scholarly journals The effect of MEP pathway and other inhibitors on the intracellular localization of a plasma membrane-targeted, isoprenylable GFP reporter protein in tobacco BY-2 cells

F1000Research ◽  
2013 ◽  
Vol 2 ◽  
pp. 170 ◽  
Author(s):  
Michael Hartmann ◽  
Andrea Hemmerlin ◽  
Elisabet Gas-Pascual ◽  
Esther Gerber ◽  
Denis Tritsch ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Intracellular Localization ◽  
Test System ◽  
Mep Pathway ◽  
Reporter Protein ◽  
Visualization System ◽  
Gfp Reporter ◽  
Carboxy Terminal ◽  
The Impact

We have established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, based on the expression of a dexamethasone-inducible GFP fused to the carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with known inhibitors like oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect the localization. During the present work, this test system has been used to examine the effect of newly designed inhibitors of the MEP pathway and inhibitors of sterol biosynthesis such as squalestatin, terbinafine and Ro48-8071. In addition, we also studied the impact of different post-prenylation inhibitors or those suspected to affect the transport of proteins to the plasma membrane on the localization of the geranylgeranylable fusion protein GFP-BD-CVIL.

scholarly journals The effect of MEP pathway and other inhibitors on the intracellular localization of a plasma membrane-targeted, isoprenylable GFP reporter protein in tobacco BY-2 cells

F1000Research ◽  
2013 ◽  
Vol 2 ◽  
pp. 170 ◽  
Author(s):  
Michael Hartmann ◽  
Andrea Hemmerlin ◽  
Elisabet Gas-Pascual ◽  
Esther Gerber ◽  
Denis Tritsch ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Intracellular Localization ◽  
Test System ◽  
Mep Pathway ◽  
Reporter Protein ◽  
Visualization System ◽  
Gfp Reporter ◽  
Carboxy Terminal ◽  
The Impact

We have established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, based on the expression of a dexamethasone-inducible GFP fused to the carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with known inhibitors like oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect the localization. During the present work, this test system has been used to examine the effect of newly designed inhibitors of the MEP pathway and inhibitors of sterol biosynthesis such as squalestatin, terbinafine and Ro48-8071. In addition, we also studied the impact of different post-prenylation inhibitors or those suspected to affect the transport of proteins to the plasma membrane on the localization of the geranylgeranylable fusion protein GFP-BD-CVIL.

scholarly journals Development of an image-based screening system for inhibitors of the plastidial MEP pathway and of protein geranylgeranylation

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 14
Author(s):  
Michael Hartmann ◽  
Elisabet Gas-Pascual ◽  
Andrea Hemmerlin ◽  
Michel Rohmer ◽  
Thomas J. Bach
Keyword(s):  
Cell Line ◽  
High Throughput Screening ◽  
Test System ◽  
Mep Pathway ◽  
Screening System ◽  
Visualization System ◽  
Microtiter Plates ◽  
Geranylgeranyl Transferase ◽  
Carboxy Terminal

We have recently established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, which involves expressing a dexamethasone-inducible GFP fused to the prenylable, carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with oxoclomazone and fosmidomycin, as well as inhibition of protein geranylgeranyl transferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect this localization. Furthermore, complementation assays with pathway-specific intermediates confirmed that the precursors for the cytosolic isoprenylation of this fusion protein are predominantly provided by the MEP pathway. In order to optimize this visualization system from a more qualitative assay to a statistically trustable medium or a high-throughput screening system, we established new conditions that permit culture and analysis in 96-well microtiter plates, followed by fluorescence microscopy. For further refinement, the existing GFP-BD-CVIL cell line was transformed with an estradiol-inducible vector driving the expression of a RFP protein, C-terminally fused to a nuclear localization signal (NLS-RFP). We are thus able to quantify the total number of viable cells versus the number of inhibited cells after various treatments. This approach also includes a semi-automatic counting system, based on the freely available image processing software. As a result, the time of image analysis as well as the risk of user-generated bias is reduced to a minimum. Moreover, there is no cross-induction of gene expression by dexamethasone and estradiol, which is an important prerequisite for this test system.

scholarly journals Development of an image-based screening system for inhibitors of the plastidial MEP pathway and of protein geranylgeranylation

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 14
Author(s):  
Michael Hartmann ◽  
Elisabet Gas-Pascual ◽  
Andrea Hemmerlin ◽  
Michel Rohmer ◽  
Thomas J. Bach
Keyword(s):  
Cell Line ◽  
High Throughput Screening ◽  
Initial Study ◽  
Test System ◽  
Mep Pathway ◽  
Screening System ◽  
Visualization System ◽  
Microtiter Plates ◽  
Geranylgeranyl Transferase

In a preceding study we have recently established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, which involves expressing a dexamethasone-inducible GFP fused to the prenylable, carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was there demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with oxoclomazone and fosmidomycin, as well as inhibition of protein geranylgeranyl transferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect this localization. Furthermore, in this initial study complementation assays with pathway-specific intermediates confirmed that the precursors for the cytosolic isoprenylation of this fusion protein are predominantly provided by the MEP pathway. In order to optimize this visualization system from a more qualitative assay to a statistically trustable medium or a high-throughput screening system, we established now new conditions that permit culture and analysis in 96-well microtiter plates, followed by fluorescence microscopy. For further refinement, the existing GFP-BD-CVIL cell line was transformed with an estradiol-inducible vector driving the expression of a RFP protein, C-terminally fused to a nuclear localization signal (NLS-RFP). We are thus able to quantify the total number of viable cells versus the number of inhibited cells after various treatments. This approach also includes a semi-automatic counting system, based on the freely available image processing software. As a result, the time of image analysis as well as the risk of user-generated bias is reduced to a minimum. Moreover, there is no cross-induction of gene expression by dexamethasone and estradiol, which is an important prerequisite for this test system.

Development of a biosensor test system with GFP reporter protein for detection of DNA damages

2006 ◽  
Vol 141 (1) ◽  
pp. 33-35 ◽  
Author(s):  
I. A. Lavrinenko ◽  
V. A. Lavrinenko ◽  
A. V. Ryabchenko ◽  
A. B. Beklemishev
Keyword(s):  
Test System ◽  
Reporter Protein ◽  
Dna Damages ◽  
Gfp Reporter

Coordination of metabolism and intracellular acid–base status: ionic regulation and metabolic consequences

1989 ◽  
Vol 67 (12) ◽  
pp. 2994-3004 ◽  
Author(s):  
Patrick J. Walsh ◽  
C. Louise Milligan
Keyword(s):  
Plasma Membrane ◽  
Intracellular Ph ◽  
Carbon Dioxide Tension ◽  
Animal Cells ◽  
Acid Base ◽  
Metabolic Compensation ◽  
Acids And Bases ◽  
Quantitative Contribution ◽  
The Impact

This review discusses the mechanisms by which animal cells regulate intracellular pH (pHi), the variations in pHi encountered in vivo, and the impact that variations in pHi (and other acid–base variables) have on metabolism. Cells regulate pHi by a combination of (i) physicochemical buffering by intracellular components; (ii) transport of acids and bases across the plasma membrane; and (iii) production and consumption of acids and bases by metabolism. Ionic transport is by far the best studied of these three mechanisms, and several specific plasma membrane exchangers (e.g., Na+–H+ exchange) are important regulators of pHi The precise quantitative contribution of the other two mechanisms to pHi regulation awaits further study. Intracellular pH variations in vivo can be substantial (i.e., up to 1 unit in some cases) and can lead to marked changes in metabolism. Furthermore, changes in carbon dioxide tension and bicarbonate concentration can also affect metabolism. Catecholamines appear to be important regulatory signals in metabolic compensation for acid–base perturbations, but in some cases acid–base disturbances may produce adaptive metabolic changes directly.

scholarly journals Utilization of green fluorescent protein as a marker for studying the expression and turnover of the monocarboxylate permease Jen1p of Saccharomyces cerevisiae

2002 ◽  
Vol 363 (3) ◽  
pp. 737-744 ◽  
Author(s):  
Sandra PAIVA ◽  
Arthur L. KRUCKEBERG ◽  
Margarida CASAL
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Reporter Protein ◽  
C Terminus ◽  
Lactate Transporter ◽  
Green Fluorescent ◽  
Endocytic Pathways

Green fluorescent protein (GFP) from Aequorea victoria was used as an in vivo reporter protein when fused to the C-terminus of the Jen1 lactate permease of Saccharomyces cerevisiae. The Jen1 protein tagged with GFP is a functional lactate transporter with a cellular abundance of 1670 molecules/cell, and a catalytic-centre activity of 123s−1. It is expressed and tagged to the plasma membrane under induction conditions. The factors involved in proper localization and turnover of Jen1p were revealed by expression of the Jen1p—GFP fusion protein in a set of strains bearing mutations in specific steps of the secretory and endocytic pathways. The chimaeric protein Jen1p—GFP is targeted to the plasma membrane via a Sec6-dependent process; upon treatment with glucose, it is endocytosed via END3 and targeted for degradation in the vacuole. Experiments performed in a Δdoa4 mutant strain showed that ubiquitination is associated with the turnover of the permease.

scholarly journals Anion channel sensitivity to cytosolic organic acids implicates a central role for oxaloacetate in integrating ion flux with metabolism in stomatal guard cells

2011 ◽  
Vol 439 (1) ◽  
pp. 161-170 ◽  
Author(s):  
Yizhou Wang ◽  
Michael R. Blatt
Keyword(s):  
Plasma Membrane ◽  
Organic Acids ◽  
Organic Acid ◽  
Stomatal Closure ◽  
Anion Channel ◽  
Guard Cells ◽  
Solute Flux ◽  
Stomatal Guard Cells ◽  
The Impact

Stomatal guard cells play a key role in gas exchange for photosynthesis and in minimizing transpirational water loss from plants by opening and closing the stomatal pore. The bulk of the osmotic content driving stomatal movements depends on ionic fluxes across both the plasma membrane and tonoplast, the metabolism of organic acids, primarily Mal (malate), and its accumulation and loss. Anion channels at the plasma membrane are thought to comprise a major pathway for Mal efflux during stomatal closure, implicating their key role in linking solute flux with metabolism. Nonetheless, little is known of the regulation of anion channel current (ICl) by cytosolic Mal or its immediate metabolite OAA (oxaloacetate). In the present study, we have examined the impact of Mal, OAA and of the monocarboxylic acid anion acetate in guard cells of Vicia faba L. and report that all three organic acids affect ICl, but with markedly different characteristics and sidedness to their activities. Most prominent was a suppression of ICl by OAA within the physiological range of concentrations found in vivo. These findings indicate a capacity for OAA to co-ordinate organic acid metabolism with ICl through the direct effect of organic acid pool size. The findings of the present study also add perspective to in vivo recordings using acetate-based electrolytes.

scholarly journals Using a mouse-adapted A/HK/01/68 influenza virus to analyse the impact of NS1 evolution in codons 196 and 231 on viral replication and virulence

2020 ◽  
Vol 101 (6) ◽  
pp. 587-598
Author(s):  
Sebastian Eiden ◽  
Ronald Dijkman ◽  
Roland Zell ◽  
Jonas Fuchs ◽  
Georg Kochs
Keyword(s):  
Amino Acids ◽  
Viral Replication ◽  
Seasonal Influenza ◽  
Influenza Viruses ◽  
Coding Region ◽  
Fitness Advantage ◽  
Interferon Induction ◽  
Carboxy Terminal ◽  
The Impact

Seasonal influenza viruses circulating between 1918 and 2009 harboured two prevalent genetic variations in the NS1 coding region. A glutamic acid (E)-to-lysine (K) exchange at position 196 was reported to diminish the capacity of NS1 to control interferon induction. Furthermore, alterations at position 231 determine a carboxy-terminal extension of seven amino acids from 230 to 237 residues. Sequence analyses of NS1 of the last 90 years suggest that variations at these two positions are functionally linked. To determine the impact of the two positions on viral replication in vivo, we used a mouse-adapted variant of A/Hong Kong/01/68 (maHK68) (H3N2). maHK68 encodes an NS1 of 237 amino acids with lysine at position 196. A panel of recombinant maHK68 viruses was generated encoding NS1 variants that differed at positions 196 and 231. Our analyses showed a clear effect of the K-196-to-E exchange on interferon induction and virus virulence. These effects were further modulated by the loss of the seven-amino-acid extension. We propose that the combination of NS1 E-196 with the short C-terminal variant conferred a fitness advantage that is reflected by increased virulence in vivo. Notably, this particular NS1 constellation was observed for the pandemic 1918 H1N1 virus.

scholarly journals Effects of four extenders on the quality of frozen semen in Arabian stallions

2019 ◽  
Vol 12 (1) ◽  
pp. 34-40 ◽  
Author(s):  
Mohaammed Saad Alamaary ◽  
Haron Wahid ◽  
Mohamed Ali ◽  
Mark Wen Han Hiew ◽  
Lawan Adamu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Integrity ◽  
Plasma Membrane Integrity ◽  
Frozen Semen ◽  
Before And After ◽  
Semen Preservation ◽  
The Impact

Aim: Different types of extenders have a variety of components which show the tolerance effect on sperm protection during freezing procedures. In the present study, we have examined the impact of the extenders HF-20 and Tris, which were locally manufactured, and they are competing with commercial extenders INRA Freeze® (IMV Technologies, France) and EquiPlus Freeze® (Minitube, Germany) on the quality of horses frozen semen. Materials and Methods: A total of 15 ejaculates from three healthy stallions were collected and cryopreserved in the same environment. Each semen sample collected was divided into four equal parts and processed. All samples were analyzed before and after freezing for motility, viability, plasma membrane integrity, and morphology. Furthermore, twenty mares were inseminated using post-thawed semen. Results: There were no differences observed among all extenders in all the parameters before freezing. Sperm cryopreserved using HF-20 showed better motility, viability, and plasma membrane integrity than Tris extender. The Tris extender showed the most inferior quality of post-thawed semen between all the extenders. HF-20, INRA Freeze®, and EquiPlus Freeze® extenders revealed the same capacity of semen preservation in vitro and in vivo. Conclusion: HF-20 extender has the same quality as INRA Freeze® and EquiPlus Freeze® that can be considered as one of the best extenders for the semen cryopreservation in horses. In contrast, Tris extender needs some degree of improvement.

scholarly journals Binding of Calmodulin to the Carboxy-Terminal Region of p21 Induces Nuclear Accumulation via Inhibition of Protein Kinase C-Mediated Phosphorylation of Ser153

2005 ◽  
Vol 25 (16) ◽  
pp. 7364-7374 ◽  
Author(s):  
Aina Rodríguez-Vilarrupla ◽  
Montserrat Jaumot ◽  
Neus Abella ◽  
Núria Canela ◽  
Sonia Brun ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Intracellular Localization ◽  
Nuclear Accumulation ◽  
Wild Type ◽  
Calmodulin Binding ◽  
Kinase C ◽  
Carboxy Terminal

ABSTRACT Intracellular localization plays an important role in the functional regulation of the cell cycle inhibitor p21. We have previously shown that calmodulin binds to p21 and that calmodulin is essential for the nuclear accumulation of p21. Here, we analyze the mechanism of this regulation. We show that calmodulin inhibits in vitro phosphorylation of p21 by protein kinase C (PKC) and that this inhibition is dependent upon calmodulin binding to p21. Two-dimensional electrophoresis analysis of cells expressing the p21 wild type or p21S153A, a nonphosphorylatable mutant of p21 at position 153, indicates that Ser153 of p21 is a phosphorylable residue in vivo. Furthermore, Western blot analysis using phospho-Ser153-specific antibodies indicates that Ser153 phosphorylation in vivo is induced when PKC is activated and calmodulin is inhibited. The mutation of Ser153 to aspartate, a pseudophosphorylated residue, inhibits the nuclear accumulation of p21. Finally, whereas wild-type p21 translocates to the cytoplasm after PKC activation in the presence of calmodulin inhibitors, p21 carrying a nonphosphorylatable residue at position 153 remains in the nucleus. We propose that calmodulin binding to p21 prevents its phosphorylation by PKC at Ser153 and consequently allows its nuclear localization. When phosphorylated at Ser153, p21 is located at the cytoplasm and disrupts stress fibers.

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