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A complementary DNA for the Aequorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms.
... Efforts to map brain gene expression and regulation in living organisms to date have involved imaging reporter genes or associated enzymes using optical techniques, positron emission tomography (PET), or MRI (13)(14)(15)(16)(17). These methods are either limited to model organisms or require transgenic animals engineered to express a particular reporter gene and an exogenous contrast probe interacting with the reporter gene to produce the desired images (15,16). ...
... Strong isotope labeling of methylated DNA in the brain enables the possibility of using noninvasive isotopic detection methods. For eMRI, we chose 13 C-NMR to detect and quantify the 13 C-labeled 5mdC in genomic DNA. The 13 C-NMR and imaging using isotope-enriched molecules have been used in various basic biological studies and clinical applications (38)(39)(40)(41). ...
... The 13 C-NMR and imaging using isotope-enriched molecules have been used in various basic biological studies and clinical applications (38)(39)(40)(41). It offers several unique advantages: 1) Its nondestructive and nonradioactive nature allows for in vivo translation to humans; 2) its broad chemical-shift dispersion allows for detecting signals specifically from 13 C-5mdC in genomic DNA relative to 13 C signals from other molecules; and 3) 13 C has low natural abundance (∼1.1%), which can increase specific signal detection from the 13 C-enriched diet. We first performed a proof-of-principle in vitro experiment using synthetic DNA oligonucleotides with a well-defined number of 5mdC in the sequence. ...
Dynamic epigenetic activity is a fundamental mechanism underpinning how the brain changes its function during development and aging and in response to environmental and disease stimuli. We developed a technology called epigenetic MRI (eMRI) that enables noninvasive imaging of DNA methylation in the brain, a major epigenetic mechanism. eMRI reveals strong regional differences in global DNA methylation in pig brains, a model with stronger resemblance to human brains than are rodents. Given the noninvasive nature of eMRI, our results pave the way for a DNA-methylation imaging paradigm for living human brains. We expect eMRI to enable many studies to unravel the molecular control of brain function and disease.
... However, these small molecules lacked addressability to specific protein targets. The discovery of the Green Fluorescent Protein (see of fusing a fluorescent tag to any given protein by molecular biology (Chalfie et al, 1994). ...
... The next significant step in the history of the GFP occurred in 1992 when Douglas Prasher successfully cloned and sequenced the GFP gene (Prasher et al, 1992) and made available its cDNA sequence for the rest of the scientific community. In 1994, Chalfie used GFP as a cell marker for the first time, expressing the fluorescent protein in the prokaryote E. coli and in the neurons of the eukaryote Caenorhabditis elegans (Chalfie et al, 1994) (Figure 2) This publication showed that GFP can be used to track protein localisation and gene expression inside living organisms without any substrates other than the protein itself and without any cofactors. Roger Tsien responded to this publication by pointing at a lack of spectral diversity and between 1994 and 1998 worked with collaborators to produce a number of GFP mutants, some which were brighter and more stable, and some whose fluorescence exhibiting the yellow, cyan and blue colours complementary to the original green (Tsien, 1998). ...
... To this end, saturation mutagenesis was performed at selected locations around the chromophore, including Cys42, and mutants were The first application of AvGFP, has been to be fused to a protein of interest so that the fluorescence signal is a reporter of colocalization of the POI within the cell, confined to membranes or cell compartments. In the first application, GFP was fused to β-tubulin, a protein abundant in touch receptors of C. elegans (Chalfie et al, 1994). This application is still the primary one nowadays, yet in more sophisticated ways. ...
Since the cloning of the Green Fluorescent Protein (GFP) in 1992 fluorescent proteins (FPs) have increasingly become essential tools in cell imaging. GFP was first discovered in the jellyfish Aequorea victoria, then homologues were found in other types of organisms such as corals, sea anemones, lancelets and small crustaceans, forming the family of GFP-like FPs. A unique feature of GFP-like proteins is the autocatalytic formation of the chromophore, the light-absorbing and light-emitting part of the protein, from three consecutive amino acids located at the centre of the β-barrel structure of the protein. The family of GFP-like FPs have fluorescence emission maxima that cover the entire visible light spectrum from deep blue to far red. In the late 2000s, another type of FPs has been derived from phytochromes, a family of red-light photoreceptors that use bilins as chromophore. The interest of these is that their fluorescence excitation and emission spectra is in the near-infrared region of the light spectrum. This region is part of the so-called ‘optical window’ of living tissues, in which light absorption and scattering by haemoglobin, water and lipids is minimized and thus should be preferred for whole-body fluorescence imaging . These FPs have been called NIR FPs. In this PhD work, I have solved the crystallographic structures of three GFP-like FPs (one very bright green FP, one chromoprotein and one weakly red fluorescent FP) and three NIR FPs derived from a monomeric phytochrome. The structural information gained on the nature and environment of the chromophores has allowed me to propose explanations for their peculiar spectroscopic properties. In particular the structure of the chromoprotein revealed the existence for the first time of a chromophore which forms a covalent bond with a nearby cysteine residue, leading to a large red-shift of the UV-vis absorption maximum.
... The fluorophore of GFP is formed spontaneously by protein folding which is its most striking feature [47,60]. Once being expressed in host cells, GFP matures in three distinct kinetic steps: (i) relatively slow protein folding ( ½ = 10 min), (ii) formation of imidazolinone (cyclization) by the nucleophilic attack of the amide of Gly67 on the carbonyl of residue 65 ( ½ = 3 min), (iii) and oxidation of the chromophore ( ½ > 19 min) (Figure 4) [61,62]. ...
... Fluorescent proteins might have a photoprotective role for cnidarian organisms such as corals or jellyfishes . More than 30 years after the first characterization on a protein level, Chalfie et al. could express functional GFP in Escherichia coli , enabling the application of GFP as a fluorescent probe to a broad scientific field . ...
... This EGFP version differs from the common variant by Cormack et al. in T65C and I167T and has its excitation and emission peaks at 479 nm and 507 nm, respectively . , EGFP , and sfGFP . Differing residue positions among the aligned proteins are marked red. ...
A large number of soluble and membrane-associated proteins form multimeric structures with two, three, or more identical subunits. These protein oligomers are involved in numerous cellular processes, including signal transmission and gene regulation. The question of whether a protein interacts as a dimer, trimer, or higher oligomer, i.e. its stoichiometry, is essential to understand its functionality.
There is a variety of methods to characterize the stoichiometry of proteins. However, the determination of the oligomeric state of proteins is still challenging. There are methods that use Förster resonance energy transfer between homotypic fluorophores (homo-FRET). For these techniques, it is necessary that oligomerizing proteins are labelled with a fluorescent tag. If the proteins form oligomers, homo-FRET between fluorescent labels leads to the partial depolarization of the emitted light, in dependence of the stoichiometry. The extent of measured depolarization is quantified by the fluorescence anisotropy.
In this study, we fused α-helical coiled-coil peptides in fusion proteins with a green fluorescent protein (GFP) moiety. To determine the oligomeric state of the coiled-coil fusion proteins via homo-FRET, we tested two approaches for their applicability, both making use of the fluorescence anisotropy in the steady-state. One of the approaches requires steady-state data and additional parameters gained from time-resolved experiments. The other approach is based solely on the steady-state anisotropy upon fractional photobleaching of the GFP moieties. For the latter technique, a suitable theoretical model was generated to calculate the oligomeric state. With the help of these methods, the stoichiometry of a number of model proteins could be determined accurately up to the trimer. This thesis thus provides a framework to evaluate experiments which reliably differentiate between monomer, dimer, trimer, and higher oligomer via homo-FRET.
When studying photobleached GFP fusion proteins, we further found that the irradiation of GFP with blue high-intensity light leads to the degradation of the protein. This light-induced degradation was observed for two different GFP variants. Two cleavage sites could be identified more specifically, most clearly a fragmentation site vicinal to the fluorophore, at the Cα atom of residue 65. It is expected that insights gained in this study broaden future applications of GFP and provide a new perspective on the behavior of the GFP fluorophore under extremely intense irradiation.
... The advantages of C. elegans as a model system of aging ( Figure 4) are also highly relevant for studies of NDs. In addition, the transparent body makes it possible to use fluorescent reporters (Chalfie et al., 1994) for in vivo visualization of the neuron(s) of interest or the whole neuronal network. The CEP neurons for example, are mechanosensory neurons responding to the neurotransmitter dopamine. ...
... In C. elegans dopaminergic neurons were identified already in 1975 (Sulston et al., 1975). Later, it was proposed as a PD model given its approachability, conservation of genes and pathways and available techniques and methods (Wintle and Van Tol, 2001), for example the possibility of using fluorescent proteins in the neuronal circuit of choice (Chalfie et al., 1994). The health state of dopaminergic neurons was initially addressed in neurotoxicity studies using 6-hydroxydopamine (6-OHDA) or 1-methyl-4phenylpyridinium (MPP+) (Nass et al., 2002;Braungart et al., 2004). ...
Since its introduction as a genetic model organism, Caenorhabditis elegans has yielded insights into the causes of aging. In addition, it has provided a molecular understanding of mechanisms of neurodegeneration, one of the devastating effects of aging. However, C. elegans has been less popular as an animal model to investigate DNA repair and genomic instability, which is a major hallmark of aging and also a cause of many rare neurological disorders. This article provides an overview of DNA repair pathways in C. elegans and the impact of DNA repair on aging hallmarks, such as mitochondrial dysfunction, telomere maintenance, and autophagy. In addition, we discuss how the combination of biological characteristics, new technical tools, and the potential of following precise phenotypic assays through a natural life-course make C. elegans an ideal model organism to study how DNA repair impact neurodegeneration in models of common age-related neurodegenerative diseases.
... GFP is a small protein of about 27 kDa consisting of 238 amino acids (aa) derived from the jellyfish Aequorea victoria . It is intrinsically fluorescent, emitting a brilliant green light when exposed to ultraviolet or blue light, due to a chromophore formed from a maturation reaction of three specific aa at the center of the protein (Ser65, Tyr66, and Gly67) [4,5]. ...
... By comparing the marker bands, it is possible to determine that the stronger and better-defined bands correspond to protein(s) with molecular weights slightly higher than 25 kDa. Given that this value is very close to that found in the bibliography for eGFP (27 kDa) , it can be concluded that this protein has been present since the beginning of the chromatography (sample G) in relevant quantities until the post-dialysis moment, where the presence of only one band of its molecular weight revealed that it was correctly isolated from the remaining proteins (sample L). This qualitative analysis corroborated the purity results previously described and presented in Table 4. ...
Protein Engineering is a highly evolved field of engineering aimed at developing proteins for specific industrial, medical, and research applications. Here, we present a practical teaching course to demonstrate fundamental techniques used to express, purify and analyze a recombinant protein produced in Escherichia coli—the enhanced green fluorescent protein (eGFP). The methodologies used for eGFP production were introduced sequentially over six laboratory sessions and included (i) bacterial growth, (ii) sonication (for cell lysis), (iii) affinity chromatography and dialysis (for eGFP purification), (iv) bicinchoninic acid (BCA) and fluorometry assays for total protein and eGFP quantification, respectively, and (v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for qualitative analysis. All groups were able to isolate the eGFP from the cell lysate with purity levels up to 72%. Additionally, a mass balance analysis performed by the students showed that eGFP yields up to 46% were achieved at the end of the purification process following the adopted procedures. A sensitivity analysis was performed to pinpoint the most critical steps of the downstream processing.
... Genetic engineering techniques create organisms producing genetically encoded fluorescent proteins by fusing a DNA sequence carrying the information for synthesizing a protein to a DNA sequence carrying the information for synthesizing a fluorophore (Chalfie et al. 1994;Thorn 2017). Genetically encoded protein fluorophores started with the discovery of green fluorescent protein (GFP) in jellyfish (Prasher et al. 1992) and the realization that GFP's DNA sequence could be fused to almost any protein DNA sequence (Chalfie et al. 1994). ...
... Genetic engineering techniques create organisms producing genetically encoded fluorescent proteins by fusing a DNA sequence carrying the information for synthesizing a protein to a DNA sequence carrying the information for synthesizing a fluorophore (Chalfie et al. 1994;Thorn 2017). Genetically encoded protein fluorophores started with the discovery of green fluorescent protein (GFP) in jellyfish (Prasher et al. 1992) and the realization that GFP's DNA sequence could be fused to almost any protein DNA sequence (Chalfie et al. 1994). ...
Live-cell fluorescence spectral imaging is an evolving modality of microscopy that uses specific properties of fluorophores, such as excitation or emission spectra, to detect multiple molecules and structures in intact cells. The main challenge of analyzing live-cell fluorescence spectral imaging data is the precise quantification of fluorescent molecules despite the weak signals and high noise found when imaging living cells under non-phototoxic conditions. Beyond the optimization of fluorophores and microscopy setups, quantifying multiple fluorophores requires algorithms that separate or unmix the contributions of the numerous fluorescent signals recorded at the single pixel level. This review aims to provide both the experimental scientist and the data analyst with a straightforward description of the evolution of spectral unmixing algorithms for fluorescence live-cell imaging. We show how the initial systems of linear equations used to determine the concentration of fluorophores in a pixel progressively evolved into matrix factorization, clustering, and deep learning approaches. We outline potential future trends on combining fluorescence spectral imaging with label-free detection methods, fluorescence lifetime imaging, and deep learning image analysis.
... As discussed above, labelling may be altered during sample preparation for EM. In its early stages of discovery, GFP demonstrated a good tolerance to fixatives such as formaldehyde and glutaraldehyde (Chalfie et al., 1994;Ward, 2006), although the latter fixative can cause imaging difficulties due to its high auto-fluorescence. GFP was reported to denature when dehydrated in pure, dried organic solvents (Ward, 2006). ...
Sample preparation is the novel bottleneck for high throughput correlative light and electron microscopy (CLEM). Protocols suitable for both imaging methods must therefore balance the requirements of each technique. For fluorescence light microscopy, a structure of interest can be targeted using: 1) staining, which is often structure or tissue specific rather than protein specific, 2) dye-coupled proteins or antibodies, or 3) genetically encoded fluorescent proteins. Each of these three methods has its own advantages. For ultrastructural investigation by electron microscopy (EM) resin embedding remains a significant sample preparation approach, as it stabilizes the sample such that it withstands the vacuum conditions of the EM, and enables long-term storage. Traditionally, samples are treated with heavy metal salts prior to resin embedding, in order to increase imaging contrast for EM. This is particularly important for volume EM (vEM) techniques. Yet, commonly used contrasting agents (e.g., osmium tetroxide, uranyl acetate) tend to impair fluorescence. The discovery that fluorescence can be preserved in resin-embedded specimens after mild heavy metal staining was a game changer for CLEM. These so-called in-resin fluorescence protocols present a significant leap forward for CLEM approaches towards high precision localization of a fluorescent signal in (volume) EM data. Integrated microscopy approaches, combining LM and EM detection into a single instrument certainly require such an “all in one” sample preparation. Preserving, or adding, dedicated fluorescence prior to resin embedding requires a compromise, which often comes at the expense of EM imaging contrast and membrane visibility. Especially vEM can be strongly hampered by a lack of heavy metal contrasting. This review critically reflects upon the fundamental aspects of resin embedding with regard to 1) specimen fixation and the physics and chemistry underlying the preservation of protein structure with respect to fluorescence and antigenicity, 2) optimization of EM contrast for transmission or scanning EM, and 3) the choice of embedding resin. On this basis, various existing workflows employing in-resin fluorescence are described, highlighting their common features, discussing advantages and disadvantages of the respective approach, and finally concluding with promising future developments for in-resin CLEM.
... While this protocol was successful, it imposed a significant barrier for general use, because of the need of first isolating a host insect eye color mutant that is defective in a known gene. This restriction was overcome with the discovery of green fluorescent protein (GFP) as a cell marker (Chalfie et al. 1994). GFP expression obviates the need to isolate an eye color mutant, thus contributing to great progress in the generation of transgenic insects in general. ...
The stagnation of our fight against malaria in recent years, mainly due to the development of mosquito insecticide resistance, argues for the urgent development of new weapons. The dramatic evolution of molecular tools in the last few decades led to a better understanding of parasite–mosquito interactions and coalesced in the development of novel tools namely, mosquito transgenesis and paratransgenesis. Here we provide a historical view of the development of these new tools and point to some remaining challenges for their implementation in the field.
... C. elegans worms are transparent, and therefore, individual cells and subcellular details can be visualized using Nomarski (also called differential interference contrast) optics (325). C. elegans is also very well suited for use in studies where fluorescent proteins are expressed (328,329). Because of its many useful features, C. elegans has been a powerful model of choice for eukaryotic genetic studies for decades. A famous example is the pattern of cell divisions and resulting cell lineages, which have been thoroughly investigated in this nematode model. ...
During the past three decades, mice, zebrafish, fruit flies, and Caenorhabditis elegans have been the primary model organisms used for the study of various biological phenomena. These models have also been adopted and developed to investigate the physiological roles of carbonic anhydrases (CAs) and carbonic anhydrase-related proteins (CARPs). These proteins belong to eight CA families and are identified by Greek letters: α, β, γ, δ, ζ, η, θ, and ι. Studies using model organisms have focused on two CA families, α-CAs and β-CAs, which are expressed in both prokaryotic and eukaryotic organisms with species-specific distribution patterns and unique functions. This review covers the biological roles of CAs and CARPs in light of investigations performed in model organisms. Functional studies demonstrate that CAs are not only linked to the regulation of pH homeostasis, the classical role of CAs but also contribute to a plethora of previously undescribed functions.
... The small size allows a large number of C. elegans to be cultured in very small spaces; the transparent body allows observation of cells and features throughout the body without the need to kill or dissect the animals (Sulston and Horvitz, 1977). Transparency also enables a wealth of studies in living animals utilizing fluorescent protein reporters (Chalfie et al., 1994), and many studies can be performed at the single-cell level; quick generation time and short lifespan make C. elegans a much faster and more efficient model to obtain the desired phenotype than mammalian models (Kobet et al., 2014;Kim and Park, 2020). Furthermore, body transparency and invariant cell lineage gave the opportunity to map the simple nervous system, consisting of 302 neurons, whose connectivity has been completely reconstructed and characterized (White et al., 1986). ...
Mitochondrial diseases are a group of genetic disorders characterized by dysfunctional mitochondria. Within eukaryotic cells, mitochondria contain their own ribosomes, which synthesize small amounts of proteins, all of which are essential for the biogenesis of the oxidative phosphorylation system. The ribosome is an evolutionarily conserved macromolecular machine in nature both from a structural and functional point of view, universally responsible for the synthesis of proteins. Among the diseases afflicting humans, those of ribosomal origin – either cytoplasmic ribosomes (80S) or mitochondrial ribosomes (70S) – are relevant. These are inherited or acquired diseases most commonly caused by either ribosomal protein haploinsufficiency or defects in ribosome biogenesis. Here we review the scientific literature about the recent advances on changes in mitochondrial ribosomal structural and assembly proteins that are implicated in primary mitochondrial diseases and neurodegenerative disorders, and their possible connection with metalloid pollution and toxicity, with a focus on MRPL44, NAM9 (MNA6) and GEP3 (MTG3), whose lack or defect was associated with resistance to tellurite. Finally, we illustrate the suitability of yeast Saccharomyces cerevisiae (S.cerevisiae) and the nematode Caenorhabditis elegans (C.elegans) as model organisms for studying mitochondrial ribosome dysfunctions including those involved in human diseases.
... Such lines were systematically produced in so called enhancer trap screens, in which the transgene was mobilised and progeny screened for specific expression in particular cells or tissues of interest (O'Kane and Gehring, 1987). Transgenic lines expressing fluorescent proteins in specific cell types, first applied in C. elegans (Chalfie et al., 1994), allowed easy detection of morphological deviations in the tissue of interest without staining in genetic screens. ...
Experimental embryologists working at the turn of the 19th century suggested fundamental mechanisms of development, such as localized cytoplasmic determinants and tissue induction. However, the molecular basis underlying these processes proved intractable for a long time, despite concerted efforts in many developmental systems to isolate factors with a biological role. That road block was overcome by combining developmental biology with genetics. This powerful approach used unbiased genome-wide screens to isolate mutants with developmental defects and to thereby identify genes encoding key determinants and regulatory pathways that govern development. Two small invertebrates were the pioneers: the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans. Their modes of development differ in many ways, but the two together led the way to unraveling the molecular mechanisms of many fundamental developmental processes. The discovery of the grand homologies between key players in development throughout the animal kingdom underscored the usefulness of studying these small invertebrate models for animal development and even human disease. We describe developmental genetics in Drosophila and C. elegans up to the rise of genomics at the beginning of the 21st Century. Finally, we discuss themes that emerge from the histories of such distinct organisms and prospects of this approach for the future.
... The GFP with a color palette ranging from blue to orange was created using a combination of chemically tailoring the core chromophore, showing potential applications for fluorescent color regulation and cell imaging. GFP has received notoriety in biology as a genetically encoded noninvasive luminous marker  due to its minimal cytotoxicity and strong photostability. However, the macromolecular assembly showed the highest emission quantum yield (QY), approaching 8%, which is more than 80-fold greater than the core chromophore. ...
The development of luminescent materials is critical to humankind. The Nobel Prizes awarded in 2008 and 2010 for research on the development of green fluorescent proteins and super-resolved fluorescence imaging are proof of this (2014). Fluorescent probes, smart polymer machines, fluorescent chemosensors, fluorescence molecular thermometers, fluorescent imaging, drug delivery carriers, and other applications make fluorescent polymers (FPs) exciting materials. Two major branches can be distinguished in the field: (1) macromolecules with fluorophores in their structure and (2) aggregation-induced emission (AIE) FPs. In the first, the polymer (which may be conjugated) contains a fluorophore, conferring photoluminescent properties to the final material, offering tunable structures, robust mechanical properties, and low detection limits in sensing applications when compared to small-molecule or inorganic luminescent materials. In the latter, AIE FPs use a novel mode of fluorescence dependent on the aggregation state. AIE FP intra- and intermolecular interactions confer synergistic effects, improving their properties and performance over small molecules aggregation-induced, emission-based fluorescent materials (AIEgens). Despite their outstanding advantages (over classic polymers) of high emission efficiency, signal amplification, good processability, and multiple functionalization, AIE polymers have received less attention. This review examines some of the most significant advances in the broad field of FPs over the last six years, concluding with a general outlook and discussion of future challenges to promote advancements in these promising materials that can serve as a springboard for future innovation in the field.
... Ainsi la cellule va fabriquer le fluorophore in situ et seules les cellules vivantes sont capables de transcrire ce gène en protéine fluorescente. La GFP (Green Fluorescent Protein) découverte dans la méduse Aequorea victoria [81,82] a été utilisée pour étudier la dynamique des protéines dans des systèmes vivants. Elle a une masse moléculaire de 26. ...
Les nouvelles techniques d’imagerie super-résolue ont considérablement facilité l’étude des interactions cellule/substrat. L'obtention d'une résolution axiale nanométrique est un paramètre clé pour analyser l'adhérence. Ainsi, j'ai développé un nouveau dispositif va-TIRFM (microscopie de fluorescence par réflexion totale interne à angle variable) à deux longueurs d’onde. Cette technique consiste à enregistrer une série d’images TIRF en augmentant l'angle d'incidence. Pour déterminer avec précision cet angle, j’ai développé une procédure de calibration simple et robuste. Un rapide traitement d'image permet de reconstruire la topographie de la membrane cellulaire avec une résolution axiale nanométrique et d’extraire la distribution spatiale de l’indice effectif du cortex cellulaire. Pour caractériser l’adhérence j’ai fait un double seuillage sur la hauteur et l’indice afin d’identifier les zones d’interactions spécifiques et non-spécifiques. Le va-TIRF peut également être utilisé pour extraire le potentiel d’interaction membrane/substrat. J'ai ainsi étudié l’adhérence des cellules gliales sur la fibronectine. J’ai montré un changement d'organisation de leurs points focaux d’adhérence et une modification de leur énergie de liaison, sous l’action d’antagonistes connus des intégrines α5β1 et αvβ3 et d’inhibiteurs du cytosquelette. Ces résultats montrent que l’énergie est un paramètre clé pour quantifier l’adhérence des cellules et l’action de différentes molécules.
... La GFP (pour « green fluorescent protein ») a été observée pour la première fois chez la méduse Aequorea victoria. Elle a été purifiée dans les années 60 puis clonée et utilisée comme marqueur de l'expression de gènes dans les années 90 (Shimomura et al., 1962;Prasher et al., 1992;Chalfie et al., 1994). En 2008, les travaux sur la GFP valurent le prix Nobel de chimie à Osamu Shimomura et Martin Chalfie, ainsi qu'à Roger Y. Tsien qui contribua en déclinant la palette de couleurs possibles, permettant de marquer plusieurs molécules ou cellules avec différentes couleurs (Zhang et al., 2002). ...
Ces travaux de thèse démontrent que la protocadhérine-18a (Pcdh18a), exprimée par les progéniteurs des cellules stromales, joue un rôle important dans leur migration, puis dans la formation de la première niche d’hématopoïèse définitive, appelé tissu hématopoïétique caudal chez le poisson zèbre. La genèse des filopodes et la migration de cellules stromales, exprimant une Pcdh18a tronquée dans sa partie cytoplasmique (Pcdh18a-ΔCP106), sont compromises et les connexions cellulaires sont plus durables au cours de la migration. Cela suggère que le domaine cytoplasmique de Pcdh18a affecte la régulation de l’actine-F et est impliqué dans la médiation de la répulsion cellulaire. L’expression de Pcdh18a-ΔCP106 conduit aussi à une diminution du nombre de cellules stromales, et altère la formation du plexus veineux, composant majeur de la niche, résultant en une niche hématopoïétique non fonctionnelle. Elle induit également la surexpression de la fibronectine 1b, ce qui contribue probablement à la morphogenèse anormale du plexus veineux. Enfin, nous avons découvert qu'un motif à quatre acides aminés REDV présent dans la région extracellulaire de Pcdh18a est requis pour la fixation des cellules stromales sur les vaisseaux et leur différenciation en cellules stromales périvasculaires.
... In 1994, Chalfie et al. reported that cells grew normally even in the presence of the gene coding for EGFP and suggested that EGFP was a good candidate for cell separation with fluorescently activated cells. They also considered that EGFP could be used as a vital marker to assess cell growth . Nevertheless, some studies have noted changes, including protein localization. ...
Integrating fluorescent genes including eGFP in the yeast genome is common practice for various applications, including cell visualization and population monitoring. The transformation of a commercial S. cerevisiae strain by integrating a cassette including a gene encoding an EGFP protein in the HO gene was carried out using CRISPR-Cas9 technology. Although this type of integration is often used and described as neutral at the phenotypic level of the cell, we have highlighted that under alcoholic fermentation (in a Chardonnay must), it has an impact on the exometabolome. We observed 41 and 82 unique biomarkers for the S3 and S3GFP strains, respectively, as well as 28 biomarkers whose concentrations varied significantly between the wild-type and the modified strains. These biomarkers were mainly found to correspond to peptides. Despite similar phenotypic growth and fermentation parameters, high-resolution mass spectrometry allowed us to demonstrate, for the first time, that the peptidome is modified when integrating this cassette in the HO gene.
... The nematode Caenorhabditis elegans is a useful model organism for understanding unsolved complex biological questions, such as nervous system function and developmental properties. In addition, C. elegans has also been used to establish many biological methods, such as fluorescent imaging, RNAi, and genome-editing techniques . It is suitable as the first animal to demonstrate cutting-edge X-ray measurements for single molecular observations in vivo. ...
The dynamic properties of protein molecules are involved in the relationship between their structure and function. Time-resolved X-ray observation enables capturing the structures of biomolecules with picometre-scale precision. However, this technique has yet to be implemented in living animals. Here, we examined diffracted X-ray blinking (DXB) and diffracted X-ray tracking (DXT) to observe the dynamics of a protein located on intestinal cells in adult Caenorhabditis elegans. This in vivo tissue-specific DXB was examined at temperatures from 20 °C to −10 °C for a recombinant ice-binding protein from Antarctomyces psychrotrophicus (AnpIBP) connected with the cells through a transmembrane CD4 protein equipped with a glycine-serine linker. AnpIBP inhibits ice growth at subzero temperatures by binding to ice crystals. We found that the rotational motion of AnpIBP decreases at −10 °C. In contrast, the motion of the AnpIBP mutant, which has a defective ice-binding ability, did not decrease at −10 °C. The twisting and tilting motional speeds of AnpIBPs measured above 5 °C by DXT were always higher than those of the defective AnpIBP mutant. These results suggest that wild-type AnpIBP is highly mobile in solution, and it is halted at subzero temperatures through ice binding. DXB and DXT allow for exploring protein behaviour in live animals with subnano resolution precision.
... C. elegans have a translucent body making it easy to visualize the movement of fluorescently labeled proteins within them. This is especially important when studying the developmental processes of the nematode, or screening for mutations affecting function (Chalfie et al., 1994). Another characteristic of C. elegans, proving them to be a valuable research tool, is their short and rapid life cycle. ...
Caenorhabditis elegans (C. elegans) is being widely explored as an in vivo model to study the effects of food bioactives. These nematodes are largely advantageous over other in vivo models as they are relatively inexpensive, have a short generation time, and have a completely sequenced genome, among other advantages. C. elegans is a commonly used model to study diseases such as Alzheimer's and Parkinson's disease; however, researchers are finding they can also give insight into the health promoting effect of food-derived bioactive compounds. As consumers become more aware of the health benefits of the foods that they consume, the study of bioactive properties of foods and food constituents is becoming an important source of information. This review focuses on the advantages of using C. elegans as a model such as their short lifespans, high level of gene conservation relative to humans, and large number of progenies per reproductive cycle. They are also easily manipulated in order to perform controlled experiments on synchronous populations. Through review of recent literature, it is clear that C. elegans can be used to study a range of food derived compounds such as bioactive peptides, phenolic compounds, carbohydrates, and lipids. This review also provides information on potential challenges associated with working with this nematode. These challenges include the need for a sterile environment, potential inaccuracy when determining if the nematodes are dead, and the simplicity of the organism making it not suitable for all studies.
... In addition, the analysis of synaptic connections by serial electron microscopy has led to the reconstruction of the complete neural wiring diagram (White et al., 1986;Cook et al., 2019), which allows the identification of precise neural circuits that control specific behaviors and physiological processes. Moreover, C. elegans transparency permits the visualization of the morphology and activity of specific cells using transgenic reporters and genetically encoded calcium indicators (Chalfie et al., 1994;Kerr and Schafer, 2006;Chung et al., 2013) in freely moving animals. Given the striking conservation in neuronal function throughout the animal kingdom, C. elegans offers the possibility to provide fundamental insights into nervous system (NS) aging. ...
Due to the increase in life expectancy worldwide, age-related disorders such as neurodegenerative diseases (NDs) have become more prevalent. Conventional treatments comprise drugs that only attenuate some of the symptoms, but fail to arrest or delay neuronal proteotoxicity that characterizes these diseases. Due to their diverse biological activities, imidazole rings are intensively explored as powerful scaffolds for the development of new bioactive molecules. By using C. elegans, our work aims to explore novel biological roles for these compounds. To this end, we have tested the in vivo anti-proteotoxic effects of imidazolium salts. Since NDs have been largely linked to impaired antioxidant defense mechanisms, we focused on 1-Mesityl-3-(3-sulfonatopropyl) imidazolium (MSI), one of the imidazolium salts that we identified as capable of improving iron-induced oxidative stress resistance in wild-type animals. By combining mutant and gene expression analysis we have determined that this protective effect depends on the activation of the Heat Shock Transcription Factor (HSF-1), whereas it is independent of other canonical cytoprotective molecules such as abnormal Dauer Formation-16 (DAF-16/FOXO) and Skinhead-1 (SKN-1/Nrf2). To delve deeper into the biological roles of MSI, we analyzed the impact of this compound on previously established C. elegans models of protein aggregation. We found that MSI ameliorates β-amyloid-induced paralysis in worms expressing the pathological protein involved in Alzheimer’s Disease. Moreover, this compound also delays age-related locomotion decline in other proteotoxic C. elegans models, suggesting a broad protective effect. Taken together, our results point to MSI as a promising anti-proteotoxic compound and provide proof of concept of the potential of imidazole derivatives in the development of novel therapies to retard age-related proteotoxic diseases.
... Cell labelling with reporter genes such as green or red fluorescent proteins (eGFP, DsRed, mCherry) could provide an attractive option to trace transplanted cells  . The expression of these reporter genes generates easily measurable signal suitable for cell monitoring and changes in signal intensity can indicate cell death or proliferation. ...
Stem cell therapy has great potential for replacing beta-cell loss in diabetic patients. However, a key obstacle to cell therapy’s success is to preserve viability and function of the engrafted cells. While several strategies have been developed to improve engrafted beta-cell survival, tools to evaluate the efficacy within the body by imaging are limited. Traditional labeling tools, such as GFP-like fluorescent proteins, have limited penetration depths in vivo due to tissue scattering and absorption. To circumvent this limitation, a near-infrared fluorescent mutant version of the DrBphP bacteriophytochrome, iRFP720, has been developed for in vivo imaging and stem/progenitor cell tracking. Here, we present the generation and characterization of an iRFP720 expressing human induced pluripotent stem cell (iPSC) line, which can be used for real-time imaging in various biological applications. To generate the transgenic cells, the CRISPR/Cas9 technology was applied. A puromycin resistance gene was inserted into the AAVS1 locus, driven by the endogenous PPP1R12C promoter, along with the CAG-iRFP720 reporter cassette, which was flanked by insulator elements. Proper integration of the transgene into the targeted genomic region was assessed by comprehensive genetic analysis, verifying precise genome editing. Stable expression of iRFP720 in the cells was confirmed and imaged by their near-infrared fluorescence. We demonstrated that the reporter iPSCs exhibit normal stem cell characteristics and can be efficiently differentiated towards the pancreatic lineage. As the genetically modified reporter cells show retained pluripotency and multilineage differentiation potential, they hold great potential as a cellular model in a variety of biological and pharmacological applications.
... . In recent decades, the gene-encoding green fluorescent protein (GFP), originally isolated from the jellyfish Aequorea victoria , has been widely used as an effective molecular marker in many prokaryotes and eukaryotes without damaging cell activities. For example, GFP was expressed in Trichoderma species, which elucidated their interactions with Pythium ultimum, invasion of the hyphae and sclerotia of Rhizoctonia solani [4,20], and penetration of the plant parasitic nematode Globodera pallida . ...
Clonostachys rosea is an important mycoparasite, with great potential for controlling nu�merous plant fungal diseases. Understanding the mechanisms and modes of action will assist the development and application of this biocontrol fungus. In this study, the highly efficient C. rosea 67-1 strain was marked with the green fluorescent protein (GFP), and the transformant possessed the same biological characteristics as the wild-type strain. Fungal interactions with Botrytis cinerea during co-culture and encounter on tomato leaves were assessed by fluorescence confocal and electron
microscopy. The results indicated that once the two fungi met, the hyphae of C. rosea grew alongside those of B. cinerea, then attached tightly to the host and developed special structures, via which the
biocontrol fungus penetrated the host and absorbed nutrients, eventually disintegrating the cells of the pathogen. Mycoparasitism to B. cinerea was also observed on tomato leaves, suggesting that
C. rosea can colonize on plants and act following the invasion of the pathogenic fungus.
Since the first fluorescent proteins (FPs) were identified and isolated over fifty years ago, FPs have become commonplace yet indispensable tools for studying the constitutive secretory pathway in live cells. At the same time, genetically encoded chemical tags have provided a new use for much older fluorescent dyes. Innovation has also produced several specialized methods to allow synchronous release of cargo proteins from the endoplasmic reticulum (ER), enabling precise characterization of sequential trafficking steps in the secretory pathway. Without the constant innovation of the researchers who design these tools to control, image, and quantitate protein secretion, major discoveries about ER-to-Golgi transport and later stages of the constitutive secretory pathway would not have been possible. We review many of the tools and tricks, some 25 years old and others brand new, that have been successfully implemented to study ER-to-Golgi transport in intact and living cells.
The advancement of super-resolution imaging (SRI) relies on fluorescent proteins with novel photochromic properties. Using light, the reversibly switchable fluorescent proteins (RSFPs) can be converted between bright and dark states for many photocycles and their emergence has inspired the invention of advanced SRI techniques. The general photoswitching mechanism involves the chromophore cis-trans isomerization and proton transfer for negative and positive RSFPs and hydration–dehydration for decoupled RSFPs. However, a detailed understanding of these processes on ultrafast timescales (femtosecond to millisecond) is lacking, which fundamentally hinders the further development of RSFPs. In this review, we summarize the current progress of utilizing various ultrafast electronic and vibrational spectroscopies, and time-resolved crystallography in investigating the on/off photoswitching pathways of RSFPs. We show that significant insights have been gained for some well-studied proteins, but the real-time “action” details regarding the bidirectional cis-trans isomerization, proton transfer, and intermediate states remain unclear for most systems, and many other relevant proteins have not been studied yet. We expect this review to lay the foundation and inspire more ultrafast studies on existing and future engineered RSFPs. The gained mechanistic insights will accelerate the rational development of RSFPs with enhanced two-way switching rate and efficiency, better photostability, higher brightness, and redder emission colors.
The ability to tune the optoelectronic properties of quantum dots (QDs) makes them ideally suited for the use as fluorescence sensing probes. The vast structural diversity in terms of the composition and size of QDs can make designing a QD for a specific sensing application a challenging process. Quantum chemical calculations have the potential to aid this process through the characterization of the properties of QDs, leading to their in silico design. This is explored in the context of QDs for the fluorescence sensing of dopamine based upon density functional theory and time-dependent density functional theory (TDDFT) calculations. The excited states of hydrogenated carbon, silicon, and germanium QDs are characterized through TDDFT calculations. Analysis of the molecular orbital diagrams for the isolated molecules and calculations of the excited states of the dopamine-functionalized quantum dots establish the possibility of a photoinduced electron-transfer process by determining the relative energies of the electronic states formed from a local excitation on the QD and the lowest QD → dopamine electron-transfer state. The results suggest that the Si165H100 and Ge84H64 QDs have the potential to act as fluorescent markers that could distinguish between the oxidized and reduced forms of dopamine, where the fluorescence would be quenched for the oxidized form. The work contributes to a better understanding of the optical and electronic behavior of QD-based sensors and illustrates how quantum chemical calculations can be used to inform the design of QDs for specific fluorescent sensing applications.
Light induces non-equilibrium time evolving molecular phenomena. The computational modeling of photo-induced processes in large systems, embedded in complex environments (i.e., solutions, proteins, materials), demands for a quantum and statistical mechanic treatment to achieve the required accuracy in the description of both the excited-state energy potentials and the choice of the initial conditions for dynamical simulations. On the other hand, the theoretical investigation on the atomistic scale of times and sizes of the ultrafast photo-induced reactivity and non-equilibrium relaxation dynamics right upon excitation requests tailored computational protocols. These methods often exploit hierarchic computation schemes, where a large part of the degrees of freedom are required to be treated explicitly to achieve the right accuracy. Additionally, part of the explicit system needs to be treated at ab initio level, where density functional theory, using hybrid functionals, represents a good compromise between accuracy and computational cost, when proton transfers, non-covalent interactions, and hydrogen bond dynamics play important roles. Thus, the modeling strategies presented in this review stress the importance of hierarchical quantum/molecular mechanics with effective non-periodic boundary conditions and efficient phase-sampling schemes to achieve chemical accuracy in ultrafast time-resolved spectroscopy and photo-induced phenomena. These approaches can allow explicit and accurate treatment of molecule/environment interactions, including also the electrostatic and dispersion forces of the bulk. At the same time, the specificities of the different case studies of photo-induced phenomena in solutions and biological environments are highlighted and discussed, with special attention to the computational and modeling challenges.
While the model organism Caenorhabditis elegans has been used by cell biologists since the 1970s, it has only recently been more widely adopted by the toxicology community. Although the worm's genome was published in 1998, the development of cheaper and more rapid sequencing technology has been instrumental in confirming that the human and C. elegans genomes share approximately 80% homology. This homology reinforces further supports using this model organism as an in vivo model for human toxicant exposure. The goal of this chapter is to provide information about the worm's development and reproductive cycles, outline pathways common to worms and humans, present endpoints for assessing development and reproduction, and provide examples of developmental or reproductive toxicants previously studied in C. elegans. This approach is designed to help scientists familiar with C. elegans gain a better understanding of how to incorporate toxicology into their research, and provide toxicologists with tools to enhance their use of these worms in developmental and reproductive toxicity studies.
The spectral properties of fluorescent proteins (FPs) depend on the protein environment of the chromophore (CRO). A deeper understanding of the CRO – environment interactions in terms of FPs spectral characteristics will allow for a rational design of novel markers with desired properties. Here, we are taking a step towards achieving this important goal. With the time‐dependent density functional theory (TDDFT), we calculate one‐ and two‐photon absorption (OPA and TPA) spectra for 5 green FPs (GFPs) and 3 yellow FPs (YFPs) differing in amino acid sequence. The goal is to reveal a role of: (i) electrostatic interactions, (ii) hydrogen‐bonds (h‐bonds), and (iii) h‐bonds together with distant electrostatic field in absorption spectra tuning. Our results point to design hypothesis towards FPs optimised for TPA‐based applications. Both h‐bonds and electrostatic interactions co‐operate in enhancing TPA cross‐section (σ TPA ) for the S 0 ‐>S 1 transition in GFPs. Furthermore, it seems that details of h‐bonds network in the CRO’s vicinity influences σ TPA response to CRO – environment electrostatic interactions in YFPs. We postulate that engineering FPs with more hydrophilic CRO’s environment can lead to greater σ TPA . We also find that removing h‐bonds formed with the CRO’s phenolate leads to TPA enhancement for transition to higher excited states than S 1 . Particularly Y145 and T203 residues are important in this regard.
Fluorescent proteins (FPs) have gained much attention over the last few decades as powerful tools in bioimaging since the discovery of green fluorescent protein (GFP) in the 1960s. The mechanism of FP bioluminenscence has been well-studied, and new variants with improved photophysical properties are being constantly generated. In this review, a brief history of GFP along with its biogenesis is first provided. Next, the fluorescent and quenching mechanism governing the photophysical property of GFP is elaborated. Most importantly, we seek to introduce the expanding family of FP derivatives that mimics the chromophore core structure of FPs. Multiple physical and chemical strategies have been discussed to minimize the inherent fluorescence quenching effect of FP derivatives. Finally, we briefly overview the biological application of FP derivatives, with a focus on fluorescent RNA aptamer and recently reported protein aggregation detection probes. Through citing and discussing the most important works in this field, this review aims to provide a general photophysical understanding regarding the luminescence phenomenon of GFP and its derivatives, as well as chemical strategies to design functional FP derivatives.
Microscopic analysis of molecules and physiology in living cells and systems is a powerful tool in life sciences. While in vivo subcellular microscopic analysis of healthy and diseased human organs remains impossible, zebrafish larvae allow studying pathophysiology of many organs using in vivo microscopy. Here, we review the potential of the larval zebrafish pancreas in the context of islets of Langerhans and Type 1 diabetes. We highlight the match of zebrafish larvae with the expanding toolbox of fluorescent probes that monitor cell identity, fate and/or physiology in real time. Moreover, fast and efficient modulation and localization of fluorescence at a subcellular level, through fluorescence microscopy, including confocal and light sheet (single plane illumination) microscopes tailored to in vivo larval research, is addressed. These developments make the zebrafish larvae an extremely powerful research tool for translational research. We foresee that living larval zebrafish models will replace many cell line‐based studies in understanding the contribution of molecules, organelles and cells to organ pathophysiology in whole organisms. Zebrafish larvae have organs that resemble human organs, including a pancreas with an islet of Langerhans, that can be dynamically modulated and tracked in vivo. Combined with genetically‐encoded fluorescent sensors, spatiotemporal controlled modulation and revolutionary microscopy, beta cell pathophysiology in the context of Type 1 diabetes can be analyzed in situ in an unprecedented manner.
C. elegans offer a unique opportunity for understanding computation in neural networks. This is largely due to their relatively compact neural network for which a wiring diagram is available. Recent advances in genetic tools for interrogating neural activity (e.g., optogenetics) make C. elegans particularly compelling as they can be expressed in many different combinations in target individual neurons. Thus, the prospect to decipher principles underlying functionality in neural networks largely depends on the ease by which transgenic animals can be generated. Traditionally, to generate transgenic animals one would inject a plasmid containing the gene of interest under the regulation of the cell- or lineage-specific promoter. This often requires laborious cloning steps of both the gene and the promoter. The Hobert lab has developed a simpler protocol in which linear PCR fragments can be injected to generate transgenic animals. Relying on this PCR fusion-based method, here we provide a detailed protocol that we have optimized for expressing various genetically encoded calcium indicators and optogenetic tools in individual or sets of neurons. We use these simple procedures to generate multiple constructs within a very short time frame (typically 1-2 days).
Genetically encoded fluorescent reporters take advantage of C. elegans' transparency to allow non-invasive, in vivo observation, and recording of physiological processes in intact animals. Here, we discuss the basic microscope components required to observe, image, and measure fluorescent proteins in live animals for students and researchers who work with C. elegans but have limited experience with fluorescence imaging and analysis.
Although neutral luminescent radicals have arisen much attention recently, their applications in fluorescence imaging are rarely reported due to their insolubility in water and weak luminescence in polar solvents. Herein, we encapsulated a luminescent radical and its precursor into nanoparticles (NPs) by an amphiphilic polymer matrix, DSPE-PEG2000, to avoid aggregation-caused quenching and poor solubility in water to further achieve its application in cell-imaging. The obtained radical NPs exhibited strong red emission, excellent stability and remarkable biocompatibility under physiological conditions. The radical NPs were incubated with live HCT116 cells and showed good cellular uptake, thus making fluorescence imaging possible in vitro. The results confirm the feasibility of stable neutral luminescent radical as a promising candidate for the application in fluorescence imaging.
Fluorescent imaging, especially in living tissue, has become a key method in modern life sciences, with the development of new tools for sample preparation, imaging, and data analysis continuously advancing our understanding of biological principles. Here, we present our strategy for in vivo imaging of the Arabidopsis shoot apical meristem (SAM), a central structure in plant development. We implement simplifications to previously published workflows and present a novel approach to subsequentially image the meristem from multiple angles at high resolution. This tool may represent a valuable resource for shoot meristem-centered research in general, but also for studies on plasmodesmata or intercellular connectivity within the SAM: via the analysis of fluorescently labeled plasmodesmata-localized proteins, via the tracing of fluorescent dyes, via analyzing the cell-to-cell mobility of fluorescently labeled proteins, but also via the analysis of morphological features of meristematic cells in mutants or upon perturbation of symplastic connectivity.
The process of mapping neuroanatomy at multiple scales is defined as neurocartography and has the ultimate goal of revealing a complete wiring diagram of synaptic connections. Neurocartography is an important pursuit because brain function is intricately linked to neuroanatomy, similar to how the function of a protein depends on its structure. Here, we developed new light microscopy approaches for neurocartography at both microscale and nanoscale levels to map single neuron morphologies and synaptic connectivity respectively. Spatially sparse labeling of neurons has been necessary when studying neuron morphologies to minimize overlap and avoid ambiguities during reconstructions. We developed two strategies to overcome this limitation and achieve spatially dense labeling. First, we used a multicolor genetic labeling (Brainbow) approach to stochastically express fluorescent proteins in a spatially dense population of neurons, facilitating reconstruction of single neuron morphologies. We extend the Brainbow viral toolbox by 1) introducing 12 fluorescent proteins in the form of 6 new viruses, 2) using a membrane and cytoplasmic dual labeling strategy, and 3) adopting the AAV.PHP.eB capsid to systemically induce expression and better control color diversity. Second, we used expansion microscopy to increase confocal imaging resolution by physically magnifying brain samples. We developed a multi-round immunostaining Expansion Microscopy (miriEx) protocol that enables multiplexed protein detection at multiple imaging resolutions. We then combined Brainbow with miriEx to simultaneously map morphology, molecular markers, and connectivity in the same brain section. We define the derivation of these properties from hyperspectral fluorescent channels as spectral connectomics, a light microscopy based approach towards mapping neuroanatomy and connectivity with molecular specificity. We applied our multimodal profiling strategy to directly link inhibitory neuron cell types with their network morphologies. Furthermore, we showed that correlative Brainbow and endogenous synaptic machinery immunostaining can be used to define putative synaptic connections between spectrally unique neurons, as well as map putative inhibitory and excitatory inputs. We envision that spectral connectomics can be applied routinely in neurobiology labs to gain insights into normal and pathophysiological neuroanatomy across multiple animals and time points. We hope that the light microscopy approaches developed in this dissertation will facilitate the extraction of new biological insights from neurocartography.
The production and purification are the first steps required in any functional or structural study of a protein of interest. In the case of membrane proteins, these tasks can be difficult due to low expression levels and the necessity to extract them from their membrane environment. This chapter describes a convenient method based on GFP tagged to the membrane protein to facilitates these steps. Production is carried out in the yeast S. cerevisiae and purification steps are carried out and monitored taking advantage of an anti-GFP nanobody. We show how GFP can be a very helpful tool for controlling the correct addressing of the protein and for probing and optimizing purification. These methods are described here for producing and purifying CaCdr1p, an ABC exporter conferring multiantifungal resistance to C. albicans. This purification method can be amenable to any other GFP-tagged protein.
Kavitäts-Exziton-Polaritonen (Polaritonen) sind hybride Quasiteilchen, die sich aufgrund starker Kopplung von Halbleiter-Exzitonen mit Kavitätsphotonen ausbilden. Diese Quasiteilchen weisen eine Reihe interessanter Eigenschaften auf, was sie einerseits für die Grundlagenforschung, andererseits auch für die Entwicklung neuartiger Bauteile sehr vielversprechend macht. Bei Erreichen einer ausreichend großen Teilchendichte geht das System in den Exziton-Polariton-Kondensationszustand über, was zur Emission von laserartigem Licht führt. Organische Halbleiter als aktives Emittermaterial zeigen in diesem Kontext großes Potential, da deren Exzitonen neben großen Oszillatorstärken auch hohe Bindungsenergien aufweisen. Deshalb ist es möglich, unter Verwendung organischer Halbleiter selbst bei Umgebungsbedingungen äußerst stabile Polaritonen zu erzeugen. Eine wichtige Voraussetzung zur Umsetzung von integrierten opto-elektronischen Bauteilen basierend auf Polaritonen ist der kontrollierte räumliche Einschluss sowie die Realisierung von frei konfigurierbaren Potentiallandschaften. Diese Arbeit beschäftigt sich mit der Entwicklung und der Untersuchung geeigneter Plattformen zur Erzeugung von Exziton-Polaritonen und Polaritonkondensaten in hemisphärischen Mikrokavitäten, in die organische Halbleiter eingebettet sind.
For neurons, especially those with long axons, the forceful transport of mitochondria, vesicles, and other cytoplasmic components by cytoskeletal motors is vital. Defects in cytoplasmic transport machinery cause a degradation of signaling capacity that is most severe for neurons with the longest axons. In humans, with motor axons up to a meter long, even a mild mutation in one copy of the gene that codes for kinesin-1, the primary anterograde axonal transport motor, can cause spastic paraplegia and other distal neuropathies. To address questions about the molecular mechanisms of organelleOrganelles movement, we turned to Drosophila as a model system, because it offered rigorous genetic and molecular approaches to the identification and inhibition of specific elements of transport machinery. However, methods for direct observation of organelleOrganelles transport were largely lacking. We describe here an approach that we developed for imagingImaging the transport behaviors of specific organellesOrganelles in the long motor axonsAxons of larvae. It is straightforward, the equipment is commonly available, and it provides a powerful tool for studying the contributions of specific proteinsProteins to organelleOrganelles transport mechanisms.
α-Isocyanoacetamide derivatives were first converted to oxazolines under newly developed conditions. These oxazolines were used as precursors of α-isocyanoacrylamides, which could lead to various 2-arylated imidazolones. In addition, it was shown that both oxazolines and their precursors could efficiently lead to imidazolones unsubstituted at C2 in a very rapid, one-pot transformation, thus paving the way toward unprecedented routes to imidazolones, having a good tolerance to different substitution patterns.
Neurons are brain cells that can represent and store information, and they help our bodies to respond to events happening around us. How is information represented, processed and stored by neurons? Which neurons are activated while we perform a specific behavior? These are fundamental scientific questions. One important experimental approach to answering these questions is to record the activity of neurons inside the brain. In 2001, scientists developed an approach that uses light and a fascinating protein that becomes brightly fluorescent when neurons are active. This engineered protein, called GCaMP, responds to the amount of calcium inside the neurons considering that once a neuron is active, calcium inside it increases. GCaMP is now commonly used in laboratories all over the world to study neurons activity. In this article, we explain how this tool works and what makes it so useful for studying the brain.
The green fluorescent protein (GFP) shows a vivid model to design and fabricate fluorescent materials, however, a simple and generic strategy has not been really achieved to mimic the GFP system. Here we report a strategy to prepare nanoscale micelles acting like the “nano-can” in GFP, enabling a group of non-luminous amines to fluoresce. The fluorescent systems can be fabricated by mixing two kinds of commercially available non-conjugated precursors, one is amphiphilic ring-type compound for micelle, the other is amine for chromophore. Our results suggest that the nanoscale micelles provide ideal confined domains, within which amine compounds can form effective chromophores. The micelle-triggered method is generalizable to various kinds of precursors, and the emission can be tuned at different colors upon altering precursors. The presented strategy will be a starting point for a new luminescent research area in which we pay more attention on the environment than chromophore.
Fluorescent proteins (FPs) have become an essential tool for biological research. Since the isolation and description of GFP, hundreds of fluorescent proteins have been discovered and created with various characteristics. The excitation of these proteins ranges from ultra‐violet (UV) up to near infra‐RED (NIR). Using conventional cytometry with each detector assigned to each fluorochrome, great care must be taken when selecting the optimal bandpass filters to minimalize the spectral overlap. In the last eight years, several companies have released full spectrum flow cytometers which eliminates the need to change optical filters for analysing FPs. This addressed at least part of the problem however, the laser wavelengths in commercial instruments are generally not ideal for all fluorescent proteins yet do allow the separation of at least 6 FPs. Another technical challenge is to have convenient single color controls. If four different FPs are being used in an experiment, single color controls will be needed to compensate or unmix the data. In the case of cultured cells this will involve having each of the FPs expressed in cell lines separately with a parental cell line expressing none. In the case of in vivo experiments, colonies of animals may need to be maintained expressing each FP along with a wildtype animal. This represents a considerable expense and inconvenience. An appealing alternative is to produce and purify FPs and covalently couple to polystyrene microspheres. Such microspheres are ready to use and can be stored at 4°C for months or even years without any deterioration in fluorescence. The same procedure can be used to couple antibodies to these particles. Here we describe this procedure which can be executed in any lab without any special equipment or skills.
We describe a dominant behavioral marker, rol‐6(su‐1006), and an efficient microinjection procedure which facilitate the recovery of Caenorhabditis elegans transformants. We use these tools to study the mechanism of C.elegans DNA transformation. By injecting mixtures of genetically marked DNA molecules, we show that large extrachromosomal arrays assemble directly from the injected molecules and that homologous recombination drives array assembly. Appropriately placed double‐strand breaks stimulated homologous recombination during array formation. Our data indicate that the size of the assembled transgenic structures determines whether or not they will be maintained extrachromosomally or lost. We show that low copy number extrachromosomal transformation can be achieved by adjusting the relative concentration of DNA molecules in the injection mixture. Integration of the injected DNA, though relatively rare, was reproducibly achieved when single‐stranded oligonucleotide was co‐injected with the double‐stranded DNA.
The structure and connectivity of the nervous system of the nematode Caenorhabditis elegans has been deduced from reconstructions of electron micrographs of serial sections. The hermaphrodite nervous system has a total complement of 302 neurons, which are arranged in an essentially invariant structure. Neurons with similar morphologies and connectivities have been grouped together into classes; there are 118 such classes. Neurons have simple morphologies with few, if any, branches. Processes from neurons run in defined positions within bundles of parallel processes, synaptic connections being made en passant. Process bundles are arranged longitudinally and circumferentially and are often adjacent to ridges of hypodermis. Neurons are generally highly locally connected, making synaptic connections with many of their neighbours. Muscle cells have arms that run out to process bundles containing motoneuron axons. Here they receive their synaptic input in defined regions along the surface of the bundles, where motoneuron axons reside. Most of the morphologically identifiable synaptic connections in a typical animal are described. These consist of about 5000 chemical synapses, 2000 neuromuscular junctions and 600 gap junctions.
Plasmid vectors are described that allow cloning of target DNAs at sites where they will be minimally transcribed by Escherichia coli RNA polymerase but selectively and actively transcribed by T7 RNA polymerase, in vitro or in E. coli cells. Transcription is controlled by the strong φ10 promoter for T7 RNA polymerase, and in some cases by the Tφ transcription terminator. The RNA produced can have as few as two foreign nucleotides ahead of the target sequence or can be cut by RNase III at the end of the target sequence. Target mRNAs can be translated from their own start signals or can be placed under control of start signals for the major capsid protein of T7, with the target coding sequence fused at the start codon or after the 2nd, 11th or 260th codon for the T7 protein. The controlling elements are contained on small DNA fragments that can easily be removed and used to create new expression vectors.
A simple technique for rapidly killing all or part of single neurons consists of filling the cell with Lucifer Yellow CH and irradiating all or part of it with intense blue light. Such treatment kills the irradiated part of the cell within a few minutes. Adjacent cells are not affected.
The number of nongonadal nuclei in the free-living soil nematode Caenorhabditis elegans increases from about 550 in the newly hatched larva to about 810 in the mature hermaphrodite and to about 970 in the mature male. The pattern of cell divisions which leads to this increase is essentially invariant among individuals; rigidly determined cell lineages generate a fixed number of progeny cells of strictly specified fates. These lineages range in length from one to eight sequential divisions and lead to significant developmental changes in the neuronal, muscular, hypodermal, and digestive systems. Frequently, several blast cells follow the same asymmetric program of divisions; lineally equivalent progeny of such cells generally differentiate into functionally equivalent cells. We have determined these cell lineages by direct observation of the divisions, migrations, and deaths of individual cells in living nematodes. Many of the cell lineages are involved in sexual maturation. At hatching, the hermaphrodite and male are almost identical morphologically; by the adult stage, gross anatomical differences are obvious. Some of these sexual differences arise from blast cells whose division patterns are initially identical in the male and in the hermaphrodite but later diverge. In the hermaphrodite, these cells produce structures used in egg-laying and mating, whereas, in the male, they produce morphologically different structures which function before and during copulation. In addition, development of the male involves a number of lineages derived from cells which do not divide in the hermaphrodite. Similar postembryonic developmental events occur in other nematode species.
Many cnidarians utilize green-fluorescent proteins (GFPs) as energy-transfer acceptors in bioluminescence. GFPs fluoresce in vivo upon receiving energy from either a luciferase-oxyluciferin excited-state complex or a Ca(2+)-activated phosphoprotein. These highly fluorescent proteins are unique due to the chemical nature of their chromophore, which is comprised of modified amino acid (aa) residues within the polypeptide. This report describes the cloning and sequencing of both cDNA and genomic clones of GFP from the cnidarian, Aequorea victoria. The gfp10 cDNA encodes a 238-aa-residue polypeptide with a calculated Mr of 26,888. Comparison of A. victoria GFP genomic clones shows three different restriction enzyme patterns which suggests that at least three different genes are present in the A. victoria population at Friday Harbor, Washington. The gfp gene encoded by the lambda GFP2 genomic clone is comprised of at least three exons spread over 2.6 kb. The nucleotide sequences of the cDNA and the gene will aid in the elucidation of structure-function relationships in this unique class of proteins.
Mutants of the mec-7 beta-tubulin gene of Caenorhabditis elegans lack the large diameter 15-protofilament microtubules normally found only in the set of six touch receptor neurons. Both a mec-7-lacZ reporter gene and affinity-purified anti-mec-7 antibodies were used to show that mec-7 is expressed primarily in the touch neurons. These data are consistent with a possible instructive role for the mec-7 tubulin in determining microtubule protofilament number. The antibodies and the mec-7-lacZ transgene were also used to examine mec-7 expression in mutants affecting the generation, differentiation or maintenance of the touch neurons. Decreased expression was observed in mutants of unc-86 and mec-3, genes that encode transcription factors essential for touch receptor neuron generation and differentiation, respectively.
As a prerequisite for the activation of MPF, the cdc2 protein kinase must undergo tyrosine dephosphorylation. Genetic studies have demonstrated that the cdc25 protein activates the cdc2 protein kinase once DNA replication has been completed. We have produced the cdc25 protein in bacteria and shown that it activates MPF in Xenopus extracts. In extracts that normally cannot enter mitosis owing to inhibition of DNA synthesis, the addition of active cdc25 protein efficiently elicits the mitotic state by inducing premature dephosphorylation of tyrosine on the cdc2 protein. The cdc25-dependent activation reaction can be reconstituted in a partially purified system lacking ATP. These biochemical experiments demonstrate that the cdc25 protein actively drives tyrosine dephosphorylation of the cdc2 protein and offer the prospect for characterizing the individual factors that regulate the activation of MPF during the progression from S phase to mitosis.
We describe a series of plasmid vectors which contain modular features particularly useful for studying gene expression in eukaryotic systems. The vectors contain the Escherichia coli beta-galactosidase (beta Gal)-encoding region (the lacZ gene) flanked by unique polylinker segments on the 5' and 3' ends, and several combinations of a variety of modules: a selectable marker (an amber suppressor tRNA), a translational initiation region, a synthetic intron segment, the early polyadenylation signal from SV40, and 3' regions from two nematode genes. A segment encoding the nuclear localization peptide from the SV40 T antigen is incorporated into many of the constructs, leading to beta Gal accumulation in nuclei, which can facilitate identification of producing cells in complex tissues. To make functional beta Gal fusions to secreted proteins, we constructed plasmids with an alternate module encoding a synthetic transmembrane domain upstream from lacZ. This domain is designed to stop transfer of secreted proteins across the membrane during secretion, allowing the beta Gal domain of the fusion polypeptide to remain in the cytoplasm and thus function in enzymatic assays. We have used the vectors to analyze expression of several genes in the nematode Caenorhabditis elegans, and have demonstrated in these studies that lacZ can be expressed in a wide variety of different tissues and cell types. These vectors should be useful in studying gene expression both in C. elegans and in other experimental systems.
A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme,
isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed
at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified.
Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments
up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule
present only once in a sample of 10(5) cells.
In the nematode Caenorhabditis elegans, microtubules with 15 protofilaments are a specialized feature of six touch-receptor neurons; microtubules found in other C. elegans neurons have 11 protofilaments. Mutations in the gene mec-7 result in touch-insensitive animals whose touch cells lack the 15-protofilament microtubules. We have characterized 54 mutations in the mec-7 gene. The absence of mec-7 activity results selectively in the recessive loss of touch sensitivity. Partial loss-of-function alleles result in a partial loss of touch sensitivity. Dominant mutations, which are isolated at an unusually high proportion, may encode abnormal products. We have cloned the mec-7 gene; it encodes a beta-tubulin which is 90-93% identical to vertebrate beta-tubulin. Our results are consistent with the hypothesis that tubulin heterogeneity contributes to the formation of structurally and functionally distinct sets of microtubules.
The mec-3 gene is essential for proper differentiation of the set of six touch receptor neurons in C. elegans. In mutants lacking mec-3 activity, the touch receptors express none of their unique differentiated features and appear to be transformed into other types of neurons. We cloned the mec-3 gene by transposon tagging and showed that a mec-3 mutant can be rescued by germ line transformation using a 5.6 kb genomic DNA fragment. In a strain in which transforming mec-3 DNA is present in about 50 copies per haploid genome, additional cells express a mec-3-dependent phenotype. The putative coding sequence of mec-3 contains a homeobox, suggesting that the mec-3 protein specifies the expression of touch cell differentiation by binding to DNA and regulating transcription of genes that encode the differentiated features of these cells.
The unique properties of firefly luciferase and the cloning of the gene for this enzyme have spawned a number of novel applications of this protein. We summarize a few of these applications including its use as a reporter gene, as a model for the study of protein import into peroxisomes, and as a component of a heterologous gene expression system.
The initial outgrowth of developing neuronal processes can be affected by a number of extrinsic interactions. Cell-cell interactions are also important in a later stage of neuronal outgrowth when processes grow into the region of their targets. The correct positioning of the process of a postembryonic sensory neuron, the touch cell AVM of the nematode Caenorhabditis elegans, at its synaptic targets requires the presence of a pair of embryonic interneurons, the BDU cells. These cells receive synapses from AVM but do not participate in the touch reflex circuit. Therefore, the AVM-BDU synapses may be required to stabilize the association between these cells and assist in the guidance of the AVM processes through a mature neuropil.
A gene expression system based on bacteriophage T7 RNA polymerase has been developed. T7 RNA polymerase is highly selective for its own promoters, which do not occur naturally in Escherichia coli. A relatively small amount of T7 RNA polymerase provided from a cloned copy of T7 gene 1 is sufficient to direct high-level transcription from a T7 promoter in a multicopy plasmid. Such transcription can proceed several times around the plasmid without terminating, and can be so active that transcription by E. coli RNA polymerase is greatly decreased. When a cleavage site for RNase III is introduced, discrete RNAs of plasmid length can accumulate. The natural transcription terminator from T7 DNA also works effectively in the plasmid. Both the rate of synthesis and the accumulation of RNA directed by T7 RNA polymerase can reach levels comparable with those for ribosomal RNAs in a normal cell. These high levels of accumulation suggest that the RNAs are relatively stable, perhaps in part because their great length and/or stem-and-loop structures at their 3' ends help to protect them against exonucleolytic degradation. It seems likely that a specific mRNA produced by T7 RNA polymerase can rapidly saturate the translational machinery of E. coli, so that the rate of protein synthesis from such an mRNA will depend primarily on the efficiency of its translation. When the mRNA is efficiently translated, a target protein can accumulate to greater than 50% of the total cell protein in three hours or less. We have used two ways to deliver active T7 RNA polymerase to the cell; infection by a lambda derivative that carries gene 1, or induction of a chromosomal copy of gene 1 under control of the lacUV5 promoter. When gene 1 is delivered by infection, very toxic target genes can be maintained silent in the cell until T7 RNA polymerase is introduced, when they rapidly become expressed at high levels. When gene 1 is resident in the chromosome, even the very low basal levels of T7 RNA polymerase present in the uninduced cell can prevent the establishment of plasmids carrying toxic target genes, or make the plasmid unstable.(ABSTRACT TRUNCATED AT 400 WORDS)
The jellyfish Aequorea emits green light whereas the photoprotein aequorin extracted from the same organism emits blue light when Ca2+ is added. Because the photogenic cells contain a green fluorescent protein (GFP) in addition to aequorin, an energy transfer from the light emitter of aequorin to GFP has been postulated. In the present study, GFP has been purified, crystallized, and partially characterized and an energy transfer in vitro from aequorin to this protein has been demonstrated. GFP was found to consist of several kinds of isomeric proteins, of which two kinds predominated. After separation, both kinds evinced the same absorption maxima at 280, 400, and 480 nm, fluorescence emission maximum at 508-509 nm, and fluorescence quantum yield of 0.72 when excited at 470 nm. Addition of Ca2+ to aequorin solutions containing a relatively low concentration of GFP resulted in luminescence close to that from aequorin alone (λmax 472 nm). When GFP and aequorin were initially coadsorbed on DEAE-cellulose or DEAE-Sephadex, however, luminescence on adding Ca2+ was green (λmax 509 nm) closely corresponding to the in vivo luminescence. The protein-protein energy transfer is considered to involve a Förster-type mechanism in vivo as well as in vitro.
Methods are described for the isolation, complementation and mapping of mutants of Caenorhabditis elegans, a small free-living nematode worm. About 300 EMS-induced mutants affecting behavior and morphology have been characterized and about one hundred genes have been defined. Mutations in 77 of these alter the movement of the animal. Estimates of the induced mutation frequency of both the visible mutants and X chromosome lethals suggests that, just as in Drosophila, the genetic units in C. elegans are large.
Many (but not all) of the bioluminescent systems in coelenter-ates involve energy transfer from an excited product molecule of the calcium activated photoprotein to a second species, the green fluorescent protein, with emission at 508 nm from its excited state. Although all the luminescent coelen-terates studied possess photoproteins, not all of them have the green fluorescent protein. This green fluorescent molecule is localized in the luminescent cells; they can thus be easily distinguished by fluorescence microscopy. The active components occur in subcellular particles; these have been isolated in an active form by homogenization in isotonic (to sea water) salt solutions.
Touch sensitivity in the nematode Caenorhabditis elegans is mediated by a set of six sensory neurons, the microtubule cells, of well-characterized anatomy and connectivity. The normal touch response is eliminated when these cells are killed by laser microsurgery. The identification of the microtubule cells as the mediators of touch sensitivity allows us to examine the effects of mutations on the development and differentiation of these cells. Forty-two touch-insensitive mutants have been isolated. These fall into 13 complementation groups. Mutations in five of the complementation groups have recognizable effects on the microtubule cells. These phenotypes include alterations of characteristic cellular ultrastructure, absence of neuronal process growth, and the absence of the cell (either by alterations in the patterns of cell division that give rise to the cells or by degeneration or death of existing cells). Because it is likely that we are approaching saturation of genes affecting primarily the microtubule cells, there appear to be relatively few genes that affect the growth and function of this class of cells and no others.
The green-fluorescent proteins (GFP) are a unique class of proteins involved in bioluminescence of many cnidaria. The GFPs serve as energy-transfer acceptors, receiving energy from either a luciferase-oxyluciferin complex or a Ca(2+)-activated photoprotein, depending on the organism. Upon mechanical stimulation of the organism, GFP emits green light spectrally identical to its fluorescence emission. These highly fluorescent proteins are unique due to the nature of the covalently attached chromophore, which is composed of modified amino acid residues within the polypeptide. This report describes the characterization of the Aequorea victoria GFP chromophore which is released as a hexapeptide upon digestion of the protein with papain. The chromophore is formed upon cyclization of the residues Ser-dehydroTyr-Gly within the polypeptide. The chromophore structure proposed here differs from that described by Shimomura [(1979) FEBS Lett. 104, 220] in a number of ways.