Name: purification of the membrane compartment for endoplasmic reticulum-associated degradation of exogenous antigens in cross-presentation
Uploaded: 2017-08-21T14:00:00.000+00:00
Duration: 12 min 48 s
Description: 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

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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L., Kyritsis, C., Tamp&#233;, R., Cresswell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+phagosomes+in+dendritic+cells+form+a+cellular+compartment+sufficient+for+cross+presentation+of+exogenous+antigens.">Early phagosomes in dendritic cells form a cellular compartment sufficient for cross presentation of exogenous antigens.</a> Proc Natl Acad Sci U S A. 100 (22), 12889-12894 (2003).</li><li>Saveanu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IRAP+identifies+an+endosomal+compartment+required+for+MHC+class+I+cross-presentation.">IRAP identifies an endosomal compartment required for MHC class I cross-presentation.</a> Science. 325 (5937), 213-217 (2009).</li><li>Zehner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+translocon+protein+Sec61+mediates+antigen+transport+from+endosomes+in+the+cytosol+for+cross-presentation+to+CD8(+)+T+cells.">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>

Gli autori non hanno nulla a rivelare.

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

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

Large-Scale Purificazione di suino o bovino fotorecettore Outer Segmenti per fagocitosi saggi su cellule retiniche dell&#39;epitelio pigmentato

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

Ricerca

Immunologia e infezioni

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

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Un metodo del rendimento efficiente e di alta per l&#39;isolamento di mouse dendritiche sottopopolazioni cellulari

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolamento di Salmonella typhimurium-contenente fagosomi dai macrofagi

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.

Studiare le dinamiche di Organelle nelle cellule B durante la formazione immunitaria di sinapsi

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both ...

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In particular, small extracellular vesicles (sEVs) act as nano-carriers transferring natural antigens, which are critical in host immunomodulation and parasite survival. This article presents a step-by-step protocol to isolate sEVs from larval stage cultures of Echinococcus granulosus and analyzes their uptake by dendritic cells obtained from murine bone marrow, which acquire adhesion and antigen presentation capacity during their maturation after one week of in vitro culture. This article provides comprehensive information for generating, purifying, and quantifying sEVs using ultracentrifugation alongside parallel analyses of dynamic light scattering and transmission electron microscopy. Additionally, a detailed experimental protocol is outlined for isolating and cultivating mouse bone marrow cells and driving their differentiation into dendritic cells using Flt3L. These dendritic cells can present antigens to na&#239;ve T cells, thereby modulating the type of immune response in vivo. Thus, alternative protocols, including confocal microscopy and flow cytometry analysis, are proposed to check the acquired maturational phenotype of dendritic cells previously exposed to parasitic sEVs. Finally, it is worth noting that the described protocol can be applied as a whole or in individual parts to carry out parasite in vitro culture, isolate extracellular vesicles, generate bone marrow-derived dendritic cell cultures, and perform uptake assays with these cells.

Here, we describe in vitro culture conditions, isolation, and increased generation of extracellular vesicles (EVs) from Echinococcus granulosus. The small EVs were characterized by dynamic light scattering and transmission electron microscopy. The uptake by bone marrow-derived dendritic cells and their phenotypic modulation were studied using confocal microscopy and flow cytometry.

Isolamento e captazione delle cellule dendritiche di piccole vescicole extracellulari da Echinococcus granulosus

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In ...

Intralumenal Vesicles and Multivesicular Bodies

Intraluminal vesicles (ILVs) are small vesicles 50-80 nm in diameter formed during the maturation of early endosomes. A specialized endosome containing numerous ILVs is called a multivesicular body (MVB). ILVs contain internalized molecules such as antigens, nucleic acids, proteins, and metabolites. Some of these molecules are released from the MVBs inside exosomes and are transported to other cells. Other MVBs contain molecules that are retained in the ILVs and are later degraded within the cell.
MVBs were first observed in neuronal cells in 1955 using electron microscopy. MVBs have a diameter of 400-500 nm, are spherical, and were initially thought to be a form of the late endosome; however, recent studies have shown that MVBs are an intermediate form of these organelles between the early and the late endosomes.
ILV formation takes place when a ubiquitin-tagged protein enters the cell in an endocytic vesicle and fuses with the early endosome. As the early endosome matures, the endosomal membrane bends inwards to form ILVs. An ESCRT protein complex mediates ILV formation. Phosphatidylinositol 3-phosphate (PtdIns3P) is also required for ILV formation. PtdIns3P acts as a docking site for ESCRTs for the bending of the endosomal membrane.
The ESCRT complex and the ATPase, Vps4, required for ILV formation can be hijacked by enveloped retroviruses that infect host cells. Normally, ILVs are formed inside maturing endosomes with the help of ESCRTs within the cell, and the ubiquitin-tagged proteins are recognized and sorted into ILVs. In contrast, retroviruses use the ESCRTs to be released outside the cell. For example, the human immunodeficiency virus (HIV) codes for proteins, such as Gag, NC-p2, and Tat, that require ubiquitination for export. This ubiquitination is essential because ESCRTs and Vps4 recognize and sort only ubiquitin-tagged proteins into viral buds. Once the viral particles bud off from an infected cell, they are released into the extracellular space to infect other host cells.

Vescicole intraluminali e corpi multivescicolari

Intraluminal vesicles (ILVs) are small vesicles 50-80 nm in diameter formed during the maturation of early endosomes. A specialized endosome containing ...

Didattica

JoVE Core

Cell Biology

Unit 1

Endocytosis and Exocytosis

TERMINI E CONCETTI CHIAVE

Receptor Downregulation in MVBs

Multivesicular bodies (MVBs) are mature endosomes that sort ubiquitinated proteins and then fuse with lysosomes to degrade the sorted proteins. Epidermal growth factor (EGF) and its receptor (EGFR) form a complex that can be internalized through endocytosis, sorted into an MVB, and later degraded.
The EGFR can initiate signaling pathways that&#160; lead to cell proliferation, migration, and differentiation. Overexpression of EGFR&#160; stimulates cells to proliferate. Excessive&#160; EGFR activation may result in cancer; therefore, EGFR expression must be tightly regulated. The degradation of the EGF-EGFR complex through the endocytic pathway is a means of downregulating EGFR and preventing its overexpression.
EGF binding to EGFR induces EGFR dimerization. EGFR is ubiquitinated by a ubiquitin ligase. Ubiquitination triggers the endocytosis of the dimerized EGF-EGFR complex. Once inside the cell, the complex is first sorted into the early endosome. As the early endosome matures, ESCRT proteins, or endosomal sorting complexes required for transport, translocate&#160; the EGFR-EGF complex into intraluminal vesicles (ILVs). Several ILVs are formed inside the maturing endosome, eventually becoming multivesicular bodies (MVBs). MVBs containing the EGF-EGFR complex then fuses with a lysosome for degradation of the EGFR-EGF complex.
Similar to EGFR, cytokine receptors are also internalized and downregulated by MVBs. Ubiquitinated receptor-cytokine complexes are endocytosed and later delivered to MVBs for degradation. The degradation weakens the cytokine signaling. In the case of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF&#593;) or interleukin-1 (IL-1), any defect in ubiquitination, endocytosis, or degradation of the receptor-cytokine complex through MVB can cause enhanced or sustained signaling. This may lead to inflammatory diseases like Crohn&#39;s disease or rheumatoid arthritis.

Sottoregolazione del recettore nelle MVB

Multivesicular bodies (MVBs) are mature endosomes that sort ubiquitinated proteins and then fuse with lysosomes to degrade the sorted proteins. Epidermal ...

Export of Misfolded Proteins out of the ER

After folding, the ER assesses the quality of secretory and membrane proteins. The correctly folded proteins are cleared by the calnexin cycle for transport to their final destination, while misfolded proteins are held back in the ER lumen. The ER chaperones attempt to unfold and refold the misfolded proteins but sometimes fail to achieve the correct native conformation. Such terminally misfolded proteins are then exported to the cytosol by ER-associated degradation or ERAD pathway for degradation.
Different modes of ERAD pathways
Hrd1 is a ubiquitin ligase and a retro-translocation channel for misfolded polypeptides. It complexes with another membrane protein, Hrd3, to form the core ERAD machinery. The other players associated with these core proteins depend upon the topology of the misfolded protein. The retrotranslocation complex depends on the luminal lectin chaperones to identify misfolded proteins. Studies in the yeast system suggest the presence of ERAD-C, ERAD-L, and ERAD-M pathways to export misfolded proteins from various parts of the ER. The target proteins of the ERAD-L and ERAD-M pathways use the Hrd1 channel to exit the ER lumen and membrane, respectively, while the ERAD-C pathway utilizes the Doa10p ubiquitin ligase complex to degrade the cytoplasmic domains of the ER membrane proteins. Sometimes, these pathways can overlap as some misfolded proteins need translocation machinery from two different ERAD pathways to exit the ER. The mammalian ERAD machinery is more complex than yeast and has multiple proteins that export and mark proteins for proteasomal degradation. Eventually, all ERAD pathways converge due to their dependence on a cytoplasmic AAA-ATPase to provide the mechanical force to extract proteins through the membrane.
Degradation of non-glycosylated proteins
The ER has another lectin-independent system that monitors and clears the non-glycosylated proteins from ER. It consists of the resident ER chaperone, BiP protein, and the ER-localized DnaJ family members (ERdjs). ER stress induces the production of HERP, a ubiquitin-like membrane protein, which assembles with other ERAD proteins like the Hrd1 and AAA-ATPase, forming a complex. BiP uses some ERdj proteins as cofactors to recruit non-glycosylated misfolded proteins to the HERP complex for retrotranslocation and ubiquitination of the misfolded protein. HERP is associated with the proteasome on the cytosolic side, where it delivers the misfolded proteins for degradation.

Esportazione di proteine mal ripiegate dal pronto soccorso

After folding, the ER assesses the quality of secretory and membrane proteins. The correctly folded proteins are cleared by the calnexin cycle for ...

Transmembrane Transport in Endoplasmic Reticulum and Peroxisomes

Antigen Presenting Cells

The immune system is a complex network of cells and molecules that protects the body from foreign invaders. T cells, a type of white blood cell, play a crucial role in this process. They recognize and attack foreign substances, such as pathogens, that enter the body.
T cells require the help of antigen-presenting cells (APCs), which process foreign antigens into smaller fragments that can be recognized by T cells. These APCs are highly specialized cells that efficiently internalize antigens through phagocytosis or receptor-mediated endocytosis.
Dendritic cells, a type of professional APC, are particularly efficient at antigen processing and presentation. They are highly mobile and can be found in tissues first exposed to foreign antigens, such as the skin, mucosal surfaces, and lymphoid organs.
Macrophages may also function as APCs, and are found in all tissues. They can engulf and process foreign antigens and release cytokines, which activate T cells and other immune cells, thereby coordinating the immune response.
B cells are another type of APC that can present antigens to T cells. When an antigen is presented to a B cell, it activates the B cell to produce antibodies against the antigen. This activation process involves the binding of the antigen to the B cell receptor, followed by a series of molecular signaling events that produce antibodies.
When an APC engulfs an antigen, it breaks into smaller fragments and combines them with major histocompatibility complex (MHC) molecules. The antigen-MHC complex is then transported to the APC&#39;s plasma membrane, where it is presented to a T cell.
The appropriate T cell receptor recognizes the processed antigen complex, triggering an immune response against the foreign invader. This process is crucial for protecting the body from infection and disease.

Cellule presentanti l'antigene

Antigen-presenting cells, also known as APCs, process foreign antigens, such as fragments from pathogens, for recognition by T cells.
When an APC engulfs ...

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

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