Visualization ofToxoplasma gondiistage conversion by expression of stage-specific dual fluorescent proteins

Parasitology ◽  
2009 ◽  
Vol 136 (6) ◽  
pp. 579-588 ◽  
Author(s):  
A. UNNO ◽  
K. SUZUKI ◽  
T. BATANOVA ◽  
S.-Y. CHA ◽  
H.-K. JANG ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Host Cells ◽  
Live Cells ◽  
Green Fluorescence ◽  
Red Fluorescence ◽  
Genes Encoding ◽  
Transgenic Parasite ◽  
P38 Mapk Inhibitor ◽  
Green Fluorescent

SUMMARYTo recognize the stage conversion ofToxoplasma gondiibetween tachyzoite and bradyzoite in live host cells, a transgenicT. gondiiline, which expressed stage-specific red and green fluorescence, was constructed.T. gondiiPLK strain tachyzoites were stably transformed with genes encoding red fluorescent protein (DsRed Express) and green fluorescent protein (GFP) under the control of tachyzoite-specific SAG1 and bradyzoite-specific BAG1 promoters, respectively. The resulting transgenic parasite was designated PLK/DUAL. When PLK/DUAL was cultured in pH 7·0 medium, the PLK/DUAL zoites expressed red fluorescence, but no detectable levels of green fluorescence were observed. The PLK/DUAL zoites reacted with anti-SAG1 antibody, but not anti-BAG1 antiserum. When PLK/DUAL was cultured under high pH conditions, or in the presence of the p38 MAPK inhibitor SB202190, a small number of zoites expressed green fluorescence and were BAG1 positive. C57BL/6J mice were infected with PLK/DUAL tachyzoites. During the acute and reactivating phase, zoites expressed red fluorescence. However, green fluorescence was not detectable. By contrast, latent cysts expressed green fluorescence. The stage-specific dual fluorescence of PLK/DUAL facilitates identification of the parasitic stage in live cells, with the advantage that fixation or immunostaining is not required.

scholarly journals CRISPR/Cas9-mediated genome editing in a reef-building coral

2018 ◽  
Vol 115 (20) ◽  
pp. 5235-5240 ◽  
Author(s):  
Phillip A. Cleves ◽  
Marie E. Strader ◽  
Line K. Bay ◽  
John R. Pringle ◽  
Mikhail V. Matz
Keyword(s):  
Genome Editing ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Mutant Gene ◽  
Mutation Induction ◽  
Induced Mutations ◽  
Important Species ◽  
Ribonucleoprotein Complexes ◽  
Genes Encoding ◽  
Green Fluorescent

Reef-building corals are critically important species that are threatened by anthropogenic stresses including climate change. In attempts to understand corals’ responses to stress and other aspects of their biology, numerous genomic and transcriptomic studies have been performed, generating a variety of hypotheses about the roles of particular genes and molecular pathways. However, it has not generally been possible to test these hypotheses rigorously because of the lack of genetic tools for corals. Here, we demonstrate efficient genome editing using the CRISPR/Cas9 system in the coral Acropora millepora. We targeted the genes encoding fibroblast growth factor 1a (FGF1a), green fluorescent protein (GFP), and red fluorescent protein (RFP). After microinjecting CRISPR/Cas9 ribonucleoprotein complexes into fertilized eggs, we detected induced mutations in the targeted genes using changes in restriction-fragment length, Sanger sequencing, and high-throughput Illumina sequencing. We observed mutations in ∼50% of individuals screened, and the proportions of wild-type and various mutant gene copies in these individuals indicated that mutation induction continued for at least several cell cycles after injection. Although multiple paralogous genes encoding green fluorescent proteins are present in A. millepora, appropriate design of the guide RNA allowed us to induce mutations simultaneously in more than one paralog. Because A. millepora larvae can be induced to settle and begin colony formation in the laboratory, CRISPR/Cas9-based gene editing should allow rigorous tests of gene function in both larval and adult corals.

scholarly journals Microtubule-Independent Motility and Nuclear Targeting of Adenoviruses with Fluorescently Labeled Genomes

2001 ◽  
Vol 75 (5) ◽  
pp. 2421-2434 ◽  
Author(s):  
Jolanta B. Glotzer ◽  
Anne-Isabelle Michou ◽  
Adam Baker ◽  
Mediyha Saltik ◽  
Matt Cotten
Keyword(s):  
Fluorescent Protein ◽  
Host Cells ◽  
Live Cells ◽  
Wild Type ◽  
Nuclear Targeting ◽  
Microtubule Network ◽  
Entry Pathway ◽  
Green Fluorescent ◽  
Affect Gene Expression ◽  
Intracellular Motility

ABSTRACT A novel adenovirus system for analyzing the adenovirus entry pathway has been developed that contains green fluorescent protein bound to the encapsidated viral DNA (AdLite viruses). AdLite viruses enter host cells and accumulate around the nuclei and near the microtubule organizing centers (MTOC). In live cells, individual AdLite particles were observed trafficking both toward and away from the nucleus. Depolymerization of microtubules during infection prevented AdLite accumulation around the MTOC; however, it did not abolish perinuclear localization of AdLite particles. Furthermore, depolymerization of microtubules did not affect AdLite motility and did not affect gene expression from wild-type adenovirus and adenovirus-derived vectors. These data revealed that adenovirus intracellular motility and nuclear targeting can be supported by a mechanism that does not rely on the microtubule network.

scholarly journals Genetically-encoded fluorescent biosensor for rapid detection of protein expression

2020 ◽  
Author(s):  
Matthew G Eason ◽  
Antonia T Pandelieva ◽  
Marc M Mayer ◽  
Safwat T Khan ◽  
Hernan G Garcia ◽  
...  
Keyword(s):  
Protein Expression ◽  
Real Time ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Live Cells ◽  
Spatiotemporal Resolution ◽  
E Coli ◽  
Fluorescent Biosensor ◽  
Green Fluorescent

Fluorescent proteins are widely used as fusion tags to detect protein expression in vivo. To become fluorescent, these proteins must undergo chromophore maturation, a slow process with a half-time of 5 to >30 min, which causes delays in real-time detection of protein expression. Here, we engineer a genetically-encoded fluorescent biosensor to enable detection of protein expression within seconds in live cells. This sensor for transiently-expressed proteins (STEP) is based on a fully matured but dim green fluorescent protein in which pre-existing fluorescence increases 11-fold in vivo following the specific and rapid binding of a protein tag (Kd 120 nM, kon 1.7 x 10^5 M-1s-1). In live E. coli cells, our STEP biosensor enables detection of protein expression twice as fast as the use of standard fluorescent protein fusions. Our biosensor opens the door to the real-time study of short-timescale processes in research model animals with high spatiotemporal resolution.

scholarly journals Tracking RNA with light: selection, structure, and design of fluorescence turn-on RNA aptamers

2019 ◽  
Vol 52 ◽  
Author(s):  
Robert J. Trachman ◽  
Adrian R. Ferré-D'Amaré
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Structural Diversity ◽  
Rna Aptamers ◽  
Live Cells ◽  
Rna Molecules ◽  
Turn On ◽  
Green Fluorescent ◽  
Fluorescence Turn On

AbstractFluorescence turn-on aptamers,in vitroevolved RNA molecules that bind conditional fluorophores and activate their fluorescence, have emerged as RNA counterparts of the fluorescent proteins. Turn-on aptamers have been selected to bind diverse fluorophores, and they achieve varying degrees of specificity and affinity. These RNA–fluorophore complexes, many of which exceed the brightness of green fluorescent protein and their variants, can be used as tags for visualizing RNA localization and transport in live cells. Structure determination of several fluorescent RNAs revealed that they have diverse, unrelated overall architectures. As most of these RNAs activate the fluorescence of their ligands by restraining their photoexcited states into a planar conformation, their fluorophore binding sites have in common a planar arrangement of several nucleobases, most commonly a G-quartet. Nonetheless, each turn-on aptamer has developed idiosyncratic structural solutions to achieve specificity and efficient fluorescence turn-on. The combined structural diversity of fluorophores and turn-on RNA aptamers has already produced combinations that cover the visual spectrum. Further molecular evolution and structure-guided engineering is likely to produce fluorescent tags custom-tailored to specific applications.

scholarly journals A genetically-encoded toolkit of functionalized nanobodies against fluorescent proteins for visualizing and manipulating intracellular signalling

2019 ◽  
Author(s):  
David L. Prole ◽  
Colin W. Taylor
Keyword(s):  
Secretory Pathway ◽  
Fluorescent Dyes ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Cell Signalling ◽  
Intracellular Signalling ◽  
Live Cells ◽  
Green Fluorescent Proteins ◽  
Membrane Contact Sites ◽  
Green Fluorescent

AbstractBackgroundIntrabodies enable targeting of proteins in live cells, but it remains a huge task to generate specific intrabodies against the thousands of proteins in a proteome. We leverage the widespread availability of fluorescently labelled proteins to visualize and manipulate intracellular signalling pathways in live cells by using nanobodies targeting fluorescent protein tags.ResultsWe generated a toolkit of plasmids encoding nanobodies against red and green fluorescent proteins (RFP and GFP variants), fused to functional modules. These include fluorescent sensors for visualization of Ca2+, H+ and ATP/ADP dynamics; oligomerizing or heterodimerizing modules that allow recruitment or sequestration of proteins and identification of membrane contact sites between organelles; SNAP tags that allow labelling with fluorescent dyes and targeted chromophore-assisted light inactivation; and nanobodies targeted to lumenal sub-compartments of the secretory pathway. We also developed two methods for crosslinking tagged proteins: a dimeric nanobody, and RFP-targeting and GFP-targeting nanobodies fused to complementary hetero-dimerizing domains. We show various applications of the toolkit and demonstrate, for example, that IP3 receptors deliver Ca2+ to the outer membrane of only a subset of mitochondria, and that only one or two sites on a mitochondrion form membrane contacts with the plasma membrane.ConclusionsThis toolkit greatly expands the utility of intrabodies for studying cell signalling in live cells.

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

2019 ◽  
Author(s):  
Jeffrey Chang ◽  
Matthew Romei ◽  
Steven Boxer
Keyword(s):  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Unit Cell Size ◽  
Chlorine Substituent ◽  
Gfp Chromophore ◽  
The One ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

<p>Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of <i>cis</i> and <i>trans</i> rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the <i>trans</i> state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.</p><p> </p><p> </p><p> </p><p> </p><p> </p><p> </p> <p> </p>

scholarly journals tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

2021 ◽  
Author(s):  
Y. Bousmah ◽  
H. Valenta ◽  
G. Bertolin ◽  
U. Singh ◽  
V. Nicolas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy ◽  
Live Cell ◽  
Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

scholarly journals Probing chemotaxis activity inEscherichia coliusing fluorescent protein fusions

2018 ◽  
Author(s):  
Clémence Roggo ◽  
Jan Roelof van der Meer
Keyword(s):  
Escherichia Coli ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Bacterial Chemotaxis ◽  
Ph Sensitive ◽  
Flagellar Motors ◽  
Receptor Interactions ◽  
Split Egfp ◽  
Green Fluorescent

ABSTRACTChemotaxis is based on ligand-receptor interactions that are transmitted via protein-protein interactions to the flagellar motors. Ligand-receptor interactions in chemotaxis can be deployed for the development of rapid biosensor assays, but there is no consensus as to what the best readout of such assays would have to be. Here we explore two potential fluorescent readouts of chemotactically activeEscherichia colicells. In the first, we probed interactions between the chemotaxis signaling proteins CheY and CheZ by fusing them individually with non-fluorescent parts of a ‘split’-Green Fluorescent Protein. Wild-type chemotactic cells but not mutants lacking the CheA kinase produced distinguishable fluorescence foci, two-thirds of which localize at the cell poles with the chemoreceptors and one-third at motor complexes. Cells expressing fusion proteins only were attracted to serine sources, demonstrating measurable functional interactions between CheY~P and CheZ. Fluorescent foci based on stable split-eGFP displayed small fluctuations in cells exposed to attractant or repellent, but those based on an unstable ASV-tagged eGFP showed a higher dynamic behaviour both in the foci intensity changes and the number of foci per cell. For the second readout, we expressed the pH-sensitive fluorophore pHluorin in the cyto- and periplasm of chemotactically activeE. coli. Calibrations of pHluorin fluorescence as a function of pH demonstrated that cells accumulating near a chemo-attractant temporally increase cytoplasmic pH while decreasing periplasmic pH. Both readouts thus show promise as proxies for chemotaxis activity, but will have to be further optimized in order to deliver practical biosensor assays.IMPORTANCEBacterial chemotaxis may be deployed for future biosensing purposes with the advantages of its chemoreceptor ligand-specificity and its minute-scale response time. On the downside, chemotaxis is ephemeral and more difficult to quantitatively read out than, e.g., reporter gene expression. It is thus important to investigate different alternative ways to interrogate chemotactic response of cells. Here we gauge the possibilities to measure dynamic response in theEscherichia colichemotaxis pathway resulting from phosphorylated CheY-CheZ interactions by using (unstable) split-fluorescent proteins. We further test whether pH differences between cyto- and periplasm as a result of chemotactic activity can be measured with help of pH-sensitive fluorescent proteins. Our results show that both approaches conceptually function, but will need further improvement in terms of detection and assay types to be practical for biosensing.

Subcellular redistribution of the renal betaine transporter during hypertonic stress

2003 ◽  
Vol 285 (5) ◽  
pp. C1091-C1100 ◽  
Author(s):  
Stephen A. Kempson ◽  
Vaibhave Parikh ◽  
Lixuan Xi ◽  
Shaoyou Chu ◽  
Marshall H. Montrose
Keyword(s):  
Plasma Membrane ◽  
Posttranscriptional Regulation ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Live Cells ◽  
Hypertonic Medium ◽  
Hypertonic Stress ◽  
Antibody Staining ◽  
Renal Inner Medulla ◽  
Green Fluorescent

The betaine transporter (BGT1) protects cells in the hypertonic renal inner medulla by mediating uptake and accumulation of the osmolyte betaine. Transcriptional regulation plays an essential role in upregulation of BGT1 transport when renal cells are exposed to hypertonic medium for 24 h. Posttranscriptional regulation of the BGT1 protein is largely unexplored. We have investigated the distribution of BGT1 protein in live cells after transfection with BGT1 tagged with enhanced green fluorescent protein (EGFP). Fusion of EGFP to the NH2 terminus of BGT1 produced a fusion protein (EGFP-BGT) with transport properties identical to normal BGT1, as determined by ion dependence, inhibitor sensitivity, and apparent Km for GABA. Confocal microscopy of EGFP-BGT fluorescence in transfected Madin-Darby canine kidney (MDCK) cells showed that hypertonic stress for 24 h induced a shift in subcellular distribution from cytoplasm to plasma membrane. This was confirmed by colocalization with anti-BGT1 antibody staining. In fibroblasts, transfected EGFP-BGT caused increased transport in response to hypertonic stress. The activation of transport was not accompanied by increased expression of EGFP-BGT, as determined by Western blotting. Membrane insertion of EGFP-BGT protein in MDCK cells began within 2-3 h after onset of hypertonic stress and was blocked by cycloheximide. We conclude that posttranscriptional regulation of BGT1 is essential for adaptation to hypertonic stress and that insertion of BGT1 protein to the plasma membrane may require accessory proteins.

Sign in / Sign up

Export Citation Format

Share Document