Les auteurs remercient René Scriwanek pour l&#39;aide à la préparation de la figure. FR est soutenu par ECHO (700.59.003), ALW Open Program (821.02.017 822.02.014 et), la coopération DFG-NWO (DN82-303) et ZonMw VICI (016.130.606) de subventions.

Une. Préparation des sphéroplastes <ol><li> Incuber la levure pendant une nuit à 30 ° C dans 10 ml du milieu approprié, déterminé par la conception de l&#39;expérience. </li><li> Le lendemain, mesurer la densité optique de la culture à 600 nm (DO 600) à l&#39;aide d&#39;un photomètre, Diluer les cellules dans le même milieu de culture jusqu&#39;à une DO 600 de 0,2 à 0,4 et de les cultiver à une phase de croissance exponentielle à moins que la conception de l&#39; expérience nécessite une condition différente. Les cellules sont en phase exponentielle lorsque la culture présente une DO 600 de 1-2. </li><li> Recueillir 10 OD 600 équivalents de cellules par centrifugation à 3500 g pendant 5 min dans un tube de 50 ml. Après centrifugation, éliminer le surnageant. </li><li> Remettre en suspension le culot de cellules dans 5 ml de 100 mM de PIPES (pH 9,6), dithiothréitol 10 mM et incubé à 30 ° C pendant 10 min. </li><li> Recueillir à nouveau les cellules par centrifugation à 3500 xg pendant 5 min. Jeter le surnageant. </li><li> Remettre en suspension les cellules in 5 ml de milieu (déterminées par la conception de l&#39;expérience) contenant 1 M de sorbitol et de 5 mg d&#39;enzyme lytique, et incuber le mélange à 30 ° C sous agitation douce pendant 30 min. </li><li> Centrifuger la suspension de cellules à 300 xg pendant 5 min pour recueillir la fraction de culot, ce qui correspond à des sphéroplastes. Jeter le surnageant. </li><li> Reprendre les sphéroplastes dans 960 pi de glace médias froid (moyen déterminé par la conception de l&#39;expérience) contenant 1 M de sorbitol et transférer le mélange dans un froid de glace de 2 ml microtube. </li></ol> 2. Nanogold absorption <ol><li> Avec une pipette, mélanger délicatement la sphéroplaste avec 4 nmol de particules chargées positivement Nanogold remises en suspension dans 40 pi d&#39;eau. Le volume final du mélange doit être de 1 ml. </li><li> Placer la suspension cellulaire obtenue sur de la glace pendant 15 min. </li><li> Transférer la suspension à la température ambiante et incuber pendant le temps nécessaire pour permettre Nanogold internalisation par endocytosis. REMARQUE: Une absorption 5 min sera principalement conduire à l&#39;étiquetage des vésicules d&#39;endocytose et compartiments endosomes précoces, tandis que l&#39;absorption de 15 min permettra de marquer le système endosomal ensemble (début et fin des endosomes). De plus longues durées d&#39;incubation (plus de 30 min) permettront également d&#39;étiqueter la vacuole. REMARQUE: Des expériences de marquage pulse-chase ne peuvent pas être effectuées avec cette méthode. Par conséquent, lors de l&#39;étiquetage pour l&#39;analyse de LE, Nanogold sera également disponible sur le PM et l&#39;INEE. </li></ol> 3. Fixation et de section <ol><li> Arrêter l&#39;absorption d&#39;Nanogold en ajoutant 1 ml de la double fixateur de force [paraformaldéhyde à 4% (PFA), 0,4% de glutaraldéhyde (GA) dans 0,1 M de tampon PHEM (20 mM de PIPES, 50 mM de HEPES, pH 6,9, mM EGTA 20, 4 mM de MgCl 2)], contenant 1 M de sorbitol à la suspension cellulaire. Gardé le tube à température ambiante. </li><li> Retourner doucement manuellement les tubes de micocentrifuge plusieurs fois pendant 30 min (ce qui permettra de garder les sphéroplastes danssuspension) et puis centrifuger deux fois à 1700 g pendant 25 sec. </li><li> Remplacer le fixateur en éliminant le surnageant et en ajoutant 1 ml de frais fixateur de force standard (2% PFA, 0,2% GA dans 0,1 M de tampon MEHP) contenant 1 M de sorbitol et incuber pendant 2 heures à température ambiante sur une roue tournant lentement. </li><li> Traiter les cellules de cryo-découpe comme décrit précédemment 6. REMARQUE: Pour les procédures de mise en valeur d&#39;absorption de Nanogold et d&#39;argent, le traitement à l&#39;acide périodique décrit dans le protocole indiqué n&#39;a pas à être effectué. Le traitement à l&#39;acide périodique est de mieux perméabiliser la paroi de la cellule 26, mais cette structure a été éliminée au cours de la génération de sphéroplastes. </li><li> Couper 50 cryosections minces nm à -120 ° C avec un couteau de diamant à sec en utilisant un ultramicrotome UCT comme décrit précédemment 8 et placez-les sur le carbone revêtu Formvar 50 mailles des grilles de nickel. </li></ol> 4. Amélioration Argent pour Nanogold particules Visualisation NOTE: La procédure pour préparer les mélanges de réaction est essentiellement celle indiquée par le fabricant. Adaptations pratiques utilisant ce protocole, rend la procédure de mise en valeur de l&#39;argent efficace et fiable sur cryosections. <ol><li> Retirez le kit d&#39;augmentation de l&#39;argent du congélateur et décongeler dans un incubateur à 37 ° ou de bain d&#39;eau. Lors de la décongélation, placer le kit dans un incubateur à 24 ° C placé dans une pièce sombre jusqu&#39;à utilisation (voir 4.7). </li><li> Placer la plaque de chauffage dans la chambre noire et le réchauffer jusqu&#39;à la température finale de 24 ° C. </li><li> Couvrez le dessus de la plaque chauffante avec le protecteur de surface, côté brillant vers le haut, et le fixer avec une bande. </li><li> Placez le Parafilm sur le Benchkote et marquer les bords avec un marqueur noir pour être en mesure de voir les bords de Parafilm dans l&#39;obscurité. </li><li> Cassette d&#39;un thermomètre à la partie supérieure de la Benchkote pour surveiller la température de la plaque chauffante. Ajuster progressivement la température si pas 24 ° C. </li><li> PlaCE le tube de 50 ml avec de l&#39;eau distillée deux fois et le kit d&#39;augmentation de l&#39;argent à l&#39;intérieur. </li><li> Remplir les petites boîtes de Pétri avec de l&#39;eau distillée deux fois, préchauffé à 37 ° C. Placez les grilles dans l&#39;eau, un spécimen de côté vers le bas, pendant 30 min. </li><li> Répétez cette étape 4.7 une fois de plus. </li><li> Transférer les grilles dans la zone de stockage et de les amener à la chambre noire. </li><li> Rincer à nouveau les grilles en les faisant passer (avec le côté de l&#39;échantillon vers le bas) sur plusieurs gouttes d&#39;eau distillée deux fois à 24 ° C placés sur le Parafilm qui ont été fixés sur la plaque chauffante. </li><li> Assurez-vous que neuf doubles gouttes d&#39;eau distillée sont prêts supplémentaires sur le Parafilm, pour le rinçage après la réaction d&#39;amplification de l&#39;argent. </li><li> Éteindre la lumière et assurez-vous que toutes les lumières sont éteintes. Allumer la lumière rouge sur. </li><li> Sortez les solutions A et B de la trousse de mise en valeur de l&#39;argent de l&#39;incubateur à 24 ° C. </li><li> Mettre six premières gouttes de la solution A puis 6 gouttes de la solution B dans un 1,5 ml microtube, et bien mélanger avec une pipette Pasteur en verre en évitant de faire des bulles. La solution est assez épaisse et il doit être mélangé manuellement avec une pipette avant de finalement vortex brièvement. </li><li> Mettez les solutions A et B dans l&#39;incubateur à 24 ° C et sortir la solution de C. </li><li> Ajouter 6 gouttes de la solution de C au mélange A / B. Puis mélanger à nouveau d&#39;abord avec une pipette Pasteur puis par vortex, éviter de faire des bulles. </li><li> Utiliser immédiatement le mélange final en plaçant les gouttes (~ 20 ul / goutte) sur le Parafilm. </li><li> Avec une pince à épiler, propres anti-magnétiques, placer les grilles sur le mélange (par ex., Argent améliorant la réaction) pendant 6 à 15 min en fonction de la mise en valeur (en taille des particules d&#39;or ie.) Souhaité être obtenu. </li><li> Retirez les grilles du mélange et de les transmettre sur le dessus de l&#39;eau distillée deux gouttes à 24 ° C pour les lavages. Tout d&#39;abord, utiliser successivement rapidement 6 gouttes puis 3 gouttes avec 7 min temps d&#39;incubation par goutte, Avant d&#39;allumer la lumière. </li><li> Passez à l&#39;étape suivante, soit l&#39;étiquetage immuno-or ou membrane coloration 27 afin de visualiser le résultat d&#39;une enquête de EM. </li></ol>

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The authors have nothing to disclose.

Immuno-microscopie électronique est une technique qui permet de combiner la localisation des protéines à la résolution ultrastructurale des transporteurs et des organites où ces protéines résident. Cela est particulièrement important lorsque l&#39;on étudie le système levure endosomique parce que ses compartiments apparaissent comme des structures ponctuées en microscopie de fluorescence. Il est donc difficile de les distinguer. Pour cette raison, l&#39;utilisation d&#39;une sonde détectable par microscopie ...

Le système de l&#39;endosome est un appareil de tri de la membrane majeur qui joue plusieurs rôles cruciaux cellulaires y compris le trafic de l&#39;enzyme lysosomale récepteurs de tri et le recyclage de la membrane plasmique (PM) 1,2 récepteurs. Les endosomes sont divisés en trois compartiments, à savoir différentes. les endosomes précoces (EE), les endosomes tardifs (LE) et les endosomes de recyclage. Cette classification est basée sur le temps nécessaire pour que la matière endocytose pour les atteindre, sur des protéines marqueurs spécifiques et de leur morphologie. Membranes, soit protéiques et lipidiques bicouches, intériorisées de la PM peuvent soit être livrés à lysosomes via les endosomes de dégradation ou être recyclés. Membranes sont également transportés vers les endosomes de l&#39;appareil de Golgi et de même, soit continuer à les lysosomes ou être récupérées vers l&#39;appareil de Golgi. En outre, les protéines peuvent être triés dans des vésicules luminales herbe vers l&#39;intérieur à partir de la membrane de l&#39;endosome limitatif, un processus qui conduit à la formation d&#39;une sous-catégorie de LE, les corps multivésiculaires. Le système de l&#39;endosome de levure est relativement moins complexe que celle des cellules eucaryotes élevées. endosomes de levure sont divisés en EE et LE. Par contraste avec les cellules de mammifères ne contiennent pas les endosomes de recyclage, mais également des organites apparentés aux lysosomes spécifiques d&#39;un tissu. Par conséquent, ils ont un réseau moins complexe de voies de trafic endosomes 3,4. Par conséquent levure a représenté et représente un système expérimental avantageux d&#39;étudier quelques-uns des principes trafic membranaire sous-jacente dans le système endosomal encore. Cet avantage est renforcé par le fait que de nombreux gènes impliqués dans les voies de endosomes ont été initialement isolé avec des écrans génétiques dans la levure 5. Bien que le système endosomal de levure dans le type sauvage et des cellules mutantes a été étudiée en utilisant des approches de microscopie par fluorescence et biochimiques, son enquête au niveau ultrastructural a seulement étéminime. Analyses morphologiques sont particulièrement pertinentes dans la levure, car la plupart des organites endosomes sont détectés comme des structures ponctuées par microscopie à fluorescence, qui rendent difficile leur identification sans équivoque 6. Malheureusement, seul un nombre limité d&#39;antisérums reconnaissant des marqueurs protéiques de levure endosomes travaille en immuno-microscopie électronique (IEM) Préparations 7-10. Pour certaines protéines, ce problème a été contourné par le marquage endogène du gène d&#39;intérêt et l&#39;utilisation d&#39;un anticorps reconnaissant l&#39;étiquette à détecter 7,11,12. Souvent, cependant, les protéines sont indétectables par IEM en raison de leurs faibles niveaux d&#39;expression. Leur surexpression n&#39;est pas une solution parce que cette approche peut induire mis-localisations et / ou des modifications dans les organites morphologie / fonctions. Ainsi, le marquage des compartiments endocytaires avec une sonde détectable par microscopie électronique EM est une option efficace. Il s&#39;agit d&#39;une solution optimale en particulier si le probe entre dans la voie d&#39;endocytose de manière dépendante du temps, ce qui permet de savoir quand il va marquer un organite spécifique 6. L&#39;absorption de Nanogold chargé positivement par sphéroplastes de levure (c&#39;est à dire. Levure où la paroi cellulaire a été éliminé par voie enzymatique) a été utilisée avec succès pour identifier le endosomes de levure compartiments 10. Ces particules se lient fortement aux lipides chargés négativement composant les membranes biologiques. Ainsi, les associés de Nanogold chargés positivement avec le PM, pénètre dans la cellule par endocytose et passe à travers la EE LE et avant d&#39;atteindre la vacuole. Ces petites particules d&#39;or, cependant, n&#39;ont pas une taille appropriée pour être vu par EM. Pour les rendre visibles, leur taille peut être agrandie par des réactions chimiques qui conduisent à la déposition de l&#39;argent ou de l&#39;or de la sonde de l&#39;or de 13 à 15. Nous avons mis au point et appliqué avec succès une approche IEM basé sur la méthode Tokuyasu pour effectuer subcellulaireétudie la localisation 8,16. Cette méthode permet d&#39;effectuer l&#39;étiquetage immunologique sur des préparations de levure avec une excellente résolution de la morphologie 8,17-24. Nous avons également mis en place une procédure combinant ce protocole IEM avec l&#39;étiquetage de Nanogold du système levure endosomique compartiments 6. En utilisant cette approche, nous avons caractérisé morphologiquement différentes sous-classes de endosomes et ultrastructuralement examiné mutants avec un trafic endosomal défaut 6,25. De plus, nous avons démontré que cet étiquetage de Nanogold peut être combiné avec marquages ​​immunomarquage offrant la possibilité d&#39;explorer la distribution d&#39;une protéine d&#39;intérêt sur les différentes sous-populations de endosomes. Nous présentons ici les modalités d&#39;étiquetage du système levure endosomique avec Nanogold chargé positivement est pratiquement réalisée.

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nanogold labeling of the yeast endosomal system for ultrastructural analyses

Endosomes sont l&#39;un des principaux membrane tri des points de contrôle dans les cellules eucaryotes et ils régulent le recyclage ou la destruction des protéines principalement de la membrane plasmique et l&#39;appareil de Golgi. En conséquence, le système de l&#39;endosome joue un rôle central dans le maintien de l&#39;homéostasie des cellules, et des mutations dans les gènes appartenant à ce réseau interconnectés par des organelles de transport vésiculaire, provoquent des pathologies graves telles que le cancer et les troubles neurobiologiques. Il est donc de l&#39;intérêt premier de comprendre les mécanismes qui sous-tendent la biogenèse et l&#39;organisation du système endosomal. La levure Saccharomyces cerevisiae a joué un rôle essentiel dans cette tâche. Pour étiqueter spécifiquement et analyser au niveau ultrastructural le système endosomal de cet organisme modèle, nous présentons ici un protocole détaillé pour l&#39;absorption d&#39;Nanogold chargé positivement par les sphéroplastes suivie par la visualisation de ces particules par une réaction d&#39;amplification de l&#39;argent. Cette méthode est également une valeur tropl pour l&#39;examen morphologique des mutants avec des défauts dans le trafic endosomal. En outre, il n&#39;est pas seulement applicable pour les examens ultrastructuraux mais il peut aussi être combiné avec des marquages ​​immunomarquage investigations protéine de localisation.

Selon le protocole présenté, la morphologie du système de levure peut être endosome accès par transmission EM. Figure 1 montre différents types de compartiments endosomiques qui ont été atteints et, par conséquent marquées avec Nanogold. Le Nanogold d&#39;argent améliorée peut être clairement vu que des particules denses d&#39;électrons. Les paramètres optimaux établis pour la réaction d&#39;amplification de l&#39;argent permet d&#39;avoir des particules d&#39;or dans une gamme de taill...

Watch this Scientific Journal Video about Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses at JoVE.com

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

Levure, Saccharomyces cerevisiae, a été un organisme modèle clé pour identifier et étudier les gènes qui régissent la biogenèse et les fonctions du système endosomal. Nous présentons ici un protocole détaillé pour le marquage spécifique des endosomes pour les études ultrastructurales.

Nanogold étiquetage du système levure endosomique pour les analyses ultrastructurales

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

nanogold-labeling-yeast-endosomal-system-for-ultrastructural

Research

JoVE Journal

Biology

10.2K vues.  University Medical Center Utrecht.  Levure, Saccharomyces cerevisiae, a été un organisme modèle clé pour identifier et étudier les gènes qui régissent la biogenèse et les fonctions du système endosomal. Nous présentons ici un protocole détaillé pour le marquage spécifique des endosomes pour les études ultrastructurales. 

Vidéo: Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

Une technique rapide pour la visualisation de Live cellules de levure immobilisées

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

Recherche

Biologie

Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform routine activities, but prosthetic suspensions using traditional socket technology can prove to be cumbersome and uncomfortable for a person with limb loss.  Moreover, for those with high proximal amputations, limited residual limb length may prevent exoprosthesis attachment all together. Osseointegrated implant technology is a novel operative procedure which allows firm skeletal attachment between the host bone and an implant. Preliminary results in European amputees with osseointegrated implants have shown improved clinical outcomes by allowing direct transfer of loads to the bone-implant interface.  Despite the apparent advantages of osseointegration over socket technology, the current rehabilitation procedures require long periods of restrictive load bearing prior which may be reduced with expedited skeletal attachment via electrical stimulation.  The goal of the osseointegrated intelligent implant design (OIID) system is to make the implant part of an electrical system to accelerate skeletal attachment and help prevent periprosthetic infection. To determine optimal electrode size and placement, we initiated proof of concept with computational modeling of the electric fields and current densities that arise during electrical stimulation of amputee residual limbs.  In order to provide insure patient safety, subjects with retrospective computed tomography scans were selected and three dimensional reconstructions were created using customized software programs to ensure anatomical accuracy (Seg3D and SCIRun) in an IRB and HIPAA approved study.  These software packages supported the development of patient specific models and allowed for interactive manipulation of electrode position and size. Preliminary results indicate that electric fields and current densities can be generated at the implant interface to achieve the homogenous electric field distributions   required to induce osteoblast migration, enhance skeletal fixation and may help prevent periprosthetic infections.  Based on the electrode configurations experimented with in the model, an external two band configuration will be advocated in the future.

There is a need to develop alternative prosthesis attachment due to limb loss attributed to vascular occlusive diseases and trauma. The goal of the work is to introduce an osseointegrated intelligent implant design system to increase skeletal fixation and reduce periprosthetic infection rates for patients needing osseointegrated technology.

Analyses Bioelectric d&#39;un système ostéo-intégrés Implant Intelligent Design pour amputés

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

Marquage fluorescent Drosophile</emOuvrages d&#39;art> Coeur

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Les études génétiques des protéines humaines réparation de l&#39;ADN utilisant de la levure comme système modèle

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

Split-ubiquitine membrane à base de levure double hybride (MYTH) Système: Un outil puissant pour identifier les interactions entre protéines

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

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 ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

Le processus de monstre vert pour la génération de souches de levure portant la délétion de gènes multiples

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Bourgeonnement de levure extraction et purification des protéines pour l&#39;étude de la fonction, Interactions, et des modifications post-traductionnelles

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

Inverser levure système à deux hybrides pour identifier les mammifères Nuclear Receptor résidus qui interagissent avec des ligands et / ou antagonistes

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

Applications de pHluorin pour Quantitative, Kinetic et à haut débit Analyse des endocytose dans la levure bourgeonnante

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...