Name: spatial and temporal control of t cell activation using a photoactivatable agonist
Uploaded: 2018-04-25T13:00:00.000+00:00
Duration: 7 min 48 s
Description: Watch this Scientific Journal Video about Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist at JoVE.com

Biz üye Huse laboratuarının tavsiye ve yardım için teşekkür ederim. ABD Ulusal Sağlık (M.H. için R01-AI087644) ve P30-CA008748 Memorial Sloan-Kettering Kanser Merkezi için Enstitüleri tarafından desteklenen.

1. uyarıcı cam yüzeylerin hazırlanması
<ol><li>Kat 8-şey odalı coverglass biotinylated Poli-L-lizin (Bio-PLL) ile 1:500 distile, deiyonize su (GKD2O) seyreltilmiş. (RT) oda sıcaklığında 30 dk için kuluçkaya.</li>
	<li>H2ile O. yıkama</li>
	<li>Kuru RT. 2 h için</li>
	<li>Blok biyo-PLL yüzeyler RT., 30 dk için engelleme arabellek (HEPES arabelleğe alınmış serum [10 mM HEPES pH 7.4, 150 mM NaCl], %2 BSA) ile kaplı<ol>
<li>Poli-L-lizin hydrobromide 10 mM NaPO4, pH 8,5 1 ml 5 mg geçiyoruz.</li>
		<li>125 μmol (0.55 mg) NHS-biotin (DMSO 100 mg/mL stokta) ekleyin ve reaksiyon pH kontrol edin. PH 8,5 altında ise 4 N NaOH pH 8,5 ve 9.5 arasında yükseltmek için 2 μL ekleyin.</li>
		<li>30 dk için girdap.</li>
		<li>20 mM Tris pH 8.0 çözünmüş 100 mM glisin 50 μL ile reaksiyon gidermek.</li>
		<li>Tam güç 10 min için spin ve süpernatant aktarmak için yeni bir tüp. Not: Bio-PLL kalitesi genellikle seri iki kat dilutions bir 96-Peki ELISA plaka üzerine malzemenin kaplama ve streptavidin kuluçka alkalen fosfataz ile birleştiğinde sonra biotin içerik miktarının önceki hazırlıklar için karşılaştırılır.</li>
	</ol>
</li>
	<li>Engelleme arabellek kaldırın. Kuyu kuru izin vermez. Streptavidin (100 µg/mL engelleme arabelleği) ekleyin. 4 ° C'de 1 h için kuluçkaya</li>
	<li>HBS içinde yıkayın. Doldurun ve HBS kuyulardan kaldırma odası slayt wells 4 - 5 kez, tersine çevirin. Kuyu kuru izin vermez.</li>
	<li>Biotinylated pMHC ligandlar ve adezyon molekülleri için yüzey ekleyin. Not: MCC ve OVA Orto-nitrobenzyl ekleyerek photocaged koruma grupları (Örneğin, nitrophenylethyl (NPE) veya nitroveratryloxycarbonyl (NVOC)) anahtar lysines ε-amino grubuna bağlı olabilir (mm, K7 OVA içinde K12). Photocaged mm ve OVA ticari satıcılardan temin edilebilir. Sulu tampon rekonstitüsyon sonra bacterially ifade-Ek ve kurulan yaklaşımlar11,12kullanarak H2-Kb refolded.</li>
	<li>Photoactivatable mm ya da OVA peptid doğrulama<ol>
<li>NVOC korumalı peptidler el bir UV lamba ile UV irradiating tarafından decage ( Tablo malzemelerigörmek) RT., 20 dk</li>
		<li>1 h için nabız 1.0 x 105 ZPT nonstimulatory peptid (negatif kontrol, örneğin Hb), 1 µM ile unirradiated photoactivatable peptid, radyoaktif photoactivatable peptid ve agonist peptid (pozitif kontrolü, örneğin, mm) 37 ° C'de bir gecede</li>
		<li>5 C. ifade T hücreleri uyarmak için C7/2B4/ve TCR, CH12 veya CH27 B hücre kullanın. OT1 T hücreleri uyarmak için RMA-s veya EL4 Timoma hücreleri kullanmak.</li>
		<li>T hücrelerinde eşit sayıda pulsed ZPT karıştırın 96-şey yuvarlak alt plaka 200 son ses seviyesinde µL. 12-24 h 37 ° C'de kuluçkaya.</li>
		<li>Supernatants her kuyudan kurtarmak ve IL-2 tarafından ELISA streptavidin horseradish peroksidaz Renkölçer algılama ile analiz. Not: Tüm peptidler tarafından electrospray kütle spektrometresi MHC kompleksi refolding önce caging durumunu teyit şiddetle tavsiye edilir.</li>
	</ol>
</li>
	<li>Katlama ve arıtma MHC ışığa maruz kalma en aza indirmek için gerçekleştirin. Örneğin, alüminyum folyo reaksiyonlar katlama sarın ve UV lambalı jel filtrasyon Kromatografi taşımak. Not: Bu photoactivatable mm-ben-Ek bulundu oldu ve OVA - H2-Kb neden UV bağımsız T hücre aktivasyonu, büyük olasılıkla eksik fonksiyonel caging nedeniyle yüksek yoğunluklu. Bu nedenle, photoactivatable pMHC nonstimulatory pMHC (hemoglobin64-76 peptid (Hb) içerene.g. -Ek ) bir 10-30 x fazlalığı immobilizasyon adımı sırasında seyreltilmiş. Bu sinyal gürültü oranı sonraki görüntüleme deneme artırır.</li>
	<li>Photoactivate CD4 için+ T hücreleri, topluca biotinylated Hb-ben-Ek (3 µg/mL), biotinylated photoactivatable mm-ben-Ek (0.1 µg/mL) ve biotinylated antikor sınıf karşı karışımı kullanın ben MHC H2-Kk (0,5 µg / mL). anti-H2-Kk antikor 5 C. teşvik eder. H2-Kkcam yüzey harekete geçirmek gören olmadan yaymak için hızlı C7/2B4/ve T hücreleri.</li>
	<li>CD8 için+ T hücreleri, kullanın biotinylated KAVYDFATL peptid taşıyan H2-Db karışımı (1 µg/mL), biotinylated photoactivatable OVA-H2-Kb (0.1 µg/mL) ve ekstrasellüler etki alanı adezyon molekülün ICAM-1 (2 µg/mL böcek hücre kültür13tarafından üretilen). ICAM-1 T hücre yüzeyinde integrin LFA1 meşgul olarak yakın kişi oluşumu teşvik eder.</li>
	<li>Tüm protein karışımları kuluçka 1s RT at veya en az 2 h 4 ° C'de ardından engelleme arabellekte uygulamak Not: Önceden belirlenen6~ 8000 µm2 başına olmak bu tür yüzeylerde Moleküler yoğunluğu oldu. Th yüzeyinde, yoğunluğu biotinylated proteinin ~ 267 molekülleri µm2decaging önce başına photoactivatable pMHC ~1/30 gösteren göz önüne alındığında olacaktır.</li>
	<li>Adım olduğu gibi 1,6 yıkama ve HBS içinde kullanıma hazır kadar bırakın.</li>
	<li>200.000 CD4 eklemek+ veya CD8+ T hücreleri uygun TCR her kuyuya ifade ve hücreleri 15dk için 37 ° C'de bağlı kalmak izin. Hücreleri bağlı ve yüzeyde yaymak, onlar photoactivation ve görüntüleme için hazırız. Not: Floresan problar Imaging ile efektör T hücrelerinin Retroviral iletim ayrıntı elswhere6,7' açıklanmıştır. Kalsiyum sinyal aynı zamanda kalsiyum hassas boya Fluo-46ile yüklenen untransduced T hücreleri kullanarak ele. Ayrıca photoactivatable pMHC decaging neden olmaktadır UV aralığındaki uyarma gerektirdiğinden ratiometric boya Fura-2 tavsiye edilmez.</li>
</ol>2. resim alma
<ol><li>Resim alma için bir UV uyumlu 150 X objektif lens ile outfitted ters bir TIRF mikroskobu kullanın. UV ışınlatayım 100 bir W civa lamba (HBO) bağlı bir dijital diyafram sistemi kullanarak kullanıcı tanımlı bölgeler. UV ışığı bu lamba 400 nm uzun pası ayna kullanarak örnek üzerine doğrudan.</li>
	<li>Görüntü analiz yazılımı yerelleştirilmiş photoactivation ve hızlandırılmış satın alma için kullanın. Çoğu deneylerde probları 488 kullanarak her iki yeşil ve kırmızı kanalı izlemek nm ve 561 nm uyarma lazerler, anılan sıraya göre. Lazer ışığı da iletir (decaging etkinleştirmek için) UV aralığında bir dual-bant dikroik ayna kullanarak örnek üzerine yönlendirilir. Lütfen Malzeme tablo ve şekil 6 mikroskop yapılandırma hakkında daha fazla bilgi için bkz:.</li>
	<li>T hücreleri içeren odası slayt takma sonra ayarları gerektiği gibi TIRF veya epifluorescence aydınlatma elde etmek için ayarlayın. Live modda floresan probe(s) ilgi ifade eden hücrelerin bir alan seçin. Mikron çaplı bölgeler photoactivation altında yazılım denetimini kullanarak tek tek hücreler için kurmak.</li>
	<li>Hızlandırılmış edinme başlar. Genellikle, 80 timepoints, 5 zaman aralığı ile iktisap her zaman noktası arasındaki s. Bu sıralı 488 için gereğinden fazla zaman bırakır nm ve çift renk deneyler durumunda 563 nm Etkilenmeler.</li>
	<li>10 timepoints sonra dijital diyafram açarak seçili bölgeler için 1-1.5 s çekim photoactivate.</li>
	<li>Zaman atlamalı tamamlandıktan sonra hücre yeni bir alan seçin ve işlemi yineleyin.</li>
</ol>3. veri analizi
Not: İmmobilize ligandlar yerelleştirilmiş photoactivation yanıt ve hücre iskeleti remodeling etkinlikler sinyal, kantitatif analiz için kullanılabilecek iyi tanımlanmış, sabit bir uyarıcı bölge oluşturur. İletişim kuralları genellikle içerir floresan yoğunluğu radyasyonlu bölgedeki miktarının veya Radyoaktif bölge konumsal bir bitiş noktası olarak mesafe ölçümleri için kullanarak analiz (e.g. centrosome için kutuplaşma değerlendirmek için Radyoaktif bölge). Her iki analiz protokolleri aşağıda açıklanmıştır. Çeşitli etkileşimli resim analiz programları yoğunluğu ve sonra ek işleme ve analiz için çıktı olabilir mesafe ölçümleri yapmak için kullanılabilir.
<ol><li>
Floresan yoğunluğu
	<ol>
<li>Hücre (FIb) dışında bir arka plan bölgesinin floresan yoğunluğu (FI) belirlemek. Bu arka plan düzeltme için kullanılacaktır.<ol>
<li>Bir mikron büyüklüğünde kare bölgesi dışında hücre ve bir maske yapmak. Floresan yoğunluğu belirlemek için analiz tıklatın | Maske istatistikleri. Demek floresan yoğunluğu seçin ve değerleri vermek. Not: Yordam ayrıntıları (örneğin 3.1.1.1) Slidebook yazılımlar için de kullanılır (bkz. Tablo malzeme). Diğer yazılım paketlerini kullanarak bu protokolünü (örn., FIJI) biraz farklı olacaktır.</li>
		</ol>
</li>
		<li>Her zaman için photoactivated bölgedeki FI belirlemek.<ol>
<li>Maske yapıldı. photoactivated bölge vurgulamak için seçin. Floresan yoğunluğu belirlemek için analiz tıklatın | Maske istatistikleri. Demek floresan yoğunluğu seçin ve değerleri vermek.</li>
		</ol>
</li>
		<li>Bölgede ölçüm FI değerleri FI kökenli çıkarma. O zaman, düzeltilmiş FI ölçümler ortalama tarafından bölerek normalize, arka plan FI photoactivation önce ilk 9 kare düzeltildi. ΔF/F = ((FI-FIb)/mean(FI1-9)-FIb)). Not: ΔF/F bir fonksiyonu olarak grafiğini çizin.</li>
	</ol>
</li>
	<li>
Mesafe
	<ol>
<li>X ve y koordinatları photoactivated Bölge Merkezi.<ol>
<li>X ve y koordinatları photoactivated bölgesinin merkezinin photoactivated yapıldı bölge vurgulamak için maske seçin. Photoactivation bölgesinin vurgulandığında, analiz seçin | Maske istatistikleri | Alan Merkezi ve değerleri vermek.</li>
		</ol>
</li>
		<li>X ve y koordinatları için her zaman için ilgi soruşturma floresan. Bu genellikle el ile veya otomatikleştirilmiş parçacık izleme elde edilir.<ol>
<li>X ve y belirlemek için faiz, floresan probe koordinatlarını Manuel parçacık izleme seçin ve faiz floresan sondası zamanla'yı tıklatın. Bir kez tüm zaman Puan izleniyor, analiz seçmek | Maske istatistikleri | Alan Merkezi ve değerleri vermek.</li>
		</ol>
</li>
		<li>Floresan protein ilgi ve her zaman noktası denklemi kullanarak photoactivated Bölge Merkezi arasındaki uzaklığı hesaplamak: mesafe √ = ((x2- x1)2+ (y2-y1)2), hangi x2 ve y2 vardır1 faiz ve x ve y1 floresan protein koordinatlarını photoactivated Bölge Merkezi koordinatlar.</li>
		<li>Grafik zamanın bir fonksiyonu olarak mesafe.</li>
	</ol>
</li>
</ol>

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Son yıllarda, ışık spatiotemporally kontrollü harekete geçirmek-in hücresel süreçler için mükemmel bir araç olarak ortaya çıkmıştır. Çeşitli yöntemler geliştirilmiştir, her ilişkili avantajları ve dezavantajları. İmmobilize, hücre dışı ligandlar decaging üzerinde dayanır, burada açıklanan sistem hızlı, hücre altı, polarize sinyal yanıt analiz için idealdir. Bu yaklaşım yukarıda açıklandığı gibi T hücrelerinin oluşumunda IS incelemek için uygulanmıştır. Ayrıca, diğer...

T lenfositler (T hücreleri) hücresel bağışıklık hücre yüzeyine MHC bağlamında görüntülenen antijenik peptidler tanıyarak merkezi bir rol oynamaktadır. TCR tarafından aracılık ettiği, Antijen tanıma saf T hücre farklılaşma sürücüler ve cytolytic ve iletişimsel tepkileri efektör nüfus tarafından teslim teşvik etmektedir. TCR nişan da hücresel mimarisi dramatik değişiklikler neden olmaktadır. Birkaç dakika içinde T hücreleri taktımı immünolojik synapse (IS)1,2olarak bilinen bir polarize arayüz şekillendirme (APC), antijen sunan hücrenin kenarını üzerine. Is yönlü yayın sitokinler veya sitotoksik T lenfositler (CTL), APC yok litik proteinler durumunda etkinleştirerek T hücre efektör yanıt-e doğru potentiates.
TCR nişan pMHC tarafından bağlayıcı sonuçta sağlam sinaptik sitoiskeleti2/ remodeling teşvik T harekete geçirmek için hücreleri (LAT), dahil olmak üzere birden çok akış aşağı adaptör moleküllerin hızlı fosforilasyon neden olmaktadır. Kortikal ipliksi aktin (F-aktin) T hücre APC yüzeye yayılan sürücüler ve IS çevre ve tükenmesi Merkezi'nden F-aktin birikimi ile karakterize bir annüler yapıda giderir. F-aktin halka oluşumu sıkı hale gelmesini Mikrotubul organizasyon Merkezi (MTOC, T hücreleri centrosome olarak da bilinir) için hemen altında arayüzü merkezi bir konuma eşleşen. Her iki olay ilk Antijen tanıma dakika içinde oluşur ve hangi sonraki etkinleştirme olayları ve efektör yanıt ortaya mimari içerik oluşturun.
Is oluşumu çalışmaya, çeşitli labs APC immobilize TCR ligandlar içerir veya kendi ligandlar3,4içerir bir lipid bilayer destekler bir cam yüzey tarafından değiştirilir yaklaşımlar geliştirdi. T hücreleri tarafından toplam iç yansıma Floresans mikroskobu (TIRF) ya da yüksek çözünürlüklü çalışmalar erken T hücre aktivasyonu ve IS oluşumu etkinleştirme confocal mikroskobu görüntüsü bu yüzeylerde IS benzeri kişiler oluşturur.
Bu yaklaşımlar tam olarak birleştirilmiş IS mükemmel görselleştirme için izin vermiş olsa da, sinyal aşağıdaki TCR:pMHC ligasyonu çoğunu TCR harekete geçirmek doğru bir şekilde takip olayların sırasını belirlemek için çabalarını zorlaştıran saniye içinde oluşur . Bu sorunu aşmak için bir photoactivation yaklaşım, photoactivatable pMHC TCR harekete geçirmek5,6,7kronolojik zamanmekansal kontrolünü elde etmek için kullanılan geliştirilmiştir. Bu sistemde, T hücreleri içeren ultraviyole (UV) ışık ile ışınlanmış kadar TCR için uyarıcı sigara photoactivatable pMHC cam yüzeyler eklenir. T hücre yüzeyin mikron büyüklüğünde bölgenin UV ışınlama T hücre tarafından kabul edilebilir bir uyarıcı bölgesi oluşturmak photocage kaldırır. Sonraki sinyal olaylar ve hücre iskeleti remodeling sonra genetik olarak kodlanmış floresan gazetecilere kullanarak izlenir. Antijenik peptidler, sınıf II MHC-Ek ve sınıf bağlamında ben MHC H2-Kb, anılan sıraya göre sunulur, güve sitokrom c88-103 (mm) ve ovalbumin257-264 (OVA), iki photoactivatable sürümü olmuştur (şekil 1) geliştirdi. Bu her iki CD4 analizini sağlar+ T hücreleri belirli mm-ben-Ek (5 C. ifade etmek için C7, 2B4, ya da ve TCRs) ve CD8+ T hücreleri belirli OVA-H2-Kb (OT1 TCR ifade).
Son on yıl içinde TCR photoactivation ve düşsel yaklaşımı erken TCR adımları sinyal kesin Kinetik kurmak ve polarize hücre iskeleti remodeling5, yöneten moleküler yolları tanımlamak için kullanılmıştır 6 , 7 , 8 , 9 , 10. Örneğin, tahlil bu centrosome belirlemede etkili oldu APC doğru hale gelmesini lipid ikinci haberci diacylglycerol IS at merkezli yerelleştirilmiş bir degrade tarafından aracılık. Bu metodoloji yüksek çözünürlüklü görüntü analizi T hücre işlevinin talep uygulamaları için değerli olmaya devam edecektir tahmin edilmektedir.

<table><tbody><tr><td>Nunc Lab-Tek Chambered Coverglass</td><td>Thermofischer Scientific</td><td>155361</td><td></td></tr><tr><td>Poly-L-lysine hydrobromide</td><td>Sigma-Aldrich</td><td>P2636</td><td>Will need to make Biotinylated Poly-L-Lysine</td></tr><tr><td>EZ-Link NHS-Biotin</td><td>Thermofischer Scientific</td><td>20217</td><td>Will need to make Biotinylated Poly-L-Lysine</td></tr><tr><td>Streptavidin</td><td>Thermofischer Scientific</td><td>434301</td><td></td></tr><tr><td>BirA-500: BirA biotin-protein ligase standard reaction kit</td><td>Avidity</td><td>BirA500</td><td>Will be used to biotinylate proteins</td></tr><tr><td>Biotinylated Hb I-E&lt;sup&gt;K&amp;nbsp;&lt;/sup&gt;</td><td></td><td></td><td>For protein folding, see reference 6. For biotinylation, use BirA kit</td></tr><tr><td>Biotinylated NPE-MCC I-E&lt;sup&gt;K&amp;nbsp;&lt;/sup&gt;</td><td>Anaspec</td><td></td><td>Custom NPE-MCC (H-ANERADLIAYL-K(Nvoc)-QATK-OH) can be purchased from Anaspec</td></tr><tr><td>Biotinylated &amp;alpha;H2-K&lt;sup&gt;k&lt;/sup&gt; antibody</td><td>BD Biosciences</td><td>553591</td><td></td></tr><tr><td>Biotinylated NPE-OVA H2-K&lt;sup&gt;b&lt;/sup&gt;</td><td>Anaspec</td><td></td><td>Custom NPE-OVA (H-SIINFE-K(Nvoc)-L-OH) can be purchased from Anaspec</td></tr><tr><td>Biotinylated KAVY H2-D&lt;sup&gt;b&lt;/sup&gt;&amp;nbsp;</td><td>Anaspec</td><td></td><td>Custom synthesized protein (KAVYDFATL) can be purchased from Anaspec</td></tr><tr><td>Biotinylated ICAM1&amp;nbsp;</td><td></td><td></td><td>For protein folding, see reference in protocol. For biotinylation, use BirA kit</td></tr><tr><td>Hand held UV lamp</td><td>UVP</td><td>UVGL-25</td><td>Lamp is held &amp;lt; 1 cm from the sample.&amp;nbsp; 30 s of 365 light is sufficient for detectable decaging, 20 min for quantitative decaging.</td></tr><tr><td>Olympus IX-81 OMAC TIRF system.</td><td>Olympus</td><td></td><td>Additional information about the imaging system can be found in &lt;strong class=&quot;xfig&quot;&gt;Figure 6&lt;/strong&gt;</td></tr><tr><td>Mosaic digital diaphragm</td><td>Andor</td><td></td><td></td></tr><tr><td>Slidebook software</td><td>Intelligent Imaging Innovations</td><td></td><td></td></tr></tbody></table>

spatial and temporal control of t cell activation using a photoactivatable agonist

T lenfositler TCR etkinleştirme aşağıdaki birkaç dakika içinde meydana gelen hızlı, polarize işaret içinde meşgul. Bu oluşumu immünolojik sinaps, T hücre aktivasyonu düzenleyen ve directionally efektör yanıt hedefleyen bir sterotiplenmiş hücre-hücre kavşak neden olmaktadır. Bu süreçlerin etkin bir şekilde çalışmak için hızlı, polarize yanıt-e doğru esir alma için uyarlanmış bir düşsel yaklaşımı gereklidir. Bu iletişim kuralı ultraviyole ışığa maruz kadar yani uyarıcı olmayan bir photoactivatable peptid-Binbaşı MHC kompleksi (pMHC) dayalı böyle bir sistem açıklar. Hedeflenen Bu reaktif videomicroscopy deneyler sırasında decaging TCR etkinleştirme kesin kronolojik zamanmekansal kontrol ve yüksek çözünürlüklü Imaging sonraki hücresel yanıtları toplam iç yansıma (TIRF) tarafından izleme sağlar. Bu yaklaşım da genetik ve farmakolojik pertürbasyon stratejileri ile uyumludur. Bu TCR sinyal immünolojik synapse altında yatan polarize hücre iskeleti yapıların oluşumu için bağlantı iyi tanımlanmış moleküler yolları Meclisi sağlar.

Photoactivation ve düşsel yaklaşımı gözlem ve hızlı, polarize sinyal yanıtlar facile miktar sağlar. Çoğaltılamaz yeteneklerini göstermek için işte TCR kaynaklı DAG birikimi ve centrosome hale gelmesini arasında kronolojik zamanmekansal korelasyon inceleyerek bir deney. 5C. C7 T hücre patlamaların retrovirally ile 2 floresan gazetecilere transduced: GFP (C1-GFP) ve RFP tübülin centrosome izlemek için tandem C1 etki alanlarından protein kinaz C-θ içeren bir DAG Biy...

Watch this Scientific Journal Video about Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist at JoVE.com

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

T hücre aktivasyonu Photoactivatable Agonist kullanarak kayma ve Temporal kontrolünü

Bu iletişim kuralı T lenfosit photoactivatable peptid-MHC, T hücre aktivasyonu hassas kronolojik zamanmekansal kontrol etkinleştirme kullanarak etkinleştirmek için bir görüntüleme tabanlı yöntemi açıklar.

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological ...

spatial-temporal-control-t-cell-activation-using-photoactivatable

Research

JoVE Journal

Immunology and Infection

6.2K Görüntüleme.  Memorial Sloan-Kettering Cancer Center. Bu iletişim kuralı T lenfosit photoactivatable peptid-MHC, T hücre aktivasyonu hassas kronolojik zamanmekansal kontrol etkinleştirme kullanarak etkinleştirmek için bir görüntüleme tabanlı yöntemi açıklar.

Video: Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events1, 2. Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time3. In order to gain deeper understanding of their roles in diverse cellular functions at the molecular level, the next step should be perturbation of protein activities at a precise subcellular location and timing. 
To achieve this goal, we have developed a method for light-induced, spatio-temporally controlled activation of small GTPases by combining two techniques: (1) rapamycin-induced FKBP-FRB heterodimerization and (2) a photo-caging method of rapamycin. With the use of rapamycin-mediated FKBP-FRB heterodimerization, we have developed a method for rapidly inducible activation or inactivation of small GTPases including Rac4, Cdc424, RhoA4 and Ras5, in which rapamycin induces translocation of FKBP-fused GTPases, or their activators, to the plasma membrane where FRB is anchored. For coupling with this heterodimerization system, we have also developed a photo-caging system of rapamycin analogs. A photo-caged compound is a small molecule whose activity is suppressed with a photocleavable protecting group known as a caging group. To suppress heterodimerization activity completely, we designed a caged rapamycin that is tethered to a macromolecule such that the resulting large complex cannot cross the plasma membrane, leading to virtually no background activity as a chemical dimerizer inside cells6. Figure 1 illustrates a scheme of our system. With the combination of these two systems, we locally recruited a Rac activator to the plasma membrane on a timescale of seconds and achieved light-induced Rac activation at the subcellular level6.

A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.

Yaşayan hücreler içinde Subselüler Düzeyinde ve Seconds Zaman ölçeği üzerinde Küçük GTPaz Faaliyet uzay-zamansal Manipülasyon

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

Biyomühendislik

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Bir Optogenetic kontrol etmek ve hücre-hücre etkileşimleri gen ifade desenleri analiz yöntemi

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

Genetik

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

Gerçek zamanlı, TCR Eşzamanlı Görüntüleme ve İlişkili Sinyal Proteinler için TIRF Mikroskopi Tekniği

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

İmmünoloji ve Enfeksiyon

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

T hücre gerçek zamanlı Canlı Görüntüleme Kompleks Oluşum Sinyal

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

Canlı Zebra balığı Embriyolar iki-Foton-Tabanlı fotoaktivasyon

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Biyoloji

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

Iltihaplı Dermisinde içinde görüntüleme CD4 + T hücre Geçiş Göç

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Nöronlar bir Genetiği kodlanmış doğal olmayan Amino Asit kullanarak Nöronal Protein Optik Kontrol

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Nörobilim

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.

This protocol describes the use of single chain MHC class I complexes to investigate molecular interactions in human CD8+ T cell activation: generation of engineered antigen presenting cells expressing single chain constructs, culture of human CD8+ T cell clone and T cell activation experiments.

Tek zincir MHC Co-agonism insan CD8 + T hücre aktivasyonu içinde araştırmak için teknoloji kullanımı

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist ...

Interrogating Individual Autoreactive Germinal Centers by Photoactivation in a Mixed Chimeric Model of Autoimmunity

Autoimmune diseases present a significant health burden. Fundamental questions regarding the development and progression of autoimmune disease remain unanswered. One requirement for advancements in our understanding of the underlying disease mechanisms and cellular dynamics is the precise coupling of the microanatomical location of cell subsets with downstream molecular or functional analyses; a goal that has traditionally been difficult to achieve. The development of stable photoactivatable biological fluorophores and their integration into reporter strains has recently enabled precise microanatomical labeling and tracking of cellular subsets in murine models. Here, we describe how the ability to analyze autoreactive lymphocytes from single germinal centers may help to provide novel insights into autoimmunity, using the combination of a novel chimeric model of autoimmunity with a photoactivatable reporter as an example. We demonstrate a procedure for generating mixed chimeras with spontaneous autoreactive germinal centers populated by lymphocytes carrying a photoactivatable green fluorescent protein reporter. Using in vivo labeling strategies, single germinal centers can be visualized in explanted lymphoid tissues and their cellular constituents photoactivated by two-photon microscopy. Photoactivated lymphocytes from single germinal centers can then be analyzed or sorted flow cytometrically, as single cells or in bulk, and may be subjected to additional downstream molecular and functional analyses. This approach may directly be applied to provide renewed insights in the field of autoimmunity, but the procedure for generating bone marrow chimeras and the photoactivation procedure may additionally find broad application in studies of infectious diseases and tumor metastases.

This protocol describes the generation of mixed murine bone marrow chimeras with spontaneous autoimmune germinal centers, in which autoreactive lymphocytes carry a photoactivatable green fluorescent protein (PA-GFP) reporter. This provides the ability to link cellular location in tissues with downstream molecular and functional analyses.

Bireysel Autoreactive Germinal merkezleri Photoactivation otoimmünite, karışık Chimeric modeli tarafından sorguya

Autoimmune diseases present a significant health burden. Fundamental questions regarding the development and progression of autoimmune disease remain ...

<meta charset="utf8"></head><body>Işık Ayarlı Allosterik Anahtar Kullanılarak Enzim Aktivitesinin Optogenetik Kontrolü

Spatiotemporal Control of Protein Activity through Optogenetic Allosteric Regulation

Optogenetics offers the potential for mimicking complex spatiotemporal control of enzyme activity down to a subcellular resolution. However, most optogenetic approaches often face significant challenges in integrating multiple capabilities in a single tool applicable to a wide range of target proteins. Achieving precise control over ON/OFF kinetics, ensuring minimum leakiness in the dark, and demonstrating efficient performance in mammalian cells with subcellular precision are some of the most common challenges faced in this field. A promising solution lies in the application of rationally designed light-sensitive domains to allosterically control protein activity. Using that strategy, we generated an optogenetic method combining all the desired features. The approach involves the incorporation of the Light-regulated allosteric switch module (LightR) in the target protein to regulate enzyme activity using blue (465 nm) light. The LightR domain is generated by linking two Vivid (VVD) photoreceptor domains, creating a light-sensitive clamp that can be incorporated into a small flexible loop within the catalytic domain of an enzyme. In its dark state, LightR clamp is open, thus distorting the enzyme&#39;s catalytic domain and inactivating it. Upon exposure to blue light, the LightR domain closes and restores the catalytic domain&#39;s structure and enzyme activity. In this manuscript, we discuss design strategies&#160;to generate a light-regulated protein kinase and demonstrate its control by blue light, reversibility, kinetics, and precise regulation at the subcellular level, enabling tight spatiotemporal precision. Utilizing Src tyrosine kinase as a model, we showcase a protocol for effectively regulating LightR-Src kinase activity. We also demonstrate LightR applicability across different enzyme classes, expanding the utility of the tool system in addressing mechanistic questions of signaling pathways in different diseases.

<meta charset="utf8"></head><body>Optogenetik Allosterik Regülasyon Yoluyla Protein Aktivitesinin Uzay-Zamansal Kontrolü

This protocol describes the application of an engineered blue-light-activated allosteric switch (LightR) domain for reversible spatiotemporal control of protein activity. Utilizing Src tyrosine kinase as a model, this study offers an elaborate protocol for developing and characterizing light-regulated Src (LightR-Src). It demonstrates the versatility of this approach across enzyme classes.