Studying the Cell Biology of Apicomplexan Parasites Using Fluorescent Proteins

2004 ◽  
Vol 10 (5) ◽  
pp. 568-579 ◽  
Author(s):  
Marc-Jan Gubbels ◽  
Boris Striepen
Keyword(s):  
Protein Trafficking ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Biological Processes ◽  
Organelle Biogenesis ◽  
Apicomplexan Parasites ◽  
Experimental Protocols ◽  
Green Fluorescent

The ability to transfect Apicomplexan parasites has revolutionized the study of this important group of pathogens. The function of specific genes can be explored by disruption of the locus or more subtly by introduction of altered or tagged versions. Using the transgenic reporter gene green fluorescent protein (GFP), cell biological processes can now be studied in living parasites and in real time. We review recent advances made using GFP-based experiments in the understanding of protein trafficking, organelle biogenesis, and cell division inToxoplasma gondiiandPlasmodium falciparum. A technical section provides a collection of basic experimental protocols for fluorescent protein expression inT. gondii. The combination of thein vivomarker GFP with an increasingly diverse genetic toolbox forT. gondiiopens many exciting experimental opportunities, and emerging applications of GFP in genetic and pharmacological screens are discussed.

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.

Cellular Uptake of Carbon Nanotubes Conjugated to Semiconductor Quantum Dots for Breast Cancer Imaging and Treatment Applications

2010 ◽  
Author(s):  
Kristen A. Zimmermann ◽  
Jianfei Zhang ◽  
Harry Dorn ◽  
Christopher Rylander ◽  
Marissa Nichole Rylander
Keyword(s):  
Carbon Nanotubes ◽  
Quantum Dots ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Properties ◽  
Breast Cancer Imaging ◽  
Fluorescent Markers ◽  
Green Fluorescent

Carbon nanotubes (CNTs) are attractive materials for early detection, treatment, and imaging of cancer malignancies; however, they are limited by their inability to be monitored in vitro and in vivo [1]. Unlabeled CNTs are difficult to distinguish using elemental analysis because they are composed entirely of carbon, which is also characteristic of cellular membranes. Although some single walled nanotubes (SWNT) have been found to exhibit fluorescent properties, not all particles in a single batch fluoresce [2]. Additionally, these emissions may be too weak to be detected using conventional imaging modalities [3]. Incorporating fluorescent markers, such as fluorescent proteins or quantum dots, allows the non-fluorescent particles to be visualized. Previously, fluorophores, such as green fluorescent protein (GFP) or red fluorescent protein (RFP), have been used to visualize and track cells or other particles in biological environments, but their low quantum yield and tendency to photobleach generate limitations for their use in such applications.

scholarly journals Red Fluorescent Protein (DsRed) as a Reporter inSaccharomyces cerevisiae

2001 ◽  
Vol 183 (12) ◽  
pp. 3791-3794 ◽  
Author(s):  
Fernando Rodrigues ◽  
Martijn van Hemert ◽  
H. Yde Steensma ◽  
Manuela Côrte-Real ◽  
Cecı́la Leão
Keyword(s):  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Green Fluorescent Proteins ◽  
Red Fluorescent Protein ◽  
Amino Terminal ◽  
Carboxyl Terminal ◽  
Dsred Protein ◽  
Green Fluorescent

ABSTRACT We describe the utilization of a red fluorescent protein (DsRed) as an in vivo marker for Saccharomyces cerevisiae. Clones expressing red and/or green fluorescent proteins with both cytoplasmic and nuclear localization were obtained. A series of vectors are now available which can be used to create amino-terminal (N-terminal) and carboxyl-terminal (C-terminal) fusions with the DsRed protein.

scholarly journals Flavin Mononucleotide-Based Fluorescent Reporter Proteins Outperform Green Fluorescent Protein-Like Proteins as Quantitative In Vivo Real-Time Reporters

2010 ◽  
Vol 76 (17) ◽  
pp. 5990-5994 ◽  
Author(s):  
Thomas Drepper ◽  
Robert Huber ◽  
Achim Heck ◽  
Franco Circolone ◽  
Anne-Kathrin Hillmer ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Real Time ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Flavin Mononucleotide ◽  
Fluorescence Signal ◽  
Reporter Protein ◽  
Reporter Proteins ◽  
Green Fluorescent

ABSTRACT Fluorescent proteins of the green fluorescent protein (GFP) family are commonly used as reporter proteins for quantitative analysis of complex biological processes in living microorganisms. Here we demonstrate that the fluorescence signal intensity of GFP-like proteins is affected under oxygen limitation and therefore does not reflect the amount of reporter protein in Escherichia coli batch cultures. Instead, flavin mononucleotide (FMN)-binding fluorescent proteins (FbFPs) are suitable for quantitative real-time in vivo assays under these conditions.

scholarly journals Fluorescence-based whole plant imaging and phenomics

2019 ◽  
Author(s):  
Stephen B. Rigoulot ◽  
Tayler M. Schimel ◽  
Jun Hyung Lee ◽  
Holly Brabazon ◽  
Kerry A. Meier ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Low Noise ◽  
Color Palette ◽  
Plant Canopies ◽  
Whole Plant ◽  
Library Screen ◽  
Green Fluorescent

SummaryReverse genetics approaches have revolutionized plant biology and agriculture. Phenomics has the prospect of bridging plant phenotypes with genes, including transgenes, to transform agricultural fields1. Genetically-encoded fluorescent proteins (FPs) have transformed studies in gene expression, protein trafficking, and plant physiology. While the first instance of plant canopy imaging of green fluorescent protein (GFP) was performed over 20 years ago2, modern phenomics has largely ignored fluorescence as a transgene indicator despite the burgeoning FP color palette currently available to biologists3–5. Here we show a new platform for standoff imaging of plant canopies expressing a wide variety of FP genes in leaves. The platform, the fluorescence-inducing laser projector (FILP), uses a low-noise camera to image a scene illuminated by compact diode lasers of various colors and emission filters to phenotype transgenic plants expressing multiple constitutive or inducible FPs. Of the 20 FPs screened, we selected the top performing candidates for standoff phenomics at ≥ 3 m using FILP in a laboratory-based laser range. Included in demonstrated applications is the performance of an osmotic stress-inducible synthetic promoter selected from a high throughput library screen. While FILP has unprecedented versatility as a laboratory platform, we envisage future iterations of the system for use in automated greenhouse or even drone-fielded versions of the platform for crop screening.

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 CFP and YFP, but Not GFP, Provide Stable Fluorescent Marking of Rat Hepatic Adult Stem Cells

2008 ◽  
Vol 2008 ◽  
pp. 1-9 ◽  
Author(s):  
Rouzbeh R. Taghizadeh ◽  
James L. Sherley
Keyword(s):  
Stem Cells ◽  
Time Scales ◽  
Cell Biology ◽  
Adult Stem Cells ◽  
Cell Function ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Fluorescent Reporter ◽  
Green Fluorescent

The stable expression of reporter genes in adult stem cells (ASCs) has important applications in stem cell biology. The ability to integrate a noncytotoxic, fluorescent reporter gene into the genome of ASCs with the capability to track ASCs and their progeny is particularly desirable for transplantation studies. The use of fluorescent proteins has greatly aided the investigations of protein and cell function on short-time scales. In contrast, the obtainment of stably expressing cell strains with low variability in expression for studies on longer-time scales is often problematic. We show that this difficulty is partly due to the cytotoxicity of a commonly used reporter, green fluorescent protein (GFP). To avoid GFP-specific toxicity effects during attempts to stably mark a rat hepatic ASC strain and, therefore, obtain stable, long-term fluorescent ASCs, we evaluated cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), in addition to GFP. Although we were unable to derive stable GFP-expressing strains, stable fluorescent clones (up to 140 doublings) expressing either CFP or YFP were established. When fluorescently marked ASCs were induced to produce differentiated progeny cells, stable fluorescence expression was maintained. This property is essential for studies that track fluorescently marked ASCs and their differentiated progeny in transplantation studies.

scholarly journals A bright and high-performance genetically encoded Ca2+ indicator based on mNeonGreen fluorescent protein

2020 ◽  
Author(s):  
Landon Zarowny ◽  
Abhi Aggarwal ◽  
Virginia M.S. Rutten ◽  
Ilya Kolb ◽  
Ronak Patel ◽  
...  
Keyword(s):  
High Performance ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Fluorescent Proteins ◽  
Model Organisms ◽  
Green Fluorescent Proteins ◽  
Comparable Performance ◽  
Green Fluorescent

AbstractGenetically encodable calcium ion (Ca2+) indicators (GECIs) based on green fluorescent proteins (GFP) are powerful tools for imaging of cell signaling and neural activity in model organisms. Following almost two decades of steady improvements in the Aequorea victoria GFP (avGFP)-based GCaMP series of GECIs, the performance of the most recent generation (i.e., GCaMP7) may have reached its practical limit due to the inherent properties of GFP. In an effort to sustain the steady progression towards ever-improved GECIs, we undertook the development of a new GECI based on the bright monomeric GFP, mNeonGreen (mNG). The resulting indicator, mNG-GECO1, is 60% brighter than GCaMP6s in vitro and provides comparable performance as demonstrated by imaging Ca2+ dynamics in cultured cells, primary neurons, and in vivo in larval zebrafish. These results suggest that mNG-GECO1 is a promising next-generation GECI that could inherit the mantle of GCaMP and allow the steady improvement of GECIs to continue for generations to come.

scholarly journals An endogenous green fluorescent protein–photoprotein pair in Clytia hemisphaerica eggs shows co-targeting to mitochondria and efficient bioluminescence energy transfer

Open Biology ◽  
2014 ◽  
Vol 4 (4) ◽  
pp. 130206 ◽  
Author(s):  
Cécile Fourrage ◽  
Karl Swann ◽  
Jose Raul Gonzalez Garcia ◽  
Anthony K. Campbell ◽  
Evelyn Houliston
Keyword(s):  
Energy Transfer ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Green Light ◽  
Resonance Energy ◽  
Green Fluorescent Proteins ◽  
Parallel Acquisition ◽  
Green Fluorescent

Green fluorescent proteins (GFPs) and calcium-activated photoproteins of the aequorin/clytin family, now widely used as research tools, were originally isolated from the hydrozoan jellyfish Aequora victoria . It is known that bioluminescence resonance energy transfer (BRET) is possible between these proteins to generate flashes of green light, but the native function and significance of this phenomenon is unclear. Using the hydrozoan Clytia hemisphaerica , we characterized differential expression of three clytin and four GFP genes in distinct tissues at larva, medusa and polyp stages, corresponding to the major in vivo sites of bioluminescence (medusa tentacles and eggs) and fluorescence (these sites plus medusa manubrium, gonad and larval ectoderms). Potential physiological functions at these sites include UV protection of stem cells for fluorescence alone, and prey attraction and camouflaging counter-illumination for bioluminescence. Remarkably, the clytin2 and GFP2 proteins, co-expressed in eggs, show particularly efficient BRET and co-localize to mitochondria, owing to parallel acquisition by the two genes of mitochondrial targeting sequences during hydrozoan evolution. Overall, our results indicate that endogenous GFPs and photoproteins can play diverse roles even within one species and provide a striking and novel example of protein coevolution, which could have facilitated efficient or brighter BRET flashes through mitochondrial compartmentalization.

scholarly journals Long-term time-lapse multimodal intravital imaging of regeneration and bone-marrow-derived cell dynamics in skin

TECHNOLOGY ◽  
2013 ◽  
Vol 01 (01) ◽  
pp. 8-19 ◽  
Author(s):  
Benedikt W. Graf ◽  
Eric J. Chaney ◽  
Marina Marjanovic ◽  
Steven G. Adie ◽  
Michael De Lisio ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Great Promise ◽  
Cell Dynamics ◽  
Registration Method ◽  
Long Time ◽  
Green Fluorescent

A major challenge for translating cell-based therapies is understanding the dynamics of cells and cell populations in complex in vivo environments. Intravital microscopy has shown great promise for directly visualizing cell behavior in vivo. However, current methods are limited to relatively short imaging times (hours), by ways to track cell and cell population dynamics over extended time-lapse periods (days to weeks to months), and by relatively few imaging contrast mechanisms that persist over extended investigations. We present technology to visualize and quantify complex, multifaceted dynamic changes in natural deformable skin over long time periods using novel multimodal imaging and a non-rigid image registration method. These are demonstrated in green fluorescent protein (GFP) bone marrow (BM) transplanted mice to study dynamic skin regeneration. This technology provides a novel perspective for studying dynamic biological processes and will enable future studies of stem, immune, and tumor cell biology in vivo.

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