Il lavoro in laboratorio Duennwald è sostenuto da finanziamenti della American Federation for Aging Research (lontano), la Fondazione malattia ereditaria (HDF) e la Fondazione William Wood.

1. Espressione di tossici PolyQ-espansione proteine ​​nel lievito Una analisi sistematica ha stabilito la precisa sequenza aminoacidica di una proteina polyQ espansione che è necessaria per produrre tossicità in lievito 7. Questo tossico polyQ-espansione proteina contiene un amino-terminale FLAG-tag seguita da 17 amminoacidi dalla sequenza originale della proteina huntingtina, una regione polyQ, e un terminale carbossilico di fusione ad una proteina fluorescente (GFP sia o CFP, vedi Figura 1 a). L&#39;espressione di proteine ​​con espansioni PolyQ glutamines di 46 o più (ad esempio 72 e 103 glutamines) produce tossicità in lievito quando espresso sotto il controllo del inducibile e relativo forte promotore GAL1 7, 9. Come descritto in precedenza, tossicità polyQ in lievito è solo apparente in cellule che portano il Rnq1p proteina prionica nella sua conformazione, [RNQ +], ad esempio il ceppo di lievito W303 9. Il tossico polyQ-espansione proteina recapitulates aspetti centrali della polyQ-biologia nel lievito, come polyQ lunghezza-dipendente di tossicità (vedi sotto) e aggregazione (Figura 1 b). In particolare, tossici polyQ di espansione delle proteine ​​non uccidono le cellule di lievito immediatamente, ma piuttosto mettere in pericolo o arrestare il ciclo cellulare e devision cellulare, in modo da rallentare o inibire la crescita di colonie di lievito su piastre o colture liquide (i nostri dati non pubblicati). 2. Potenziali problemi con le cellule di lievito Esprimendo tossici PolyQ-espansione Proteine Le cellule di lievito che esprimono le proteine ​​tossiche in caso contrario di espansione polyQ possono non mostrare alcun difetto di crescita del 9. La natura genetica di questi soppressori di polyQ-tossicità non sembra basarsi su semplici mutazioni mendeliani e quindi possono verificarsi con frequenza relativamente elevata (i nostri risultati non pubblicati). Noi ipotizziamo che questi soppressori spontanei sono causati dalla polimerizzazione dei prioni non identificati che funzionano in modo simile a [RNQ +] nella determinazione toxicity.Thes polyQdi e soppressori spontanee di tossicità polyQ può compromettere il buon esito di qualsiasi esperimento che mira a caratterizzare la tossicità polyQ o per identificare e caratterizzare modificatori di tossicità polyQ. Al fine di evitare il verificarsi frequente di questi soppressori spontanee, seguire le misure precauzionali descritte di seguito, che hanno dimostrato di essere molto efficace: <ol><li> Utilizzare le cellule di lievito fresco per gli esperimenti di tossicità. Non conservare il lievito per lunghi periodi di tempo. Spesso recuperare colonie di lievito fresco da scorte congelate. </li><li> Spesso monitorare l&#39;espressione e aggregazione di tossici polyQ-espansione proteine ​​mediante microscopia a fluorescenza. </li><li> Conservare le cellule di lievito in media che reprimere l&#39;espressione della tossicità tossico polyQ in ogni momento (cioè in terreni selettivi contenenti glucosio come fonte di carbonio unica) Prima di eseguire le misure della tossicità. </li><li> Utilizzare almeno tre trasformanti indipendenti per ogni polyQ-tossicità esperimento. &lt;/ Li&gt; </ol> 3. Saggi di crescita <ol><li> Per ciascuna condizione sperimentale, inoculare una colonia ciascuno da tre trasformanti indipendenti di cellule di lievito che ospitano il tossico polyQ-espansione proteina in 3 ml di mezzo di lievito selettivi con glucosio come unica fonte di carbonio. </li><li> Incubare queste colture notte a 30 ° C. In queste condizioni, le cellule non sono esprimere la proteina polyQ-espansione perché il glucosio nel mezzo reprime la loro espressione. Non lasciate che questi overgrow culture; mantenere l&#39;OD 600 (assorbimento di luce di lunghezza d&#39;onda 600 nm) delle culture notte inferiore a 1. </li><li> Noi abitualmente utilizzano tre diversi test per controllare i difetti di crescita di cellule di lievito che esprimono il tossico polyQ espansione delle proteine: </li></ol> 3,1 crescita su piastre <ol><li> Diluire le colture di lievito durante la notte (cresciuto con 220 scuotimento rpm) ad un OD 600 di 0.0005 (vale a dire una diluizione di 1:1000 a = 0,5 OD600 cultura) in selettiva meØ 7 contenente glucosio. </li><li> Uniformemente distribuito 50 microlitri (con conseguente ca. 700 colonie per piastra) di ogni cultura diluito su una piastra (diametro 10 cm) con terreno selettivo contenente glucosio come unica fonte di carbonio e una piastra con terreno selettivo contenente galattosio come unica fonte di carbonio. </li><li> Incubare le piastre per tre o quattro giorni a 30 ° C. Dopo l&#39;incubazione, fotografare ciascuna piastra e contare il numero di colonie sul glucosio e il numero di colonie sulle piastre galattosio. In condizioni ideali, non ci dovrebbero essere o solo poche colonie sulla piastra galattosio quando si utilizzano cellule di lievito che esprime una altamente tossico polyQ espansione proteina (103q, Figura 2a). </li></ol> 3.2 saggi Spotting Questo saggio è più quantitativa di saggio placcatura descritto sopra e può così rivelare anche sottili differenze di tossicità polyQ con lo stesso esperimento sulla stessa piastra. <ol><li> Diluire il overnculture ight coltivate in terreno con glucosio ad una OD 600 di 0,1. </li><li> Pipettare 200 ul di queste culture diluiti in sterili a 96 pozzetti e preparare cinque diluizioni seriali di cinque volte in acqua sterile utilizzando una pipetta multicanale. </li><li> Utilizzando un frogger, (chiamato anche spotter, con 8 x 6 perni per il trasferimento di cellule in sospensione) trasferire le sospensioni cellulari su piastre contenenti terreni selettivi con glucosio come unica fonte di carbonio e piastre contenenti terreno selettivo con galattosio come unica fonte di carbonio. </li><li> Lasciare le piastre asciugare prima incubati a 30 ° C per tre o quattro giorni. </li><li> Dopo l&#39;incubazione, scattare fotografie di ogni disco (Figura 2b). </li></ol> 3,3. Monitoraggio tossicità polyQ nel lievito da una crescita di colture liquide Questo protocollo è il più quantitativa (OD600 numeri) dei tre saggi descritti qui e può anche rilevare differenze molto sottili di tossicità polyQ. Il suddetto occurrence di soppressori spontanee, comunque, potenzialmente in grado di produrre risultati fuorvianti. Pertanto consigliamo di combinare questo test con almeno uno dei due saggi placcatura sopra descritti. Noi proponiamo di utilizzare lo strumento BioscreenC per questi esperimenti. Il BioscreenC è uno strumento che misura automaticamente la densità ottica delle colture di lieviti a 100-pozzetti mentre incubate a temperature definite con agitazione definito. Altri metodi per la coltivazione di cellule di lievito e misurare la loro densità ottica possono anche applicare. <ol><li> Lavare le cellule di lievito da un supporto minimo contenente glucosio come unica fonte di carbonio per tre volte in 3 ml di acqua sterile. </li><li> Diluire le culture notte coltivate in terreno con uno di galattosio OD 600 di 0,1. </li><li> Riempire ciascun pozzetto della piastra di ben 100 BioscreenC con 300 ml delle colture di lieviti. </li><li> Aprire il programma Easy Experiment Bioscreen. Determinare il numero di campioni che si desidera controllare (compresi gli spazi e le medie solocontrolli), impostare la temperatura a 30 ° C, impostare la durata degli esperimenti a 3 giorni, impostare gli intervalli di misura per 15 minuti, impostare il filtro a 600 nm / Brown, e impostare la modalità di agitazione a 15 secondi prima di ogni misurazione forza media. </li><li> Lo strumento BioscreenC e il software in dotazione produrrà fogli di calcolo Excel di ogni punto dati ricavati durante l&#39;esperimento. </li><li> Preparare curve di crescita con Excel per ciascun campione e confrontare la crescita di diversi campioni (figura 2c). Una descrizione dettagliata di analisi dei dati ottenuti da esperimenti BioscreenC è già stata fornita 10. </li></ol> 4. Risultati rappresentativi <img alt="Figura 1" src="/files/ftp_upload/3461/3461fig1.jpg" /> Figura 1. Il modello di lievito polyQ. a) Rappresentazione schematica del tossico polyQ espansione delle proteine. b) la microscopia a fluorescenza che mostra cellule di lievito che esprimono una breve, non tossico polyQcellule proteina espansione (25q, pannello di sinistra) e lievito che esprime una lunga, polyQ tossico-espansione proteina (103q, pannello di destra). <img alt="Figura 2" src="/files/ftp_upload/3461/3461fig2.jpg" /> Figura 2. Risultati rappresentativi dei saggi di crescita di cellule di lievito che esprimono polyQ-espansione proteine. a) saggio placcatura. Circa 700 cellule di lievito sono state ripartite su piastre e incubate per tre giorni a 30 ° C. Il pannello superiore mostra una piastra contenente terreno di glucosio, cioè l&#39;espressione del tossico polyQ-espansione proteina (103q) non viene indotta. Il pannello inferiore mostra una piastra di lievito contenente mezzo galattosio, ossia l&#39;espressione del tossico polyQ-espansione proteina viene indotta. Si noti che nell&#39;esperimento qui, non soppressore spontanea si è verificato. B) Individuazione del dosaggio. Cinque serie di cinque diluizioni di cellule di lievito ospitare sia un non-proteina tossica polyQ (25q) o un tossico polyQ espansione Protein (103q) sono stati depositati su piastre di glucosio che reprimono la espressione delle proteine ​​(pannello sinistro) o su piastre galattosio che inducono la loro espressione. Le piastre sono state poi incubate per tre giorni a 30 ° C. c) esperimento BioscreenC. La crescita di colture di cellule di lievito che esprimono sia una proteina non-tossico polyQ (25q) o tossica polyQ-espansione proteina (103q) sono stati controllati dal strumento BioscreenC. Le condizioni sperimentali e l&#39;analisi degli esperimenti BioscreenC sono descritti nel testo principale.

<ol>
	<li>Soto, C., Estrada, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+misfolding+and+neurodegeneration.">Protein misfolding and neurodegeneration.</a> Arch. Neurol. 65, 184-189 (2008).</li><li>Ross, C. A., Tabrizi, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huntington's+disease:+from+molecular+pathogenesis+to+clinical+treatment.">Huntington's disease: from molecular pathogenesis to clinical treatment.</a> Lancet Neurol. 10, 83-98 (2011).</li><li>Zoghbi, H. Y., Orr, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamine+repeats+and+neurodegeneration.">Glutamine repeats and neurodegeneration.</a> Annu. Rev. Neurosci. 23, 217-247 (2000).</li><li>Meriin, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytosis+machinery+is+involved+in+aggregation+of+proteins+with+expanded+polyglutamine+domains.">Endocytosis machinery is involved in aggregation of proteins with expanded polyglutamine domains.</a> FASEB J. 21, 1915-1925 (2007).</li><li>Giorgini, F., Guidetti, P., Nguyen, Q., Bennett, C. S., Muchowski, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genomic+screen+in+yeast+implicates+kynurenine+3-monooxygenase+as+a+therapeutic+target+for+Huntington+disease.">A genomic screen in yeast implicates kynurenine 3-monooxygenase as a therapeutic target for Huntington disease.</a> Nat. Genet. 37, 526-5231 (2005).</li><li>Duennwald, M. L., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+ERAD+and+ER+stress+are+early+and+specific+events+in+polyglutamine+toxicity.">Impaired ERAD and ER stress are early and specific events in polyglutamine toxicity.</a> Genes Dev. 22, 3308-3319 (2008).</li><li>Duennwald, M. L., Jagadish, S., Muchowski, P. J., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flanking+sequences+profoundly+alter+polyglutamine+toxicity+in+yeast.">Flanking sequences profoundly alter polyglutamine toxicity in yeast.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 11045-1150 (2006).</li><li>Duennwald, M. L., Jagadish, S., Giorgini, F., Muchowski, P. J., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+network+of+protein+interactions+determines+polyglutamine+toxicity.">A network of protein interactions determines polyglutamine toxicity.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 11051-116 (2006).</li><li>Meriin, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huntington+toxicity+in+yeast+model+depends+on+polyglutamine+aggregation+mediated+by+a+prion-like+protein+Rnq1.">Huntington toxicity in yeast model depends on polyglutamine aggregation mediated by a prion-like protein Rnq1.</a> J. Cell. Biol. 157, 997-1004 (2002).</li><li>Murakami, C., Kaeberlein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+Yeast+Chronological+Life+Span+by+Outgrowth+of+Aged+Cells.">Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells.</a> J. Vis. Exp. (27), e1156-e1156 (2009).</li><li>Gitler, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beer+and+bread+to+brains+and+beyond:+can+yeast+cells+teach+us+about+neurodegenerative+disease.">Beer and bread to brains and beyond: can yeast cells teach us about neurodegenerative disease.</a> Neurosignals. 16, 52-62 (2008).</li><li>Giorgini, F., Muchowski, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+genetic+modifiers+of+amyloid+toxicity+in+yeast.">Screening for genetic modifiers of amyloid toxicity in yeast.</a> Methods Enzymol. 412, 201-222 (2006).</li><li>Ehrnhoefer, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+tea+(-)-epigallocatechin-gallate+modulates+early+events+in+huntingtin+misfolding+and+reduces+toxicity+in+Huntington's+disease+models.">Green tea (-)-epigallocatechin-gallate modulates early events in huntingtin misfolding and reduces toxicity in Huntington's disease models.</a> Hum. Mol. Genet. 15, 2743-2751 (2006).</li><li>Cashikar, A. G., Duennwald, M., Lindquist, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chaperone+pathway+in+protein+disaggregation.+Hsp26+alters+the+nature+of+protein+aggregates+to+facilitate+reactivation+by+Hsp104.">A chaperone pathway in protein disaggregation. Hsp26 alters the nature of protein aggregates to facilitate reactivation by Hsp104.</a> J. Biol. Chem. 280, 23869-2375 (2005).</li></ol>

Non ci sono conflitti di interesse dichiarati.

Questo manoscritto descrive tre approcci complementari sperimentali per misurare polyQ-tossicità nel lievito organismo modello basato sulla crescita ridotta di cellule di lievito che esprimono tossici polyQ-espansione proteine. Il lavoro nel lievito ha offerto profonde intuizioni sui meccanismi cellulari e molecolari di base di misfolding e la sua tossicità conseguente, compreso il misfolding e la tossicità del polyQ-espansione proteine ​​9,11,12. Esperimenti basati sui protocolli presentati qui hanno ...

<table border="1"><tbody><tr><td> Nome dello strumento </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Frogger (6x8 pin) </td><td> V &amp; P scientifico, San Diego </td><td> VP 407 AH </td></tr><tr><td> BioscreenC </td><td> Growthcurves USA </td><td> 5101370 </td></tr><tr><td> 100-pozzetti a nido d&#39;ape </td><td> Growthcurves USA </td><td> 9602550 </td></tr></tbody></table>

growth assays to assess polyglutamine toxicity in yeast

Misfolding è associato con molte malattie umane, in particolare malattie neurodegenerative, quali morbo di Alzheimer, morbo di Parkinson, malattia di Huntington e 1. Malattia di Huntington (HD) è causata dall&#39;espansione anormale di uno (polyQ) all&#39;interno della regione poliglutammina huntingtina proteina. Il polyQ-espansa proteina huntingtina raggiunge una conformazione aberrante (cioè misfolds) e provoca tossicità cellulare 2. Almeno altri otto malattie neurodegenerative sono causate da polyQ di espansioni, compresa la spinocerebellare Atassie e la malattia di Kennedy 3. Il lievito organismo modello ha facilitato intuizioni significative nella base cellulare e molecolare di polyQ-tossicità, incluso l&#39;impatto dei fattori intra-ed inter-molecolari di polyQ-tossicità, e l&#39;individuazione di percorsi cellulari che sono in pericolo in cellule che esprimono polyQ espansione proteine ​​3-8. Importantely, molti aspetti della polyQ-tossicità che sono stati trovati nel lievito sono state riprodotte in altri sistemi sperimentali e in qualche misura nei campioni provenienti da pazienti HD, dimostrando così l&#39;importanza del modello di lievito per la scoperta dei meccanismi di base che sottendono polyQ-tossicità. Un modo diretto e relativamente semplice per determinare polyQ-tossicità in lievito è misurare difetti di crescita di cellule di lievito che esprimono proteine ​​polyQ-espansione. Questo manoscritto descrive tre approcci complementari sperimentale per determinare polyQ-tossicità in lievito misurando la crescita di cellule di lievito che esprimono proteine ​​polyQ-espansione. I primi due approcci sperimentali monitorare la crescita del lievito sui piatti, il terzo approccio controlla la crescita di colture di lievito liquido utilizzando lo strumento BioscreenC. Inoltre, questo manoscritto descrive le difficoltà sperimentali che possono verificarsi durante la manipolazione modelli polyQ lievito e delinea le strategie che vi aiuteranno a evitare oridurre al minimo queste difficoltà. I protocolli descritti qui possono essere utilizzate per identificare e caratterizzare percorsi genetici e piccole molecole che modulano polyQ-tossicità. Inoltre, i saggi descritti possono servire come modelli per le analisi accurate della tossicità causata da altre proteine ​​mal ripiegate malattie associate a modelli lievito.

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

Questo manoscritto descrive tre protocolli complementari per valutare la tossicità di poliglutamina (polyQ)-espansione proteine ​​nel lievito<em&gt; Saccharomyces cerevisiae</em&gt;. Questi protocolli possono essere facilmente modificati per controllare la tossicità di altre proteine ​​mal ripiegate in lievito.

I saggi di crescita per valutare la tossicità poliglutamina nel lievito

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

growth-assays-to-assess-polyglutamine-toxicity-in-yeast

Research

JoVE Journal

Biology

13.5K Views.  Boston Biomedical Research Institute. Questo manoscritto descrive tre protocolli complementari per valutare la tossicità di poliglutamina (polyQ)-espansione proteine ​​nel lievito Saccharomyces cerevisiae. Questi protocolli possono essere facilmente modificati per controllare la tossicità di altre proteine ​​mal ripiegate in lievito.

Video: 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.

Identificazione dei fenotipi inibizione della crescita indotta da Espressione di effettori batterica di tipo III nel lievito

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 ...

Ricerca

Biologia

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 ...

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.

A lungo termine del test di tossicità letale con il crostaceo 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.

Metodi di valutazione subcellulari Compartimenti di muscoli in<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.

Analisi comparativa di ormone umano della crescita nel siero Utilizzando SPRI, Nano-SPRI e saggi 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: Metodo su vasta scala per la quantificazione multiparametrica della crescita del lievito

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.

Effetto di proteine fluorescenti su Fusion partner utilizzando saggi di tossicità di Polyglutamine in lievito

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.

Medio-throughput Screening Test per la valutazione degli effetti sulla Ca2 +-segnalazione e reazione acrosomiale in sperma umano

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.

Esame della dinamica nucleare del lievito di fissione mitotico e meiotico da fluorescenza microscopia a celle vive

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.

Misurazione della tossicità del Peptide RAN in C. elegans

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