Gli autori ringraziano René Scriwanek per l&#39;assistenza alla preparazione della figura. FR è sostenuto da ECHO (700.59.003), ALW programma aperto (821.02.017 e 822.02.014), la cooperazione DFG-NWO (DN82-303) e ZonMw VICI (016.130.606) sovvenzioni.

1. Spheroplast Preparazione <ol><li> Incubare il lievito notte a 30 ° C in 10 ml di mezzo appropriato determinato dal disegno dell&#39;esperimento. </li><li> Il giorno dopo, misurare la densità ottica della coltura a 600 nm (OD 600) usando un fotometro, Diluire le cellule nello stesso terreno di coltura ad una OD 600 di 0,2-0,4 e farle crescere ad una fase di crescita esponenziale meno che la progettazione del esperimento richiede una condizione diversa. Le cellule sono in fase esponenziale quando la coltura ha un diametro esterno 600 di 1-2. </li><li> Raccogliere 10 OD 600 equivalenti di cellule mediante centrifugazione a 3500 xg per 5 minuti in un tubo da 50 ml. Dopo la centrifugazione, scartare il surnatante. </li><li> Risospendere il pellet di cellule in 5 ml di tubi 100 mM (pH 9,6), ditiotreitolo 10 mM e incubate a 30 ° C per 10 min. </li><li> Raccogliere nuovamente le cellule per centrifugazione a 3.500 xg per 5 min. Scartare il surnatante. </li><li> Risospendere le cellule in 5 ml di terreno (determinato dal disegno dell&#39;esperimento) contenente 1 M sorbitolo e 5 mg di enzima litico, e incubare la miscela a 30 ° C con delicata agitazione per 30 min. </li><li> Centrifugare la sospensione cellulare a 300 xg per 5 minuti per raccogliere la frazione di pellet, che corrisponde alle sferoplasti. Scartare il surnatante. </li><li> Risospendere le sferoplasti in 960 ml di ghiaccio freddo supporti (medio determinato dal disegno dell&#39;esperimento) contenente 1 M sorbitolo e trasferire il composto in una ghiacciata 2 ml provetta. </li></ol> 2. Nanogold assorbimento <ol><li> Usando una pipetta, mescolare delicatamente il spheroplast con 4 nmol di particelle cariche positivamente nanogold risospese in 40 ml di acqua. Il volume finale della miscela deve essere di 1 ml. </li><li> Posizionare la sospensione cellulare ottenuta in ghiaccio per 15 min. </li><li> Trasferire la sospensione a temperatura ambiente ed incubare per il tempo necessario a consentire nanogold internalizzazione via endocytosis. NOTA: A 5 min assorbimento sarà principalmente portare l&#39;etichettatura di vescicole e dei primi comparti endosomiali, mentre un 15 min assorbimento permetterà di contrassegnare l&#39;intero sistema endosomiale (precoce e endosomi tardivi). Tempi di incubazione più lungo (più di 30 min), consentiranno inoltre di etichettare vacuolo. NOTA: esperimenti di etichettatura Pulse-chase non possono essere eseguite con questo metodo. Pertanto, quando l&#39;etichettatura per l&#39;analisi di LE, nanogold anche essere trovate in PM e INEE. </li></ol> 3. Fissazione e sezionamento <ol><li> Interrompere l&#39;assorbimento nanogold con l&#39;aggiunta di 1 ml di doppio fissativo forza [4% paraformaldeide (PFA), 0,4% glutaraldeide (GA) in 0.1 M tampone PHEM (20 TUBI mm, 50 HEPES mM, pH 6,9, 20 mM EGTA, MgCl 4 mM 2)] contenente 1 M sorbitolo alla sospensione cellulare. Mantenuto il tubo a temperatura ambiente. </li><li> Capovolgere delicatamente manualmente le provette micocentrifuge più volte durante 30 minuti (questo permetterà di mantenere le sferoplasti insospensione) e centrifugare due volte a 1700 xg per 25 sec. </li><li> Sostituire il fissativo scartando il surnatante e con l&#39;aggiunta di 1 ml di fissativo fresco forza standard (2% PFA, 0,2% GA in 0.1 M tampone PHEM) contenente 1 M sorbitolo e incubare per 2 ore a temperatura ambiente su una ruota lenta rotazione della camera. </li><li> Elaborare le cellule per crio-sezionamento come descritto in precedenza 6. NOTA: Per le procedure di valorizzazione nanogold assorbimento e d&#39;argento, il trattamento con acido periodico descritto nel protocollo indicato è di non essere effettuati. Il trattamento con acido periodico è permeabilize meglio la parete cellulare 26 ma questa struttura è stata eliminata durante la generazione di sferoplasti. </li><li> Tagliare 50 criosezioni sottili nm a -120 ° C con la lama di diamante a secco utilizzando un UCT ultramicrotomo come descritto in precedenza 8 e metterli su carbone Formvar rivestito 50 maglie griglie di nichel. </li></ol> 4. Miglioramento argento per Nanogold particelle Visualizzazione NOTA: La procedura per preparare le miscele di reazione è fondamentalmente quello indicato dal costruttore. Adeguamenti pratici che utilizzano questo protocollo, rende la procedura di valorizzazione argento efficace e affidabile su criosezioni. <ol><li> Rimuovere il kit di potenziamento argento dal freezer e scongelare in un incubatore a 37 ° C oa bagnomaria. Quando scongelati, posizionare il kit in un incubatore a 24 ° C collocato in una stanza buia fino al momento dell&#39;uso (vedi 4.7). </li><li> Posizionare la piastra di riscaldamento in camera oscura e riscaldarlo alla temperatura finale di 24 ° C. </li><li> Coprire la parte superiore della piastra di riscaldamento con la protezione di superficie, lato lucido, e fissarlo con un nastro. </li><li> Posizionare il Parafilm sul Benchkote e segnare i bordi con un pennarello nero per essere in grado di vedere i bordi parafilm nel buio. </li><li> Nastro un termometro sulla parte superiore della Benchkote per monitorare la temperatura della piastra riscaldante. Gradualmente regolare la temperatura se non 24 ° C. </li><li> PlaCE Il tubo da 50 ml con acqua bidistillata e il kit di miglioramento argento all&#39;interno. </li><li> Riempire le piccole piastre di Petri con acqua bidistillata, pre-riscaldato a 37 ° C. Posizionare le griglie in acqua, provino lato negativo, per 30 min. </li><li> Ripetere questo passo 4.7 ancora una volta. </li><li> Trasferire le griglie nel box di stoccaggio e portarli alla camera oscura. </li><li> Risciacquare nuovamente le griglie facendoli passare (con il lato campione giù) su alcune gocce di acqua bidistillata a 24 ° C immessi sul parafilm che è stato fissato sulla piastra di riscaldamento. </li><li> Assicurarsi che 9 supplementare doppio gocce d&#39;acqua distillata sono pronti sul Parafilm, per il risciacquo dopo la reazione valorizzazione argento. </li><li> Spegnere la luce e assicurarsi che tutte le luci siano spente. Accendere la luce rossa. </li><li> Estrarre le soluzioni A e B del kit potenziamento argento dal termostato 24 ° C. </li><li> Mettere primi 6 gocce di soluzione A e poi 6 gocce di soluzione B in un 1,5 ml provetta e mescolare bene con una pipetta Pasteur di vetro evitando di fare le bolle. La soluzione è abbastanza spessa e deve essere miscelato manualmente con una pipetta prima di finalmente vortex brevemente. </li><li> Mettere le soluzioni A e B di nuovo in incubatrice 24 ° C ed estrarre la soluzione C. </li><li> Aggiungere 6 gocce della soluzione C al mix A / B. Poi mescolare ancora prima con una pipetta Pasteur e poi nel vortex, evitare di fare bolle. </li><li> Utilizzare immediatamente la miscela finale inserendo (~ 20 l / drop) gocce sul Parafilm. </li><li> Con un pulito, pinzette anti-magnetico, posizionare le griglie sulla miscela (ad es., Argento migliorando reazione) da 6 a 15 minuti a seconda della valorizzazione (ie. Dimensione delle particelle d&#39;oro) voluto da ottenere. </li><li> Rimuovere le griglie dalla miscela e trasferirli sulla superficie dell&#39;acqua bidistillata scende a 24 ° C per lavaggi. In primo luogo, utilizzare in rapida successione 6 gocce e poi 3 gocce con 7 min tempo di incubazione per goccia, Prima di accendere le luci. </li><li> Procedere con il passaggio successivo, sia l&#39;etichettatura immuno-oro o marcatura di membrana 27, al fine di visualizzare il risultato di un&#39;inchiesta EM. </li></ol>

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The authors have nothing to disclose.

Immuno-microscopia elettronica è una tecnica che permette di combinare localizzazione di proteine ​​con la risoluzione ultrastrutturale dei vettori e organelli dove queste proteine ​​risiedono. Ciò è particolarmente importante quando si studia il sistema lievito endosomiale perché i suoi comparti appaiono come strutture puntuate in microscopia a fluorescenza. È pertanto difficile distinguerle. Per questo motivo l&#39;uso di una sonda rilevabile da EM e immettendo il percorso endocitosi in modo dipendente da...

Il sistema endosomal è un importante apparato di smistamento membrana che svolge molteplici ruoli cellulari cruciali, tra cui il traffico di recettori smistamento enzima lisosomiale e il riciclaggio di membrana plasmatica (PM) recettori 1,2. Endosomi sono divisi in tre diversi comparti, vale a dire. gli endosomi precoci (EE), alla fine degli endosomi (LE) e le endosomi di riciclo. Questa classificazione si basa sul tempo impiegato materiale endocitosi raggiungerli, sulle proteine ​​marker specifiche e sulla loro morfologia. Membrane, ovvero proteine ​​e lipidi bistrati, interiorizzate dal PM possono sia essere consegnati ai lisosomi via endosomi per la degradazione o essere riciclati. Le membrane sono trasportati endosomi dal Golgi e similmente, sia continuare ad lisosomi o essere recuperate indietro ai Golgi. Inoltre, le proteine ​​possono essere filtrate in vescicole luminali erba verso l&#39;interno dalla membrana endosomal limitante, un processo che porta alla formazione diuna sottocategoria di LE, i corpi multivescicolari. Il sistema endosomal lievito è relativamente meno complessa di quella delle cellule eucariotiche superiori. Endosomi lievito sono suddivisi in EE e LE. In contrasto con cellule di mammifero non contengono endosomi di riciclo ma anche organelli lisosomi legati tessuto-specifici. Di conseguenza, essi hanno una rete meno complessa di endosomal rotte del narcotraffico 3,4. Pertanto lievito ha rappresentato e rappresenta un sistema sperimentale vantaggiosa per studiare alcuni dei principi traffico membrana sottostante nel sistema endosomal. Questo vantaggio è accentuato dal fatto che numerosi geni coinvolti nelle vie endosomali sono stati inizialmente isolato con schermi genetici nel lievito 5. Mentre il sistema endosomiale lievito wild type e cellule mutanti è stata ampiamente studiata utilizzando approcci biochimici e di microscopia a fluorescenza, la sua indagine a livello ultrastrutturale è stato solominima. Analisi morfologiche sono particolarmente rilevanti nel lievito perché la maggior parte degli organelli endosomiali vengono rilevati come strutture puntuate mediante microscopia a fluorescenza, che rendono difficile la loro identificazione univoca 6. Purtroppo solo un numero limitato di antisieri riconoscere proteine ​​marker lievito endosomali sta lavorando in immuno-microscopia elettronica (IEM) preparazioni 7-10. Per alcune proteine, questo problema è stato aggirato dalla codifica endogeno del gene di interesse e l&#39;impiego di un anticorpo che riconosce il tag di rilevarla 7,11,12. Spesso, tuttavia, le proteine ​​sono rilevabili da IEM causa dei loro livelli di espressione bassi. La loro sovraespressione non è una soluzione, perché questo approccio può indurre mis-localizzazioni e / o alterazioni nei organelli morfologia / funzioni. Così l&#39;etichettatura dei compartimenti endocitici con una sonda rilevabile mediante microscopia elettronica EM è un&#39;opzione efficace. Questa è una soluzione ottimale soprattutto se il prOBE sta entrando nel percorso endocitosi in modo dipendente dal tempo, che permette di conoscere quando segnerà un organello specifico 6. L&#39;assorbimento di nanogold carica positiva da sferoplasti lievito (vale a dire. Lievito in cui la parete cellulare è stato rimosso enzimaticamente) è stato utilizzato con successo per identificare la endosomiale lievito scomparti 10. Queste particelle si legano fortemente ai lipidi carichi negativamente che compongono le membrane biologiche. Così i soci nanogold cariche positivamente con il PM, penetra nella cellula per endocitosi e passa attraverso l&#39;EE e LE prima di raggiungere il vacuolo. Queste piccole particelle d&#39;oro, tuttavia, non hanno dimensioni adeguate per essere visto da EM. Per renderle visibili, le loro dimensioni possono essere allargata mediante reazioni chimiche che portano alla deposizione di argento o oro intorno alla sonda oro 13-15. Abbiamo sviluppato e applicato con successo un approccio IEM basato sul metodo Tokuyasu effettuare subcellularelocalizzazione studia 8,16. Questo metodo consente di eseguire etichettatura immunogold su preparazioni di lievito con una risoluzione eccellente della morfologia 8,17-24. Abbiamo anche stabilito una procedura che combina questo protocollo IEM con l&#39;etichettatura nanogold del sistema lievito endosomale compartimenti 6. Usando questo approccio abbiamo morfologicamente caratterizzato diverse sottoclassi di endosomi e ultrastrutturalmente esaminato mutanti con un traffico endosomiale difetto 6,25. Inoltre, abbiamo dimostrato che questa etichettatura nanogold può essere combinato con etichettature immunogold fornendo la possibilità di esplorare la distribuzione di una proteina di interesse sulle diverse sottopopolazioni endosomi. Qui vi presentiamo come è praticamente eseguito l&#39;etichettatura del sistema lievito endosomiale con nanogold carica positiva.

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nanogold labeling of the yeast endosomal system for ultrastructural analyses

Endosomi sono uno dei principali membrana ordinamento posti di blocco nelle cellule eucariotiche e regolano il riciclaggio o la distruzione delle proteine ​​per lo più dalla membrana plasmatica e il Golgi. Di conseguenza il sistema endosomal svolge un ruolo centrale nel mantenimento dell&#39;omeostasi cellulare, e mutazioni in geni appartenenti a questa rete di organelli interconnessi da trasporto vescicolare, causano gravi patologie tra cui il cancro e disturbi neurobiologici. E &#39;quindi di primaria importanza per comprendere i meccanismi alla base della biogenesi e l&#39;organizzazione del sistema endosomal. Il lievito Saccharomyces cerevisiae è stato fondamentale in questo compito. Per etichettare e analizzare a livello ultrastrutturale sistema endosomal di questo organismo modello specifico, presentiamo qui un protocollo dettagliato per la nanogold assorbimento carica positiva da sferoplasti seguite dalla visualizzazione di queste particelle attraverso una reazione valorizzazione argento. Questo metodo è anche un prezioso troppol per l&#39;esame morfologico di mutanti con difetti nel traffico endosomal. Inoltre, non è solo applicabile per esami ultrastrutturali ma può anche essere combinato con etichettature immunogold delle indagini localizzazione della proteina.

Secondo il protocollo presentato, la morfologia del sistema lievito endosome può essere accesso da EM trasmissione. Figura 1 mostra diversi tipi di compartimenti endosomiali che sono stati raggiunti e quindi etichettati con nanogold. Il nanogold-argento avanzato può essere chiaramente visto come elettroni particelle dense. Le impostazioni ottimali stabiliti per la reazione valorizzazione argento permette avente particelle di oro in una gamma omogenea dimensioni tra 5 e 15 nm, che non interferisce con ...

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

Lievito di birra, Saccharomyces cerevisiae, è stata un organismo modello chiave per identificare e studiare geni che regolano la biogenesi e le funzioni del sistema endosomal. Qui vi presentiamo un protocollo dettagliato per l&#39;etichettatura specifico dei comparti endosomiali per gli studi ultrastrutturali.

Nanogold Etichettatura del Sistema lievito endosomiale per le analisi ultrastrutturali

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 Views.  University Medical Center Utrecht.  Lievito di birra, Saccharomyces cerevisiae, è stata un organismo modello chiave per identificare e studiare geni che regolano la biogenesi e le funzioni del sistema endosomal. Qui vi presentiamo un protocollo dettagliato per l'etichettatura specifico dei comparti endosomiali per gli studi ultrastrutturali. 

Video: 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

Una tecnica rapida per la visualizzazione di Live cellule di lievito immobilizzato

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

Ricerca

Biologia

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.

Analisi di un Sistema di bioelettrica Osteointegrata Intelligent Design impianto per amputati

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.

Etichette fluorescenti di Drosophila Cuore Strutture

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.

Studi genetici di proteine ​​umane di riparazione del DNA Utilizzando lievito come sistema modello

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-Ubiquitina membrana a base di lievito Due-Hybrid (MITO) Sistema: un potente strumento per identificare interazioni proteina-proteina

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.

Modificato lievito-sistema del doppio ibrido per identificare proteine ​​che interagiscono con il fattore di crescita progranulina

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.

Il processo di Green Monster per la generazione di ceppi di lieviti trasporto gene delezioni multiple

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.

Germogliamento lievito proteine ​​estrazione e purificazione per lo Studio della funzione, interazioni e modificazioni post-traduzionali

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.

Reverse lievito sistema Two-ibrida per identificare mammiferi Nuclear Receptor residui che interagiscono con leganti e / o antagonisti

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.

Le domande di pHluorin per quantitativa, Cinetica e high-throughput analisi di endocitosi in germogliamento lievito

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