scholarly journals An in vivo translation-reporter system for the study of protein synthesis in zebrafish embryos

2018 ◽  
Author(s):  
Inês Garcez Palha ◽  
Isabelle Anselme ◽  
Sylvie Schneider-Maunoury ◽  
François Giudicelli
Keyword(s):  
Protein Synthesis ◽  
Protease Inhibitor ◽  
Fluorescent Protein ◽  
Mrna Translation ◽  
Transgenic Zebrafish ◽  
Zebrafish Embryos ◽  
Reporter System ◽  
Control Of Gene Expression ◽  
A Cell

ABSTRACTControl of gene expression at the translation level is increasingly regarded as a key feature in many biological processes. Simple, inexpensive, and reliable procedures to visualise sites of protein production are required to allow observation of the spatiotemporal patterns of mRNA translation at subcellular resolution. We present a method, named SPoT (for Subcellular Patterns of Translation), developed upon the original TimeStamp technique (Lin et al., 2008), consisting in the expression of a fluorescent protein fused to a tagged, self-cleavable protease domain. Addition of a cell-permeable protease inhibitor instantly stabilizes newly produced, tagged protein allowing to distinguish recently synthesized protein from more ancient one. After a brief protease inhibitor treatment, the ratio of tagged vs non-tagged forms is highest at sites where proteins are the most recent, i.e. sites of synthesis. Therefore, by comparing tagged and non-tagged protein it is possible to spotlight sites of translation. By specifically expressing the SPoT cassette in neurons of transgenic zebrafish embryos, we reveal sites of neuronal protein synthesis in diverse cellular compartments during early development.

scholarly journals An in vivo translation-reporter system for the study of protein synthesis in zebrafish embryos

Biology Open ◽  
2018 ◽  
Vol 7 (12) ◽  
pp. bio039362
Author(s):  
Inês Garcez Palha ◽  
Isabelle Anselme ◽  
Sylvie Schneider-Maunoury ◽  
François Giudicelli
Keyword(s):  
Protein Synthesis ◽  
Zebrafish Embryos ◽  
Reporter System

Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells

Blood ◽  
2010 ◽  
Vol 116 (6) ◽  
pp. 909-914 ◽  
Author(s):  
Enid Yi Ni Lam ◽  
Christopher J. Hall ◽  
Philip S. Crosier ◽  
Kathryn E. Crosier ◽  
Maria Vega Flores
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Fluorescent Protein ◽  
Living Organism ◽  
Time Lapse ◽  
Dorsal Aorta ◽  
Transgenic Zebrafish ◽  
Hematopoietic Stem ◽  
Green Fluorescent

Abstract Blood cells of an adult vertebrate are continuously generated by hematopoietic stem cells (HSCs) that originate during embryonic life within the aorta-gonad-mesonephros region. There is now compelling in vivo evidence that HSCs are generated from aortic endothelial cells and that this process is critically regulated by the transcription factor Runx1. By time-lapse microscopy of Runx1-enhanced green fluorescent protein transgenic zebrafish embryos, we were able to capture a subset of cells within the ventral endothelium of the dorsal aorta, as they acquire hemogenic properties and directly emerge as presumptive HSCs. These nascent hematopoietic cells assume a rounded morphology, transiently occupy the subaortic space, and eventually enter the circulation via the caudal vein. Cell tracing showed that these cells subsequently populated the sites of definitive hematopoiesis (thymus and kidney), consistent with an HSC identity. HSC numbers depended on activity of the transcription factor Runx1, on blood flow, and on proper development of the dorsal aorta (features in common with mammals). This study captures the earliest events of the transition of endothelial cells to a hemogenic endothelium and demonstrates that embryonic hematopoietic progenitors directly differentiate from endothelial cells within a living organism.

Use of an in Vitro Protein Synthesizing System to Test the Mode of Action of Chloracetamides

Weed Science ◽  
1980 ◽  
Vol 28 (3) ◽  
pp. 334-340 ◽  
Author(s):  
Luanne M. Deal ◽  
J. T. Reeves ◽  
B. A. Larkins ◽  
F. D. Hess
Keyword(s):  
Protein Synthesis ◽  
Sand Culture ◽  
Leucine Incorporation ◽  
Insoluble Protein ◽  
In Vitro System ◽  
A Cell ◽  
Protein Incorporation ◽  
Vitro Protein

The effects of chloracetamides on protein synthesis were studied both in vivo and in vitro. Four chloracetamide herbicides, alachlor [2-chloro-2′,6′-diethyl-N-(methoxymethyl)acetanilide], metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide], CDAA (N–N-diallyl-2-chloroacetamide), and propachlor (2-chloro-N-isopropylacetanilide) were tested for inhibition of [3H]-leucine incorporation into protein. Incorporation of3H-leucine into trichloroacetic acid (TCA)-insoluble protein was inhibited in oat (Avena sativaL. ‘Victory’) seedlings grown in sand culture and treated 12 h at 1 × 10−4M with these chloracetamides. The herbicides were also tested in a cell-free protein synthesizing system containing polyribosomes purified from oat root cytoplasm. These herbicides had no effect on the rates of polypeptide elongation nor on the synthesis of specific polypeptides when herbicides (1 × 10−4M) were added directly to the system. Polypeptide formation was inhibited 89% when 1 × 10−4M cycloheximide was added during translation. Cytoplasmic polyribosomes were isolated from oat roots treated 12 h with 1 × 10−4M herbicide. Translation rates and products were not altered when these polyribosomes were added to the in vitro system. Protein synthesis is inhibited when tested in an in vivo system; however, the inhibition does not occur during the translation of mRNA into protein.

A novel regulatory element between the human FGA and FGG genes

2012 ◽  
Vol 108 (09) ◽  
pp. 427-434 ◽  
Author(s):  
Richard J. Fish ◽  
Marguerite Neerman-Arbez
Keyword(s):  
Gene Cluster ◽  
Fluorescent Protein ◽  
Regulatory Element ◽  
Luciferase Reporter ◽  
Transgenic Zebrafish ◽  
Regulatory Sequences ◽  
Gene Promoters ◽  
Cvd Risk ◽  
Enhancer Activity

SummaryHigh circulating fibrinogen levels correlate with cardiovascular disease (CVD) risk. Fibrinogen levels vary between people and also change in response to physiological and environmental stimuli. A modest proportion of the variation in fibrinogen levels can be explained by genotype, inferring that variation in genomic sequences that regulate the fibri-nogen genes (FGA, FGB and FGG) may affect hepatic fibrinogen production and perhaps CVD risk. We previously identified a conserved liver enhancer in the fibrinogen gene cluster (CNC12), between FGB and FGA. Genome-wide Chromatin immunoprecipitation-sequencing (ChIP-seq) demonstrated that transcription factors which bind fibrinogen gene promoters also interact with CNC12, as well as two potential fibrinogen enhancers (PFE), between FGA and FGG. Here we show that one of the PFE sequences has potent hepatocyte enhancer activity. Using a luciferase reporter gene system, we found that PFE2 enhances minimal promoter- and FGA promoter-driven gene expression in hepatoma cells, regardless of its orientation with respect to the promoters. A region within PFE2 bears a short series of conserved nucleotides which maintain enhancer activity without flanking sequence. We also demonstrate that PFE2 is a liver enhancer in vivo, driving enhanced green fluorescent protein expression in transgenic zebrafish larval livers. Our study shows that combining public domain ChIP-seq data with in vitro and in vivo functional tests can identify novel fibrinogen gene cluster regulatory sequences. Variation in such elements could affect fibrinogen production and influence CVD risk.

scholarly journals Cardioluminescence in transgenic zebrafish embryos: a calcium imaging tool to study drug effects and pathological modeling

2021 ◽  
Author(s):  
Manuel Vicente ◽  
Jussep Salgado-Almario ◽  
Michelle M. Collins ◽  
Antonio Martinez-Sielva ◽  
Masafumi Minoshima ◽  
...  
Keyword(s):  
Signal To Noise Ratio ◽  
Light Output ◽  
Drug Effects ◽  
Study Drug ◽  
Transgenic Zebrafish ◽  
Zebrafish Embryos ◽  
Cardiovascular Research ◽  
Imaging Tool ◽  
Adrenergic Blocker

The zebrafish embryo has emerged as an excellent model in cardiovascular research. The existing techniques to monitor Ca2+ in the heart based on fluorescent Ca2+ biosensors are limited due to phototoxicity and photobleaching. To overcome these issues, we have used bioluminescence. We generated a transgenic line expressing GFP-Aequorin in the heart, Tg(cmlc2:GA), and optimized an in vivo aequorin reconstitution protocol to improve the luminescence capacity. This allowed imaging Ca2+ in long duration recordings in embryos of 3 to 5 days post-fertilization. The analogs diacetyl h-coelenterazine and f-coelenterazine enhanced the light output and signal-to-noise ratio from the embryos. With this cardioluminescence model, we monitored the time-averaged Ca2+ levels and beat-to-beat Ca2+ oscillations. Changes in Ca2+ levels were observed by incubation with BayK8644, an L-type Ca2+ channel agonist, the channel blocker nifedipine, and β-adrenergic blocker propranolol. Treatment of zebrafish embryos with terfenadine for 24 hours has been proposed as a model of heart failure. Tg(cmlc2:GA) embryos treated with terfenadine showed a 2:1 atrioventricular block and a decrease in the ventricular Ca2+ levels.

Laser-induced gene expression in specific cells of transgenic zebrafish

Development ◽  
2000 ◽  
Vol 127 (9) ◽  
pp. 1953-1960 ◽  
Author(s):  
M.C. Halloran ◽  
M. Sato-Maeda ◽  
J.T. Warren ◽  
F. Su ◽  
Z. Lele ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Gene ◽  
Transgenic Zebrafish ◽  
Hsp70 Promoter ◽  
Expression Of Genes ◽  
Transgenic Lines ◽  
Transgenic Embryos ◽  
Green Fluorescent

Over the past few years, a number of studies have described the generation of transgenic lines of zebrafish in which expression of reporters was driven by a variety of promoters. These lines opened up the real possibility that transgenics could be used to complement the genetic analysis of zebrafish development. Transgenic lines in which the expression of genes can be regulated both in space and time would be especially useful. Therefore, we have cloned the zebrafish promoter for the inducible hsp70 gene and made stable transgenic lines of zebrafish that express the reporter green fluorescent protein gene under the control of a hsp70 promoter. At normal temperatures, green fluorescent protein is not detectable in transgenic embryos with the exception of the lens, but is robustly expressed throughout the embryo following an increase in ambient temperature. Furthermore, we have taken advantage of the accessibility and optical clarity of the embryos to express green fluorescent protein in individual cells by focussing a sublethal laser microbeam onto them. The targeted cells appear to develop normally: cells migrate normally, neurons project axons that follow normal pathways, and progenitor cells divide and give rise to normal progeny cells. By generating other transgenic lines in which the hsp70 promoter regulates genes of interest, it should be possible to examine the in vivo activity of the gene products by laser-inducing specific cells to express them in zebrafish embryos. As a first test, we laser-induced single muscle cells to make zebrafish Sema3A1, a semaphorin that is repulsive for specific growth cones, in a hsp70-sema3A1 transgenic line of zebrafish and found that extension by the motor axons was retarded by the induced muscle.

P0053A METHOD FOR THE ISOLATION OF FLUORESCENT GLOMERULI FROM ZEBRAFISH LARVAE

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Anna Iervolino ◽  
Tim Lange ◽  
Florian Siegerist ◽  
Maximilian Schindler ◽  
Giovambattista Capasso ◽  
...  
Keyword(s):  
Glomerular Filtration ◽  
Fluorescent Protein ◽  
Transgenic Zebrafish ◽  
Renal Tubules ◽  
Glomerular Filtration Barrier ◽  
Podocyte Injury ◽  
Zebrafish Larvae ◽  
Filtration Barrier ◽  
Glomerular Damage

Abstract Background and Aims The zebrafish is a powerful animal model to study the glomerular morphology and the function of the permselectivity of the glomerular filtration barrier. Since zebrafish larvae develop quickly and can be bred to transparency, in vivo observation of these animals is possible. At 48 hours post fertilization (dpf), zebrafish develop a single filtering glomerulus which is attached to a pair of renal tubules. Like in mammals, the glomerular filtration barrier consists of a fenestrated endothelium, the glomerular basement membrane (GBM) and interdigitating podocyte foot processes bridged by a molecularly conserved slit diaphragm. By the use of genetically modified zebrafish strains with fluorescently labeled podocytes, it is possible to study alterations of the glomerulus during the development of renal disease directly in vivo and in vitro. As an injury model we used the nitroreductase/metronidazole (NTR/MTZ) zebrafish line to induce podocyte apoptosis and detachment from the GBM. Moreover, treatment of these larvae with MTZ induces glomerular injury that mimics focal segmental glomerulosclerosis (FSGS). The aim of our study was to establish a glomeruli isolation method which allows us to identify deregulation of miRNAs and mRNAs in the injured glomeruli by sequencing. Method The transgenic zebrafish strain Cherry (Tg(nphs2:Eco.nfsB-mCherry); mitfaw2/w2; mpv17a9/a9) which expresses the prokaryotic enzyme nitroreductase (NTR) fused to mCherry, a red fluorescent protein, under the control of the podocyte-specific podocin (nphs2) promoter in a transparent zebrafish strain, was used. The NTR/MTZ is a model of cell ablation to mimic podocyte injury. The prodrug MTZ (80 µM) is converted into a cytotoxin by NTR leading to a dose-dependent apoptosis exclusively in NTR-expressing podocytes. To induce podocyte injury, we treated Cherry larvae at 4 days post fertilization with MTZ (80 µM) freshly dissolved in 0.1% DMSO-E3 medium for 48 hours. Control larvae were treated with 0.1% DMSO-E3 medium. The treatment was stopped by a MTZ washout at 6 dpf. In order to perform the miRNA and mRNA sequencing on glomeruli isolated from MTZ-treated and control larvae we tried to establish a method to obtain total RNA samples of good quality. For this purpose, three different approaches were tested and validated: 1) Sieving method, 2) Fluorescence-Activated Cell Sorting method (FACS), and 3) manual isolation of glomeruli by using a micropipette. Results Zebrafish larvae developed a glomerular damage similar to FSGS after MTZ-treatment. MTZ-treated larvae showed severe pericardial edema, a reduction of the nephrin and podocin expression, proteinuria and an increased mortality rate at 8 dpf. After many tests we showed that glomeruli isolation using the sieving method and FACS were not efficient due to contaminations with other organs (sieving) and a loss of a large amount of cells per sample (FACS), respectively. Samples of the required quality for sequencing resulted only from the manual glomeruli isolation. Conclusion Here we describe methods to isolate fluorescent glomeruli from transgenic zebrafish larvae. For our studies, we used the NTZ/MTR kidney disease model in order to identify mRNAs and miRNAs regulated in response to glomerular damage. This technique will further allow to screen for healing drugs in high-throughput experiments.

scholarly journals Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

2015 ◽  
Vol 113 (3) ◽  
pp. 497-502 ◽  
Author(s):  
Marie-Aude Plamont ◽  
Emmanuelle Billon-Denis ◽  
Sylvie Maurin ◽  
Carole Gauron ◽  
Frederico M. Pimenta ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Labeling ◽  
Activation Mechanism ◽  
Photoactive Yellow Protein ◽  
Reversible Binding ◽  
A Cell ◽  
Rapid Labeling ◽  
Green Fluorescent

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

scholarly journals In Vivo Indicators of Cytoplasmic, Vacuolar, and Extracellular pH Using pHluorin2 in Candida albicans

mSphere ◽  
2017 ◽  
Vol 2 (4) ◽  
Author(s):  
Hélène Tournu ◽  
Arturo Luna-Tapia ◽  
Brian M. Peters ◽  
Glen E. Palmer
Keyword(s):  
Candida Albicans ◽  
Intracellular Ph ◽  
Fungal Pathogen ◽  
Fluorescent Protein ◽  
Extracellular Ph ◽  
Adaptive Responses ◽  
Ph Homeostasis ◽  
Content Type ◽  
A Cell

ABSTRACT Candida albicans is an opportunistic fungal pathogen that colonizes the reproductive and gastrointestinal tracts of its human host. It can also invade the bloodstream and deeper organs of immunosuppressed individuals, and thus it encounters enormous variations in external pH in vivo. Accordingly, survival within such diverse niches necessitates robust adaptive responses to regulate intracellular pH. However, the impact of antifungal drugs upon these adaptive responses, and on intracellular pH in general, is not well characterized. Furthermore, the tools and methods currently available to directly monitor intracellular pH in C. albicans, as well as other fungal pathogens, have significant limitations. To address these issues, we developed a new and improved set of pH sensors based on the pH-responsive fluorescent protein pHluorin. This includes a cytoplasmic sensor, a probe that localizes inside the fungal vacuole (an acidified compartment that plays a central role in intracellular pH homeostasis), and a cell surface probe that can detect changes in extracellular pH. These tools can be used to monitor pH within single C. albicans cells or in cell populations in real time through convenient and high-throughput assays. Environmental or chemically induced stresses often trigger physiological responses that regulate intracellular pH. As such, the capacity to detect pH changes in real time and within live cells is of fundamental importance to essentially all aspects of biology. In this respect, pHluorin, a pH-sensitive variant of green fluorescent protein, has provided an invaluable tool to detect such responses. Here, we report the adaptation of pHluorin2 (PHL2), a substantially brighter variant of pHluorin, for use with the human fungal pathogen Candida albicans. As well as a cytoplasmic PHL2 indicator, we describe a version that specifically localizes within the fungal vacuole, an acidified subcellular compartment with important functions in nutrient storage and pH homeostasis. In addition, by means of a glycophosphatidylinositol-anchored PHL2-fusion protein, we generated a cell surface pH sensor. We demonstrated the utility of these tools in several applications, including accurate intracellular and extracellular pH measurements in individual cells via flow cytometry and in cell populations via a convenient plate reader-based protocol. The PHL2 tools can also be used for endpoint as well as time course experiments and to conduct chemical screens to identify drugs that alter normal pH homeostasis. These tools enable observation of the highly dynamic intracellular pH shifts that occur throughout the fungal growth cycle, as well as in response to various chemical treatments. IMPORTANCE Candida albicans is an opportunistic fungal pathogen that colonizes the reproductive and gastrointestinal tracts of its human host. It can also invade the bloodstream and deeper organs of immunosuppressed individuals, and thus it encounters enormous variations in external pH in vivo. Accordingly, survival within such diverse niches necessitates robust adaptive responses to regulate intracellular pH. However, the impact of antifungal drugs upon these adaptive responses, and on intracellular pH in general, is not well characterized. Furthermore, the tools and methods currently available to directly monitor intracellular pH in C. albicans, as well as other fungal pathogens, have significant limitations. To address these issues, we developed a new and improved set of pH sensors based on the pH-responsive fluorescent protein pHluorin. This includes a cytoplasmic sensor, a probe that localizes inside the fungal vacuole (an acidified compartment that plays a central role in intracellular pH homeostasis), and a cell surface probe that can detect changes in extracellular pH. These tools can be used to monitor pH within single C. albicans cells or in cell populations in real time through convenient and high-throughput assays.

scholarly journals The hnRNA-Binding Proteins hnRNP L and PTB Are Required for Efficient Translation of the Cat-1 Arginine/Lysine Transporter mRNA during Amino Acid Starvation

2009 ◽  
Vol 29 (10) ◽  
pp. 2899-2912 ◽  
Author(s):  
Mithu Majumder ◽  
Ibrahim Yaman ◽  
Francesca Gaccioli ◽  
Vladimir V. Zeenko ◽  
Chuanping Wang ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Binding Protein ◽  
Mrna Translation ◽  
Amino Acid Starvation ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Polypyrimidine Tract Binding Protein ◽  
Global Protein Synthesis

ABSTRACT The response to amino acid starvation involves the global decrease of protein synthesis and an increase in the translation of some mRNAs that contain an internal ribosome entry site (IRES). It was previously shown that translation of the mRNA for the arginine/lysine amino acid transporter Cat-1 increases during amino acid starvation via a mechanism that utilizes an IRES in the 5′ untranslated region of the Cat-1 mRNA. It is shown here that polypyrimidine tract binding protein (PTB) and an hnRNA binding protein, heterogeneous nuclear ribonucleoprotein L (hnRNP L), promote the efficient translation of Cat-1 mRNA during amino acid starvation. Association of both proteins with Cat-1 mRNA increased during starvation with kinetics that paralleled that of IRES activation, although the levels and subcellular distribution of the proteins were unchanged. The sequence CUUUCU within the Cat-1 IRES was important for PTB binding and for the induction of translation during amino acid starvation. Binding of hnRNP L to the IRES or the Cat-1 mRNA in vivo was independent of PTB binding but was not sufficient to increase IRES activity or Cat-1 mRNA translation during amino acid starvation. In contrast, binding of PTB to the Cat-1 mRNA in vivo required hnRNP L. A wider role of hnRNP L in mRNA translation was suggested by the decrease of global protein synthesis in cells with reduced hnRNP L levels. It is proposed that PTB and hnRNP L are positive regulators of Cat-1 mRNA translation via the IRES under stress conditions that cause a global decrease of protein synthesis.

Sign in / Sign up

Export Citation Format

Share Document