Le travail dans le laboratoire Duennwald est pris en charge par des subventions de la Fédération américaine pour recherche sur le vieillissement (AFAR), la Fondation des maladies héréditaires (HDF) et la Fondation William Wood.

1. Expression des toxiques polyQ-expansion protéines chez la levure Une analyse systématique a mis en place la séquence d&#39;acides précise aminés d&#39;une protéine polyQ-expansion qui est nécessaire pour produire une toxicité chez la levure 7. Ce toxique polyQ-dilatation protéine contient un groupe amino-terminal de FLAG-tag suivie par 17 acides aminés de la séquence originale de la protéine huntingtine, une région polyQ, et une fusion carboxy-terminale à une protéine fluorescente (GFP ou l&#39;autre ou PCP, voir figure 1a). L&#39;expression de protéines avec des expansions polyQ de 46 glutamines ou plus (par exemple 72 et 103 glutamines) produit une toxicité chez la levure lorsqu&#39;il est exprimé sous le contrôle de l&#39;inductible et solide par rapport promoteur GAL1 7, 9. Comme décrit précédemment, la toxicité polyQ dans la levure n&#39;est qu&#39;apparente dans les cellules qui transportent l&#39;Rnq1p protéine dans sa conformation prion, [RNQ +], par exemple, la souche de levure W303 9. Le r protéine toxique polyQ-expansionecapitulates aspects centraux de la polyQ-biologie dans la levure, telles que polyQ dépendant de la longueur de toxicité (voir ci-dessous) et d&#39;agrégation (Figure 1 b). Notamment, toxiques polyQ-expansion protéines ne tuent pas les cellules de levure immédiatement; ils sont plutôt nuire ou d&#39;arrêter le cycle cellulaire et devision cellule, ce qui ralentit ou inhiber la croissance de colonies de levures sur des plaques ou des cultures liquides (nos données non publiées). 2. Problèmes potentiels avec cellules de levure exprimant toxiques polyQ-expansion protéines Les cellules de levure exprimant les protéines toxiques d&#39;expansion polyQ contraire ne peut pas montrer tout défaut une croissance de 9. La nature génétique de ces suppresseurs de polyQ-toxicité ne semble pas être fondée sur de simples mutations mendéliennes et ne peut donc se produire avec une fréquence relativement élevée (nos résultats non publiés). Nous pensons que ces suppresseurs spontanés sont causées par le durcissement des prions non identifiés qui fonctionnent de façon similaire à [RNQ +] pour déterminer toxicity.Thes polyQe suppresseurs spontanés de toxicité polyQ peut mettre en péril la réussite de toute expérience qui vise à caractériser la toxicité polyQ ou pour identifier et caractériser des modificateurs de la toxicité polyQ. Afin d&#39;éviter l&#39;apparition fréquente de ces suppresseurs spontanés, suivez les mesures de précaution décrites ci-dessous, qui se sont avérées très efficaces: <ol><li> Utiliser des cellules de levure fraîche pour des expériences de toxicité. Ne pas stocker la levure pendant de longues périodes de temps. Foire de récupérer des colonies de levures fraîches en provenance de stocks congelés. </li><li> Surveiller fréquemment l&#39;expression et l&#39;agrégation des toxiques polyQ-expansion protéines par microscopie à fluorescence. </li><li> Gardez les cellules de levure dans les médias qui répriment l&#39;expression de la toxicité toxique polyQ en tout temps (c.-à-dans des milieux sélectifs contenant du glucose comme seule source de carbone) avant de commencer toute mesures de toxicité. </li><li> Utilisez au moins trois transformants indépendants pour chaque expérience polyQ-toxicité. &lt;/ Li&gt; </ol> 3. Les analyses de croissance <ol><li> Pour chaque condition expérimentale, ensemencer une colonie de chaque de trois transformants indépendants de cellules de levure hébergeant le toxique polyQ-expansion protéines dans 3 ml de levure médias sélectifs avec du glucose comme seule source de carbone. </li><li> Incuber ces cultures la nuit à 30 ° C. Dans ces conditions, les cellules ne sont pas l&#39;expression de la protéine de polyQ-expansion parce que le glucose dans le milieu réprime leur expression. Ne laissez pas envahir ces cultures; garder la DO 600 (absorbance de la lumière de longueur d&#39;onde de 600 nm) des cultures de la nuit en dessous de 1. </li><li> Nous utilisons régulièrement trois dosages différents pour surveiller les défauts de croissance de cellules de levure exprimant la toxicité polyQ-expansion protéines: </li></ol> 3.1 Croissance sur des plaques <ol><li> Diluer les cultures de levure nuit (cultivé avec 220 secousses tpm) pour une DO 600 de 0,0005 (soit une dilution 1:1000 d&#39;une DO600 = 0,5) la culture dans sélective moidia 7 contenant du glucose. </li><li> Répartir uniformément 50 pi (résultant dans le CA. 700 colonies par plaque) de chaque culture diluée sur une plaque (10 cm de diamètre) avec un milieu sélectif contenant du glucose comme seule source de carbone et une plaque avec un milieu sélectif contenant du galactose comme seule source de carbone. </li><li> Incuber les boîtes pendant trois à quatre jours à 30 ° C. Après l&#39;incubation, de prendre des photos de chaque plaque et compter le nombre de colonies sur le glucose et le nombre de colonies sur les plaques de galactose. Dans des conditions idéales, il devrait y avoir aucun ou seulement très peu de colonies sur la plaque de galactose lors de l&#39;utilisation des cellules de levure exprimant une très toxique polyQ-expansion protéines (103 octodecies, figure 2a). </li></ol> 3.2 dosages Spotting Ce test est plus quantitative que le dosage de placage décrite ci-dessus et peut donc révéler même des différences subtiles dans la toxicité polyQ avec la même expérience sur la même plaque. <ol><li> Diluer le overnuit cultures cultivées dans un milieu avec du glucose pour une DO 600 de 0,1. </li><li> Introduire à la pipette 200 ul de ces cultures stériles dilués dans des plaques 96 puits et de préparer cinq dilutions quintuplé en série dans l&#39;eau stérile à l&#39;aide d&#39;une pipette multi-canaux. </li><li> L&#39;utilisation d&#39;un Frogger, (aussi appelé un observateur, avec 8 x 6 broches pour le transfert de cellules en suspension) transférer les suspensions cellulaires sur des plaques contenant des milieux sélectifs avec du glucose comme seule source de carbone et des plaques contenant des milieux sélectifs avec du galactose comme seule source de carbone. </li><li> Laisser les plaques sécher avant de les incuber à 30 ° C pendant trois à quatre jours. </li><li> Après l&#39;incubation, de prendre des photos de chaque plaque (Figure 2b). </li></ol> 3,3. Surveillance de la toxicité polyQ dans la levure par la croissance des cultures en milieu liquide Ce protocole est le plus quantitative (DO600 numéros) des trois essais décrits ici et peut même détecter des différences très subtiles de la toxicité des polyQ. Le précitée ocCurrence de suppresseurs spontanés, cependant, peut potentiellement produire des résultats trompeurs. Je recommande donc cet essai combinant avec au moins un des deux essais de placage décrits ci-dessus. Nous proposons d&#39;utiliser l&#39;instrument BioscreenC pour ces expériences. Le BioscreenC est un instrument qui mesure automatiquement la densité optique des cultures de levures dans 100 des plaques tout en incubés à des températures définies avec l&#39;agitation défini. D&#39;autres méthodes pour cultiver des cellules de levure et de mesurer leur densité optique peuvent également s&#39;appliquer. <ol><li> Laver les cellules de levure à partir de supports minimaux contenant du glucose comme seule source de carbone trois fois dans 3 ml d&#39;eau stérile. </li><li> Diluer les cultures de la nuit cultivées dans un milieu avec du galactose à une DO 600 de 0,1. </li><li> Remplir chaque puits de la plaque 100-bien BioscreenC avec 300 pl des cultures de levures. </li><li> Ouvrez le programme Expérience facile Bioscreen. Déterminer le nombre d&#39;échantillons que vous souhaitez surveiller (y compris les ébauches et les moyennes seulementcontrôles), régler la température à 30 ° C, régler la longueur des expériences pour 3 jours, de définir les intervalles de mesure à 15 minutes, régler le filtre à 600 nm / Brown, et définir le mode d&#39;agitation pour 15 secondes avant chaque mesure à force moyenne. </li><li> L&#39;instrument BioscreenC et le logiciel ci-joint sera de produire des feuilles de calcul Excel chaque point de données prises pendant l&#39;expérience. </li><li> Préparer les courbes de croissance avec Excel pour chaque échantillon et comparer la croissance des différents échantillons (figure 2c). Une description détaillée de l&#39;analyse des données produites par les expériences BioscreenC a été fourni avant le 10. </li></ol> 4. Les résultats représentatifs <img alt="Figure 1" src="/files/ftp_upload/3461/3461fig1.jpg" /> Figure 1. Le modèle de la levure polyQ. a) Représentation schématique de la toxicité polyQ-expansion protéines. b) La microscopie à fluorescence montrant des cellules de levure exprimant un court, non-toxique polyQcellules de protéines d&#39;expansion (25Q, panneau de gauche) et de levure exprimant un long, toxique polyQ-expansion protéines (103 octodecies, panneau de droite). <img alt="Figure 2" src="/files/ftp_upload/3461/3461fig2.jpg" /> Figure 2. Les résultats représentatifs de tests de croissance de cellules de levure exprimant polyQ-expansion protéines. a) test de placage. Environ 700 cellules de levure ont été étalés sur des plaques et incubées pendant trois jours à 30 ° C. Le panneau supérieur présente une plaque contenant milieu glucose, soit l&#39;expression de la toxicité polyQ-dilatation protéine (103 octodecies) n&#39;est pas induite. Le panneau inférieur présente une plaque de support de levure contenant du galactose, à savoir l&#39;expression de la toxicité polyQ-dilatation protéine est induite. Notez que dans l&#39;expérience décrite ici, pas de suppresseur spontanée a eu lieu. B) Repérer dosage. Cinq série de cinq dilutions de cellules de levure héberger soit un non-protéine toxique polyQ (25Q) ou une substance toxique polyQ-expansion protein (103 octodecies) ont été repérés sur des plaques de glucose qui répriment l&#39;expression des protéines (panneau de gauche) ou sur des plaques de galactose qui induisent leur expression. Les plaques ont ensuite été incubées pendant trois jours à 30 ° C. c) expérience BioscreenC. La croissance des cultures de cellules de levure exprimant soit un non-protéine toxique polyQ (25Q) ou une substance toxique polyQ-expansion protéines (103 octodecies) ont été suivis par le instrument BioscreenC. Les conditions expérimentales et l&#39;analyse des expériences BioscreenC sont décrits dans le texte principal.

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Pas de conflits d&#39;intérêt déclarés.

Ce manuscrit présente trois approches expérimentales complémentaires pour mesurer la toxicité polyQ-dans la levure organisme modèle fondé sur une croissance réduite des cellules de levure exprimant toxiques polyQ-expansion protéines. Travail dans la levure a offert un aperçu de profondes dans les mécanismes de base cellulaires et moléculaires de repliement des protéines et de sa toxicité qui a suivi, y compris le mauvais repliement et la toxicité des polyQ-expansion protéines 9,11,12. Des expé...

<table border="1"><tbody><tr><td> Nom de l&#39;instrument </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Frogger (6x8 broches) </td><td> V &amp; P scientifique, San Diego </td><td> VP 407 AH </td></tr><tr><td> BioscreenC </td><td> Growthcurves États-Unis </td><td> 5101370 </td></tr><tr><td> 100 puits ont en nid d&#39;abeille </td><td> Growthcurves États-Unis </td><td> 9602550 </td></tr></tbody></table>

growth assays to assess polyglutamine toxicity in yeast

Mauvais repliement des protéines est associée à de nombreuses maladies humaines, en particulier les maladies neurodégénératives, telles que la maladie d&#39;Alzheimer, la maladie de Parkinson, la maladie de Huntington 1. La maladie de Huntington (HD) est provoquée par l&#39;expansion de polyglutamine anormale d&#39;un (polyQ) dans la région la protéine huntingtine. La protéine huntingtine polyQ-expansé atteint une conformation aberrante (c&#39;est à dire qu&#39;il misfolds) et entraîne une toxicité cellulaire 2. Au moins huit autres maladies neurodégénératives sont causées par des polyQ-extensions, y compris le spinocérébelleuse Ataxies et la maladie de Kennedy 3. La levure organisme modèle a facilité des informations importantes sur la base cellulaire et moléculaire de la polyQ-toxicité, y compris l&#39;impact des facteurs intra-et inter-moléculaire de polyQ-toxicité, et l&#39;identification des mécanismes cellulaires qui sont déficients dans les cellules exprimant l&#39;expansion polyQ- protéines 3-8. Importantment, de nombreux aspects de polyQ-toxicité qui ont été trouvés dans la levure ont été reproduites dans d&#39;autres systèmes expérimentaux et dans une certaine mesure dans des échantillons provenant de patients HD, démontrant ainsi l&#39;importance du modèle de la levure pour la découverte des mécanismes de base qui sous-tendent polyQ-toxicité. Une manière directe et relativement simple de déterminer la toxicité polyQ-dans la levure est de mesurer les défauts de croissance de cellules de levure exprimant polyQ-expansion protéines. Ce manuscrit décrit trois approches expérimentales complémentaires pour déterminer la toxicité polyQ-dans la levure par la mesure de la croissance des cellules de levure exprimant polyQ-expansion protéines. Les deux premières approches expérimentales de surveiller la croissance des levures sur les assiettes, la troisième approche surveille la croissance des cultures de levures liquides en utilisant l&#39;instrument BioscreenC. En outre, ce manuscrit décrit les difficultés expérimentales qui peuvent survenir lors de la manipulation des modèles polyQ levure et décrit les stratégies qui vous aideront à éviter ou àminimiser ces difficultés. Les protocoles décrits ici peuvent être utilisées pour identifier et à caractériser les voies génétiques et des petites molécules qui modulent polyQ-toxicité. En outre, les tests décrits peuvent servir de modèles pour des analyses précises de la toxicité causée par d&#39;autres maladies associées à des protéines mal repliées dans les modèles de levure.

Watch this Scientific Journal Video about Growth Assays to Assess Polyglutamine Toxicity in Yeast at JoVE.com

Growth Assays to Assess Polyglutamine Toxicity in Yeast

Ce manuscrit décrit trois protocoles complémentaires pour évaluer la toxicité de polyglutamine (polyQ)-extension protéines dans la levure<em&gt; Saccharomyces cerevisiae</em&gt;. Ces protocoles peuvent être facilement modifié pour contrôler la toxicité d&#39;autres protéines mal repliées dans la levure.

Les analyses de croissance pour évaluer la toxicité de polyglutamine dans la levure

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's ...

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13.5K vues.  Boston Biomedical Research Institute. Ce manuscrit décrit trois protocoles complémentaires pour évaluer la toxicité de polyglutamine (polyQ)-extension protéines dans la levure Saccharomyces cerevisiae. Ces protocoles peuvent être facilement modifié pour contrôler la toxicité d'autres protéines mal repliées dans la levure.

Vidéo: Growth Assays to Assess Polyglutamine Toxicity in Yeast

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

Identification des phénotypes inhibition de la croissance induite par l&#39;expression de bactéries de type III Effectors dans la levure

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

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Biologie

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Mise à jour de levure-système à deux hybrides pour identifier les protéines qui interagissent avec le facteur de croissance progranuline

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

À long terme des essais de toxicité létale avec le Crustacé Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Méthodes d&#39;évaluation de Subcellular compartiments de muscle en<em&gt; C. elegans</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

Analyse comparative de l&#39;hormone de croissance dans le sérum en utilisant SPRi, Nano-SPRi et dosages ELISA

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

ODELAY: une méthode à grande échelle pour la quantification multiparamétrique de la croissance de la levure

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

Effet des protéines fluorescentes sur des partenaires de Fusion utilisant les épreuves de toxicité Polyglutamine chez la levure

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

Moyen-throughput Screening tests pour l’évaluation des effets sur Ca2 +-signalisation et Acrosome Reaction en sperme humain

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Examen de la dynamique nucléaire de levure de fission mitotique et méiotique par fluorescence Microscopie à cellules vivantes

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

Mesurer RAN Peptide Toxicity à C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...