Questo lavoro è supportato da Takasaki Università di salute e del benessere.

1. crescere cellule e aggiunta di antigeni esogeni <ol><li>preparare bOVA utilizzando un contrassegno di biotina-della proteina kit seguendo il produttore &#39; protocollo s. 
	Nota: Normalmente, bOVA contiene biotina di 2 M per 1 M OVA mediamente.</li>
	<li>DC2.4 crescere le cellule in RPMI-1640 completate con 2 mM L-Glutammina, piruvato di sodio di 1 mM, 0,1 mM aminoacidi non essenziali, 100 U/mL penicillina-streptomicina, 55 mM 2-mercaptoetanolo, 10 mM HEPES (pH 7.5) e 10% siero fetale del vitello (d'ora in avanti RPMI) a 37 ° C in 5 % CO 2 in un incubatore (d'ora in avanti senza menzione, le cellule vengono incubate a questa condizione). È anche possibile utilizzare DMEM completati con 10% siero fetale di vitello, 3,7 g/L NaHCO 3 (d'ora in avanti DMEM) al posto di RPMI.</li>
	<li>Un giorno prima di purificazione, dividere le celle in RPMI a 1 x 10 5 cellule/mL in una piastra di coltura del tessuto. Evitare di mantenere le cellule in uno stato confluente. DC2.4 cellule possono altamente incorporare antigeni esogeni fino a uno stato semi-confluente, ma perdono rapidamente la capacità dopo aver raggiunto uno stato di sovra-confluenti.</li>
	<li>Prima the DC2.4 cellule sono semi-confluenti, aggiungere 250 µ g/mL di bOVA per 1 x 10 6 cellule/mL e incubare per 2-4 h. 
	Nota: Durante gli esperimenti a valle, tutti i buffer e i reagenti sono conservati a 4 ° C se non diversamente indicato.</li>
</ol> 2. Preparazione dei microsomi <ol><li>raccolto le cellule DC2.4 di pipettaggio delicato dalla piastra di tessuto in una nuova provetta conica 50 mL.</li>
	<li>Centrifugare a 1.000 x g per 5 min, 4 ° C e sfilare con cautela il mezzo con bOVA aspirazione.</li>
	<li>Lavare le cellule DC2.4 due volte con 40 mL di tampone PBS (1,37 mM NaCl, 8,1 mM Na 2 HPO 4, 2,68 mM KCl, 1,47 mM KH 2 PO 4) mediante centrifugazione a 1.000 x g per 5 min, 4 ° C e scartare il supernatante ogni volta dall'aspirazione.</li>
	<li>Risospendere il pellet in passaggio 2.3 in 1-2 mL (1/20-1/10 di volume di terreno di coltura) del mezzo di omogeneizzazione (0,25 M saccarosio, 1 mM EDTA, 10 mM HEPES-NaOH [pH 7.4]) con volume di 1/1.000 di cocktail di inibitore di proteasi e trasferimento del sedimento DC2.4 di celle in un ghiacciata lisata omogeneizzatore.</li>
	<li>Frantumare le cellule DC2.4 sedimento delicatamente da 10-20 colpi con l'omogeneizzatore lisata ghiacciata. 
	Nota: Durante la fase di rottura, l'omogeneizzatore lisata è raffreddato su Ice.</li>
	<li>Trasferire la sospensione cellulare-interrotto ad un nuovo tubo conico da 15 mL.</li>
	<li>Aggiungere 8-9 mL di terreno di omogeneizzazione a totale di 10 mL e centrifugare la provetta conica per 10 min a 2.000 x g e 4 ° C.</li>
	<li>Trasferire il surnatante in una nuova provetta conica 15 mL per rimuovere cellule ininterrotte e nuclei e centrifugare la provetta conica ancora per 10 min a 2.000 x g e 4 ° C.</li>
	<li>Trasferire il surnatante in un nuovo ultracentrifugazione provetta e centrifugare per 45 min a 100.000 x g e 4 ° C.</li>
	<li>Aspirare il supernatante e risospendere il sedimento con attenzione in 1-2 mL di terreno di omogeneizzazione con volume di 1/1.000 di cocktail di inibitore di proteasi pipettando la soluzione su e giù più volte per rendere una frazione del microsoma per esperimenti a valle.</li>
	<li>Trasferire la frazione del microsoma in una provetta da 5 mL fondo tondo nuovo. 
	Nota: Dopo questo passaggio, è possibile arricchire i microsomi oggettive di centrifugazione in gradiente di densità iodixanol secondo il produttore &#39; protocollo s, rimuovere aspecifici microsomi. Le frazioni di picco per antigeni esogeni sono raccolti e poi sottoposto al passaggio successivo di purificazione (passaggio 3).</li>
</ol> 3. Purificazione dei microsomi con bOVA ERAD che subiscono <ol><li>volume aggiuntivo 1/100 di fresco SA-magnetico perline alla frazione del microsoma di passo 2.11 nei 5 mL fondo tubo tondo. 
	Nota: Prima di questo passaggio, è possibile pre-cancellare la frazione del microsoma di controllo magnetico perline per ridurre contaminazioni dei microsomi aspecifici.</li>
	<li>Delicatamente mescolare bene e ruotare il tubo del fondo tondo lentamente per 30 min a 4 ° C.</li>
	<li>Aggiungere 2-3 mL di terreno di omogeneizzazione a 4 mL totale nella provetta fondo tondo e miscelare bene.</li>
	<li>Mettete il tubo fondo tondo su un supporto magnetico e incubare per 10 min a 4 ° C. Poiché le perline associate alle vescicole sono attratti dal magnete, microsfere marrone si accumulano gradualmente alla parete del tubo più vicina al magnete.</li>
	<li>Con il tubo attentamente restanti nello stand magnetico, scartare i surnatanti di aspirazione per rimuovere le vescicole non associate.</li>
	<li>Lavare i branelli magnetici vincolati alle vescicole due volte con 5 mL di medium di omogeneizzazione magnetico riposare per 10 min a 4 ° C e scartare il surnatante ogni volta accuratamente aspirando. 
	Nota: Senza il supporto magnetico, purificazione tramite le centrifugazioni a 2.000 x g per 10 min, 4 ° C è disponibile anche.</li>
	<li>Risospendere i branelli magnetici associati alle vescicole attentamente nel medium di omogeneizzazione 100 µ l pipettando la soluzione su e giù più volte.</li>
	<li>Trasferire le vescicole sedimento in una microprovetta nuova come il microsoma purificato per esperimenti a valle. 
	Nota: In genere, 50 µ l del microsoma frazione contenente 5-20 µ g proteine da 1 x 10 7 cellule può essere isolato.</li>
</ol> 4. Analisi dei microsomi del purificato perle <ol><li>risospendere il magnetico associati alle vescicole da passo 3.8 da 100-200 µ l di tampone TNE (pH a 20 mM Tris-HCl 7.4, 150 mM NaCl, 0,5 M EDTA, 1% Nonidet P-40) con volume di 1/1.000 di cocktail di inibitore della proteasi invece il mezzo di omogeneizzazione.</li>
	<li>Trasferire il lisato dal punto 4.1 una nuova microprovetta 1,5 mL.</li>
	<li>Determinare la concentrazione di proteina di fase 4.2 utilizzando un kit di test BCA per il produttore &#39; protocollo s. 
	Nota: In genere, 5-10 µ l lisato dalla fase 4.2 è sufficiente per determinare la concentrazione nella proteina.</li>
	<li>Trasferire il lisato dalla fase 4.2 (2 µ g di proteina per la macchiatura d'argento e 10 µ g di proteina per macchiarsi occidentale) in nuove microprovette.</li>
	<li>Mettere le microprovette su un blocco di calore alle proteine 95 ° C e far bollire in 1x tampone di gel-caricamento SDS (100 mM Tris-HCl a pH 6.8, bromofenolo 200 mM dithiothreitol, 4% SDS, 0,2%, 20% glicerolo) per 5 min.</li>
	<li>Centrifugare le microprovette per 10 min a 21.500 x g e 4 ° C. raccogliere i surnatanti in nuove microprovette per rimuovere le frazioni insolubili.</li>
	<li>Analizzare le proteine risolte nel passaggio 4.6 (2 µ g) di SD-pagina standard.</li>
	<li>Visualizzare le bande proteiche dall'argento che macchia usando la macchiatura d'argento kit in seguito il produttore &#39; protocollo s.</li>
	<li>Analizzare le proteine risolte nel passaggio 4.6 (10 µ g) di SD-pagina standard e il Western blotting. Utilizzare il reagente (SA-HRP) per il produttore &#39; s protocollo e visualizzare da fluorography.</li>
</ol> 5. In Vitro Ricostituzione di ERAD ubiquitinazione di bOVA usando purificato microsomi <ol><li>trasferimento purificato microsomi (5-10 µ g di proteine) dal passaggio 3.8 con un 50% del volume del reticulocyte lysato (RL), 1 x tampone di reazione (50 mM Tris-HCl pH 7.4, 3 mM ATP, 0,5 mM MgCl 2) e 12:02 ubiquitina etichetta a bandiera in una nuova microprovetta. Il volume finale di questo esperimento è 20-40 µ l.</li>
	<li>Incubare la provetta per 2 ore a 37 ° C. 
	Nota: Se la quantità di bOVA ubiquitinata nel passaggio 5.12 è troppo piccola per rilevare mediante Western blotting, aggiungere 10 µM di MG132 o 2 µM di lactacistina per inibire l'elaborazione di bOVA l'ubiquitinate da proteosomi.</li>
	<li>Fermare la reazione inserendo nella microprovetta su ghiaccio.</li>
	<li>Risolvere i microsomi aggiungendo 500 µ l di tampone TNE con volume di 1/1.000 di cocktail di inibitore di proteasi.</li>
	<li>Centrifugare la provetta per 10 min a 21,500 x g e a 4 ° C. raccogliere i surnatanti in una nuova microprovetta per rimuovere le frazioni insolubili, che contengono ubiquitinate bOVA in DC2.4 prima del dosaggio di ubiquitinazione in vitro.</li>
	<li>Trasferire il surnatante ad una nuova microprovetta e aggiungere volume 1/100 di nuove perline SA-magnetico (solitamente 5 µ l per 500 µ l di surnatanti).</li>
	<li>Delicatamente mescolare bene e ruotarlo nella microprovetta lentamente per 30 min a 4 ° C.</li>
	<li>Centrifugare la provetta per 10 min a 21.500 x g e 4 ° C. scartare il surnatante dall'aspirazione a recuperare la bOVA e bOVA ubiquitinate associato con i branelli magnetici SA.</li>
	<li>Lavare due volte i branelli magnetici SA raccolti con 1 mL di tampone TNE mediante centrifugazione per 10 min a 21.500 x g e 4 ° C. scartare il supernatante ogni volta dall'aspirazione.</li>
	<li>Bollire i branelli magnetici SA in 1x tampone per 5 min a 95 ° C su un calore-blocco per risolvere le proteine purificate mediante i branelli magnetici SA di caricamento del gel di SDS.</li>
	<li>Centrifugare la provetta per 10 min a 21.500 x g e 4 ° C. raccogliere i surnatanti in una nuova microprovetta per rimuovere le frazioni insolubili.</li>
	<li>Analizzare i branelli magnetici SA vincolati alle proteine standard SD-PAGE e Western blotting. L'uso di anticorpi (Anti-Flag, anti-multi-Ub e anti-mouse IgG-HRP) e il reagente (SA-HRP) è per il produttore &#39; protocollo s e visualizzate da fluorography.</li>
</ol>

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<li>Zehner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+translocon+protein+Sec61+mediates+antigen+transport+from+endosomes+in+the+cytosol+for+cross-presentation+to+CD8%28%2B%29+T+cells%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">The translocon protein Sec61 mediates antigen transport from endosomes in the cytosol for cross-presentation to CD8(+) T cells.</a> Immunity. 42 (5), 850-863 (2015).</li>
</ol>

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Negli studi precedenti di CP, gli antigeni esogeni incorporati accumulato nell'area riservata del tardo endosoma o ER da microscopia immunofluorescente16,30,31,32. Si stima che ERAD-come trasporto e la lavorazione di antigeni esogeni sono effettuate in queste aree specialistiche dell'ER o endosoma tardivo, come il compartimento cellulare è stato identificato da saccarosio o centrifugazione su ...

Il MHC sono molecole sono espresse sulla superficie di tutte le cellule nucleate, con brevi peptidi antigenici derivati da antigeni endogeni, che vengono elaborati dal sistema ubiquitina-proteasoma nel cytosol1. Dopo l'elaborazione, peptidi antigenici sono trasportati nel lume del reticolo endoplasmatico (ER) del trasportatore di peptidi TAP. Nel lume dell'ER, una serie di specifiche chaperoni assistere il caricamento del peptide e il corretto ripiegamento del MHC I complessi. Questa serie di molecole è chiamato il complesso peptide-caricamento (PLC), che indica che l'ER è un vano centrale per peptide caricamento su MHC I2. Dopo peptide di caricamento, il MHC sono molecole vengono trasportati alla superficie delle cellule e svolgono un ruolo chiave nel sistema immunitario adattativo come auto-marcatori e consente il CD8+ linfociti T citotossici (CTL) per rilevare le cellule tumorali o agenti infettivi di antigenica peptidi da proteine non-sé3.
In cellule (APC) presentanti l'antigene, peptidi antigenici da antigeni esogeni sono anche presentata al MHC I4,5,6,7,8 tramite CP, che è trasportato principalmente fuori da DCs9,10,11. CP è essenziale sia per l'attivazione di ingenuo CD8+ T cellule e memoria CD8+ T cells in anti-infettivi e anti-tumorali CTLs12,13e nel mantenimento della tolleranza immunitaria inattivando di self-acting cellule T naive14,15. Il CP svolge molti ruoli importanti nel sistema immunitario adattativo, tuttavia i meccanismi molecolari della CP devono ancora essere descritti dettagliatamente. Gli studi precedenti di CP ha rivelato che antigeni esogeni sono stati localizzati in ER e l'endosoma e sono stati elaborati dal ERAD, suggerendo che antigeni esogeni sono trasportati dall'endosoma al pronto soccorso per ERAD-come elaborazione e peptide caricamento16 . Tuttavia, raccogliendo la prova indica che il caricamento del peptide di CP è effettuato non in ER, ma piuttosto in compartimenti endocitosi non classici, che hanno anche caratteristiche distintive del ER (Figura 1)17,18 ,19,20,21. Per evitare la degradazione dei precursori del peptide antigenico dall'alta attività di aminopeptidasi22 nel citosol, elaborazione e caricamento in CP peptide si verifica nella zona prossimale di questi comparti endocitosi non classici (Figura 1). Anche se le caratterizzazioni di questi comparti endocitosi sono controverse, non ci sono nessun esistenti specifiche molecole diverse da antigeni esogeni in questo comparto.
ERAD è una via cellulare, che in particolare rimuove le proteine misfolded dall'ER. Nella via ERAD, proteine misfolded vengono retrogradely trasportate attraverso la membrana ER al citoplasma ed elaborate dal sistema ubiquitina-proteasoma23,24,25. Quando le molecole di grandi dimensioni, quali le proteine, vengono trasportate attraverso il doppio strato lipidico, queste molecole passano attraverso un apparato molecolare chiamato complesso in un Traslocone, come il complesso di Sec61 e Derlin complesso l'ER26, e il complesso Tom e Tim il i mitocondri27. Quando esogenicamente aggiunto gli antigeni vengono trasportati attraverso la membrana dell'ER, essi devono penetrare bilayer del lipido in complesso con translocons, come il complesso di Sec61. Il metodo qui descritto purificato la vescicola mirata utilizzando queste molecole di membrana-penetrante come marcatori per i compartimenti endocitosi.
Il metodo qui descritto è un nuovo protocollo di purificazione di vescicola utilizzando il DC-come cella linea DC2.428 e biotinilati ovoalbumina (bOVA) come un antigene esogeno. I compartimenti endocitosi sono stati purificati da streptavidina (SA)-biglie magnetiche utilizzando il bOVA membrana-penetrante come un creatore. In questo microsoma purificato, alcuni esogenicamente aggiunto bOVA era ancora conservata nelle frazioni della membrana ma sono stato trasportato all'esterno del microsoma e poi ubiquitinate e trattato in vitro29. Questo microsoma purificata contenuta non solo proteine endocitiche vano, ma anche proteine ER-residente per ERAD e il peptide caricamento complesso; suggerendo che il compartimento cellulare è il vano endocitico prospettico per CP29. Questo protocollo non è dipenda dal tipo di antigeni esogeni e vale anche per altri sottoinsiemi di DC e altri tipi di cellule, quali i macrofagi, linfociti B e cellule endoteliali, per chiarire il meccanismo molecolare preciso di DCs per CP abile.

<table><tbody><tr><td>RPMI 1640</td><td>gibco by life technologies</td><td>11875-093</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Equitech bio</td><td>SFB30</td><td></td></tr><tr><td>Sodium pyruvate</td><td>gibco by life technologies</td><td>11360-070</td><td></td></tr><tr><td>MEM non-essential amino acids</td><td>gibco by life technologies</td><td>11140-050</td><td></td></tr><tr><td>HEPES</td><td>gibco by life technologies</td><td>15630-080</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>gibco by life technologies</td><td>21985-023</td><td></td></tr><tr><td>L-glutamine</td><td>gibco by life technologies</td><td>25030-164</td><td></td></tr><tr><td>Penisicillin-Sreptomycin</td><td>gibco by life technologies</td><td>15140-122</td><td></td></tr><tr><td>DMEM</td><td>gibco by life technologies</td><td>12100-46</td><td></td></tr><tr><td>OVA</td><td>SIGMA</td><td>A5503</td><td></td></tr><tr><td>Biotin-protein labelling kit</td><td>Thermo Fisher Scientific</td><td>F6347</td><td></td></tr><tr><td>MG-132</td><td>Santa Cruz Biotechnology</td><td>201270</td><td></td></tr><tr><td>lactacystin</td><td>SIGMA</td><td>L6785</td><td></td></tr><tr><td>Dounce homogenizer</td><td>IUCHI</td><td>131703</td><td></td></tr><tr><td>protease inhibitor cocktails</td><td>SIGMA</td><td>P8340</td><td></td></tr><tr><td>iodixanol</td><td>Cosmo bio</td><td>1114542</td><td></td></tr><tr><td>SA-magnetic beads</td><td>New England Biolabs</td><td>201270</td><td></td></tr><tr><td>control magnetic beads</td><td>Chemagen</td><td>M-PVA012</td><td></td></tr><tr><td>magnetic stand</td><td>BD Biosciences</td><td>552311</td><td></td></tr><tr><td>BCA protein assay kit</td><td>Thermo Fisher Scientific</td><td>23225</td><td></td></tr><tr><td>silver staining kits</td><td>Cosmo bio</td><td>423413</td><td></td></tr><tr><td>Reticulocyte Lysate</td><td>Promega</td><td>1730714</td><td></td></tr><tr><td>Flag-tagged ubiquitin</td><td>SIGMA</td><td>U5382</td><td></td></tr><tr><td>anti-ovalbumin (OVA,mouse)</td><td>Antibody Shop</td><td>HYB 094-06</td><td></td></tr><tr><td>ant-multi-ubiquitin (mouse)</td><td>MBL</td><td>D058&amp;minus;3</td><td></td></tr><tr><td>anti-Flag (mouse)</td><td>SIGMA</td><td>F3165</td><td></td></tr><tr><td>trypsin</td><td>SIGMA</td><td>85450C</td><td></td></tr></tbody></table>

purification of the membrane compartment for endoplasmic reticulum-associated degradation of exogenous antigens in cross-presentation

Le cellule dendritiche (DCs) sono altamente capace di elaborare e presentare antigeni esogeni interiorizzati su classe di istocompatibilità (MHC) sono molecole conosciuto anche come cross-presentazione (CP). CP svolge un ruolo importante non solo nella stimolazione dell'ingenuo CD8+ T cellule e memoria CD8+ T cellule per infettive e immunità del tumore ma anche nell'inattivazione di self-acting le cellule T naive di anergia delle cellule T o l'eliminazione delle cellule T. Sebbene il meccanismo molecolare critico del CP rimane essere delucidato, raccogliendo la prova indica che antigeni esogeni vengono elaborati tramite associati al reticolo endoplasmatico degradazione (ERAD) dopo l'esportazione da Tronchetti endocitosi scomparti. Fino a poco tempo, caratterizzazioni di questi comparti endocitosi sono stati limitati perché non c'erano specifici marcatori molecolari diversi da antigeni esogeni. Il metodo qui descritto è un nuovo protocollo di isolamento della vescicola, che permette per la purificazione di questi comparti endocitosi. Utilizzando questo microsoma purificato, abbiamo ricostituito il ERAD-come trasporto, ubiquitinazione e l'elaborazione del antigene esogeno in vitro, suggerendo che il sistema ubiquitina-proteasoma elaborato l'antigene esogeno dopo l'esportazione da questo compartimento cellulare. Questo protocollo possa essere applicato ad altri tipi di cella per chiarire il meccanismo molecolare di CP.

Per delucidare il meccanismo molecolare di CP, è necessario identificare i compartimenti cellulari, dove antigeni esogeni subiscono ERAD-come trasporto e la lavorazione. Mentre le osservazioni da microscopia di immunofluorescenza o da microscopia elettronica identificato il compartimento cellulare dove antigeni esogeni accumulato16,17,18,19</s...

Watch this Scientific Journal Video about Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation at JoVE.com

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Il metodo qui descritto è un nuovo protocollo di isolamento della vescicola, che permette per la purificazione dei compartimenti cellulari dove antigeni esogeni vengono elaborati dal reticolo endoplasmatico-collegata di degradazione in cross-presentazione.

Purificazione del compartimento di membrana per la degradazione del reticolo endoplasmatico-collegata di antigeni esogeni in Cross-presentazione

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

purification-membrane-compartment-for-endoplasmic-reticulum

Research

JoVE Journal

Immunology and Infection

8.2K Views.  Takasaki University of Health and Welfare. Il metodo qui descritto è un nuovo protocollo di isolamento della vescicola, che permette per la purificazione dei compartimenti cellulari dove antigeni esogeni vengono elaborati dal reticolo endoplasmatico-collegata di degradazione in cross-presentazione.

Video: Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

Purificazione e aggregazione della proteina precursore dell&#39;amiloide dominio intracellulare

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

Ricerca

Medicina

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

Reconstitution of Msp1 Extraction Activity with Fully Purified Components

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function depends on a robust quality control system to maintain protein homeostasis (proteostasis). Declines in mitochondrial proteostasis have been linked to cancer, aging, neurodegeneration, and many other diseases. Msp1 is a AAA+ ATPase anchored in the outer mitochondrial membrane that maintains proteostasis by removing mislocalized tail-anchored proteins. Using purified components reconstituted into proteoliposomes, we have shown that Msp1 is necessary and sufficient to extract a model tail-anchored protein from a lipid bilayer. Our simplified reconstituted system overcomes several of the technical barriers that have hindered detailed study of membrane protein extraction. Here, we provide detailed methods for the generation of liposomes, membrane protein reconstitution, and the Msp1 extraction assay.

Here, we present a detailed protocol for reconstitution of Msp1 extraction activity with fully purified components in defined proteoliposomes.

Ricostituzione dell'attività estrattiva msp1 con componenti completamente purificati

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function ...

Biochimica

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca2+ in the ambient buffer. Subsequent depletion of Ca2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation.

Solid-supported, protein-free, double phospholipid bilayer membranes (DLBM) can be transformed into complex and dynamic lipid nanotube networks and can serve as 2D bottom-up models of the endoplasmic reticulum.

Formazione spontanea e riorganizzazione delle reti di Nanotube del lipido artificiale come un modello Bottom-Up per il reticolo endoplasmico

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense ...

Bioingegneria

Purification of the Sarco-Endoplasmic Reticulum Ca2+-ATPase from Rabbit Muscle

Some P-type ATPases, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), are inherently labile membrane proteins that require specific physicochemical conditions during purification to obtain them with high purity and structural quality and in a catalytically active form. The disaccharide trehalose is a compatible solute that is synthesized and accumulated in high concentrations in the yeast cytoplasm to stabilize the membranes and proteins. The use of trehalose as an additive in the protocol for the purification of plasma membrane H+-ATPase results in a high-quality preparation, the hexameric structure of which is shown by biochemical analytical methods. Trehalose can, therefore, be used as a stabilizing additive for the purification of membrane proteins (P-ATPases). This protocol describes the modification of the classical protocol for SERCA purification by subjecting SERCA to centrifugation on a trehalose concentration gradient. The inclusion of this carbohydrate led to the purification of SERCA in a catalytically active form with high purity and, importantly, in a stable form. Partial biochemical characterization of the purified SERCA (SDS-PAGE, enzyme kinetics, FITC labeling, circular dichroism spectroscopy) showed that the enzyme is suitable for functional and structural studies. The use of trehalose in the purification protocol of P-type ATPases and other labile membrane (and cytosolic) proteins is suggested.

This protocol describes an improved SERCA purification method, which includes the disaccharide trehalose in the final centrifugation step. This carbohydrate stabilizes proteins under harsh conditions. The purified SERCA was catalytically active and displayed high purity, making it suitable for structural and functional studies.

Purificazione del reticolo sarco-endoplasmatico Ca2+-ATPasi dal muscolo del coniglio

Some P-type ATPases, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), are inherently labile membrane proteins that require specific ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

Visualizzazione di mRNA reticolo endoplasmatico localizzate in cellule di mammifero

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Biologia

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

Visualizzazione del reticolo endoplasmatico sottodomini in cellule in coltura

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Protein abundance reflects the rates of both protein synthesis and protein degradation. This article describes the use of cycloheximide chase followed by western blotting to analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast).

Cicloesimide Analisi Chase di degradazione delle proteine ​​in<em&gt; Saccharomyces cerevisiae</em

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein ...

The Endoplasmic Reticulum

The endoplasmic reticulum or ER makes up for more than half of the membranes in a cell and accounts for 10% of total cell volume. It is also the primary protein and lipid synthesis factory for most cell organelles, such as the Golgi apparatus, lysosomes, secretory vesicles, and the plasma membrane. Despite being the most extensive and functionally complex subcellular organelle, ER was the last to be discovered. After years of deliberation, Keith Porter and George Palade in the year 1954, produced the first high-resolution electron microscope images to affirm the presence of ER in the eukaryotic cell.
The ER membranes were identified as sites for protein synthesis and crucial for intracellular transport using radio-labeled and fluorescent-labeled amino acids. The isolation of ER is a tricky task as it forms an intricate mesh with other subcellular organelles. However, on cell homogenization, the disrupted ER membranes reseal into small closed vesicles called microsomes. These vesicles form a microsystem capable of sustaining all ER-related functions like protein and lipid synthesis, calcium signaling, and glycosylation. Subcellular fractionation is the best and often-used technique for the purification of these membranes. When separating using a sucrose gradient, the rough ER microsomes sediments at a higher density than smooth ER microsomes.
The ER network in a cell is dynamic. It is constantly shape-shifting along with the cytoskeleton to bolster mechanical support for the cell structure. Despite the distinct cisternae and tubule morphologies of the ER membrane, interconversion between the two is possible and is governed by the expression of membrane proteins. The ER network rearranges by tubule growth, retraction, and fusion of adjacent ER-ER membranes.
As mentioned above, the ER network is required for optimal overall cellular health. Disruption of ER morphology is linked to pathological conditions, including neurological disorders like Alzheimer&#39;s disease, hereditary spastic paraplegia, and viral infections like hepatitis C virus, and dengue virus.

Il reticolo endoplasmatico

The endoplasmic reticulum or ER makes up for more than half of the membranes in a cell and accounts for 10% of total cell volume. It is also the primary ...

Didattica

JoVE Core

Cell Biology

Unit 1

Transmembrane Transport in Endoplasmic Reticulum and Peroxisomes

TERMINI E CONCETTI CHIAVE

ER Retrieval Pathway

In the secretory pathway, vesicles transport proteins from one cellular compartment to another in forward transport to deliver the protein to its correct location. Occasionally, misfolded proteins and incorrect proteins escape their original compartments, and a retrieval pathway is used to return the escaped proteins to their original compartment.
The ER uses many checkpoints to prevent the entry of incorrectly folded or a resident protein as cargo onto a transport vesicle. These mechanisms include cargo selectivity of the receptors, aggregation of functionally similar proteins, and retention sequences on resident proteins.
Biochemical studies characterizing the cargo-sorting receptors have shown that they are ligand-specific and bind only at specific pH (pH 5.0 in yeast and mammals). Functionally similar ER-resident can proteins aggregate to form large complexes that cannot be loaded onto transport vesicles, thus avoiding the accidental escape of these proteins from the organelle. Additionally, the membrane-spanning domain of several ER and Golgi proteins contain a retention sequence that marks these proteins for staying back in the ER, so receptors do not bind them.
If an ER-resident protein escapes these checkpoints and gets loaded onto a Golgi-bound vesicle, a specific amino acid sequence called a retrieval signal helps the protein get recognized and transported back to the ER. The receptors involved in retrieval mechanisms recognize the retrieval signals at the carboxyl terminus of soluble proteins of the ER and in the cytoplasmic domain of some ER and Golgi membrane proteins. The receptors selectively capture such proteins and package them in vesicles that transport them back to the ER.
Two well-characterized retrieval signals are the carboxy-terminal tetrapeptide KDEL and KKXX found in many ER lumen resident proteins. BiP, a molecular chaperone, plays a vital role in preventing the aggregation of misfolded proteins. Interaction between BiP and unfolded proteins is mediated by a substrate-binding domain and a nucleotide-binding domain for ATPase activity. KDEL receptor in the post-ER compartments such as ERGIC and Golgi binds with the carboxyl-terminal retrieval signal of BiP and returns BiP to the ER via COPI vesicles.

Percorso di recupero del ER

In the secretory pathway, vesicles transport proteins from one cellular compartment to another in forward transport to deliver the protein to its correct ...

Purificazione del compartimento di membrana per la degradazione del reticolo endoplasmatico-collegata di antigeni esogeni in Cross-presentazione

Riepilogo

Esplora Altri Video

Purificazione e aggregazione della proteina precursore dell'amiloide dominio intracellulare

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

Ricostituzione dell'attività estrattiva msp1 con componenti completamente purificati

Formazione spontanea e riorganizzazione delle reti di Nanotube del lipido artificiale come un modello Bottom-Up per il reticolo endoplasmico

Purificazione del reticolo sarco-endoplasmatico Ca²⁺-ATPasi dal muscolo del coniglio

Visualizzazione di mRNA reticolo endoplasmatico localizzate in cellule di mammifero

Visualizzazione del reticolo endoplasmatico sottodomini in cellule in coltura

Cicloesimide Analisi Chase di degradazione delle proteine in

Il reticolo endoplasmatico

Percorso di recupero del ER

Purificazione del compartimento di membrana per la degradazione del reticolo endoplasmatico-collegata di antigeni esogeni in Cross-presentazione

Riepilogo

Esplora Altri Video

Purificazione e aggregazione della proteina precursore dell'amiloide dominio intracellulare

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

Ricostituzione dell'attività estrattiva msp1 con componenti completamente purificati

Formazione spontanea e riorganizzazione delle reti di Nanotube del lipido artificiale come un modello Bottom-Up per il reticolo endoplasmico

Purificazione del reticolo sarco-endoplasmatico Ca2+-ATPasi dal muscolo del coniglio

Visualizzazione di mRNA reticolo endoplasmatico localizzate in cellule di mammifero

Visualizzazione del reticolo endoplasmatico sottodomini in cellule in coltura

Cicloesimide Analisi Chase di degradazione delle proteine ​​in

Il reticolo endoplasmatico

Percorso di recupero del ER

Purificazione del reticolo sarco-endoplasmatico Ca²⁺-ATPasi dal muscolo del coniglio

Cicloesimide Analisi Chase di degradazione delle proteine in