scholarly journals Real-time measurement of E2:ERα transcriptional activity in living cells

2019 ◽  
Author(s):  
Manuela Cipolletti ◽  
Stefano Leone ◽  
Stefania Bartoloni ◽  
Claudia Busonero ◽  
Filippo Acconcia
Keyword(s):  
Plasma Membrane ◽  
Real Time ◽  
Cell Biology ◽  
Transcriptional Activity ◽  
Receptor Activation ◽  
Living Cells ◽  
Membrane Localization ◽  
Dependent Manner ◽  
Plasma Membrane Localization ◽  
The Impact

AbstractKinetic analyses of diverse physiological processes have the potential to unveil new aspects of the molecular regulation of cell biology at temporal levels. 17β-estradiol (E2) regulates diverse physiological effects by binding to the estrogen receptor α (ERα), which primarily works as a transcription factor. Although many molecular details of the modulation of ERα transcriptional activity have been discovered including the impact of receptor plasma membrane localization and its relative E2-evoked signalling, the knowledge of real-time ERα transcriptional dynamics in living cells is lacking. Here, we report the generation of MCF-7 and HeLa cells stably expressing a modified luciferase under the control of an E2-sensitive promoter, which activity can be continuously monitored in living cells and show that E2 induces a linear increase in ERα transcriptional activity. Ligand-independent (e.g., epidermal growth factor) receptor activation was also detected in a time-dependent manner. Kinetic profiles of ERα transcriptional activity measured in the presence of both receptor antagonists and inhibitors of ERα plasma membrane localization reveals a biphasic dynamic of receptor behaviour underlying novel aspects of receptor-regulated transcriptional effects. Finally, analysis of the rate of the dose-dependent E2 induction of ERα transcriptional activity demonstrates that low doses of E2 induce an effect identical to that determined by high concentrations of E2 as a function of the duration of hormone administration. Overall, we present the characterization of sensitive stable cell lines where to study the kinetic of E2 transcriptional signaling and to identify new aspects of ERα function in different physiological or pathophysiological conditions.

The C2 domains of classical/conventional PKCs are specific PtdIns(4,5)P2-sensing domains

2007 ◽  
Vol 35 (5) ◽  
pp. 1046-1048 ◽  
Author(s):  
S. Corbalán-García ◽  
M. Guerrero-Valero ◽  
C. Marín-Vicente ◽  
J.C. Gómez-Fernández
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Living Cells ◽  
Model Membranes ◽  
Membrane Localization ◽  
Dependent Manner ◽  
C2 Domain ◽  
C2 Domains ◽  
Local Densities

The C2 domains of cPKCs [classical/conventional PKCs (protein kinase Cs)] bind to membranes in a Ca2+-dependent manner and thereby act as cellular Ca2+ effectors. Recent findings have demonstrated that the C2 domain of cPKCs interacts specifically with PtdIns(4,5)P2 through its polybasic cluster located in the β3–β4-strands, this interaction being critical for the membrane localization of these enzymes in living cells. In addition, these C2 domains exhibit higher affinity to bind PtdIns(4,5)P2 than any other polyphosphate phosphatidylinositols. It has also been shown that the presence of PtdIns(4,5)P2 in model membranes decreases the Ca2+ concentration required for classical C2 domains to bind them. Overall, the studies reviewed here suggest a new mechanism of membrane docking by the C2 domains of cPKCs in which the local densities of phosphatidylserine and PtdIns(4,5)P2 on the inner leaflet of the plasma membrane are sufficient to drive Ca2+-activated membrane docking during a physiological Ca2+ signal.

scholarly journals A Dibasic Motif in the Tail of a Class XIV Apicomplexan Myosin Is an Essential Determinant of Plasma Membrane Localization

2000 ◽  
Vol 11 (4) ◽  
pp. 1385-1400 ◽  
Author(s):  
Christine Hettmann ◽  
Angelika Herm ◽  
Ariane Geiter ◽  
Bernd Frank ◽  
Eva Schwarz ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Fluorescent Protein ◽  
Gliding Motility ◽  
Site Directed Mutagenesis ◽  
Membrane Localization ◽  
Membrane Association ◽  
Dependent Manner ◽  
Plasma Membrane Localization ◽  
Arginine Residues

Obligate intracellular parasites of the phylum Apicomplexa exhibit gliding motility, a unique form of substrate-dependent locomotion essential for host cell invasion and shown to involve the parasite actin cytoskeleton and myosin motor(s). Toxoplasma gondii has been shown to express three class XIV myosins, TgM-A, -B, and -C. We identified an additional such myosin, TgM-D, and completed the sequences of a related Plasmodium falciparum myosin, PfM-A. Despite divergent structural features, TgM-A purified from parasites bound actin in an ATP-dependent manner. Isoform-specific antibodies revealed that TgM-A and recombinant mycTgM-A were localized right beneath the plasma membrane, and subcellular fractionation indicated a tight membrane association. Recombinant TgM-D also had a peripheral although not as sharply defined localization. Truncation of their respective tail domains abolished peripheral localization and tight membrane association. Conversely, fusion of the tails to green fluorescent protein (GFP) was sufficient to confer plasma membrane localization and sedimentability. The peripheral localization of TgM-A and of the GFP-tail fusion did not depend on an intact F-actin cytoskeleton, and the GFP chimera did not localize to the plasma membrane of HeLa cells. Finally, we showed that the specific localization determinants were in the very C terminus of the TgM-A tail, and site-directed mutagenesis revealed two essential arginine residues. We discuss the evidence for a proteinaceous plasma membrane receptor and the implications for the invasion process.

scholarly journals Lipid binding by the N-terminal motif mediates plasma membrane localization of Bordetella effector protein BteA

2021 ◽  
pp. 100607
Author(s):  
Ivana Malcova ◽  
Ladislav Bumba ◽  
Filip Uljanic ◽  
Darya Kuzmenko ◽  
Jana Nedomova ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Binding ◽  
Membrane Localization ◽  
Effector Protein ◽  
Plasma Membrane Localization

scholarly journals Palmitoylation of Sindbis Virus TF Protein Regulates Its Plasma Membrane Localization and Subsequent Incorporation into Virions

2016 ◽  
Vol 91 (3) ◽  
Author(s):  
Jolene Ramsey ◽  
Emily C. Renzi ◽  
Randy J. Arnold ◽  
Jonathan C. Trinidad ◽  
Suchetana Mukhopadhyay
Keyword(s):  
Plasma Membrane ◽  
Sindbis Virus ◽  
Molecular Mechanisms ◽  
Wild Type Virus ◽  
Membrane Localization ◽  
Clear Understanding ◽  
Wild Type ◽  
Plasma Membrane Localization ◽  
C Terminus ◽  
N Terminus

ABSTRACT Palmitoylation is a reversible, posttranslational modification that helps target proteins to cellular membranes. The alphavirus small membrane proteins 6K and TF have been reported to be palmitoylated and to positively regulate budding. 6K and TF are isoforms that are identical in their N termini but unique in their C termini due to a −1 ribosomal frameshift during translation. In this study, we used cysteine (Cys) mutants to test differential palmitoylation of the Sindbis virus 6K and TF proteins. We modularly mutated the five Cys residues in the identical N termini of 6K and TF, the four additional Cys residues in TF's unique C terminus, or all nine Cys residues in TF. Using these mutants, we determined that TF palmitoylation occurs primarily in the N terminus. In contrast, 6K is not palmitoylated, even on these shared residues. In the C-terminal Cys mutant, TF protein levels increase both in the cell and in the released virion compared to the wild type. In viruses with the N-terminal Cys residues mutated, TF is much less efficiently localized to the plasma membrane, and it is not incorporated into the virion. The three Cys mutants have minor defects in cell culture growth but a high incidence of abnormal particle morphologies compared to the wild-type virus as determined by transmission electron microscopy. We propose a model where the C terminus of TF modulates the palmitoylation of TF at the N terminus, and palmitoylated TF is preferentially trafficked to the plasma membrane for virus budding. IMPORTANCE Alphaviruses are a reemerging viral cause of arthritogenic disease. Recently, the small 6K and TF proteins of alphaviruses were shown to contribute to virulence in vivo. Nevertheless, a clear understanding of the molecular mechanisms by which either protein acts to promote virus infection is missing. The TF protein is a component of budded virions, and optimal levels of TF correlate positively with wild-type-like particle morphology. In this study, we show that the palmitoylation of TF regulates its localization to the plasma membrane, which is the site of alphavirus budding. Mutants in which TF is not palmitoylated display drastically reduced plasma membrane localization, which effectively prevents TF from participating in budding or being incorporated into virus particles. Investigation of the regulation of TF will aid current efforts in the alphavirus field searching for approaches to mitigate alphaviral disease in humans.

A novel microfluidic microelectrode chip for a significantly enhanced monitoring of NPY-receptor activation in live mode

Lab on a Chip ◽  
2017 ◽  
Vol 17 (24) ◽  
pp. 4294-4302 ◽  
Author(s):  
Franziska D. Zitzmann ◽  
Heinz-Georg Jahnke ◽  
Felix Nitschke ◽  
Annette G. Beck-Sickinger ◽  
Bernd Abel ◽  
...  
Keyword(s):  
Real Time ◽  
Microfluidic Chip ◽  
Microelectrode Array ◽  
Fem Simulation ◽  
Receptor Activation ◽  
Living Cells ◽  
Real Time Monitoring ◽  
Non Invasive ◽  
Npy Receptor ◽  
Simulation Based

We present a FEM simulation based step-by-step development of a microelectrode array integrated into a microfluidic chip for the non-invasive real-time monitoring of living cells.

scholarly journals Systemic Platelet-activating Factor Receptor Activation Augments Experimental Lung Tumor Growth and Metastasis

2014 ◽  
Vol 7 ◽  
pp. CGM.S14501 ◽  
Author(s):  
Patrick C. Hackler ◽  
Sarah Reuss ◽  
Raymond L. Konger ◽  
Jeffrey B. Travers ◽  
Ravi P. Sahu
Keyword(s):  
Lung Cancer ◽  
Tumor Growth ◽  
Cancer Progression ◽  
Lung Tumor ◽  
Platelet Activating Factor ◽  
Receptor Activation ◽  
Dependent Manner ◽  
Melanoma Tumor ◽  
Tumor Growth And Metastasis ◽  
The Impact

Pro-oxidative stressors including cigarette smoke (CS) generate novel lipids with platelet-activated factor-receptor (PAF-R) agonistic activity mediate systemic immunosuppression, one of the most recognized events in promoting carcinogenesis. Our previous studies have established that these oxidized-PAF-R-agonists augment murine B16F10 melanoma tumor growth in a PAF-R-dependent manner because of its effects on host immunity. As CS generates PAF-R agonists, the current studies sought to determine the impact of PAF-R agonists on lung cancer growth and metastasis. Using the murine Lewis Lung Carcinoma (LLC1) model, we demonstrate that treatment of C57BL/6 mice with a PAF-R agonist augments tumor growth and lung metastasis in a PAF-R-dependent manner as these findings were not seen in PAF-R-deficient mice. Importantly, this effect was because of host rather than tumor cells PAF-R dependent as LLC1 cells do not express functional PAF-R. These findings indicate that experimental lung cancer progression can be modulated by the PAF system.

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