Bu eser Takasaki Üniversitesi Sağlık ve refah tarafından desteklenir.

1. büyüyen hücreler ve ayrıca eksojen antijenleri bir biotin proteinli etiketleme kullanarak <ol><li>hazırla bOVA kit üretici takip &#39; s protokolü. 
	Not: Normalde, ortalama 1 M OVA başına 2 M biotin bOVA içerir.</li>
	<li>RPMI-1640 2 mM L-glutamin, 1 mM sodyum pyruvate, 0,1 mM gerekli olmayan amino asitler, 100 U/mL penisilin-streptomisin, 55 mM 2-mercaptoethanol, 10 mM HEPES (pH 7.5) ve 5'te 37 ° C'de % 10 fetal buzağı serum (bundan sonra RPMI) ile takıma hücrelerde DC2.4 büyümek % CO 2 oksijen kuluçka (bundan sonra söz, hücreleri bu durum inkübe). DMEM % 10 fetal buzağı serum, 3.7 g/L NaHCO 3 (bundan sonra DMEM) yerine RPMI ile takıma kullanmak mümkündür.</li>
	<li>Bir gün önce arıtma, 1 x 10 5 hücre/ml bir doku kültürü plaka RPMI hücreleri bölün. Konfluent State hücreleri tutarak kaçının. DC2.4 hücreleri son derece eksojen antijenleri yarı Konfluent devlet kadar dahil ama hızla bir aşırı birleşmesi durumunda ulaştıktan sonra yeteneği kaybetmek.</li>
	<li>Daha önce DC2.4 hücreleri yarı Konfluent, 250 µg/mL 1 x 10 6 hücre/mL için bOVA ekleyin ve 2-4 h için kuluçkaya 
	Not: aşağı akım deneyler sırasında tüm arabellekleri ve Kimyasalları 4 ° C'de aksi belirtilmediği sürece tutulur.</li>
</ol> 2. Microsomes hazırlanması <ol><li>DC2.4 hücreler yumuşak doku tabaktan bir yeni 50 mL konik tüp içine pipetting tarafından hasat.</li>
	<li>5 dk, 4 ° C için 1000 x g, santrifüj ve bOVA ile orta aspirasyon tarafından dikkatli bir şekilde çıkarın.</li>
	<li>DC2.4 hücrelerle iki kez PBS arabelleği (1.37 mM NaCl, 8.1 mM Na 2 HPO 4, 2.68 mM KCl, 1,47 mM KH 2 PO 4) tarafından Santrifüjü 1000 x g 5 min, 4 ° C için de 40 mL yıkayın ve süpernatant aspirasyon her zaman atın.</li>
	Homojenizasyon orta (0.25 M Sükroz, 1 mM EDTA, 10 mM HEPES-NaOH [pH 7.4]) ile 1/1000 hacmi proteaz inhibitörü kokteyller ve transfer resuspended DC2.4 <li>resuspend Pelet adım 2.3 içinde 1 - 2 mL (1/20-1/10 cilt kültür ortamının) hücreleri içine bir buz gibi Dounce homogenizer.</li>
	<li>Bozmaya resuspended DC2.4 hücreleri nazikçe tarafından 10-20 vuruş ile buz gibi Dounce homogenizer. 
	Not: buz üzerinde Dounce homogenizer bozulma adımı sırasında soğutmalı</li>
	<li>Yeni bir 15 mL konik tüp hücre kesintiye süspansiyon transfer.</li>
	<li>10 mL toplam 8-9 mL homojenizasyon orta ekleyin ve konik tüp 2000 g x ve 4 10 dk santrifüj kapasitesi ° C.</li>
	<li>Süpernatant kırılmamış hücreleri ve hücre çekirdeği kaldırmak için bir yeni 15 mL konik tüp aktarmak ve konik tüp 10 min 2.000 g x ve 4 için tekrar santrifüj kapasitesi ° C.</li>
	<li>İçine a yeni ultrasantrifüj tüp ve 100.000 g x ve 4 45 dk santrifüj süpernatant transfer ° C.</li>
	<li>Süpernatant Aspire edin ve yukarı ve aşağı microsome kesir aşağı akım deneyler için yapmak için birkaç kez çözüm pipetting tarafından dikkatle içinde homojenizasyon orta 1-2 mL 1/1000 birim proteaz inhibitörü kokteyller ile Pelet resuspend.</li>
	<li>Transfer microsome kesir bir yeni 5 mL yuvarlak alt tüpüne. 
	Not: bu adımdan sonra iodixanol yoğunluk gradient Santrifüjü üreticiye göre tarafından objektif microsomes zenginleştirmek mümkün &#39; s protokolü, non-spesifik microsomes kaldırmak için. Tepe fraksiyonları eksojen antijenleri için toplanan ve sonra arıtma (Adım 3) sonraki adıma tabi.</li>
</ol> 3. Microsomes arıtma bOVA ile geçiren ERAD <ol><li>Ekle 1/100 birim taze SA-manyetik boncuklar 5 mL. adımda 2.11 microsome kısmını yuvarlak alt tüp. 
	Not: bu adımdan önce microsome kesir contaminations belirsiz microsomes azaltmak için denetim manyetik boncuklar tarafından önceden temizlemek mümkün mü.</li>
	<li>Hafifçe karıştırın ve yavaş yavaş 30 dk için yuvarlak alt tüp 4'te döndürmek ° C.</li>
	<li>2-3 mL homojenizasyon orta 4 mL toplam yuvarlak alt tüp içine ekleyin ve hafifçe karıştırın.</li>
	<li>Yuvarlak alt tüp manyetik bir stand üzerine yerleştirin ve 4 ° C'de 10 dakika için kuluçkaya Veziküller için bağlı boncuk mıknatıs için ilgi vardır beri kahverengi mikro-boncuk mıknatıs için en yakın tüp duvar için yavaş yavaş birikir.</li>
	<li>Tüp ile dikkatli aspirasyon tarafından ilişkisiz veziküller kaldırmak için supernatants atmak kalan manyetik tribünde.</li>
	<li>Yıkama manyetik boncuklar veziküller homojenizasyon orta 5 mL ile iki kez için manyetik tarafından bağlı 4 ° C'de 10 dakika stand ve süpernatant her zaman dikkatli aspirasyon tarafından atın. 
	Not: Manyetik stand, arıtma centrifugations 10 dk, 2.000 x g de tarafından olmadan 4 ° C de mevcuttur.</li>
	<li>Manyetik boncuklar bağlı veziküller dikkatle içinde 100 µL homojenizasyon çevre için yukarı ve aşağı birkaç kez çözüm pipetting resuspend.</li>
	<li>Aşağı akım deneyler için saf microsome olarak yeni bir microtube resuspended veziküller transfer. 
	Not: Genellikle, 5-20 µg proteinler 1 x 10 7 hücreleri içeren 50 µL microsome kesir ayrılmış olabilir.</li>
</ol> 4. Saf Microsomes analizini veziküller bağlı <ol><li>Resuspend manyetik boncuklar adım 3.8 TNE arabellek 100-200 µL tarafından (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0,5 M EDTA, %1 Nonidet P-40) 1/1000 birim proteaz inhibitörü kokteyller ile homojenizasyon orta yerine.</li>
	<li>Lysate 4.1 adımından yeni bir 1.5 mL microtube transfer.</li>
	<li>Üretici başına BCA tahlil kit kullanarak adım 4.2 protein konsantrasyonu belirlemek &#39; s protokolü. 
	Not: Genellikle 5-10 µL 4.2 adımından lysate protein konsantrasyonu belirlemek için yeterlidir.</li>
	<li>Lysate adım 4.2 (2 µg gümüş boyama için protein ve protein Western Blot için 10 µg) yeni mikrotüpler transfer.</li>
	<li>1 SDS jel-yükleme arabellek x 95 ° C ve kaynatın proteinleri ısı bloğunda mikrotüpler koymak (100 mM Tris-HCl pH 6.8, 200 mM dithiothreitol, % 4 SDS, %0,2 bromophenol mavi, % 20 gliserol) 5 dakika süreyle</li>
	<li>Mikrotüpler 21,500 g x ve 4 10 dk santrifüj kapasitesi ° C. toplamak supernatants yeni mikrotüpler çözünmez kesirler kaldırmak için.</li>
	<li>Adım 4.6 (2 µg) standart SD-sayfa tarafından çözümlenen proteinleri analiz.</li>
	<li>Protein bantları gümüş boyama kullanarak boyama gümüş tarafından kit üreticinin görselleştir &#39; s protokolü.</li>
	<li>Adım 4.6 (10 µg) standart SD-sayfa ve Western Blot tarafından çözümlenen proteinleri analiz. Üreticinin reaktif (SA-HRP) kullanmak &#39; s protokol ve fluorography tarafından görselleştirmek.</li>
</ol> 5. Vitro Sulandırma bOVA kullanarak saf Microsomes ERAD Ubiquitination <ol><li>Transfer saf microsomes (5-10 µg protein) adım 3.8 Retikülosit lysate (RL), % 50 hacmi ile reaksiyon arabellek (50 mM Tris pH 7.4, 3 mM ATP, 0,5 mM MgCl x 1 2) ve 0 pM 2 bayrak öğesini Ubikuitin yeni bir microtube içinde. 20-40 µL bu deneyin son birimdir.</li>
	<li>Incubate microtube 2 h 37 ° c 
	Not: ubiquitinated bOVA 5.12 adımda miktarı Western Blot tarafından tespit etmek için çok küçük ise, MG132 10 µM veya lactacystin proteasomes tarafından ubiquitinated bOVA işlenmesini engellemek için 2 µM ekleyin.</li>
	<li>Buz üzerinde microtube yerleştirerek reaksiyonu durdurmak</li>
	<li>Microsomes 500 µL TNE arabellek proteaz inhibitörü kokteyller hacmi 1/1.000 ile ekleyerek çözümlemek.</li>
	<li>Microtube 21,50, 10 dk santrifüj kapasitesig ve 4 x 0 ° c ubiquitinated bOVA DC2.4 in vitro ubiquitination tahlil önce içeren çözünmez kesirler kaldırmak için yeni bir microtube içine supernatants toplamak.</li>
	<li>Süpernatant için yeni bir microtube transfer ve yeni SA-manyetik boncuklar (genellikle 5 µL supernatants 500 µL için) 1/100 birim ekleme.</li>
	<li>Hafifçe karıştırın ve 4'te microtube 30 dk için yavaş yavaş döndürmek ° C.</li>
	<li>Microtube 21,500 g x ve 4 10 dk santrifüj kapasitesi ° C. atma süpernatant bOVA ve ubiquitinated bOVA kurtarmak için aspirasyon tarafından bağlı SA-manyetik boncuklar ile.</li>
	<li>21,500 g x ve 4 10 dk aralıklarla tarafından iki kez toplanan SA-manyetik boncuklar TNE arabelleği 1 mL ile yıkayın ° C. atma süpernatant her zaman aspirasyon.</li>
	<li>Arabellek arıtılmış proteinler tarafından SA-manyetik boncuklar gidermek için bir ısı blokta 95 ° C'de 5 min için yükleme SDS jel x 1 SA-manyetik boncuklar kaynatın.</li>
	<li>Microtube 21,500 g x ve 4 10 dk santrifüj kapasitesi ° C. çözünmez kesirler kaldırmak için yeni bir microtube supernatants toplamak.</li>
	<li>Standart SD-sayfa ve Western Blot proteinler için bağlı SA-manyetik boncuklar analiz. Antikorlar (Anti-bayrak, anti-multi-Ub ve anti-fare IgG-HRP) ve reaktif (SA-HRP) üreticisidir &#39; s Protokolü ve fluorography tarafından görüntülenmiştir.</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>

Yazarlar ifşa gerek yok.

CP önceki çalışmalarda, immünfloresan mikroskobu16,30,31,32tarafından geç endosome veya ER kısıtlı alana eklenen eksojen antijenleri birikmiş. Hücresel yuvası Sükroz veya iodixanol yoğunluk gradient Santrifüjü kullanarak tanımlandığı gibi bu ERAD benzeri taşıma ve işleme eksojen antijenleri ER veya geç endosome, bu özel alanlarda yapılmaktadır olduğunu tahmin edilme...

MHC ı molekülleri ile kısa antijenik peptidler sitozol1Ubikuitin-proteasome sistemi tarafından işlenen endojen antijenleri türetilen tüm çekirdekli hücrelerin yüzeyinde ifade. İşlendikten sonra antijenik peptidler endoplazmik retikulum (ER) Lümen DOKUNUN peptid taşıyıcı tarafından taşınmaktadır. ER Lümen peptid yükleme ve doğru katlanır MHC ben karmaşık bir dizi belirli chaperones yardım. Molekülleri bu dizi acil servis merkezi a bölme için yükleme MHC ben2peptid olduğunu belirten peptid-yükleme karmaşık (PLC) denir. Yükleme, peptid MHC sonra ben moleküller hücre yüzeyine taşınır ve bir anahtar edinilmiş bağışıklık sistemi olarak kendini işaretleri ve etkinleştirir CD8+ sitotoksik T lenfositler (CTL) kanser hücreleri veya enfeksiyöz ajanlar algılamaya tarafından antijenik rolü peptidler olmayan kendine proteinler3.
Hücreleri (APC) sunulması antijen, eksojen antijenleri üzerinden antijenik peptidler de sunulan MHC ben esas olarak yapılan4,5,6,7,8 üzerinden CP, dışarı DCs9,10,11tarafından. CP esastır saf CD8+ T aktivasyonu için hem de hücreleri ve bellek CD8+ T hücreleri içine CTL'ler12,13Anti-bulaşıcı ve anti-tumoral ve immün tolerans bakım ihracı, kendi kendine oyunculuk saf T hücreleri14,15. CP moleküler mekanizmaları henüz ayrıntılı olarak tarif edilebilir ancak CP edinilmiş bağışıklık sisteminde birçok önemli rol oynar. Eksojen antijenleri ve acil servis ve endosome hem de lokalize ERAD, eksojen antijenleri ERAD benzeri işleme ve16 yükleme peptid acil servise endosome taşınmaktadır düşündüren tarafından işlenmiş olan CP önceki çalışmaların ortaya . Ancak, CP peptid yüklenmesini serviste değil ama da Ayrıca ER (şekil 1)17,18 farklı özelliklere sahip çok klasik olmayan endositik bölmeleri, gerçekleştirilir biriken kanıtlar gösteriyor ,19,20,21. Antijenik peptid öncüleri bozulma aminopeptidaz yüksek faaliyet tarafından önlemek için bu klasik olmayan endositik bölmeleri (şekil 1) proksimal bölgede22 sitozol, işleme ve peptid CP içinde yükleme oluşur. Endositik bu bölmeleri karakterizasyonu tartışmalı olmasına rağmen bu yerde eksojen antijenlere başka hiçbir varolan belirli moleküller vardır.
ERAD özellikle misfolded proteinler ER kaldırır hücresel bir yoludur. ERAD yolu misfolded proteinler retrogradely sitoplazma için ER membran aracılığıyla taşınır ve ubiquitin-proteasome sistemi23,24,25tarafından işlenen. Proteinler gibi büyük moleküllerin lipid bilayer ile taşınmaktadır zaman, bu moleküller Derlin ER26ve Tom karmaşık içinde karmaşık ve Tim ve Sec61 karmaşık gibi bir translocon kompleksi olarak adlandırılan bir moleküler aparatı ile başarılı mitokondri27. Ne zaman exogenously eklendi antijenleri ER membran taşınır, lipid bilayer ile translocons, Sec61 kompleksi gibi kompleks içinde nüfuz gerekir. Burada açıklanan yöntemi endositik salonlar için işaretleyici olarak bu zarı delici moleküller kullanarak hedeflenen vezikül saf.
Burada açıklanan yöntemi eksojen bir antijen DC benzeri hücre satırı DC2.428 ve biotinylated ovalbumin (bOVA) kullanarak yeni bir vezikül arıtma protokoldür. Endositik bölmeleri streptavidin (SA) tarafından saf-membran-Penetran bOVA bir üreticisi kullanarak manyetik boncuklar. Bu saf microsome, bazı exogenously bOVA hala membran kesirler korundu ama dışında microsome ve sonra ubiquitinated taşınan ve işlenen ekledi vitro29. Bu saf microsome ERAD ve karmaşık yükleme peptid için hem de ER yerleşik proteinler endositik yuvası özel proteinler yer alan; hücresel yuvası CP29potansiyel endositik yuvası olduğunu düşündüren. Bu iletişim kuralı eksojen antijenleri türüne bağlı değildir ve ayrıca diğer DC alt kümeleri ve DCs hassas moleküler mekanizması için yeterli CP netleştirmek için diğer hücre tipleri, endotel hücreleri, makrofajlar ve B hücreleri gibi için geçerlidir.

<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

Dendritik hücreler (DC) işleme ve MHC sınıf (MHC) üzerine içselleştirilmiş eksojen antijen sunan son derece yetenekli ben moleküller olarak da bilinen çapraz-sunum (CP). CP çalış önemli bir rol sadece saf CD8 uyarılması+ T hücreleri ve bellek CD8+ T hücreleri için bulaşıcı ve tümör bağışıklığı ama aynı zamanda kendiliğinden hareket inactivation saf T hücreleri tarafından T hücre anerji veya T hücre silme. Her ne kadar aydınlatılmamıştır için CP kritik moleküler mekanizması kalır, biriken kanıtlar eksojen antijenleri endoplazmik retikulum ilişkili bozulması ile (ERAD) klasik olmayan ihracat sonra endositik işlenir gösterir bölmeleri. Çünkü eksojen antijenlere başka hiçbir belirli moleküler işaretleri vardı yakın zamana kadar bu endositik bölmeleri karakterizasyonu sınırlıydı. Burada açıklanan endositik bu bölmeleri arıtma için izin verir yeni bir vezikül yalıtım Protokolü yöntemidir. Bu saf microsome kullanarak, biz ERAD benzeri taşıma, ubiquitination ve eksojen antijen vitro, işlenmesi Ubikuitin-proteasome sistemi ihracat bundan sonra eksojen antijen işleme düşündüren yeniden düzenlendi hücresel bölme. Bu iletişim kuralı daha fazla CP moleküler mekanizması netleştirmek için diğer hücre tipleri için uygulanabilir.

CP moleküler mekanizması aydınlatmak için nerede eksojen antijenleri ERAD benzeri taşıma ve işleme tabi hücresel kompartmanlarda tanımlamak gereklidir. Gözlemler immünfloresan mikroskopi veya elektron mikroskobu nerede eksojen antijenleri16,17,18,19birikmiş hücresel yuvası tespit ederken, 30</su...

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

Burada açıklanan nerede eksojen antijenleri endoplazmik retikulum ilişkili düşmesine çapraz-sunum tarafından işlenen hücresel kompartmanlarda arıtma için izin verir yeni bir vezikül yalıtım Protokolü yöntemidir.

Membran yuvası arıtma için çapraz-sunudaki eksojen antijenleri endoplazmik retikulum ilişkili bozulma

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 Görüntüleme.  Takasaki University of Health and Welfare. Burada açıklanan nerede eksojen antijenleri endoplazmik retikulum ilişkili düşmesine çapraz-sunum tarafından işlenen hücresel kompartmanlarda arıtma için izin verir yeni bir vezikül yalıtım Protokolü yöntemidir.

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.

Retina Pigment Epitel Hücreleri üzerinde Fagositoz Tahliller için Domuz veya Sığır fotoreseptör dış Kesimleri Büyük Ölçekli Arıtma

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

İmmünoloji ve Enfeksiyon

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.

Msp1 Ayıklama Etkinliğinin Tamamen Saflaştırılmış Bileşenlerle Yeniden Uzlaştırılması

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

Biyokimya

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.

Fare Dendritik Hücre Alt Grupları İzolasyonunda bir Verimli ve Yüksek Verim Yöntem

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.

Salmonella typhimuriumyalıtım-makrofajlar üzerinden Phagosomes içeren

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.

Immün SYNAPSE oluşumu sırasında B hücrelerinde organelle dinamikleri eğitimi

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.

Echinococcus granulosus'tan Küçük Hücre Dışı Veziküllerin İzolasyonu ve Dendritik Hücre Alımı

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.

İntralümenal Veziküller ve Multiveziküler Cisimler

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

Education

JoVE Core

Hücre Biyolojisi

Unit 1

Endositoz ve Ekzositoz

KEY TERMS AND CONCEPTS

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.

MVB'lerde Reseptör Aşağı Regülasyonu

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.

Yanlış Katlanmış Proteinlerin Acil Servislerden Dışa Aktarılması

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

Endoplazmik Retikulum ve Peroksizomlarda Transmembran Taşıma

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.

Antijen Sunan Hücreler

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