Yazarlar, yararlı tartışmalar için Eleanor Fleming ve Jessica Chen teşekkür etmek istiyorum. Finansman Ulusal Alerji ve Bulaşıcı Hastalıklar Enstitüsü (NIH-NIAID) Ödülü R15AI079685 ve Araştırma Kurumu Cottrell Koleji Bilim Ödülü verildi. Howard Hughes Tıp Enstitüsü ve Staley fon Ek öğrenci destek sağlanmıştır.

1. Büyük Unilamellar lipid veziküller (luvs) hazırlanması <ol><li> Hücre zarı, 19 tahlil için taklit olarak hizmet vermeye luvs hazırlayın . </li><li> Mix fosfatidilkolin (POPC 760,10 g / mol), phosphatidylglycerol (POPG. 770,99 g / mol), 5-dimethylaminonaphthalene-1-Sülfonil 50 kloroform çözünmüş phosphatidylethanolamine (DNS-PAPA, 994,350 g / mol) (Avanti Polar lipidler) : 45:5 oranı. , Her test deney ve kontrol luvs ayrı bir hazırlık gerektirir gibi iki lipit kek şişeleri hazırlanmış olmalıdır. Toplam lipid (0,188 mg POPC, 0.169 mg POPG, ve 0.019 mg DNS-PAPA) 0.376 mg, genellikle tek bir translokasyon deneme için yeterli veziküller yapmak için yeterli. </li><li> N 2 gaz akışı kullanarak Kloroform buharlaşır. </li><li> 9-14 saatlik bir vakum desikatörde lipid pasta kurulayın. </li><li> Bir hisse senedi 10 mM HEPES tampon solüsyonu (10 mM HEPES, 238,3 g / mol, 45 mM NaCl, 58,44 g / mol, 1 mM EDTA, 372,24 g / mol pH 7.4) hazırlayın. HEPES tamponu st4 ° C az ORed </li><li> Yukarıda hazırlanan 10 mM HEPES tamponu 0.4 mM domuz tripsin (23.3 kg / mol) bir stok solüsyonu hazırlayın. Tripsin, -20 ° C&#39;de saklanır ve uygulamada, genellikle kullanımdan önce en fazla 2 donma-çözülme döngüsünden tabi değildir. </li><li> 4.0 mM Bowman-Birk tripsin inhibitörü (8 kg / mol) -20 ° C&#39;de 10 mM HEPES tampon çözelti ve mağaza stok solüsyonu hazırlayın. Kullanımda, bu hisse aylık doldurulan ve tekrar yapılmadan önce genellikle en fazla 3 donma / çözülme döngüsü uğramaz. </li><li> 0.2 mM 0.4 mM tripsin stok ve kurutulmuş lipid pastası 10 mM HEPES tampon çözelti eşit hacimde ekleyerek tripsin çözüm sulandırın deneysel multilamellar veziküller (MLVs). 0.376 mg toplam lipid tek bir örnek için, tripsin çözüm ve HEPES tamponu 150 mcL 150 mcL bir karışımı yeterlidir. </li><li> 0.2 mM tripsin ve 2.0 mM Bowman-Birk tripsin inhibitörü ile bir çözüm 0 eşit hacimde ekleyerek sulandırın kontrol MLVs.4 mM tripsin hisse senedi ve 4.0 mM tripsin inhibitörü kurutulmuş lipid kek. 0.376 mg toplam lipid tek bir örnek için, tripsin çözüm ve tripsin inhibitörü 150 mcL 150 mcL bir karışımı yeterlidir. </li><li> Cam şişe 1.7 ml mikrosantrifüj tüp Transfer MLV çözümler. </li><li> Sıvı azot 5 donma / çözülme döngüsü Konu MLV çözümleri. </li><li> Lipid ekstruder (Avanti Polar lipidler) HEPES tamponu her Teflon ekstruder parça nemlendirilmiş iki filtre destekleri koyarak hazırlayın. Iki teflon ekstruder adet arasında 0,1 mikron gözenekleri (whatman, Birleşik Krallık) ile nuclepore parça kazınmış membran yerleştirin ve metal ekstruder teneke kutu içinde ekstruder adet yerleştirin. Metal üst ekstruder spreyi sıkma ve tutucu yerleştirmeden önce iki ekstruder parçaları üzerinde çörek ayırıcı yerleştirin. </li><li> Vezikül örnek kaybını önlemek için 250 mcL cam şırınga kullanarak 500 mcL 10 mM HEPES tamponu ile ekstruder içinde boşluğu hacmi doldurun. &lt;/ Li&gt; <li> Ekstruder her MLV örnek yükleyin ve tekdüze unilamellar vezikül boyutu sağlamak için en az 21 kez zarından örnek itin. Luvs MLV çözüm aşağıdaki ekstrüzyon elde edilir. </li><li> Mağaza luvs 4 ° C ve 48 saat içinde kullanın. </li></ol> 2. Miktarının Konsantrasyon LUV <ol><li> LUV konsantrasyonu toplam fosfor içeriği örnek 20 belirleyerek belirlendi. </li><li> 8.9 NH 2 SO 4 oda sıcaklığında, nanopure su ve mağaza hazırlayın . </li><li> % 2.5 w / v amonyum molibdat VI (588,04 g / mol) nanopure su çözüm hazırlayın. % 10 w / v askorbik asit (176,1 g / mol) nanopure su çözüm hazırlayın. Tüp folyo ile kaplayarak ışığa maruz askorbik asit çözeltisi koruyun. 4 ° C&#39;de 2 ay kadar her iki çözümü de saklayın. </li><li> 0.65 mM fosfat tampon çözeltisi (tekbazlı susuz sodyum fosfat, 120 g / mol) hazırlayın. </li><li> Altı standart tu hazırlayınbes. Standartlar 0.0 mcL, 50 mcL (0,0325 μmoles), 100 mcL (0.065 μmoles), 175 mcL (0,114 μmoles), 250 mcL (0.163 μmoles), 350 mcL veya fosfor çözüm (0,228 μmoles) içermelidir. </li><li> Üç nüsha halinde numune tüpleri LUV hazırlayın. Her numune tüpü, yaklaşık 0,1 μmoles luvs içermelidir. Deneysel ve kontrol LUV konsantrasyonları ayrı ayrı ölçülmesi gerekir. </li><li> Bowman-Birk inhibitörü tozu herhangi bir kuru fosfor tuzlarının düzeltmek için, bir kontrol, boş hazır olmalıdır LUV. Örnek tüp LUV kontrol yerleştirildi boş 0.2 mM trypsin/2.0 mM Bowman-Birk inhibitörü çözüm aynı hacimde içermelidir. </li><li> Yeri 450 standart örnek, ve boş tüplerin her birine 8,9 NH 2 SO 4 mcL. </li><li> Az 175-210 Isı örnekleri, standartlar ve 25 dakika boşlukları ° C fırında. </li><li> Tüm tüpleri çıkarın ve soğumasını bekleyin. Tu her biri için% 30 H 2 O 2 150 mcL eklebes. </li><li> Isı örnekleri, standartlar ve boşlukları 175-210 az 30 dakika süreyle ° C </li><li> Örnekleri, standartlar ve ısı boşlukları kaldırın. Herhangi bir çözümleri henüz renksiz değilse 50 mcL H 2 O 2, 175-210 ek bir 15 dakika tüpleri ve ısı ° C ekleyin </li><li> 3.9 mL nanopure H 2 O, 0.5 mL amonyum molibdat çözüm ve her tüpe 0,5 ml askorbik asit çözeltisi ekleyin. </li><li> Kaynar su banyosunda tüm tüpler 5-7 dakika ısıtın. </li><li> Her bir standart ve örnek bir Agilent 8453 UV-Visible spektrofotometre ile 820 nm dalga boyunda absorbansı ölçülür. 0.0 mmoles fosfor içeren tüp tüm standartlara ve deney için boş olarak kullanılmalıdır örnekleri LUV. 0.2 mM trypsin/2.0 mM Bowman-Birk inhibitörü çözüm içeren tüp kontrolü için boş olarak kullanılmalıdır örnek LUV. </li><li> Lineer absorbans vs fosfor içeriği standart eğri ve toplam phosph hesaplamak için kullanılır.Orus içerik ve numunelerin konsantrasyon LUV. </li></ol> 3. Peptid Çözümler hazırlanması <ol><li> Nanopure H 2 O peptid çözülür Bu testte kullanılan peptidler açıklandığı gibi bir triptofan kalıntı içermelidir. </li><li> Peptid çözüm üç nüsha olarak 282 nm dalga boyunda absorbansı ölçülür. 5700 M -1 cm -1 triptofan molar sönüm katsayısı kullanılarak peptid konsantrasyonu hesaplayın. </li><li> 30 mcM nihai konsantrasyonu nanopure su peptit sulandırınız. -20 ° C&#39;de muhafaza </li></ol> 4. Translokasyon Niceleme <ol><li> 1.7 ml mikrosantrifüj tüpler deney ve kontrol örnekleri hazırlayın. 250 mcM son bir konsantrasyon için her çözüm için yeterli luvs ekleyin. Bu inhibitörü tripsin konsantrasyonu daha fazla 10X nihai bir konsantrasyon kadar yeterince Bowman-Birk inhibitörü çözüm ekleyin. 450 mcL nihai çözüm hacmi getirmek için yeterli 10 mM HEPES tamponu ekleyin. </li> &lt;li&gt; Starna mikro kuvars flüorometre hücre içine yerleştirin 50 mcL peptid çözüm. Luvs ve peptid arasındaki oran hiçbiri vezikül transloke olsa bile hemen hemen tüm bozulmamış peptidler FRET sinyal vermek için bir dansyl grup yeterli yakınlık olduğunu sağlamak için yeterince yüksek. Bu, benzer ve veziküller içine yerini değiştirmek peptidler için kontrol koşullarında gözlenen FRET tutarlı. </li><li> Küvet peptid çözümü için deney ya da kontrol bir örnek ekleyin. Bu 3 mcM son peptid konsantrasyonu getirmelidir. </li><li> Saniyede en az bir kez bir floresan okuma, deney ya da kontrol ek hemen sonra 25 dakikalık bir floresan kinetiği deney çözüm LUV başlayın. 280 nm dalgaboyu 525 nm dalga boyunda emisyon dalga boyu ve sıcaklık 25 ayarlayın ° C Cary Eclipse Floresans Spektrofotometre kullanarak, yüksek PMT hassasiyetini ayarlamak. </li></ol><html> 5. Kantitatif Translokasyon oranı oluşturma <ol><li> Veri toplama onbeş saniye sonra floresan okuma gibi ilk floresan (F o) tanımlayın. Bizim enstrüman düzeninde, numune eklendikten sonra, toplanan floresan verilerin ilk on beş saniye sisteme örnek odası kapanış ve diğer tedirginlikler nedeniyle karıştırma güvenilmez olduğunu bulduk. F o her zaman bir noktada göreli floresan (F / F 0) elde etmek için sonraki her okuma bölün. </li><li> Ortalama deneyi son dakikada gelen tüm göreceli floresan okuma nihai ortalama bağıl floresan elde etmek için <img alt="[F / Fo] avg" src="/files/ftp_upload/3571/3571eq1.jpg" /> . </li><li> Böl <img alt="[F / Fo] avg" src="/files/ftp_upload/3571/3571eq1.jpg" /> kontrol örnekleri için <img alt="[F / Fo] avg" src="/files/ftp_upload/3571/3571eq1.jpg" /> Deneysel örnekler için düzeltilmiş son floresan değeri elde etmek için. </li></ol>le &quot;&gt; 6 Temsilcisi Sonuçlar Şekil 2 sağlam translokasyon gösteren temsili bir peptid için bu testin sonuçlarını gösterir. Bu deneme için (siyah iz) sinyal sinyal FRET zaman içinde belirgin bir düşüş gösterir. Ancak, eksik tripsin inhibisyonu veya translokasyon yeteneği olmayan diğer faktörler nedeniyle sinyal FRET potansiyel kaybı kontrol etmek için önemlidir. Bu amaçla, her zaman da peptid ve tripsin ve Bowman-Birk tripsin inhibitörü (gri iz, Şekil 2) de içeren luvs arasındaki sinyal FRET ölçmek. Bizim ellerde, her peptit için bu kontrolü gerçekleştirmek için bir deneme çalıştırmak her zaman önemli olmuştur. Bu bize net bir şekilde lipid vezikül preparatlar veya araç gürültü, genellikle bu konsantrasyonlarda gözlemlenen nispeten zayıf floresan sinyalleri için önemli olabilir arasında herhangi bir farklılık nedeniyle sinyal herhangi bir değişiklik için doğru sağlar. Kontrol deneyimlerini önemitripsin inhibitörü içeren luvs kullanarak riment Şekil 3&#39;te gösterilmiştir verilere göre vurgulanır. Bu durumda, peptid sinyal Şekil 2 deney örneği (siyah iz) buna benzer bir miktar azalmıştır. Ancak, kontrol numune Bu peptid için daha hızlı bir düşüş gösterir, net translokasyon düşüktür. Protokolün 5. Bölüm Bu deneyde translokasyon miktarının belirlenmesi için basit bir yöntem açıklanır. Yüksek translokasyon oranları, etkin bir şekilde yerini değiştirmek peptidlerin göstergesidir; Şekil 2&#39;de gösterildiği gibi hücre delici peptid translokasyon oranı 1.16, zayıf translocating peptidler yakın 1 translokasyon oranları var. Deneyimlerimize göre 0,01 ile 0,06 aralığında, farklı vezikül preparatları ile yapılan üç bağımsız deneyler için standart hata. <img alt="Şekil 1" src="/files/ftp_upload/3571/3571fig1.jpg" /> Şekil 1. Translocat şematikiyon tahlil. Deneysel örnekleri (A) floresan dansyl PAPA ile katkılı (siyah çubuklar) ve tripsin (makas) kapsüllü içeren LUV. Tripsin inhibitörü (kırmızı daire), tripsin luvs dışında inhibe etmek için kullanılır. Luvs peptid (mor) maruz kalmaktadır. Ile Peptid dernek translocting peptidler sınır tanımayan iç tripsin karşılaşırsanız azalır sinyal (yeşil) FRET membranlar verim LUV. (B) translokasyon ilgisiz sinyal FRET azalır ölçmek için tripsin ve tripsin inhibitörü kontrol kapsüllü örnekleri LUV. <img alt="Şekil 2" src="/files/ftp_upload/3571/3571fig2.jpg" /> Şekil 2. Hücre delici peptid Temsilcisi veri antimikrobiyal pepide translocating bir temsilci, kendi kontrolü FRET sinyal ile karşılaştırıldığında önemli bir düşüş gösterir . <img alt="Şekil 3" src="/files/ftp_upload/3571/3571fig3.jpg" /> Rakam3. Denetimin önemini vurgulayan Temsilcisi veri olmayan bir translocating peptid triptik sindirim Eksik inhibisyonu, zamanla hem de deney ve kontrol sinyali FRET bir damla yol açan çözümler LUV . Deney ve kontrol izleri arasındaki fark görece küçük olduğundan, bu mutant zayıf translocating bir peptid olarak karakterize edilir.

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Çıkar çatışması ilan etti.

Burada sunulan protokol lipid veziküllerin içinde ve dışında peptidler konsantrasyon göreceli değişimi değerlendirmek için kullanılabilir. Bu değişiklikler translokasyon yeteneği ile ilgilidir. Bu protokol, ilaç taşıyıcı vektörler olarak potansiyeli olan hücre delici peptidler tanımlamak için kullanılabilir. Hücre delici peptidler ilgi büyüdükçe, nicel bir şekilde, doğrudan translokasyon olayı ölçme yöntemleri geliştirilmiş ve nasıl kullanıldığını görmek için ilginç olacak. ...

<table border="1"><tbody><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> 16:00-18:01 PG </td><td> Avanti Polar lipidler </td><td> 840457C </td></tr><tr><td> 01:18 Dansyl PE </td><td> Avanti Polar lipidler </td><td> 810330C </td></tr><tr><td> 16:00-18:01 PC </td><td> Avanti Polar lipidler </td><td> 850457C </td></tr><tr><td> Domuz tripsin </td><td> Sigma </td><td> T-0303 </td></tr><tr><td> Bowman-Birk tripsin / kimotripsin inhibitörü </td><td> Sigma </td><td> T-9777 </td></tr><tr><td> Mini-extruder </td><td> Avanti Polar lipidler </td><td> 610000 </td></tr><tr><td> Amonyum molibdat (para) </td><td> Alfa Aesar </td><td> 10811 </td></tr><tr><td> L-askorbik asit </td><td> Sigma </td><td> A1417 </td></tr><tr><td> Hidrojen Peroksit,% 30 çözüm </td><td> Mallinckrodt kimyasalları </td><td> 5240-05 </td></tr><tr><td> Hepes </td><td> Sigma </td><td> H-3375 </td></tr><tr><td> NaCl </td><td> Sigma </td><td> S-9625 &lt;/ Td&gt; </tr><tr><td> EDTA </td><td> Sigma </td><td> E5134 </td></tr><tr><td> NaH 2 PO 4 </td><td> Sigma </td><td> S-0751 </td></tr></tbody></table>

measuring peptide translocation into large unilamellar vesicles

Hücre zarları, hücre membran reseptörleri 1 yardımı olmadan kolayca çapraz peptidler aktif bir ilgi vardır . Bunların çoğu, sık sık ilaç taşıyıcı vektörler 1-3 olarak potansiyeli ile dikkat çeken hücre delici peptidler, olarak adlandırılır. Ayrıca, özellikle 4,5 membran litik olmayan mekanizmaları, hücre parçalama neden olmadan bakteri membranlar arası ve hücre içi süreçleri ile 6,7 müdahale ederek hücrelerini öldürmek ile faaliyet antimikrobiyal peptidler artan bir ilgi var . Nitekim, yazarlar, giderek hücre delici ve antimikrobiyal peptidler 1,8 arasındaki ilişkiyi işaret var . Membranı translokasyonu ve peptid yapısı ve yerini değiştirmek yeteneği arasında ilişki sürecinde bir firma anlayışı translokasyon için etkili, tekrarlanabilir testleri gerektirir. Bazı gruplar büyük unilamel içine translokasyon ölçmek için yöntemler önerdilar lipid veziküller (luvs) 9-13. Luvs bakteriyel ve ökaryotik hücre zarları için faydalı model olarak hizmet veren ve peptid floresan çalışmaları 14,15 sık sık kullanılır . Burada, ilk magainin ve buforin II 16,17 gibi antimikrobiyal peptidler, dikkate Matsuzaki ve arkadaşları tarafından geliştirilen yöntemin bizim uygulama açıklar. Bu yöntem için protokol sağlamaya ek olarak, biz de bu testte kullanarak translokasyon yeteneği rakamlarla veri analizi için basit bir yaklaşım sunuyoruz. Bu translokasyon testin diğerlerine göre avantajları membranı translokasyonu oranı hakkında bilgi vermek ve triptofan içeren peptidler, peptid özellikleri 18 değiştirebilir Ayrıca, floresan bir etiket gerektirmez bir potansiyele sahip olduğunu. Kısaca, lipid veziküller içine translokasyon yeteneği yerli triptofan artıkları ve d arasında Foster Rezonans Enerji Transferi (FRET) bir fonksiyonu olarak ölçülüransyl phosphatidylethanolamine proteinler ile ilişkili dış membran (Şekil 1) LUV. Hücre-delici peptidler luvs kapsüllenir sınır tanımayan tripsin karşılaşırsanız LUV membran ve sinyal FRET bir düşüş disassociation yol açan, bölünmüş. Translocating peptid için bu düşüş gözlenen sinyal luvs tripsin ve tripsin inhibitörü ya da lipid membranlar kendiliğinden çapraz peptid içeren tripsin luvs için maruz kaldığı hem de içeren aynı peptid gözlenen daha anlamlı olarak daha fazla FRET. Floresans bu değişiklik zamanla peptid translokasyonu doğrudan kantifikasyon sağlar.

Watch this Scientific Journal Video about Measuring Peptide Translocation into Large Unilamellar Vesicles at JoVE.com

Measuring Peptide Translocation into Large Unilamellar Vesicles

Bu protokol, büyük unilamellar lipid veziküller içine peptid translokasyonu kantitatif ölçmek için bir yöntem. Bu yöntem ayrıca membran translokasyon oranı hakkında bilgi sağlar ve çift katlı lipit katmanını etkin ve kendiliğinden geçtiğini peptidler tanımlamak için kullanılabilir.

Büyük Unilamellar Veziküller Ölçme Peptid Translokasyon

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

measuring-peptide-translocation-into-large-unilamellar-vesicles

Research

JoVE Journal

Biology

13.8K Görüntüleme.  Wellesley College. Bu protokol, büyük unilamellar lipid veziküller içine peptid translokasyonu kantitatif ölçmek için bir yöntem. Bu yöntem ayrıca membran translokasyon oranı hakkında bilgi sağlar ve çift katlı lipit katmanını etkin ve kendiliğinden geçtiğini peptidler tanımlamak için kullanılabilir.

Video: Measuring Peptide Translocation into Large Unilamellar Vesicles

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

Metagenomic Kütüphaneler Büyük Ölçekli Ekranlar

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

Biyoloji

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

Büyük Ekle Çevre Genomik Kütüphane Üretim

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

İyon Kanalları ve Fonksiyonel Analiz Mutagenez Heterologously Memeli Hücreleri cinsinden ifade edilir

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

Akış Sitometri Plazma Membran GLUT4 Translokasyon Kantitatif Ölçüm

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic development 1, 2. One of the signaling pathways involved in this regulation is the Wnt pathway, which is used many times during embryogenesis and critical for human disease3, 4, 5. Multiple molecular components of this pathway coordinately regulate signaling in a spatially-restricted manner, but the underlying mechanisms are not fully understood. Xenopus embryonic epithelial cells is an excellent system to study subcellular localization of various signaling proteins. Fluorescent fusion proteins are expressed in Xenopus embryos by RNA microinjection, ectodermal explants are prepared and protein localization is evaluated by epifluorescence. In this experimental protocol we describe how subcellular localization of Diversin, a cytoplasmic protein that has been implicated in signaling and cell polarity determination6, 7 is visualized in Xenopus ectodermal cells to study Wnt signal transduction8. Coexpression of a Wnt ligand or a Frizzled receptor alters the distribution of Diversin fused with red fluorescent protein, RFP, and recruits it to the cell membrane in a polarized fashion 8, 9. This ex vivo protocol should be a useful addition to in vitro studies of cultured mammalian cells, in which spatial control of signaling differs from that of the intact tissue and is much more difficult to analyze.

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity. An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells. This protocol will help address questions related to spatial control of signaling.

Floresan Proteinleri Polarize Translokasyon Xenopus Wnt Sinyalizasyonu Cevabı Ektoderm

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic ...

Examining BCL-2 Family Function with Large Unilamellar Vesicles

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate apoptosis1. Following cellular stress (e.g., DNA damage), the pro-apoptotic BCL-2 family effectors BAK (BCL-2 antagonistic killer 1) and/or BAX (BCL-2 associated X protein) become activated and compromise the integrity of the outer mitochondrial membrane (OMM), though the process referred to as mitochondrial outer membrane permeabilization (MOMP)1. After MOMP occurs, pro-apoptotic proteins (e.g., cytochrome c) gain access to the cytoplasm, promote caspase activation, and apoptosis rapidly ensues2.
In order for BAK/BAX to induce MOMP, they require transient interactions with members of another pro-apoptotic subset of the BCL-2 family, the BCL-2 homology domain 3 (BH3)-only proteins, such as BID (BH3-interacting domain agonist)3-6. Anti-apoptotic BCL-2 family proteins (e.g., BCL-2 related gene, long isoform, BCL-xL; myeloid cell leukemia 1, MCL-1) regulate cellular survival by tightly controlling the interactions between BAK/BAX and the BH3-only proteins capable of directly inducing BAK/BAX activation7,8. In addition, anti-apoptotic BCL-2 protein availability is also dictated by sensitizer/de-repressor BH3-only proteins, such as BAD (BCL-2 antagonist of cell death) or PUMA (p53 upregulated modulator of apoptosis), which bind and inhibit anti-apoptotic members7,9. As most of the anti-apoptotic BCL-2 repertoire is localized to the OMM, the cellular decision to maintain survival or induce MOMP is dictated by multiple BCL-2 family interactions at this membrane.
 Large unilamellar vesicles (LUVs) are a biochemical model to explore relationships between BCL-2 family interactions and membrane permeabilization10. LUVs are comprised of defined lipids that are assembled in ratios identified in lipid composition studies from solvent extracted Xenopus mitochondria (46.5% phosphatidylcholine, 28.5% phosphatidylethanoloamine, 9% phosphatidylinositol, 9% phosphatidylserine, and 7% cardiolipin)10. This is a convenient model system to directly explore BCL-2 family function because the protein and lipid components are completely defined and tractable, which is not always the case with primary mitochondria. While cardiolipin is not usually this high throughout the OMM, this model does faithfully mimic the OMM to promote BCL-2 family function. Furthermore, a more recent modification of the above protocol allows for kinetic analyses of protein interactions and real-time measurements of membrane permeabilization, which is based on LUVs containing a polyanionic dye (ANTS: 8-aminonaphthalene-1,3,6-trisulfonic acid) and cationic quencher (DPX: p-xylene-bis-pyridinium bromide)11. As the LUVs permeabilize, ANTS and DPX diffuse apart, and a gain in fluorescence is detected. Here, commonly used recombinant BCL-2 family protein combinations and controls using the LUVs containing ANTS/DPX are described.

Biochemically-defined large unilamellar vesicles (LUVs) are a convenient model system to analyze BCL-2 family interactions with immediate implications in better understanding the mitochondrial pathway of apoptosis. A method to produce LUVs, along with standard BCL-2 family protein combinations and controls to examine LUV permeabilization, are presented.

Büyük ünilamellar Veziküller ile incelenmesi BCL-2 Aile Fonksiyonu

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

Bir transmembran proteinin yeniden oluşturulması, Mikroskop ve yama kelepçe Çalışmaları Dev tek-katmanlı keseler içine Voltaj kapılı iyon kanalı, KvAP,

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows.

Peptit bağlanma floresan protein DNA ile yüzey-gergin büyük DNA moleküllerinin Görselleştirme

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating ...

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many different proteins associate with curved membranes because of their ability to sense or induce membrane curvature. Typically, these processes involve a multitude of proteins making them too complex to study quantitatively in the cell. We describe a protocol to reconstitute a curved membrane in vitro, mimicking a curved cellular structure, such as the endocytic neck. A giant unilamellar vesicle (GUV) is used as a model of a cell membrane, whose internal pressure and surface tension are controlled with micropipette aspiration. Applying a point pulling force on the GUV using optical tweezers creates a nanotube of high curvature connected to a flat membrane. This method has traditionally been used to measure the fundamental mechanical properties of lipid membranes, such as bending rigidity. In recent years, it has been expanded to study how proteins interact with membrane curvature and the way they affect the shape and the mechanics of membranes. A system combining micromanipulation, microinjection, optical tweezers, and confocal microscopy allows measurement of membrane curvature, membrane tension, and the surface density of proteins, concurrently. From these measurements, many important mechanical and morphological properties of the protein-membrane system can be inferred. In addition, we lay out a protocol of creating GUVs in the presence of physiological salt concentration, and a method of quantifying the surface density of proteins on the membrane from fluorescence intensities of labeled proteins and lipids.

Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.

Membran nanotüpler dev Unilamellar veziküller çekerek

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

Büyük gözlü manken coğrafi Atrofi, içine insan embriyonik kök hücre elde edilen retina Pigment epitel hücrelerinin subretinal nakli

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...