspase-1 processing, suggesting a role for the Asc/NLRC4/caspase-1 complex in caspase-1 cleavage. In cells deficient for Asc, NLRC4 did not assemble into discrete puncta, and pyroptosis occurred at an accelerated rate. These data indicate that Asc mediates integration of NLR components into caspase-1 processing platforms and that recruitment of NLR components into an Asc complex can dampen pyroptotic responses. Thus, a negative feedback role of complexes containing Asc may be important for regulating caspase-1-mediated responses during microbial infection.IMPORTANCECaspase-1 is a protease activated during infection that is central to the regulation of several innate immune pathways. Studies examining the macromolecular complexes containing this protein, known as inflammasomes, have provided insight into the regulation of this protease. This work demonstrates that the intracellular bacteriumLegionella pneumophilainduces formation of complexes containing caspase-1 by multiple mechanisms and illustrates that an adapter molecule called Asc integrates signals from multiple independent upstream caspase-1 activators in order to assemble a spatially distinct complex in the macrophage. There were caspase-1-associated activities such as cytokine processing and secretion that were controlled by Asc. Importantly, this work uncovered a new role for Asc in dampening a caspase-1-dependent cell death pathway called pyroptosis. These findings suggest that Asc plays a central role in controlling a distinct subset of caspase-1-dependent activities by both assembling complexes that are important for cytokine processing and suppressing processes that mediate pyroptosis.

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