Bu çalışma NIAID, NIH intramural fonu tarafından desteklenmektedir.

1. Diktiyostelyum diskodeyum kemotaktik yetkili hücrelerinin hazırlanması <ol><li> Kemoatraktan cAMP, 22 sallayarak kültürünün zengin medya D3-T hasat büyüyen hücreleri ° C kemotaktik D. diskodeyum hücreleri oluşturmak için </li><li> (DB tampon 5 mM Na 2 HPO 4, 5 mM KH 2 PO 4, 2 mM MgCl 2, ve 0,1 mM CaCl 2 içeren), besin değeri olmayan gelişim tampon hücreler iki kez yıkayın. </li><li> 2x10 7 hücre / ml yoğunlukta DB tampon hücreleri yeniden askıya. </li><li> Bir saat için 100 rpm at 22 ° C 10 ml, 250 ml cam şişe içinde hücreleri iyice çalkalayın. </li><li> 75 nM cAMP, cAMP darbeli tedavi olarak belirlenmiş bir süreç nihai bir konsantrasyon elde etmek için 6 saat boyunca her altı dakikada bir 10 ml hücre 7.5 mcM cAMP stokunun 100 ul sunun. CAMP pulsing tedavi, D. 5-6 saat sonra diskodeyum hücreleri cAMP degrade doğru yetkili kemotaktik olur. </li><li> 5 dakika için 200 g santrifüj hücreleri toplamak ve sonra DB tamponu ile tekrar süspansiyon hücreleri 2.5 mM kafein içeren ve 200 rpm&#39;de 22 sallamak ° C&#39;de 20 dakika için kemotaktik bir durum hücre basolate için. </li></ol> 2. Görünür ve manipüle kemoatraktan degrade Görüntüleme chemotaxing hücreleri <ol><li> Dolgu 1 mcM cAMP ve Alexa 594 DB tampon 0.1μg/μl az bir taze hazırlanmış 30 ul çözüm mikropipet. </li><li> Mikropipet sahibine Femtotip takın ve bir basınç kaynağı aparatları boru, Eppendorf FemtoJet sistemi bağlamak. </li><li> Istikrarlı bir degrade oluşturmak amacıyla sabit bir basınç sağlamak için mikromanipülatör motorlu bir micromanipulator (Eppendorf TransferMan NK2) mikropipet montaj takın. </li><li> Mount konfokal mikroskop 40X petrol lensin üzerinden 6 ml DB tampon ile dolu bir iyi LABTEK odasına ve görüş alanı içine alan parlak optik, merkezi Femtotip kullanın. </li><li> Basınç kaynağı açın ve cAMP / Alexa 594 karışımı bir degrade oluşturmak için 70 hPa tazminat basıncı (Adet). </li><li> CAMP ve Alexa 594 floresan 543 nm lazer çizgisi ile uyarma kullanarak istenilen konsantrasyon karışımı izlenmesi cAMP degrade gözünüzde canlandırın. </li><li> Mikromanipülatör otomatik konumlama işlevi istenilen pozisyonlar mikropipet koymak ve Pozisyonu 1 Pozisyon 2 ve 3 numaralı pozisyon için hücre maruz kaldığı degrade işlemek için onları ayarlamak için kullanın. </li></ol> 3. TAŞINMAZ nonpolarized hücre sistemi cAMP degrade algılama dahil görüntüleme sinyalleme olaylarını kolaylaştırır <ol><li> Kafein tedavi 500g az 3 dakika sonra, bir kısım hücreler ve santrifüj çıkarın. </li><li> Tamponu çıkarın ve 2.5 mM kafein içeren taze DB tamponu ile 5x10 5 hücre / ml hücreleri sulandırmak. </li><li> Iyi dört odanın her bir kuyu için bir tek iyi oda veya 0.4 ml 1 ml hücre süspansiyonu uygulayın. </li><li> 10 dakika için, dikkatlice unattached hücreleri çıkarmak ve aynı hacmi ile yerine tampon pipetle hücreleri uyması izin verin. </li><li> Istenilen hücrelerin mikroskop altında bulun ve görüntüleme başlayın. </li><li> Istikrarlı bir degrade açığa hücreleri bileşenleri sinyal dinamiklerini izlemek için tasarlanmış bir deney için, deneyler öncesinde 10 dakika süreyle 5.0 mcM (son konsantrasyon) Latrunculin B hücreleri tedavi. </li></ol> 4. Eşzamanlı izleme heterotrimeric G-protein aktivasyonu ve PIP 3 üretim <ol><li> cAMP hücreleri ortak ifade GαCFP ve YFPGβ (G hücreleri) ve hücreleri PIP 3 gösterge PH-GFP (PH hücreleri) 7 ifade geliştirmek darbeli </li><li> 1:1 oranında ve plaka bunları tek-iyi ya da 4-kuyu odaları ile bu iki tip hücre karıştırın. </li><li> Oluşturun ve 10 nm genişliği ile 464 ila 624 nm spektral aralığında Lambda Yığın Acquisition modunu kullanarak emisyon parmak izi referans eğrisi CFP, YFP ve GFP kaydetmek. </li><li> G hücreleri ve PIP 3 üretim, 464, 10 nm artışlarla ile 544 nm spektral aralığında aynı Lambda Yığın Toplama modu ile PH hücrelerde aynı anda görüntü G-protein aktivasyonu. </li><li> G. bireysel kanal içine CFP ve YFP yoğunluğu ayrı matematiksel Lambda Yığın her fluorofor katkısını hesaplamak için kaydedilmiş CFPand YFP ve emisyon parmak izlerini kullanarak Zeiss 510META yazılımı Lineer Karışmama işlevi uygular </li><li> Aynı strateji ile, matematiksel olarak PH hücreler, GFP yoğunluğunu hesaplamak için kaydedilmiş GFP ve arka plan emisyon parmak izlerini kullanarak doğrusal Karışmama fonksiyonu geçerlidir. </li></ol> 5. Temsilcisi sonuçları: <ol><li> D. mükemmel bir model sistem sosyal bir amip, D. GPCR aracılı kemotaksis diskodeyum. diskodeyum yaşam döngüsü sırasında çarpıcı bir kemotaksis sergiler. Genetik ve biyokimyasal avantajları nedeniyle, D. d güçlü bir sistem kemotaksis çalışma. </li><li> Görünür ve manipüle chemoattract alanları altında hücreler, Kemotaksis Burada, biz ilk olarak 10 mg / ml ile karıştırılarak 2 mcM cAMP seyreltme dizi cAMP konsantrasyonu ve floresan boya Alexa 594 yoğunluğu arasında doğrusal bir ilişki elde etmek için basit bir yöntem gösterir Alexa 594 (Şekil 2A). Sonra, Alexa 594 (Şekil Şekil 2B) yoğunluğunu bir degrade cAMP konsantrasyonunun kantitatif bir ölçüm kurmak, dahası, degrade görselleştirmek için kolay bir yol sağlar. </li><li> Hücre hareketi, hücre kutuplaşma ve yön algılama Kuplajsız aktin polimerizasyonu inhibitörü önceden mevcut morfolojik polarite ortadan kaldırır ve hücrelerin yönlü algılama yeteneği (Şekil 3A) korurken aynı zamanda hücre hareketi önler. Örneğin düzgün uygulanan uyarılar veya bir degrade görünür ve manipüle cAMP stimülasyonu İstihdam, giriş garanti eder. Bu yöntem, kantitatif analiz degrade algılama makine anahtar sinyal bileşenlerinin cAMP bağlı yeniden dağıtımı sağlar. Bu sinyal bileşenlerinin Ölçülen Spatiotemporal dinamikleri gradyanlar (Şekil 3B) polarize biyokimyasal tepkileri üretirken sinyalizasyon ağı tek tip uyarılara uyum nasıl elde ettiğini anlamak için izin verir. </li><li> Istikrarlı bir degrade maruz üzerine sinyalizasyon ağı chemosensing kinetiği Sistemik ölçüm yön-algılama hücreleri ilk degrade karşılaştığınızda, her bir bileşenin hücre içi kutuplaşma kurulmasına nasıl katkıda bileşenleri anlamak için sinyalizasyon dinamikleri / kinetik ölçmek için çok önemlidir. Yüksek bir tempo-uzaysal çözünürlüğü ile canlı hücre görüntüleme uygulaması, ilk olarak sabit bir cAMP gradiyenti (Şekil 4A-C) maruz hücre bifazik PIP3 üretim göstermektedir. Canlı hücre görüntüleme uygulamak, dinamikleri sistematik olarak ölçülen yön-algılama PIP3 üretim (Şekil 4D, E) cAMP stimülasyon belirli sinyalizasyon ağı. </li><li> Eşzamanlı izleme şekilde uygulanmaktadır cAMP stimülasyon üzerine heterotrimeric G-protein aktivasyonu ve PIP 3 üretim. Förster rezonans enerji transferi (kısaltılmış FRET) cAMP stimülasyon üzerine heterotrimeric G protein aktivasyonu (disosiasyon) izlemek için etkili bir yaklaşım sağlar. Burada, değişim ve PIP membran translokasyonu sırasıyla 3 probu, PH-G ve PH hücreler, GFP, FRET izleme (Şekil 5 heterotrimeric G protein aktivasyonu ve PIP 3 üretim eşzamanlı ölçümü için bir kolay kolay kabul sistemi tarif .) Bir şekilde uygulanmaktadır cAMP stimülasyon, geçici bir PIP 3 üretim tetikleyen ise, kalıcı bir G protein aktivasyonunu tetikler. </li></ol><img src="/files/ftp_upload/3128/3128fig1.jpg" alt="Şekil 1"/> Şekil 1: D. mükemmel bir model sistem GPCR aracılı kemotaksis diskodeyum. A. Programı yönlü algılama kısa bir sinyal yolağı gösterir. B. cAMP degrade D. hızlı kemotaksis indükler diskodeyum hücreleri. Hücreler PIP 3 probu, PH-GFP (Yeşil) ifade eder. Gradient Alexa 594 (Kırmızı) tarafından görüntülenmiştir. Ölçek çubuğu = 50μm. <img src="/files/ftp_upload/3128/3128fig2.jpg" alt="Şekil 2"/> Şekil 2: görünür ve manipüle chemoattract alanlar altında hücreleri Kemotaksis. Doğrusal bir ilişki bir degrade cAMP konsantrasyonu B. Kantitatif ölçüm A. Grafik 2 mcM cAMP 10 mcg / mL Alexa 594 ile karışık bir seyreltme dizi cAMP konsantrasyonu ve floresan boya Alexa 594 yoğunluğu arasında doğrusal bir ilişki gösterir. A. floresan boya Alexa 594 cAMP konsantrasyonu ve yoğunluğu <img src="/files/ftp_upload/3128/3128fig3.jpg" alt="Şekil 3"/> Şekil 3: Hücre hücre polarizasyon ve yön algılama ile motilite Kuplajsız. A. Görüntü aktin polimerizasyonu inhibitörü Latrunculin B tedavisi ile hareketsiz hücreler yönlü algılama yeteneği korumak olduğunu göstermektedir. Hücreler PIP 3 probu, PH-GFP (Yeşil) ifade eder. Gradient Alexa 594 (Kırmızı) tarafından görüntülenmiştir. B. manipüle cAMP uyarılması ve hareketsiz hücre sistemi yönlü algılama anahtar sorular adresi sağlar. Ölçek çubuğu = 10μm. <img src="/files/ftp_upload/3128/3128fig4.jpg" alt="Şekil 4"/> Şekil 4: istikrarlı bir degrade maruz kalma üzerine sinyalizasyon ağı chemosensing kinetiği Sistemik ölçümleri. A. Montaj C. C sunulan PIP 3 üretim kinetiği ölçümü için sabit bir cAMP gradiyenti (Kırmızı) maruz kaldığı hücrenin bir bifazik PIP 3 üretim (Yeşil) B. Görüntü çıkarlarının bölgeler (ROI) gösterir . Kinetik of istikrarlı bir degrade maruz kalan hücrelerde PIP 3 üretim D. Programı cAMP stimülasyon PIP 3 üretim yönlü algılama sinyalizasyon ağı gösterir. Onların kinetiği üzerine istikrarlı bir degrade maruz E aynı renk düz çizgiler sunulmuştur. <img src="/files/ftp_upload/3128/3128fig5.jpg" alt="Şekil 5"/> Şekil 5: GPCR sinyalizasyon ağları aynı anda izleme çoklu olaylar. A. Programı heterotrimeric G protein aktivasyonu ve PIP 3 üretim değişikliği ve sırasıyla PIP3 probu, G ve PH hücrelerinin PH-GFP, membran translokasyonu FRET izleme eş zamanlı ölçümü gösterir. B. Montaj G ve PH hücreleri gökkuşağı görüntüleri gösterir geçici bir PIP3 üretim tetikleyen ise, düzgün uygulanan cAMP stimülasyon, periferik hücre kalıcı bir G protein aktivasyonunu tetikler. Zaman noktalarında 4.9s, 10.2s ve 20.4s önce (0s) ve stimülasyon sonrası C. G protein aktivasyonu ve düzgün uygulanan cAMP stimülasyon üzerine PIP3 üretim Kinetik.

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Çıkar çatışması ilan etti.

Hücrelerin kemotaktik yetkili sahne ulaşma süreçleri Vahşi tip D. diskodeyum hücreleri, onu alır, yaklaşık 5 ~ 6 saat oda sıcaklığında geliştirme darbeli, hücreler de kutuplaşmış bir hücresel morfoloji ve hızlı hücre göçü (Şekil 1) görüntüleme sırasında iyi bir kemotaktik yetkili sahne onları ikna etmek için. Nabız gibi atan, sıcaklık ve farklı genetik geçmişleri cAMP konsantrasyonu gibi çeşitli faktörler, kemotaktik yetkili evre ulaşma sürec...

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar </th></tr><tr><td> D3-T Büyüme Medya </td><td> KD Tıp </td><td></td><td></td></tr><tr><td> Kafein </td><td> Sigma </td><td></td><td></td></tr><tr><td> Latrunculin B </td><td> Moleküler Probları </td><td></td><td></td></tr><tr><td> Alexa 594 </td><td> Moleküler Probları </td><td></td><td></td></tr><tr><td> cAMP </td><td> Sigma </td><td></td><td></td></tr><tr><td> ChronTrol XT programlanabilir zamanlayıcı </td><td> ChronTrol Corp </td><td></td><td></td></tr><tr><td> Miniplus 3 peristaltik pompa </td><td> Gilbson </td><td></td><td></td></tr><tr><td> Platform döner çalkalayıcı </td><td></td><td></td><td></td></tr><tr><td> FemtoJet microcapillary basınç kaynağı </td><td> Eppendorf </td><td></td><td></td></tr><tr><td> Tek ve dört iyi Lab-Tek II coverglass odaları </td><td> Nalge Nunc International </td><td></td><td></td></tr><tr><td> EKK 510 META ya da eşdeğer floresan mikroskop </td><td> Zeiss </td><td></td><td> 40X 1.3 NA veya 60X 1.4 NA yağ DIC Plan-Neofluar objektif lens </td></tr><tr><td> Olympus X81 veya eşdeğeri </td><td> Olympus </td><td></td><td> 100X 1.47 NA TIRF objektif lens gerektirir </td></tr></tbody></table>

imaging g-protein coupled receptor (gpcr)-mediated signaling events that control chemotaxis of dictyostelium discoideum

Birçok ökaryotik hücrelerin kendi ortamlarında kimyasal sinyallerin degradeler algılar ve buna göre 1 geçirebilirsiniz. Bu güdümlü hücre göçü, bağışıklık hücreleri ve nöronal hücrelerin 2, 3 desenlendirme ticareti gibi görevleri yürütmek için çeşitli hücreler için gerekli kemotaksis olarak adlandırılır . G-protein reseptörler büyük bir aile (GPCRs), in vivo 4, kemokinler olarak bilinen değişken küçük peptidler, hücre göçü doğrudan algılar . Kemotaksis araştırma nihai hedefi, bir GPCR makine duyuları kemokin Degradeleri ve kontrolleri kemotaksis için önde gelen olaylar sinyalizasyon nasıl anlamak için. Bu amaçla, biz kemoatraktan bir degrade chemoattractants, hücre hareketi uzaysal konsantrasyonlarını izlemek için gerçek zamanlı olarak görüntüleme teknikleri kullanmak, GPCR heterotrimeric G-protein aktivasyonu aracılı ve 5-8 ökaryotik hücrelerin kemotaksis alan hücre içi sinyal olaylar . Basit ökaryot organizma, Diktiyostelyum diskodeyum, lökositlerin benzer chemotaxic davranışlar gösterir, ve D. diskodeyum ökaryotik kemotaksis eğitim için önemli bir model sistem. Serbest yaşayan amipler, D. olarak diskodeyum hücrelerin zengin orta bölün. Açlıktan sonra, hücreler multicullular yapıları oluşturmak cAMP aracılı kemotaksis aracılığıyla toplu bir gelişim programına girmek. D. cAMP kemotaksis dahil birçok bileşenleri tespit edilmiştir diskodeyum. Bir GPCR cAMP bağlama (car1) Gγ ve Gβγ altbirimden 7, 9, 10 Fototerapisi heterotrimeric G-proteinleri neden olur. Gβγ altbirimden sırayla aktive PI3K 3 PIP PIP 2 hücre zarının 11-13 dönüştürme Ras etkinleştirin. PIP 3, böylece bu proteinlerin membran 14, 15 işe pleckstrin Homoloji (PH) etki ile proteinler için bağlanma yerleri olarak hizmet vermektedir . Car1 reseptörlerinin aktivasyonu 3 PIP PIP 2 16, 17 dephosphorylates PTEN, membran dernekler de kontrol eder. Moleküler mekanizmaları, evrimsel olarak nötrofil 18 gibi insan hücrelerinin kemokin GPCR aracılı kemotaksis korunmuş . D. kemotaksis çalışmak için aşağıdaki yöntemlerden mevcut diskodeyum hücreleri. 1. Kemotaktik bileşeni hücrelerinin hazırlanması. 2. CAMP degrade hücre Görüntüleme kemotaksis. 3. , G-protein heterotrimeric tek canlı hücreler bir GPCR kaynaklı aktivasyon izleme. 4. Görüntüleme kemoatraktan tetiklenen dinamik PIP 3, gerçek zamanlı olarak tek canlı hücrelerinde yanıtlar . Bizim gelişmiş görüntüleme yöntemleri, insan lökositlerin kemotaksis çalışma yapılabilir.

Watch this Scientific Journal Video about Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum at JoVE.com

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Burada, kemotaksis soruşturma için ayrıntılı canlı hücre görüntüleme yöntemleri açıklanmaktadır. Biz göç eden hücreleri olayları sinyal Spatiotemporal dinamiklerini izlemek için floresan mikroskobik yöntemler sunuyoruz. Ölçüm sinyal olayların bir GPCR sinyal ağı daha chemoattractants ve kontroller ökaryotik hücrelerin yönlü göç algılama degrade nasıl elde ettiğini anlamak için bize izin verir.

Görüntüleme G-protein Coupled Reseptör (GPCR) aracılı Sinyal Olaylar Kontrolü Kemotaksis Diktiyostelyum diskodeyum</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

imaging-g-protein-coupled-receptor-gpcr-mediated-signaling-events

Research

JoVE Journal

Biology

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

17.9K Görüntüleme.  National Institute of Allergy and Infectious Diseases, National Institutes of Health. Burada, kemotaksis soruşturma için ayrıntılı canlı hücre görüntüleme yöntemleri açıklanmaktadır. Biz göç eden hücreleri olayları sinyal Spatiotemporal dinamiklerini izlemek için floresan mikroskobik yöntemler sunuyoruz. Ölçüm sinyal olayların bir GPCR sinyal ağı daha chemoattractants ve kontroller ökaryotik hücrelerin yönlü göç algılama degrade nasıl elde ettiğini anlamak için bize izin verir.

Video: Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

Işıksal Çözülmüş İki foton Mikroskop kullanarak G Protein Coupled Reseptör Etkileşmeleri in vivo Niceleme

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Biyoloji

Quantifying Agonist Activity at G Protein-coupled Receptors

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.
Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (Kb) is much greater than that for the inactive state (Ka). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (Kobs), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (&#949;) (Figure 2).
Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the Kobs and relative efficacy of an agonist 1,2. In this report, we show how to modify this analysis to estimate the agonist Kb value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate Kb in absolute units of M-1.
Our method of analyzing agonist concentration-response curves 3,4 consists of global nonlinear regression using the operational model 5. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of Kobs and a parameter proportional to efficacy (&tau;). The estimate of &tau;Kobs of one agonist, divided by that of another, is a relative measure of Kb (RAi) 6. For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (&tau;sys). In this case, the Kb value of an agonist is equivalent to &tau;Kobs/&tau;sys 3.
Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.

A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.

G Protein-Coupled reseptörleri Agonist Aktivitesi miktarının

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

Unimoleküler Biyosensörler kullanma Canlı Hücreler Olaylar Sinyal gerçek zamanlı izlenmesi için Mikroskopi FRET

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

Erken Otofaji Etkinlikler Hücre Görüntüleme Canlı: Omegasomes ve Ötesi

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

Genetik olarak kodlanmış Molecular Probes G protein-bağlanmış alıcılar Çalışması için

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

Bir floresan bazlı Kalsiyum Mobilizasyon Deneyi G proteini ile birleştirilmiş alıcıların karakterizasyonu

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

TIRF Mikroskopi ile Tek olay ebatları da G protein-bağlanmış alıcılar üzerinden Klatrin dolayımlı endositoz görselleştirme

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular development program, during which single cells chemotax towards aggregation centers1-4. This process is dependent on the release of cyclic adenosine monophosphate (cAMP)5. cAMP is produced in waves through the concerted action of adenylate cyclase and phosphodiesterases, and binds to G protein-coupled cAMP receptors6,7. A widely used assay to analyze the mechanisms involved in the developmental cycle of the lower eukaryote Dictyostelium discoideum is based on the observation of cell aggregation in submerged conditions8,9. This protocol describes the analysis of the role of coronin A in the developmental cycle by starvation in tissue-culture plates submerged in balanced salt solution (BSS)10. Coronin A is a member of the widely conserved protein family of coronins that have been implicated in a wide variety of activities11,12. Dictyostelium cells lacking coronin A are unable to form multicellular aggregates, and this defect can be rescued by supplying pulses of cAMP, suggesting that coronin A acts upstream of the cAMP cascade10. The techniques described in these studies provide robust tools to investigate functions of proteins during the initial stages of the developmental cycle of Dictyostelium discoideum upstream of the cAMP cascade. Therefore, utilizing this aggregation assay may allow the further study of coronin A function and advance our understanding of coronin biology.

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

The social amoeba Dictyostelium discoideum undergoes a developmental transition into a multicellular organism when starved. The evolutionary conserved protein coronin A plays a crucial role in the initiation of development. Using aggregation assays as our main method, we aim to elucidate the role of coronin A in early development.

Erken Açlık Tepki Koronin A Fonksiyonu incelenmesi<em&gt; Dictyostelium discoideum</em&gt; Toplama Tahliller

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Görüntüleme G Nötrofil gibi HL60 Hücreleri Kemotaksis ve Sinyal Olaylar Reseptör aracılı Protein-çiftli

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

Protein Kalite Kontrol Sistemi incelenmesi<em&gt; D. diskodeyum</em&gt; Sıcaklık kontrollü Canlı Hücre Görüntüleme kullanarak

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...