Biz mükemmel teknik yardım için Andrea Kremsreiter teşekkür etmek istiyorum. Madison - Laboratuvarımızda çalışma NIH GM096088 ve Wisconsin Üniversitesi Vilas Trust tarafından desteklenmektedir.

1.. Maya luminometri Yöntemleri Saccharomyces cerevisiae suşu BYYT kullanın. Bu BY4741 (mata, ura3D0, his3D1, leu2D0, lys2D0, yvc1 :: HIS3, tok1 :: kanMX4) bir yvc1-tok1-türevidir. Batiza et al. 26 ve Loukin et al. 24 de tarif edildiği gibi, leu-seçilebilir aequorin-sentezleyen plasmid pEVP1/Aeq ve ura-seçilebilir rat-TRPV4 ifade eden plazmid ile p416GPDV4 hücrelerini dönüştürmek plasmid yapımı ve dönüşüm 27 kullanılması için standart yöntemler . Maya suşu ve plazmalar istek üzerine mevcuttur. <ol><li> Gece boyunca 1 M sorbitol ile takviye leu-ura-&quot;DCD&quot; Bırakma ortamı 28 (CMD-leu-ura ortamı 29 bir sülfat tükenmiş varyant), bir 30 ° C karıştırıcısı içinde post-logaritmik faza maya hücrelerinin kültürü 2 mi . Luciferi 2 uM ile desteklenmiş taze ortam içinde 1.8 ml, bu kültürlerin 200 ul inokülen coelenterazine. Çalkalamadan 24 saat daha oda sıcaklığında karanlıkta büyütün. </li><li> Kültürün ozmolarite (aşağıda) (tipik olarak en mOsM&#39;den 1400) ve diğer çözümleri bir buhar basıncı osmometresi kullanılarak ölçülür. </li><li> Tümbölen 20 tek tüp lüminometre için bir 12 mm Lüminometre tüpüne taze kültür ul. </li><li> Hipotonik şok için, arzu edilen şok derecesine bağlı olarak, 25 mM NaEGTA (pH 7.2) ya da NaMES (pH 7.2) 500-100 mM NaCl, (1,200-400 mOsm toplam ozmolaritesi) ihtiva eden bir çözelti 200 ul ekleyin. Sürekli önce lüminesans izlemek ve en az 120 saniye ozmotik downshock sonra, sadece kısa seyreltme işlemi tarafından kesildi. Göreli ışık birimi (RUL) gibi bir masaüstü bilgisayarda Lüminometre çıktı kaydettirin. </li></ol> Transgenik olmayan TRPV4 çalışma için de, biz maya suşu çok sayıda cevapları incelemek için otomatik bir sistem içinde yukarıdaki protokol bir varyantını kullandık. Çalışma of hipo-30 ya da bir mikro-luminometre kullanılarak maya deletome (4906 maya tek gen deletants of toplama) hiperosmotik 31 uyaranlara ve karşılık gelen yazılım programı ile analiz tepkiler başarılı olmuştur. 2. ve 3. Oosit Elektrofizyoloji Yöntemleri Elektrofizyoloji temel yöntemlerini 32. kullanır. PCR, vahşi tip veya mutant TRPV4 yüksek kalitede PfuUtra polimeraz kullanılarak ve pGH19 33 entegre olan açık okuma çerçevesi yükseltmek. Bu, Xenopus β-globin geni ve bir T7 RNA polimeraz promoterinin alt bölgesinin 5 &#39;ve 3&#39; UTRs arasındaki ORF kesin bir ekleme gerektirir. 3 &#39;UTR bölgesinin alt baş ve bir in vitro T7 RNA polimeraz reaksiyonunda şablonlar olarak kullanılan yapı linearize. Standart moleküler biyolojik teknikleri kullanarak 34. X&#39;den Evre V-VI oosit kullanın laevis. Hayvancılık, kısmi ovariectobenim, defolliculation ve vitellin zarf kaldırma standart işlemlerinizi 32,33,35 izleyin. Bir Drummond Nanoinject II otomatik mikropüskürtücüden kullanarak oosit başına cRAN çözümün 2-40 ng enjekte edilir. ~ Her bir deney grubu için 30 oosit enjekte edilir. TRPV4-cRNA enjeksiyonundan sonra, gentamisin (100 ug / ml) ile ND96 ortam içinde inkübe oosit ve 1 uM rutenyum 14, kırmızı. 2. İki elektrotlu voltaj-kıskaç <ol><li> Ayrı bir pipet çektirmesi ile hassas atılan 100 ul mikropipetler gelen borosilikat cam kayıt pipetler çekin. </li><li> Gerilim-ölçme ve akım enjekte pipet iki elektrodu çekin ~ 1 mikron çaplı bir ucu diyafram var çekilir. </li><li> 2 M KCI ile Dolgu elektrot 0.1-0.2 MQ direnç ile sonuçlanır. Birlikte VG-2Ax100 sanal-topraklı banyo kelepçe ile, bir Digidata 1440 sayısallaştırıcıdan aracılığıyla Geneclamp 500 amplifikatör arayüzüne bağlı HS2A headstages, onları monte edin. Kazanmak<html> pClamp10 yazılımı kullanarak veri. </li><li> 20X büyütme ile mikroskop aşamasında üzerine monte edilen bir fabrikasyon plastik odası içinde bir 1 ml&#39;lik bir banyo içinde, test edilecek oosit yerleştirin. Banyo çözümü sanal-topraklı bir 3 M KCl ağar köprü ve yakın oosit yerleştirilen ve VG-2A sanal toprak aşamaya bağlanan bir klorlu gümüş tel gereğidir. </li><li> Çözelti, giriş ve çıkış olarak plastik boru donanımları ile sürekli perfüzyon için bölmeyi oluşturmak. Giriş boru altı 50 ml şırınga gövdesi, farklı bir çözeltisi ile doldurulmuş, her bir manifold bir fabrikasyon perfüzyon sistemine bağlanır bağlayın. Herhangi bir çözeltinin ağırlık besleme hızları kontrol için -2-3 boru donanımları ile ilgili &quot;Keck&quot; rampa kenetlerle ml / min. </li><li> Mikromanipülatörler kendi headstages hem elektrotlar monte edin. Mikromanipülasyon ile oosit elektrot penetrasynların gerçekleştirin. </li></ol> 3. Doğrudan Mechanosensitivity Patch Kelepçe incelenmesi &quot;&gt; Pipet hazırlanması GΩ yapışma oluşumu ve on-hücre ya da kesilmiş modlarının oluşumu da dahil olmak üzere, yamalı kenetleme ve temel yöntem Hamill&#39;e olanlar et al. 36 ve Conn 32 bulunmaktadır. <ol><li> 98 mM KCI, 1 mM MgCI2 ve 10 mM K +-HEPES, pH 7.2 ile ucunda bir ~ 1 mikron çaplı bir delik (kabarcık sayısı 5-6) borosilikat cam pipetler doldurun. </li><li> Ayrıca, bir manometre, bir 5 ml şırıngaya bir plastik boru boyunca ve bir T-bağlantı ile yama kelepçe pipet takın. Elektrot ve manometre çıkışları hem işlemek için bir bilgisayar kullanın. 10 kHz veri Edinme ve daha sonra analiz için 1 kHz sekiz kutuplu Bessel filtre aracılığıyla oynatmak. </li><li> Farklı yamalar tekrar tekrar aynı oosit kesilebilir. Bu uygulama TRPV4 moleküler aktivitesinin incelenmesi daha verimli ve güvenilir kılar. </li></ol>

<ol>
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Yazarlar, hiçbir rakip mali çıkarlarını olmadığını beyan ederim.

Giriş bölümünde belirtildiği gibi, yaygın TRPV4 fonksiyonlarını incelemek için kullanılan yöntemler bazen tutarsızlıklara ve tartışmalar sonuçlandı. Burada açıklanan yöntemlerin iki set bazı avantajlar sunmaktadır ve mevcut yöntem tamamlayabilir. Biz TRPV4 sadece çalışmaları tarif ederken, bu buluşun metodları ve başka iyon kanalları incelemek için uzatılabilir. 1.. Maya luminometri Yöntemleri Tomurcuklanan maya lipid bileşimin <s...

TRPS (geçici reseptör potansiyeller) duyusal fonksiyonları 1,2 hizmet katyon kanallarının yedi alt familyaya oluşmaktadır. Memeliler, TRPV olarak, TRPS en vanilloid alt ailesi, altı çeşidi vardır. TRPV4 (tip 4) ısı 3,4 cevap verir, bazı kimyasallar, ozmotik şişme, ya da kayma gerilmesi. TRPV4 gen tekrar tekrar aday geni ve / veya 5-8 ifade klonlama ile izole edilmiştir. Bu ikinci yöntem, hipo-ozmolarite gen ürün tepkisini takip etti. TRPV4 çok farklı hücre tipleri 3,4 gelişimi, fizyoloji, herhangi bir patolojinin hemen hemen tüm organ ve işlevler cinsinden ifade edilir. Çarpıcı periferal nöropatiler ve / veya iskelet displazileri (iskelet gelişiminde anormallikler) 9-11 neden&gt; 50 insan otozomal dominant TRPV4 mutasyonlar. Iskelet displazisi aralığında hafif brachyomia tip 3 (tip-3 cücelik), spondylometaphyseal displazi Kozlowski tipi, seve dandisplaziler yeniden, bazı neonatal veya embriyonik ölüme neden olur. Mekanizmaların tüm görgü olası görünüyor olsa da, hiçbiri çeşitlilik, karmaşıklık, değişkenliği ve bu hastalıkların 4 zaman zaman çakışmalar açıklıyor. Diğer TRP kanalları 1 gibi, TRPV4 bir tetramerdir. Sıçan ya da insan TRPV4 olarak, her bir alt birim 871 artıkları oluşmaktadır. Bu temel öğesi altı transmembran olası voltaj kapılı K + kanallarının benzer bir şekilde düzenlenmiş olan bir heliks (S1-S6) &#39;dir. Orada, S4 S1 çevresel bir etki oluşturur ve S5 ve S6 geçirgenlik, gözenek alanı oluşturur. S5 ve TRPV4 ve S6 arasında muhtemelen katyon filtre oluşturmak için yakınsama dört hangi dizisi LDLFKLTIGMGDL, ardından kısa bir gözenek sarmal olduğunu. 470 artıklı N-terminal segmenti sitoplazmik proteinler ya da küçük ligandların bağlandığı bilinen 6 ankirin tekrarı içerir. C-terminal 152-bakiye sitoplazmik kademeli ot bağlanan diğer olası siteler arasında bir kalmodulin-bağlayıcı sekansı içerirOnun elemanları 3. TRPV4 esasen 1 anyonları hariç bir katyondur kanalıdır. Fizyolojik fonksiyonu Ca 2 + akınına uyaranlara transdüksiyonu için olsa da, bir P Ca iki değerli lehine, aynı zamanda bir Eisenman IV geçirgenlik sekansı ile diğer katyonlar geçirgen: P Na ~ 7 12. Tek kanallı iletkenliği ~ 90 pS dışa ve ~ 40 PS içe 6,13,14 de düzeltilir. Heterolojik ifade (aşağıda) akım hipo-ozmotik şişme, kayma gerilmesi, ya da sıcaklık 15 ile aktive edilebilir. Ayrıca, çoklu doymamış yağ asitleri 16,17 ve sentetik phorbal ester 18 4αPDD tarafından aktive edilir. Şu anda, her ikisi de, yüksek verim, küçük moleküllü ekran tarafından keşfedilen 10 -9 -8 10 M 20 etkili, en güçlü agonist GSK1016790A 19 ve antagonist olan GSK2193874. TRPV4 araştırma iki anahtar alanlar kafa kalır: (1) TRPV4 büyük ölçüde mechanosensitivity için çalışılmıştır bile, moleküler temeli tartışmalıdır. Bir model, hipo-osmolarite herhangi bir şekilde epoksijenaz ile asit (EET) epoxyeicosatrienoic dönüştürülür çoklu doymamış yağ asidi (PUFA), arakidonik asit (AA), üretilmesi için fosfolipaz A2 (PLA2) harekete geçirir ve EET bağlanmasını TRPV4 16 aktive tarif 17. Yine de, TRPV4 kendisi doğrudan basit bir açıklama sağlamaktadır, membran streç 14 (aşağıda) yanıt gösterilmiştir. (2) TRPV4 mutant patolojileri şaşırtıcı vardır. Temel olarak, bir hastalık kaybı, azaltılması ya da kanalı faaliyetlerinin artış nedeniyle olup olmadığını bilmek gerekir. Burada bile, edebiyat çok net değil. Birden iskelet-displazi alellerin daha yüksek bir aktiviteye sahip olduğu rapor edilirken, (kazanç fonksiyon-, GOF) 4,21, çeşitli etkinlikler (kayıp-fonksiyon, LOF) 10,22 azalma olduğu bildirildi. 14 alellerin sistematik bir çalışma için onları bulduTüm GOF edilecek mutasyonlar (aşağıda) 23. TRPV4 fonksiyon tam kaybına rağmen sadece küçük kusurları ile canlı veya bereketli nakavt fare, - bazı Lofs olan iddia trpV4-/ fenotip çelişiyor gibi görünüyor. Bu tartışmalara bir kısmı metodolojik kaynağı vardır. Laboratories farklı yöntemler, ya da bir yöntem varyantlarını kullanımı ve farklı bakarsak standartları kullanır. En sık olarak, TRPV4 geçici (HEK, CHO, HeLa) ve agonist veya hipotonik stimülasyonu üzerine iç [Ca2 +] yükselişi Ca 2 + duyarlı fluoresan boya, Fura-2 ile kayıtlıdır kültürlenmiş memeli hücrelerinde ifade edilir. Bu flüorometrik tahlil üzerinde aşırı güven 1 eleştirilmiştir. Farklı popülasyonlarda ekspresyon seviyesi, bunun içinde dağılım, hem de etkili bir boya konsantrasyonu, kontrol ve belgelenmiş gerekir. Daha güvenilir doğrudan elektrofizyolojik muayenesi olmaktır. Hatta bu gibi commonly uygulanan, sorunsuz da değildir. Kültürlenen hücrelerde ekspresyon seviyeleri kontrol edilmesi zor olduğu için, bütün hücre akımları büyük farklılıklar vardır. Akımlar küçük çünkü Ayrıca, güvenilir istatistikler genellikle pratik değil büyük çaplı, güvenmek zorunda kalacak. Patch-kelepçe muayeneler nadiren yapılmıştır. Bu tür bazı kayıtlar 16,17 istatistiksel değerlendirme zorlu hale aktivite patlamaları kümeleri göstermektedir. Daha iyi moleküler mechanosensitivity anlamak için, biz TRPV4 incelemek için iki ek sistemleri geliştirdiler. (1) uzakta olağan memeli ortaklarından TRPV4 izole etmek için, biz maya 24 tomurcuklanma sıçan TRPV4 dile getirdiler. Bu evrimsel uzak bağlamda TRPV4 Fonksiyonel ifadesi hala olağan ortaklar olmadan ozmotik kuvvet cevap verebilecek gösterdi. Maya gibi AA veya EET hiçbir PUFAların yapar, ve genom, bu Expre hiçbir PLA2&#39;nin veya epoksijenaz genleri Çünküsalgılanması da TRPV4 kuvveti duyacak için, gerekli olmadığını göstermektedir. Moleküler biyolojik en müsait ökariyotlarda TRPV4 olması da etkili ileri veya geri-genetik manipülasyonlar 25 verir. (2) TRPV4 derinlemesine biyofiziksel analizler için, Xenopus oositlerde TRPV4 ifade edilmiştir. NA (10 -10 10 -9 A) akımları alt-nA verim kültür hücreleri, aksine, bir oosit uA adlı (10 -6 -4 10 A) akımları ifade eder. Gürültü üzerine çok daha büyük sinyali daha iyi ölçümlerini ve daha güvenli karşılaştırılmasını sağlar. TRPV4, bu ifade, aynı zamanda bir kumanda kelepçesi kullanılarak tek tek moleküller olarak incelenebilir. Tek bir oosit tekrar ölçümü daha güvenilir hale tekrarlanan yama örnekleme sağlar. Bu tür çalışmalar TRPV4 kanal kendisi doğrudan membran germe kuvveti 14 ile aktive edilebilir olduğunu gösterdi. Analizler aynı zamanda 14 temsilci iskelet-displazi alellerin tüm kazanç fonksiyon-mutasyonlar olduğunu gösterdi. Bundan başka, degrBu yapısal Ca 2 + kaçağı ee iskelet hastalıkları 23 şiddetini paraleldir. Çünkü onların yenilik ve kullanışlılığı, bu iki yöntemin detaylı prosedürler TRPV4 veya benzeri kanallar gelecekteki araştırmalarda çoğaltmaları izin burada toplanıyor.

<table border="0" cellpadding="0" cellspacing="0" width="595">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vapor Pressure Osmometer</td>
			<td style="width:93px;">Wescor</td>
			<td style="width:119px;">Wescor 5500</td>
			<td style="width:167px;">Logan, UT, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sirius Single Tube Luminometer </td>
			<td style="width:93px;">Titertek-Berhold</td>
			<td style="width:119px;">Sirius </td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microplate luminometer</td>
			<td style="width:93px;">Titertek-Berthold </td>
			<td style="width:119px;">Mithras LB940</td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Luminometry software</td>
			<td style="width:93px;">Titertek-Berthold</td>
			<td style="width:119px;">MikroWin2000</td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:216px;">Patch clamp and software</td>
			<td style="width:93px;">Axon Instrument</td>
			<td style="width:119px;">HS2A headstage, VG-2Ax100 virtual grounded bath clamp, GeneClamp 500 amplifier, digidata 1440 digitizer, pClamp10 software </td>
			<td>Union City, CA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">manometer</td>
			<td style="width:93px;">Bio-Tec</td>
			<td style="width:119px;"> </td>
			<td>Winooski, VT, USA</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pipet puller</td>
			<td style="width:93px;">Sutter Instrument Co</td>
			<td style="width:119px;">Model P-97</td>
			<td>Novata, CA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microinjector</td>
			<td style="width:93px;">Drummond Scientific</td>
			<td style="width:119px;">Nanoinject II</td>
			<td>Broomall, PA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Material:</td>
			<td style="width:93px;"> </td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">luciferin coelenterazine</td>
			<td style="width:93px;">Invitrogen</td>
			<td style="width:119px;"> </td>
			<td>Carlsbad, CA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">T7 RNA polymerase reaction</td>
			<td style="width:93px;">Ambion</td>
			<td style="width:119px;">mMessage mMachine</td>
			<td>Austin, TX, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gentamicin</td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ruthenium red </td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">micropipets </td>
			<td style="width:93px;">Drummond</td>
			<td style="width:119px;">100 microliter micropipets</td>
			<td>Broomall, PA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">high-fidelity PfuUtra polymerase </td>
			<td style="width:93px;">Stratagene</td>
			<td style="width:119px;"> </td>
			<td>La Jolla, CA, USA</td>
		</tr>
	</tbody>
</table>

yeast luminometric and xenopus oocyte electrophysiological examinations of the molecular mechanosensitivity of trpv4

TRPV4 (geçici reseptör Potansiyelleri, vanilloid etti, tip 4) omurgalı dokularda yaygın olarak ifade edilir ve mekanik kuvvetlerin de dahil olmak üzere çeşitli uyaranlar tarafından aktif hale gelir. Bazı insan mutasyon TRPV4 karmaşık kalıtsal kemik veya nöronal patolojiler neden olur. Vahşi tip ya da mutant TRPV4 transgenler yaygın olarak kültürlenmiş memeli hücrelerinde ifade edilir ve Fura-2 fluorometri ile ve elektrotlar ile incelenir. Mechanosensitivity ve hastalıkların moleküler bazların mekanizması açısından, mevcut literatür kafa karıştırıcı ve tartışmalıdır. Varolan yöntemler tamamlamak için, biz TRPV4 moleküler özelliklerini incelemek için iki ek yöntemleri açıklanmaktadır. (1) Rat TRPV4 ve aequorin transgen tomurcuklanma maya içine transforme edilir. Transformant nüfusunun hipo-Osmotik şok TRPV4 kanalı üzerinden serbest nedeniyle, Ca 2 + ile aequorin kombinasyonuna lüminometrik bir sinyal verir. İşte TRPV4 her zamanki memeli ortak proteinlerin izole edilirve kendi mechanosensitivity ortaya koymaktadır. (2) TRPV4 of cRNA Xenopus oositlerine enjekte edilir. Uygun bir inkübasyon süresinden sonra, makroskopik TRPV4 mevcut bir iki elektrodlu voltaj kelepçe ile incelenir. Oosit banyosundan atıl ozmotikum uzaklaştırılması üzerine akım yükselme mechanosensitivity göstergesidir. Oositlerden mikroamper (10 A -4 -6 ila 10) akımları net quantifications ve daha emin değerlendirmeler, sonuçta kültürlenmiş hücrelerden nanoAmpere subnano-to (10 -10 A -9 10) akımları çok daha büyüktür. Tek kanal proteinlerin faaliyetlerini yansıtan mikroskobik akımlar da doğrudan doğruya-hücre ya da eksize modunda, bir yama kelepçe altında kayıtlı olabilir. Aynı oosit daha iyi veri replikasyonu sağlayan, birden fazla yama örnekleri sağlar. Yamalar uygulanan emmelerde doğrudan mechanosensitivity değerlendirmek TRPV4 etkinleştirebilirsiniz. Bu yöntemler aynı zamanda TRP kanallarının diğer tip çalışmaya faydalı olmalıdır.

Tipik luminometri kullanılarak bir deneyde, 400-1,200 mOsM&#39;den aşağı hipotonik şok, bir dizi 1400 mOsm&#39;de kültür 20 ul farklı düşük ozmotik basıncı şok çözümler 200 ul eklenmesi ile elde edilir. Maya hücreleri boş p416GPDV4 plazmid veya iyon filtre bir mutasyon (Şekil 1) 24 ile TRPV4 geni içeren bir transforme: Deneysel RLU iki negatif kontroller ile karşılaştırıldığında zaman TRPV4 aktivitesi belirgindir. Tipik iki el...

Watch this Scientific Journal Video about Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4 at JoVE.com

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

TRPV4 en incelemek için yaygın olarak kullanılan yöntemler tamamlamak için, iki yöntem tarif edilmiştir: Onun mechanosensitivity hipo-ozmotik tehdidi üzerine transgenik maya Ca 2 +-akorinden luminometri ele alınabilir. Ayrıca TRPV4-RNA incelenebilir on-hücre ya da kesilmiş modunda kelepçe bütün hücre iki elektrotlu voltaj kelepçe veya yama ile Xenopus oosit enjekte edilir.

Maya lüminometrik ve<em&gt; Xenopus</emTRPV4 Moleküler Mechanosensitivity arasında&gt; Yumurta Elektrofizyolojik Sınavları

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including ...

yeast-luminometric-xenopus-oocyte-electrophysiological-examinations

Research

JoVE Journal

Biology

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

10.0K Görüntüleme.  University of Wisconsin – Madison.  TRPV4 en incelemek için yaygın olarak kullanılan yöntemler tamamlamak için, iki yöntem tarif edilmiştir: Onun mechanosensitivity hipo-ozmotik tehdidi üzerine transgenik maya Ca 2 +-akorinden luminometri ele alınabilir. Ayrıca TRPV4-RNA incelenebilir on-hücre ya da kesilmiş modunda kelepçe bütün hücre iki elektrotlu voltaj kelepçe veya yama ile Xenopus oosit enjekte edilir. 

Video: Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

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

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Xenopus Oosit ifade İyon Kanalları Patch Kelepçe Kaydı

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

Görselleştirme RNA Yerelleştirme Xenopus Oosit

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

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 ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

Fare Oosit Mikroenjeksiyon, olgunlaşması ve ploidi Değerlendirmesi

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Barkodlu Maya Kütüphaneler Rekabet Genomik Ekranlar

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Yüksek Aktif Maya Ribozomlar Kromatografik Arıtma

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

Çoklu Gen Delesyonlar Taşıma Maya Suşlarında Nesil için Yeşil Canavar Süreci

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

<em&gt; Xenopus</emFluorometri ile&gt; Yumurta Kes-açık Vazelin Gap Gerilim-kelepçe Tekniği

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Surface Spreading and Immunostaining of Yeast Chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.

A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

Yüzey Yayma ve Maya Kromozomlar immün

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...