Bu çalışmada parçalar (SFB 1027, A6) Deutsche Forschungsgemeinschaft hibe tarafından desteklenen nazik vardı.

Not: Resim 1' de genel deneysel iş akışı genel bakış tasvir edilir.
Dikkat: RTA insanlar için çok toksiktir. Emanet laboratuvar izni S2 (Biyogüvenlik seviye 2 eşdeğeri) gereklidir. Lütfen tüm deneme sırasında eldiven.
1. Contegra ifadesi O'nun öğesini RTA Escherichia coli olarak
<ol>
	<li>E. coli hücreleri ifade plazmid pET24-RTA(onun) 6 veya standart elektroporasyon protokolleri9,10kullanarak boş vektör pET24a(+) ile dönüşümü. Boş plazmid içeren hücreler bir negatif kontrol hizmet vermektedir.</li>
	<li>Seçimden sefaloridin içeren (100 µg/mL) LB plakalar üzerinde olumlu klonların sonra RTA ifade Plazmid ve 5 mL LBkan orta (LB orta ile sefaloridin 100 µg/mL) boş vektörde içeren hücre aşılamak ve 37 ° C ve 24 saat için 220 devir/dakika, kuluçkaya.</li>
	<li>1 L LBkan orta 5 mL öncesi kültür ile ek ve hücreleri 0.8-1.0 (yaklaşık 3-4 h) OD600 gelinceye hücreleri 37 ° C ve 220 devir/dakika, kuluçkaya. Bundan sonra kültür sıcaklık 28 ° C için azaltmak ve 1 M H2O. izopropil-β-D-1-thiogalactopyranoside (IPTG) stok çözüm hazırlamak</li>
	<li>RTA ifade E. coli 1 mM son bir konsantrasyon IPTG ekleyerek teşvik.</li>
	<li>3.5 h 28 ° c ve 220 devir/dakika, hasat hücreleri tarafından Santrifüjü 10.000 x g ve 4 ° C'de 10 dakika, sonra iki kez ile 5 mL de bağlama arabellek (500 mM NaCl, 10 mM imidazole ve 20 mM KH2PO4 Pelet yıkama pH 7.4 =) 10.000 x g ve 10 dk ve resuspend Pelet 5 mL bağlama arabelleği için 4 ° C '. Not: Bu aşamada Protokolü duraklatılmış ve hücreleri 80 ° C'de için birkaç gün saklanabilir.</li>
</ol>
2. O'nun öğesini RTA benzeşme Kromatografi ile saflaştırılması
<ol>
	<li>Hücreleri aşağıdaki iletişim kuralını kullanarak buzda solüsyon içeren temizleyicide: 15 s darbe (20 mikron), 30 s Duraklat. Bu beş kez tekrarlayın.</li>
	<li>Hücre lysate, 21,000 x g ve 4 ° C'de 15 dakika santrifüj kapasitesi ve steril enjektör filtre sistemi (0.2 µm gözenek boyutu) kullanarak süpernatant filtre. Not: Hücre granül başarıyla sonicated örnekleri şeffaf kenarlıklarını göster.</li>
	<li>5 mL ile Ni2 +donatılmış bir otomatik arıtma sistemi kullanın-steril filtre E. coli süpernatant dan O'nun öğesini RTA kesir arındırmak için benzeşme sütun temel. Genel olarak, 1 mL/dk elüsyon hızını kullanın ve verimli olmayan toksin bağlayıcı ve toksin aktivite kaybı önlemek için tüm arıtma sistemi serin. Not: Verimli RTA arıtma için parametreleri Tablo 1' de listelenmiştir. Ayrıca bkz: Becker ve ark. daha fazla bilgi9için.<ol>
		<li>Kısaca, benzeşim sütun bağlama arabellek depolama arabellek kaldırmak için 20 mL ile equilibrate. Bir Ģırınga kullanarak benzeşme sütuna süpernatant steril filtre uygulayın.</li>
		<li>Sütun bağlama arabellek sütundan ilişkisiz proteinler kaldırmak için 25-35 mL ile yıkayın. 280 Absorbans UV kadar çamaşır adımı gerçekleştirmeniz nm başlangıç UV değeri yakın olduğunu.</li>
		<li>Bağlı RTA kesir elüsyon arabellek 20-35 ml elute (500 mM imidazole, 500 mM NaCl, 20 mM KH2PO4, pH 7.2 =) ve örnek buz (şekil 2A ve şekil 2B) üzerinde tutun. Not: Elüsyon RTA fraksiyonunun UV emme bir artış tarafından işaretlenir. Unutmayın ki sadece spesifik olmayan ilişkili proteinler ile kontaminasyonu önlemek için bu kesir toplamak için.</li>
		<li>Eluted örnekleri 20 µL Coomassie mavi11 veya Western blot analizi12,13 (isteğe bağlı) Boyama gerçekleştirmek için kullanın. Birincil RTA algılamak ve örnek saflık (şekil 3) doğrulamak için anti-O'nun antikorlar (1:1, 000) ve ikincil anti-mouse-IgG-HRP (1:10, 000) kullanın.</li>
		<li>Eluted RTA kesir 5 mL desalting sütun desalt ve 0, 8 M sorbitol örnek equilibrate.<ol>
			<li>Arıtma sistemi benzeşme sütun desalting sütun tarafından eski yerine koymak ve ilk sütun 0, 8 M sorbitol 20 mL ile equilibrate.</li>
			<li>Eluted RTA kesir bir şırınga ile sütun uygulanır. Sütun 0, 8 M sorbitol 100 mL ile yıkama ve UV emme (şekil 2C) artmaya başlar başlamaz desalted RTA kesir bir 15 mL tüp elute.</li>
			<li>Eluted RTA kesir 4 ° C'de depolayın Not: tuz kirlenmesini önlemek için gürültülerinden artırır doğrudan RTA kesir örnekleme durdurur. Desalting yordam parametreleri tablo 1'de listelenmiştir.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Eluted RTA kesir 10.000 x g ve 4 ° C'de 30-180 dk 10 kDa kesme spin sütun için 1-2 mL son hacmi kullanarak için konsantre ve örnek 4 ° C'de 3-4 hafta için saklayın. Dikkat: RTA etkinlik tam bir kaybına yol açar dondurma beri örnek dondurma değil.</li>
	<li>Bir geleneksel protein tayini kit kullanarak protein konsantrasyonu belirlemek. Protein konsantrasyonu 1-1.5 mg/mL aralığında olmalıdır. Not: RTA protein konsantrasyonu çok yüksek ise çökelti eğilimi (&#62; 5 mg/mL).</li>
</ol>
3. Maya dönüştürme ve hücre duvarı kaldırma
<ol>
	<li>Vahşi-türü veya seçili Maya silme mutantlar standart lityum asetat dönüştürme yöntemleri14kullanarak GFP muhabir plazmid pRS315-K28SP- GFP6 ile dönüşümü. Hücreleri lösin drop-out (d/o) üzerinde kuluçkaya olumlu klon seçim için 2-3 gün için 30 ° C'de glikoz tabak (% 2 glikoz, %1,5 agar, % 0.5 amonyum sülfat, %0,17 Maya azot Bankası (YNB) ve %0.087 d/o mix lösin olmadan).</li>
	<li>3 farklı Maya klonlar her plaka almak ve 220 devir/dakika ve 30 ° C-OD600 lösin d/o raffinose orta (%2 raffinose, % 0.5 amonyum sülfat, %0,17 YNB ve %0.087 d/o lösin mix) 100 ml klonlar aşılamak 1.0-2.0 = (2-4 x 107 hücre/mL). Not: Hücre büyümesini farklı Maya silme suşlarının farklıdır. OD600 bir Spektrofotometre ile izleyin.</li>
	<li>OD600 değerleri hesaplamak için seyreltik örnekleri OD600 0,1-0,3 = (1:5-1:10 dilutions) ve OD600 bir Spektrofotometre ölçmek. H2O lösin d/o raffionose orta karşılık gelen tutarı ile desteklenmiş başvuru olarak kullanın.</li></ol>Son olarak, OD600 resuspend 125 5 ml steril su (5 x 108 hücre/mL) =.
	<li>Santrifüj kapasitesi 10.000 x g de 5 min için 25 ° C'de hücreler, hücre iki kez 5 mL steril su ile yıkayın ve 50 mL spheroplasting arabellek hücrelerde resuspend (0,8 M sorbitol, 10 mM Tris-HCl (pH 7.5), 10 mM CaCl2, 2 mM dithiothreitol (DTT) ve 200 µg/mL litik enzim).</li>
	<li>50 mL kültür 100 devir/dakika ve 30 ° C 90 dk. isteğe bağlı için kuluçkaya: faz kontrast mikroskobu tarafından spheroplast oluşumu her 15dk izlemek. Mikroskop altında hücreler koyu şeffaf gri renk olmalıdır. Bright (refractile) hücreleri yeterince sindirilmiş, oysa hayaletler (soluk gri hücreleri küçük, varsa, iç yapısı ile) aşırı sindirilir.</li>
	<li>Spheroplast hazırlık etkinliği her örnek için kontrol edin.
	<ol>
		<li>Bitirme adım 3.5, spheroplasted 50 mL kültürünün 4 µL 496 µL spheroplasting arabellek (yaklaşık 2 × 106 spheroplasts) ile karıştırın ve santrifüj kapasitesi 400 x g de 10 dakika sonra.</li>
		<li>10 mL H2O resuspend Pelet distile, girdap örnek 30 s ve plaka 10 µL lösin d/o glikoz plakaları (% 2 glikoz, %1,5 agar, % 0.5 amonyum sülfat, %0,17 YNB ve %0.087 d/o mix lösin olmadan) örneğinin dışarı için.</li>
		<li>Hücreleri 30 ° C'de 3 gün kuluçkaya Yetişkin hücre kolonileri plaka üzerinde toplam sayısını saymak. Veri değerlendirme için 98 daha yüksek verimliliği ile sadece örnekleri kullanır (Toplam hücre koloni &#60; 40 kolonileri/plaka).</li>
	</ol>
	</li>
	<li>Santrifüj stabilize hücrelerden adım 3.5 g ve iki kez 5 mL içeren 10 dk ve yıkama hücreler için 4 ° C x 400 lösin d/o raffinose orta (0,8 M sorbitol, % 2'raffinose, % 0.5 amonyum sülfat, %0,17 YNB ve %0.087 bırakma mix lösin olmadan). 5 mL stabilize lösin d/o raffinose orta (1 x 108 hücre/mL) hücrelerde resuspend ve doğrudan hücre GFP muhabir tahlil ölçülerini (Bölüm 4) kullanın.</li>

4. GFP muhabir tahlil ölçüm 96-şey plakaları
<ol>
	<li>Tohum dışarı adım 3.7 96-şey tabak (200 µL/de) içine elde Maya hücre spheroplasts.</li>
	<li>70 µL stabilize lösin d/o raffinose orta negatif kontrol içeren eklemek (eluate bir Ni2 +-benzeşme arıtılmış lysate hücre boş vektör ifade E. coli IPTG kaynaklı hücrelerden) veya RTA 5 µM (son RTA konsantrasyon içinde saf 160 g/M'ye RTA karşılık gelen) her şey içinde.</li>
	<li>Hemen GFP ifade ikna etmek ve daha sonra ölçüm başlatmak için 30 stabilize µL galaktoz çözüm (% 30 galaktoz, 0, 8 M sorbitol) ekleyin. Not: deney başına en az 3 teknik çoğaltır gerçekleştirmek ve Maya zorlanma 3 Biyolojik çoğaltır.</li>
	<li>Numune Hazırlama (adımları 4.1-4.3), Floresans Reader'da 96-şey plaka koymak ve ölçüm başlatmak sonra. GFP Floresans algılama için gerekli ayarla 475/509 nm filtre kullanın. Ölçümler 30 ° c, 120 rpm ve sallayarak çapı 1 mm 20 h (10 dk aralıklarla ölçüm) bir zaman penceresi üzerinde gerçekleştirin. Not: Normalde tüm okuyucu sistemlerinde GFP filtre kümesi kullanılabilir. Sıcaklık, ölçüm aralıkları ve RTA konsantrasyon zaman penceresini kendi ihtiyaçları için ayarlanabilir. Temsilcisi sonuçları şekil 4' te gösterilmiştir.</li>
	<li>İsteğe bağlı: kalite kontrol ölçüm ek iç denetimlerini kullanın. Bir negatif kontrol 30 µL stabilize lösin d/o raffinose orta yerine % 30 galaktoz (GFP indüksiyon) ekleyerek hazırlayın. Buna ek olarak, 70 µL 0,8 M stabilize sorbitol G418 çözüm (300 µg/mL) ekleyin. Maya ifadede GFP engeller gibi protein çeviri inhibitörü G418 protein inhibisyonu için pozitif kontrol olarak hizmet vermektedir.</li>
	<li>Yüzde 20 h saat noktası göre aşağıdaki denklemi için göreli GFP Floresans hesaplamak (bkz. şekil 4A): <img alt="Equation" src="/files/ftp_upload/56588/56588eq1.jpg"> nerede NC negatif kontrol olduğunu
	<ol>
		<li>Alternatif olarak, her ölçüm için göreli GFP Floresans belirlemek (Ayrıca bkz: adım 4.4 Bu durumda 10 min) Yukarıdaki denklem kullanarak. Şekil 4B' de gösterildiği GFP Floresans yoğunluklarda (y ekseni) Zaman içinde (x ekseni) kurutma tarafından bir grafik oluşturun.</li>
	</ol>
	</li>
</ol>

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Yazarlar ifşa gerek yok.

Yukarıdaki Protokolü gerçekleştirirken, biz deneme başarılı bir sonuç elde etmek için aşağıdaki önerileri öneririz.
Kapaklı protein ifade için 1 mM IPTG konsantrasyonu aşmayacak şekilde önemlidir. IPTG konsantrasyonları &#62; 1 mM inhibe organizatörü kaynaklı RTA ifade ve toksin verimleri düşürmek için kurşun. Ayrıca, hücreleri 28 ° C eklenmesi vücut oluşumu, verimsiz katlama ve toksin inactivation önlemek için daha yüksek sıcaklıklarda ekili olmamalıdır...

Etkili tedavi tedaviler hala büyük ölçüde eksik olduğundan hastalarda bulaşan bakteri üreten toksin tarafından her sosyal sağlık sistemi için ciddi bir tıbbi ve mali yük özellikle temsil eder. Yeni tedavi stratejileri, tıbben a karmaşık bağımlılık yapan maddeler mekanizmaları geliştirmek için / B toksinler kolera toksini, Shiga toksin veya "Risin" gibi tam olarak uygulanmalıdır roman güçlü deneyleri üzerinde dayalı moleküler seviyede anlaşılması gerekir.
Son yıllarda, birçok araştırma A analiz girişiminde / B toksin taşımacılığında Maya ve radyoaktif toksin gibi zaman alıcı ve maliyet yoğun yöntemleri kullanarak memeli hücreleri etiketleme siRNA tabanlı tarama yanı sıra1,2 3yaklaşıyor. Bazı durumlarda, toksin kaçakçılığı Floresans mikroskobu tarafından görüntülenmeyecektir vivo içinde bireysel toksini alt birimleri fluorophores, kuantum nokta veya floresan proteinler4,5ile kimyasal ve/veya genetik kaplin sonra olmuştur. Ne yazık ki, bu değişiklikler genellikle etkin olmayan ve/veya değiştirilmiş doğal özellikleri toksinler için yol. Enzim lacZ, luciferase veya floresan proteinler gibi temel muhabir sistemlerinin kullanımı dolaylı olarak çok çeşitli bilimsel sorular cevap vermenin başka bir zarif yolu yok (örneğin GFP veya Discosoma sp. kırmızı floresan protein () dsRed)).
Bu makale, basit bir protokol, extracellularly uygulanan RTA S. cerevisiaehücre içi taşıma gereken hücresel bileşenleri tanımladığı açıklanmıştır. Böylece, dolaylı olarak GFP Floresans emisyon tarafından sonra içinde vivo RTA-aracılı protein çeviri inhibisyon ölçen bir protein biyosentezi sensör GFP tarafından takip bir N-terminal ER-Mümessillik sinyal içeren bir floresan-muhabir plazmid görür çeviri6. RTA endositoz ve/veya hücre içi ticareti olumsuz (veya olumlu) etkilenir ki vahşi-türüne göre belirli Maya silme mutant içinde durumda bu bir artış (ya da azalmak) GFP Floresans emisyon6' tespit edilebilir.
Şimdiye kadar tüm yöntemleri Maya hücreleri RTA Ulaştırma analiz RTA ER sitozol retro-translocation sürecine sınırlandı. Böyle yapay bir sistemde, bir ER alma sinyali içeren RTA bir intihar fenotip1,7' kaynaklanan bir indüklenebilir organizatörü üzerinden ifade edilir. B-alt "Risin" in birim bağlama hücre aynı şekilde bu el yazması açıklanan deneysel kurulumunda eksik ve böylece, tamamen doğal durum "Risin" holotoxin zehirlenme8, toksin taşıma plazma temsil etmiyor olsa da, membran acil servise Golgi aygıtı aracılığıyla yakından bu roman tahlil ile taklit. İlginçtir, pilot çalışmada elde edilen ilk sonuçlar RTA tarafından kullanılan ticaret yolları "Risin" holotoxin zehirlenme güzergahı ile çarpıcı benzerlikler ortaya gösterir.
Özet olarak, açıklanan yöntemi belirli bir rolü RTA endositoz seçili hücresel proteinler ve Maya kaçakçılığı belirlemek için kullanılabilir. Ayrıca, bu deneysel kurulumu kolayca diğer ribozom üretilen ve çeşitli Maya ve bakteriyel türler gibi zymocin veya Shiga toksin salgıladığı toksinler ihracı için adapte.

<table><tbody><tr><td>&lt;strong&gt;Bacterial and yeast strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; BL21 DE3(Gold)</td><td>Aligent Technologies</td><td>230130</td><td></td></tr><tr><td>&lt;em&gt;S. cerevisiae&lt;/em&gt; BY4742</td><td>Euroscarf</td><td>Y10000</td><td></td></tr><tr><td>&lt;em&gt;S. cerevisiae&lt;/em&gt; BY4742 deletion mutants</td><td>Dharmacon</td><td>YSC1054</td><td>whole collection</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Plasmids used in this protocol&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pET24a&lt;sup&gt;(+) &lt;/sup&gt;(Novagen)</td><td>Millipore</td><td>69772-3</td><td></td></tr><tr><td>pET-RTA&lt;sub&gt;(His6)&lt;/sub&gt;</td><td>Becker &lt;em&gt;et al.&lt;/em&gt; (2016)&lt;sup&gt;3&lt;/sup&gt;</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Zymolyase 20T</td><td>USBio</td><td>Z1000.250</td><td>lytic enzyme for cell wall removal</td></tr><tr><td>LB broth medium</td><td>Thermo Scientific</td><td>10855021</td><td>15 g agar was added for plate production</td></tr><tr><td>YNB</td><td>Thermo Scientific</td><td>DF0335-15-9</td><td></td></tr><tr><td>Ammonium sulfate</td><td>Sigma-Aldrich</td><td>A4418-100G</td><td></td></tr><tr><td>Yeast drop-out mix supplemts without leucine</td><td>Sigma-Aldrich</td><td>Y1376-20G</td><td></td></tr><tr><td>Agar</td><td>Sigma-Aldrich</td><td>05040-100G</td><td></td></tr><tr><td>D-glucose</td><td>Sigma-Aldrich</td><td>G8270-100G</td><td></td></tr><tr><td>DTT</td><td>Sigma-Aldrich</td><td>10197777001</td><td></td></tr><tr><td>D-raffinose</td><td>Sigma-Aldrich</td><td>83400-25G</td><td></td></tr><tr><td>D-sorbitol</td><td>Sigma-Aldrich</td><td>S1876-1KG</td><td></td></tr><tr><td>D-galactose</td><td>Sigma-Aldrich</td><td>G0750-10MG</td><td></td></tr><tr><td>G418</td><td>Thermo Scientific</td><td>11811031</td><td></td></tr><tr><td>IPTG</td><td>Sigma-Aldrich</td><td>I6758-1G</td><td></td></tr><tr><td>Imidazole</td><td>Roth</td><td>3899.1</td><td></td></tr><tr><td>PAGE ruler prestained</td><td>Fermentas</td><td>26616</td><td>protein ladder used for Western analysis</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Material for RTA purification, desalting and reader measurements&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Spectrophotometer Ultrospec 2100 pro</td><td>Amersham</td><td></td><td></td></tr><tr><td>Soniprep 150</td><td>MSE</td><td></td><td>old model, other models available</td></tr><tr><td>Fluoroskan Ascent</td><td>Thermo Scientific</td><td>5210470</td><td>old model, not available anymore</td></tr><tr><td>&amp;Auml;KTAPurifier</td><td>Thermo Scientific</td><td>28406266</td><td>Product is discontinued and replaced</td></tr><tr><td>HisTRAP HP column</td><td>GE Healthcare</td><td>17-5248-02</td><td></td></tr><tr><td>HiTRAP desalting column</td><td>GE Healthcare</td><td>11-0003-29</td><td></td></tr><tr><td>Midisart sterile filter</td><td>Sartorius</td><td>16534K</td><td>0.2 &amp;micro;m pore size</td></tr><tr><td>BCA protein assay kit</td><td>Pierce</td><td>23225</td><td></td></tr><tr><td>660 nm assay kit</td><td>Thermo Scientific</td><td>22660</td><td></td></tr><tr><td>96 well plates</td><td>Thermo Scientific</td><td>260860</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies (optional)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-Tetra-His</td><td>Qiagen</td><td>34670</td><td>primary antibody; 1:1,000 dilution</td></tr><tr><td>Anti-mouse-HRP</td><td>Sigma-Aldrich</td><td>A9044-2ML</td><td>secondary antibody, 1:10,000 dilution</td></tr></tbody></table>

a simple fluorescence-based reporter assay to identify cellular components required for ricin toxin a chain (rta) trafficking in yeast

Bakteriyel ve bitki A / B toksinler yararlanmak doğal ticaret yolları ökaryotik hücrelerde sitozol onların hücre içi isabet ulaşmak için ve en sonunda öldürmek için. Böyle A / B toksinler genellikle oluşur bir enzimatik etkin Asubunit (örneğin, "Risin" toksin (RTA)) ve bir veya daha fazla hücre için belirli bağlama toksin yüzey reseptörleri hücre için sorumlu olan Bsubunit(s), bağlama. Nasıl bizim mevcut bilgi A / B toksinler verimli endositoz ve intraselüler protein yüksek ökaryotik hücrelerde sıralama gibi temel hücresel mekanizmaları anlamaya yardımcı hücreleri bilim adamları sarhoş edici yetenekli. Tıbbi açıdan, aynı şekilde büyük toksin ticaret yolları hastalar için yeterli tedavi çözümleri bulmak veya sonunda kanser tedavisi için tedavi toksin tabanlı uygulamalar geliştirmek için tanımlamak önemlidir.
Beri genom çapında analizleri a / B toksin memeli hücrelerinde kaçakçılığı, zaman alıcı, karmaşık ve pahalı, çeşitli çalışmalarda a / B toksin taşıma Saccharomyces cerevisiaeMaya model organizma olarak gerçekleştirilen. Maya ve daha yüksek ökaryotik hücrelerin daha az karmaşık, temel hücresel süreçler olmasına rağmen çok sık Maya elde edilen sonuçlar ve vardır benzer memeli durumuna aktarılabilir.
Burada, hücre içi RTA Maya ticareti analiz etmek için hızlı ve kolay muhabir tahlil açıklayın. Yeni tahlil önemli bir avantajı sitozol sadece RTA retro-translocation endoplazmik retikulum (ER) üzerinden araştırmak için bir fırsattır, ama oldukça endositoz ve retrograd toksin acil servise plazma zarı taşıma. Tahlil geçici kullanma-in RTA toksisite Floresans emisyon yeşil flüoresan protein (GFP) aracılığıyla dolaylı ölçüm sağlar bir muhabir plazmid vivo içinde çeviri sonra. RTA verimli 28S rRNA depurination tarafından protein biyosentezi inisiyasyon önlediğinden bu tahlil konak hücre proteinleri tanımlaması değişiklikleri tespiti yoluyla hücre içi RTA ulaşım dahil Floresans emisyon sağlar.

Kabaca başarılı RTA ve sonraki GFP muhabir tahlil deney için tek adımları özetleyen Resim 1, bu el yazması açıklanan Protokolü'nün genel iş akışı gösterilmektedir. Tek tek her adım daha ayrıntılı bir açıklamasını iletişim kuralında bulunabilir. Şekil 2 bir başarılı RTA arıtma beklenen sonucu benzeşme Kromatografi (şekil 2A) ve boş vektör kontrolü (<strong class="x...

Watch this Scientific Journal Video about A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast at JoVE.com

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

Hücresel bileşenleri tanımlamak için basit Floresans tabanlı muhabir tahlil Risin toksin için Maya ticareti bir zincir (RTA) gerekli.

El yazması, bir maya tabanlı Floresans muhabir tahlil kaçakçılığı dahil ve bitki toksin Risin (RTA) bir alt birim sitotoksik süreçleri öldürmek hücresel bileşenleri tanımlamak için nasıl kullanılacağını açıklar.

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast

7.9K Görüntüleme.  Saarland University. El yazması, bir maya tabanlı Floresans muhabir tahlil kaçakçılığı dahil ve bitki toksin Risin (RTA) bir alt birim sitotoksik süreçleri öldürmek hücresel bileşenleri tanımlamak için nasıl kullanılacağını açıklar.

Video: A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

Canlı İmmobilize Maya Hücreleri Görselleştirme Hızlı Tekniği

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

Biyoloji

In Vivo Monitoring of Transcriptional Activity During Metabolic Transition Using a Bioluminescent Reporter in Yeast

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is particularly relevant for survival in fluctuating environments such as those found in beer fermentation. However, sugar transitions are an environmental variable that is challenging to predict and detect, impacting the outcome of beer fermentations. This protocol describes an in vivo system to monitor transcriptional activation associated with the glucose-to-maltose metabolic shift in Saccharomyces eubayanus that applies to different wild Saccharomyces yeast strains. 
The system employs an episomal bioluminescent transcriptional reporter for maltose metabolism, focusing on MAL32, since it provides a good readout for metabolic shifts, as studied in S. cerevisiae. For this, yeast strains were transformed with plasmids containing the MAL32 regulatory region from S. eubayanus, controlling the expression of a gene encoding for a destabilized version of firefly luciferase1, and a hygromycin resistance gene used exclusively during transformation to ensure plasmid acquisition. Following selection, transformed yeast cells can be cultured under non-selective conditions, as the episomal plasmid remains stable in culture conditions for up to 7 days. 
This system was validated under a complex sugar environment in microfermentation assays, confirming the effectiveness of the luciferase reporter in informing metabolic transitions. Samples were collected regularly and analyzed with a luminometer, providing continuous insights into yeast responses. While broadly applicable, this protocol is particularly valuable for assessing yeast performance under fermentation conditions, where metabolic changes pose a significant challenge. Additionally, this methodology can be adapted by selecting alternative promoters to explore a broader range of responses to environmental changes, allowing characterization as well as optimization of wild yeast strains for diverse industrial applications.

This protocol employs a bioluminescent reporter, allowing measurements of transcriptional activity in Saccharomyces eubayanus to monitor the glucose-to-maltose transition, enabling real-time analysis of metabolic adaptations and supporting strain optimization for industrial fermentation under diverse conditions.

In vivo (İn canlı) Mayada Biyolüminesan Raportör Kullanılarak Metabolik Geçiş Sırasında Transkripsiyonel Aktivitenin İzlenmesi

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is ...

Genetik

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Analizi<em&gt; Yersinia enterocolitica</emAna Hücreleri içine&gt; Efektör Translokasyon Beta-laktamaz Effector Fusions kullanma

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

İmmünoloji ve Enfeksiyon

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Maya İzleme Mitophagy Floresan Mikroskopi Testi Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Fisyon Maya Kantitatif Canlı Hücre Floresan mikroskopi Analizi

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

Tek Maya Hücreleri MAPK İsteyerek İfade zamansal Kantitasyonu

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

Büyüme-tabanlı Belirlenmesi ve Protein Parçalanabilirliklerinin Genetik Talepler biyokimyasal Onay<em&gt; Saccharomyces cerevisiae</em

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

Budding Maya endositozun kantitatif, kinetik ve Yüksek verim Analizi pHluorin Uygulamaları

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

Floresan proteinlerin etkisi Polyglutamine toksisite deneyleri Maya kullanılarak füzyon ortakları

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

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