Name: subcellular fractionation for erk activation upon mitochondrial-derived peptide treatment
Uploaded: 2017-09-25T15:00:00.000+00:00
Duration: 7 min 55 s
Description: Watch this Scientific Journal Video about Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment at JoVE.com

Questo lavoro è stato supportato da un Ellison/AFAR Postdoctoral Fellowship nel programma di ricerca di invecchiamento di SJK, e un premio della Fondazione Glenn e NIH concede a PC (1P01AG034906, 1R01GM 090311, 1R01ES 020812). Tutti gli autori compaiono nel film.

1. peptide trattamento alle cellule <ol><li>piastra 2 milioni di SH-SY5Y o cellule HEK293 (2 x 10 6) in 10 cm piatto e coltivano per 2 giorni</li> <li>(opzionale) il prossimo giorno, lavare le cellule con Dulbecco privo di siero &#39; s Modified Eagle media Medium (DMEM) una volta e incubare con siero DMEM gratuito durante la notte se il trattamento deve essere fatto in media liberi del siero.</li>
	<li>Il giorno 3, sciogliere S14G-humanin peptidi in 0,2 µm filtrata, acqua distillata e rinvenire come soluzione di riserva di 1mM. 
	Nota: I peptidi devono essere disciolto in solvente appropriato, che può essere determinato dalle caratteristiche di sequenza del peptide (ad es., carica complessiva) e può essere fornito dal produttore se i peptidi sono disponibili in commercio. Ad esempio, S14G-humanin, SHLP2 e SHLP6 e MOTS-c si dissolvono in acqua. Aliquotare i peptidi in un volume adeguato (ad es., 50 µ l) per evitare di congelare e scongelare i cicli. Una volta scongelato, non utilizzarli ancora.</li>
	<li>Aliquota il siero pre-riscaldato-contenente o media del siero-liberi in un conico da 50 mL tubo e aggiungere la soluzione di riserva 1 mM temperatura ambiente per trasformarlo in una concentrazione di lavoro (ad esempio, 1 µM, 10 µM).</li>
	<li>Sostituire il supporto con 6 mL di soluzione del peptide da passo 1.4. Incubare le cellule per la giusta quantità di tempo. 
	Nota: Scegliere il tempo di incubazione a seconda della condizione che attiva ERK.</li>
</ol> 2. Frazionamento subcellulare di citoplasma e nucleo <ol><li>lavare le cellule due volte con 10 mL di PBS ghiacciata, raschiare le cellule fuori la piastra in 5 mL di PBS ghiacciata e trasferire la sospensione cellulare in una provetta conica da 15 mL.</li>
	<li>Centrifugare le cellule a 500 x g per 5 min a 4 ° C.</li>
	<li>Aspirare il PBS e risospendere il pellet in 200 µ l di tampone di frazionamento ghiacciata, (pH di 10 mM HEPES = 7.6, 3 mM MgCl 2, 10mm KCl, 5% (v/v) glicerolo, 1% tensioattivi non ionici (C 13 H 22 O (C 2 H 4 O) n), inibitori delle proteasi/fosfatasi). 
	Nota: Per mantenere le proteine nel loro stato di fosforilazione, l'inibitore della fosfatasi come bene come proteasi inibitore deve essere appena aggiunto e campioni devono essere tenuti sul ghiaccio a tutti i tempi. Ripetendo il blocco e thaw cicli di campioni devono essere evitati. Aliquotare campioni in piccola quantità (ad es. 50 μg) prima del loro congelamento a -80 ° C.</li>
	<li>Incubare risospeso in ghiaccio per 15 minuti e poi Centrifugare a 250 x g per 5 min a 4 ° C.</li>
	<li>Raccogliere il surnatante come la frazione citoplasmatica e tenere il pellet per frazione nucleare.</li>
	<li>Centrifugare il surnatante per rimuovere i detriti cellulari e altri contaminanti a 18.000 x g per 10 min a 4 ° C e poi trasferire il surnatante in un nuovo tubo del microcentrifuge. Questa è la frazione di citosol.</li>
	<li>Risospendere il pellet in tampone ghiacciata di 200 μL (10 mM a pH HEPES = 7.6, 1.5 mM MgCl 2, 420 mM NaCl, 25% (v/v) glicerolo, 0,2 mM EDTA, inibitori delle proteasi/fosfatasi) e centrifugare a 250 x g per 5 min a 4 ° C</li>
	<li>Rimuovere il supernatante e risospendere il pellet in tampone di estrazione ghiacciata, nucleare 100 μL (pH di 20 mM HEPES = 7.6, 1.5 mM MgCl 2, 420 mM NaCl, 25% (v/v) glicerolo, 0,2 mM EDTA, inibitori delle proteasi/fosfatasi) e Sonicare 10 volte (5 s, 10 s sconto 30% amp.) il ghiaccio</li>
	<li>Centrifugare a 18.000 x g per 10 min a 4 ° C.</li>
	<li>Trasferire il surnatante ad un nuovo tubo del microcentrifuge. Si tratta di frazione nucleare.</li>
	<li>Quantificare la quantità di proteina usando un'analisi BCA</li></ol>
 3. frazione mitocondriale grezza <ol><li>lavare le cellule in ogni piatto di 10 cm con 10 mL di PBS freddo, aggiungere 5 mL di PBS ghiacciata e staccare le celle utilizzando un raschietto cell. 
	Nota: Utilizzare tre piatti di 10cm di cellule per ottenere una buona resa dei mitocondri per Western Blotting.</li>
	<li>Trasferire la sospensione cellulare in una provetta conica da 15 mL e combinare tutto da tre piatti di 10cm in una provetta conica da 15 ml.</li>
	<li>Centrifugare le cellule a 600 x g per 10 min a 4 ° C.</li>
	<li>Aspirare il PBS e risospendere il pellet in 1 mL di buffer di isolamento mitocondri ghiacciata (10 mM Tris-MOPS, 1 mM EGTA/Tris, 200 mM di saccarosio, regolare a pH = 7,4).</li>
	<li>Omogeneizzare le cellule con 25 colpi di un omogeneizzatore 2 mL con il pestello rivestito politetrafluoroetilene su Ice. 
	Nota: Questo passaggio è fondamentale per mantenere l'integrità mitocondriale e massimizzare la resa della frazione mitocondriale. Il numero dei tratti deve essere ottimizzato per ogni tipo di cellula. Preraffreddare l'omogeneizzatore prima di iniziare la procedura.</li>
	<li>Trasferire l'omogeneizzato a un tubo del microcentrifuge ed e centrifugare a 600 x g per 10 min a 4 ° C per rimuovere i nuclei ed ininterrotte cellule.</li>
	<li>Raccogliere il surnatante, trasferirlo in un nuovo tubo del microcentrifuge e centrifugare a 7.000 x g per 10 min a 4 ° C. 
	Nota: Il pellet è allentato, raccogliere il surnatante con cura e cercare di non disturbare il pellet.</li>
	<li>Rimuovere il supernatante, risospendere il pellet con 200 μL di tampone di isolamento mitocondri ghiacciata e trasferire la soluzione in un nuovo tubo del microcentrifuge. Centrifugare la provetta a 7.000 x g per 10 min a 4 ° C.</li>
	<li>Ripetere il punto 3.8 a lavare la pallina. Non è necessario trasferire il surnatante in un nuovo tubo del microcentrifuge in questo passaggio.</li>
	<li>Rimuovere il supernatante dal pellet lavato e risospendere il pellet contenente i mitocondri con 50 μL di tampone RIPA.</li>
	<li>Incubare la sospensione in ghiaccio per 10 min.</li>
	<li>Centrifugare la sospensione a 16.000 x g per 15 min a 4 ° C.</li>
	<li>Trasferire il surnatante ad un nuovo tubo del microcentrifuge. Questa è la frazione mitocondriale.</li>
	<li>Quantificare la quantità di proteina usando un'analisi BCA.</li>
</ol> 4. Western Blotting per fosfo-specifici proteine <ol><li>eseguire un'elettroforesi del gel di SDS-poliacrilammide (8-16% preconfezionati gel) e la proteina di trasferimento di una membrana PVDF 4.</li>
	<li>Incubare la membrana con 5% BSA in TBST (0,1% polisorbato 20) per 30 min a temperatura ambiente per bloccare i siti di legame non specifico background. 
	Nota: Per il rilevamento di proteina fosforilata, bloccare la membrana con BSA non latte. Il latte contiene caseina, una fosfoproteina abbondante, che si traduce in alto segnale aspecifico.</li>
	<li>Incubare la membrana con l'anticorpo primario (anti-fosfo-ERK1/2) a 4 ° C durante la notte.</li>
	<li>Il giorno successivo, lavare la membrana con TBST (0,1% polisorbato 20) tre volte per 5 min a temperatura ambiente.</li>
	<li>Incubare la membrana con anticorpo secondario (anti-coniglio HRP) per 1 h a temperatura ambiente.</li>
	<li>Lavare la membrana con TBST (0,1% polisorbato 20) tre volte per 5 min a temperatura ambiente.</li>
	<li>Incubare la membrana con soluzione di ECL e la membrana nell'analizzatore dell'immagine di immagine.</li>
	<li>Striscia la membrana con stripping buffer per 15 min a temperatura ambiente e lavare la membrana con TBST tre volte per 5 min.</li>
	<li>Ripetere i punti da 4.2 a 4.7 usando l'anticorpo di anti-totale ERK1/2.</li>
	<li>Per verificare la purezza di ogni frazione, eseguire SDS-PAGE ed eseguire immunoblots utilizzando anticorpi che riconoscono i marcatori per ogni vano (ad es., Liuzzi B1 per nucleo, GAPDH per cytosol e TOM20 per i mitocondri).</li>
</ol>

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Pinchas Cohen è un azionista e consulente per Kelvin CohBar, Inc., lo Yen ha servito come un consulente per CohBar, Inc.

Qui, abbiamo dimostrato che humanin peptide-ha mediato l'attivazione di ERK1/2 si verifica a due diversi tipi di cellule, e la localizzazione subcellulare di attivazione di ERK1/2 può essere diversa a seconda delle condizioni (ad es., dose di peptide, punto del tempo e delle cellule tipo). È stato dimostrato che humanin segnali attraverso due diversi recettori22,23, che possono spiegare le differenze nella segnalazione tra le due linee cellulari come p...

Gli studi emergenti indicano che peptidi mitocondriali-derivati (MDPs) svolgono i ruoli importanti nel metabolismo e nella citoprotezione1,2,3. Capire la via di trasduzione del segnale in presenza di MDPs ci dà comprensione nel meccanismo da cui MDPs modulano varie funzioni. Il primo MDP identificati, humanin, ha dimostrato di aumentare la chinasi segnale-regolata extracellulare 1/2 (ERK1/2) fosforilazione attraverso suo recettore vincolante4,5. Tuttavia, l'effetto a valle dell'attivazione di ERK1/2 è ancora aggiunto.
La cascata di ERK1/2 serve come un mediatore essenziale in una varietà di processi cellulari compreso proliferazione, migrazione cellulare, il metabolismo cellulare, sopravvivenza ed apoptosi6,7,8. Ad per orchestrare tutti questi processi cellulari distinti, l'attività e la localizzazione subcellulare di ERK1/2 sono strettamente regolate da fosfatasi e impalcatura proteine9,10. Oltre a modificazione post-traduzionale, la dinamica di marcia di ERK1/2 inoltre regola la sua segnalazione funzione, attività e specificità11,12. ERK1/2 è localizzato principalmente nel citosol13. Un insieme delle proteine di ancoraggio e impalcatura aiutano a trattenere ERK1/2 in elementi del citoscheletro, sulla superficie degli organelli, o diffusa nel citoplasma13. Stimolazione, ERK1/2 è fosforilato e viene dissociato dalle sue proteine di ancoraggio, che permette la traslocazione di ERK1/2 ad altri compartimenti subcellulari, tra cui il nucleo, mitocondri, Golgi e lisosomi14, 15 , 16.
Anche se humanin è noto per attivare la via di segnalazione di ERK1/2, l'attivazione di ERK1/2 è osservata solo nei lisati cellulari totali. Come descritto in precedenza, poiché la localizzazione subcellulare di ERK1/2 svolge un ruolo cruciale nel suo effetto a valle, l'analisi della localizzazione subcellulare e i livelli totali di fosforilata ERK1/2 è necessario fornire una comprensione globale di attivazione di ERK1/2 indotta da humanin e l'attivazione di obiettivi a valle.
Per comprendere gli organelli bersaglio di attivazione di ERK1/2, è stato effettuato il frazionamento subcellulare seguito mediante western blotting per fosforilata ERK1/2. Questo metodo può essere facilmente implementato come sfrutta i reagenti e la normale attrezzatura da laboratorio. I compartimenti subcellulari isolati sono di elevata purezza, permettendo i risultati semplicemente essere interpretati. Immunostaining di ERK1/2 può produrre risultati simili. Tuttavia, alcuni compartimenti subcellulari sono relativamente difficili da visualizzare e richiedono metodi di fissazione e permeabilizzazione speciali. ERK1/2 livelli variano in compartimenti subcellulari, e questa variazione potrebbe causare falsi positivi e falsi negativi segnali quando guardando lisati di cellule intere. Pertanto, un immunoblot utilizzando compartimenti subcellulari isolati ci dà una migliore comprensione della localizzazione di ERK1/2.
La versatilità del metodo consente modifiche del protocollo per studiare gli effetti di altri stimolanti tra cui altri MDPs o la traslocazione di altre molecole di segnalazione come STAT3. Recentemente scoperti piccoli peptidi simil-humanin (SHLPs) sono codificati dalla regione di rRNA 16S dove humanin viene codificato, e hanno proprietà simili ma distinti rispetto a HN17. Ad esempio, SHLP2 e SHLP3 attiva ERK1/2 dopo 8 h sebbene humanin attiva ERK1/2 entro 5 min Examining localizzazione subcellulare di ERK1/2 in risposta a diversi peptidi ci darà una migliore comprensione della biologia di questi peptidi. La prova emergente ha mostrato che la localizzazione subcellulare delle molecole di segnalazione svolge un ruolo cruciale nei loro effetti a valle. Per esempio, STAT3, tradizionalmente, è noto per essere localizzato principalmente nel citosol nelle cellule a riposo, e quindi trasloca nel nucleo per attivare l'espressione genica in risposta a citochine18. STAT3 inoltre trasloca ai mitocondri e regola il ciclo del TCA e ATP produzione19. Per quanto riguarda il regolamento di autofagia, diversa localizzazione subcellulare di STAT3 modula l'autofagia in vari modi20. Ad esempio, STAT3 nucleare trascrizionalmente regola geni autophagy-relative e agisce come un modulatore di autofagia. STAT3 citoplasmatica inibisce costitutivamente l'autofagia interagendo con l'autofagia molecole di segnalazione. STAT3 mitocondriale inibisce e impedisce mitophagy sopprimendo l'autofagia indotto da stress ossidativo. Pertanto, questo metodo di isolamento compartimento subcellulare è cruciale per comprendere il ruolo di altre molecole di segnalazione, nonché il ERK1/2.

<table><tbody><tr><td>p44/42 MAPK (ERK1/2)</td><td>Cell signaling</td><td>9102</td><td>Dilution 1:1,000</td></tr><tr><td>phospho-p44/42 MAPK (ERK1/2)(Thr202/Tyr204)</td><td>Cell signaling</td><td>4370</td><td>Dilution 1:1,000</td></tr><tr><td>Lamin B1</td><td>Cell signaling</td><td>12586</td><td>Nuclear Marker, Dilution 1:1,000</td></tr><tr><td>GAPDH</td><td>Cell signaling</td><td>5174</td><td>Cytoplasmic marker, Dilution 1:2,000</td></tr><tr><td>Tom20</td><td>Santa cruz</td><td>SC-17764</td><td>Mitochondria marker, Dilution 1:2,000</td></tr><tr><td>anti-Rabbit-HRP conjugated</td><td>Cell signaling</td><td>7074</td><td>Dilution 1:30,000</td></tr><tr><td>RIPA Lysis and Extraction Buffer</td><td>ThermoFisher SCIENTIFIC</td><td>89900</td><td></td></tr><tr><td>100 mm Culture Dish</td><td>ThermoFisher SCIENTIFIC</td><td>12556002</td><td></td></tr><tr><td>HNG peptide</td><td>Genescript</td><td></td><td></td></tr><tr><td>25mm sylinge filter</td><td>ThermoFisher SCIENTIFIC</td><td>09-719A</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>MgCl2</td><td>Sigma</td><td>M8266</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G9012</td><td></td></tr><tr><td>Triton X-100</td><td>ThermoFisher SCIENTIFIC</td><td>BP151-100</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>3609</td><td></td></tr><tr><td>MOPS</td><td>Sigma</td><td>M1254</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E3889</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S7903</td><td></td></tr><tr><td>Tris-base</td><td>ThermoFisher SCIENTIFIC</td><td>BP152-1</td><td></td></tr><tr><td>HCL</td><td>Sigma</td><td>H1758</td><td></td></tr><tr><td>PBS</td><td>Lonza</td><td>17-512F</td><td></td></tr><tr><td>Cell Scraper</td><td>FALCON</td><td>353085</td><td></td></tr><tr><td>Halt&amp;trade; Protease and Phosphatase Inhibitor Cocktail (100X)</td><td>ThermoFisher SCIENTIFIC</td><td>78440</td><td></td></tr><tr><td>Thomas Pestle Tissue Grinder Assemblies with Smooth Pestles</td><td>Thomas Scientific</td><td>3432S90</td><td></td></tr><tr><td>Tween-20</td><td>ThermoFisher SCIENTIFIC</td><td>BP337-500</td><td></td></tr><tr><td>BSA</td><td>ThermoFisher SCIENTIFIC</td><td>BP1600-100</td><td></td></tr><tr><td>8-16% Mini-PROTEAN TGX Precast Protein Gels</td><td>BIO RAD</td><td>4561104</td><td></td></tr><tr><td>Mini Trans-Blot Module</td><td>BIO RAD</td><td>1658030</td><td></td></tr><tr><td>Trans-Blot Turbo Transfer System</td><td>BIO RAD</td><td>1704150</td><td></td></tr><tr><td>Trans-Blot Turbo RTA Mini PVDF Transfer Kit</td><td>BIO RAD</td><td>1704272</td><td></td></tr><tr><td>Clarity Western ECL Blotting Substrates</td><td>BIO RAD</td><td>1705060</td><td></td></tr><tr><td>Restore Western blot stripping buffer</td><td>ThermoFisher SCIENTIFIC</td><td>21059</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>ThermoFisher SCIENTIFIC</td><td>11965-092</td><td></td></tr><tr><td>Sonicator, Medel: FB120</td><td>ThermoFisher SCIENTIFIC</td><td>695320-07-12</td><td></td></tr></tbody></table>

subcellular fractionation for erk activation upon mitochondrial-derived peptide treatment

Peptidi mitocondriale-derivati (MDPs) sono una nuova classe di peptidi che sono codificati da piccolo open reading frame all'interno di altri noti geni del genoma mitocondriale. MDPs hanno un'ampia varietà di effetti biologici quali proteggere i neuroni dall'apoptosi, migliorare gli indicatori metabolici e proteggere le cellule da chemioterapia. Humanin era la MDP prima di essere scoperto ed è il peptide più studiato tra la famiglia MDP. I recettori di membrana e vie di segnalazione a valle di humanin sono state caratterizzate con attenzione. MDPs aggiuntive quali MOTS-c e SHLP1-6 sono stati scoperti più di recente e i meccanismi di segnalazione devono ancora essere delucidato. Qui descriviamo un metodo di coltura basata di cella per determinare la funzione di questi peptidi. In particolare, tecniche di frazionamento cellulare in combinazione con macchiarsi occidentale consentono la determinazione quantitativa dell'attivazione e spostamento della molecola di segnalazione importante. Mentre ci sono altri metodi di frazionamento cellulare, quello qui descritto è un metodo facile e semplice. Questi metodi possono essere utilizzati più ulteriormente per delucidare il meccanismo di azione di questi peptidi e altri agenti terapeutici.

Utilizzando la procedura qui presentata, abbiamo trattato le cellule HEK293 e SH-SY5Y con 1 μM e 100 μM S14G-humanin, un potente humanin analogico21, rispettivamente, a completare la media per i periodi di tempo indicato (Figura 1A e Figura 1 B). abbiamo poi esaminato la forma totale e fosforilata di ERK1/2 al Thr202/Tyr204 da estratti di proteina ...

Watch this Scientific Journal Video about Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment at JoVE.com

Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

Questo protocollo descrive come stimolare le cellule con peptidi derivati mitocondriale e valutare la cascata di segnalazione e localizzazione di fosfo-proteine.

Frazionamento subcellulare per l'attivazione di ERK previo trattamento di Peptide derivato mitocondriale

Mitochondrial-derived peptides (MDPs) are a new class of peptides that are encoded by small open reading frames within other known genes of the ...

subcellular-fractionation-for-erk-activation-upon-mitochondrial

Research

JoVE Journal

Biology

7.8K Views.  University of Southern California. Questo protocollo descrive come stimolare le cellule con peptidi derivati mitocondriale e valutare la cascata di segnalazione e localizzazione di fosfo-proteine.

Video: Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

Assaying Protein Kinase Activity with Radiolabeled ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [&#947;-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([&#947;-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

Analizzare l&#39;attività della proteina con l&#39;ATP radioattivato

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

Ricerca

Biochimica

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

Sondaggio per mitocondriale complessa attività di cellule staminali embrionali umane

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

Biologia

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

Spaziale e temporale Analisi di attiva ERK nel<em&gt; C. elegans</em&gt; Linea germinale

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Biologia dello sviluppo

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. This method utilizes hypotonic buffers, digitonin, mechanical lysis and differential centrifugation to isolate the cytoplasm, mitochondria and plasma membrane. The process can be scaled to accommodate the needs of researchers, is inexpensive and straightforward. This method will allow researchers to determine protein localization in cells without specialized centrifuges and without the use of commercial kits, both of which can be prohibitively expensive. We have successfully used this method to separate cytosolic, plasma membrane and mitochondrial proteins in the human monocyte cell line U937.

Here, we present a protocol to isolate the plasma membrane, cytoplasm and mitochondria of U937 cells without the use of high-speed centrifugation. This technique can be used to purify subcellular fractions for subsequent examination of protein localization via immunoblotting.

Il frazionamento delle cellule delle cellule U937 in assenza di centrifugazione ad alta velocità

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. ...

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic cues. Their membranes are often juxtaposed at defined contact sites, which become hubs for the exchange of signaling molecules and membrane components1,2,3,4. The inter-organellar membrane microdomains that are formed between the endoplasmic reticulum (ER) and the mitochondria at the opening of the IP3-sensitive Ca2+ channel are known as the mitochondria associated-ER membranes or MAMs4,5,6. The protein/lipid composition and biochemical properties of these membrane contact sites have been extensively studied particularly in relation to their role in regulating intracellular Ca2+ 4,5,6. The ER serves as the primary store of intracellular Ca2+, and in this capacity regulates a myriad of cellular processes downstream of Ca2+ signaling, including post-translational protein folding and protein maturation7. Mitochondria, on the other hand, maintain Ca2+ homeostasis, by buffering cytosolic Ca2+ concentration thereby preventing the initiation of apoptotic pathways downstream of Ca2+ unbalance4,8. The dynamic nature of the MAMs makes them ideal sites to dissect basic cellular mechanisms, including Ca2+ signaling and regulation of mitochondrial Ca2+ concentration, lipid biosynthesis and transport, energy metabolism and cell survival 4,9,10,11,12. Several protocols have been described for the purification of these microdomains from liver tissue and cultured cells13,14. 
	Taking previously published methods into account, we have adapted a protocol for the isolation of mitochondria and MAMs from the adult mouse brain. To this procedure we have added an extra purification step, namely a Triton X100 extraction, which enables the isolation of the glycosphingolipid enriched microdomain (GEM) fraction of the MAMs. These GEM preparations share several protein components with caveolae and lipid rafts, derived from the plasma membrane or other intracellular membranes, and are proposed to function as gathering points for the clustering of receptor proteins and for protein&#x2013;protein interactions4,15.

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial preparations.

I mitocondri associati Membrane ER (MAMs) e glicosfingolipidi microdomini arricchiti (GEM): Isolamento dal cervello di topo

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic ...

Neuroscienze

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances, many yeast mitochondrial proteins still remain with their functions completely unknown. This protocol provides a simple and reliable method to determine the submitochondrial localization of proteins, which has been fundamental for the elucidation of their molecular functions.

Valutazione della localizzazione della proteina submitocondriale nel lievito in erba Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. Despite a broad genetic heterogeneity, germline gain-of-function mutations in key regulators of the RAS-MAPK pathway underlie the majority of the cases, and, thanks to advanced sequencing techniques, potentially pathogenic variants affecting the RAS-MAPK pathway continue to be identified. Functional validation of the pathogenicity of these variants, essential for accurate diagnosis, requires fast and reliable protocols, preferably in vivo. Given the scarcity of effective treatments in early childhood, such protocols, especially if scalable in cost-effective animal models, can be instrumental in offering a preclinical ground for drug repositioning/repurposing. 
Here we describe step-by-step the protocol for rapid generation of transient RASopathy models in zebrafish embryos and direct inspection of live disease-associated ERK activity changes occurring already during gastrulation through real-time multispectral F&#246;rster resonance energy transfer (FRET) imaging. The protocol uses a transgenic ERK reporter recently established and integrated with the hardware of commercial microscopes. We provide an example application for Noonan syndrome (NS) zebrafish models obtained by expression of the Shp2D61G. We describe a straightforward method that enables registration of ERK signal change in the NS fish model before and after pharmacological signal modulation by available low-dose MEK inhibitors. We detail how to generate, retrieve, and assess ratiometric FRET signals from multispectral acquisitions before and after treatment and how to cross-validate the results via classical immunofluorescence on whole embryos at early stages. We then describe how, via examining standard morphometric parameters, to query late changes in embryo shape, indicative of a resulting impairment of gastrulation, in the same embryos whose ERK activity is assessed by live FRET at 6 h post fertilization.

RASopathies are multisystem genetic syndromes caused by RAS-MAPK pathway hyperactivation. Potentially pathogenic variants awaiting validation emerge continuously while poor preclinical evidence limits therapy. Here, we describe our in vivo protocol to test and cross-validate RASopathy-associated ERK activation levels and its pharmacological modulation during embryogenesis by live FRET imaging in Teen-reporter zebrafish.

Un protocollo efficiente per valutare la modulazione dell'attività di ERK nei primi modelli di sindrome di Noonan del pesce zebra tramite microscopia FRET dal vivo e immunofluorescenza

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

La valutazione delle funzioni mitocondriali e la vitalità cellulare in cellule renali proteina chinasi C overexpressing isoenzimi

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

Un test funzionale per Gap giunzionale esame; Elettroporazione di cellule aderenti su indio-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

Frazionamento subcellulare per l'attivazione di ERK previo trattamento di Peptide derivato mitocondriale

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Analizzare l'attività della proteina con l'ATP radioattivato