Name: subcellular fractionation for erk activation upon mitochondrial-derived peptide treatment
Uploaded: 2017-09-25T15:00:00.000+00:00
Duration: 7 min 55 s
Description: Watch this Scientific Journal Video about Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment at JoVE.com

Bu eser bir Ellison/AFAR doktora sonrası bursu SJK için eskime araştırma programı tarafından desteklenen ve Glenn Vakfı Ödülü ve NIH verir PC için (1P01AG034906, 1R01GM 090311, 1R01ES 020812). Tüm yazarlar filmde görünür.

1. peptid tedavi hücrelere <ol><li>iki milyon SH-SY5Y plaka veya HEK293 hücrelere 10 cm (2 x 10 6) yemek ve onlar için 2 gün</li> <li>(isteğe bağlı) ertesi gün, Dulbecco serum içermeyen hücrelerle yıkama &#39; s modifiye Orta (DMEM) medya bir kez kartal ve ücretsiz DMEM gecede serum boş ortam içinde yapılması gereken tedavi ihtiyacı varsa serum ile kuluçkaya.</li>
	<li>3 günde S14G-humanin peptidler µm filtre, distile su ve 1 mM hisse senedi çözüm olarak sulandırmak 0,2 içinde erimesi. 
	Not: Peptidler peptid (örneğin, genel ücret) sıra özellikleri tarafından belirlenen ve peptidler ticari olarak kullanılabilir durumdaysa üreticisi tarafından sağlanan uygun çözücü içinde çözünmüş. Örneğin, S14G-humanin, SHLP2 ve SHLP6 ve MOTS-c suda çözülür. Aliquot peptidler döngüleri erime ve donma önlemek için bir uygun birimi (örneğin, 50 μL) içinde. Bir kez çözdürülen, bunları tekrar kullanmayın.</li>
	<li>Aliquot Önceden ısıtılmış serum içeren veya bir 50 ml konik tüp ve bir çalışma konsantrasyonu (örneğin, 1 µM, 10 µM) yapmak için oda sıcaklığında 1 mM hisse senedi çözüm eklemek serum-Ücretsiz medya.</li>
	<li>Yerine medya ile 6 mL peptid eriyik--dan adım 1.4. ve hücreleri uygun süreyi için kuluçkaya. 
	Not: kuluçka süresi ERK etkinleştiren, koşula bağlı olarak seçin.</li>
</ol> 2. Sitozol ve çekirdeği için hücre altı ayırma <ol><li>buz gibi PBS 10 mL iki kez hücrelerle yıkama, hücreleri 5 mL buz gibi PBS de plaka kapalı kazımak ve hücre süspansiyon 15 mL konik tüp içine transfer.</li>
	<li>Hücreleri, 500 x g 5 min için saat 4'te santrifüj kapasitesi ° C.</li>
	<li>PBS Aspire edin ve Pelet buz gibi ayırma arabellek 200 µL içinde yeniden askıya alma (10 mM HEPES pH 7,6, 3 mM MgCl 2, 10 mM KCl, %5 (v/v) gliserol, % 1 iyonik olmayan yüzey aktif (C 13 H 22 O (C 2 H 4 O) = n), proteaz/fosfataz inhibitörleri). 
	Not: protein fosforilasyon durumları tutmak için fosfataz inhibitörü olarak iyi proteaz inhibitörü taze eklenmelidir ve örnekleri buza hiç tutulmalıdır zaman. Donma ve erime yinelenen örnekleri döngüleri kaçınılmalıdır. Onları-80 buz gibi önce az miktarda (örneğin 50 μg) içine aliquot örnekleri ° C.</li>
	<li>15 dakika buzda yeniden askıya alınmış Pelet kuluçkaya ve 5 min için 250 x g, saat 4'te santrifüj kapasitesi ° C.</li>
	<li>Süpernatant sitoplazmik kesir olarak toplamak ve Pelet nükleer kesir için devam.</li>
	<li>Hücresel enkaz ve 18.000 x g 4 ° C'de 10 dakika için de diğer kirleri çıkarmak ve sonra süpernatant yeni bir microcentrifuge tüp içine aktarmak için süpernatant santrifüj kapasitesi. Sitozol kesir bu.</li>
	<li>Pelet 200 μL buz gibi yıkama arabellekte resuspend (10 mM HEPES pH 7,6, 1.5 mM MgCl 2, 420 mM NaCl, %25 (v/v) gliserol, 0.2 mM EDTA, proteaz/fosfataz inhibitörleri =) ve 4 ° C'de 5 min için 250 x g, santrifüj</li>
	<li>Süpernatant kaldırmak ve Pelet 100 μL buz gibi nükleer ayıklama arabellekte resuspend (20 mM HEPES pH 7,6, 1.5 mM MgCl 2, 420 mM NaCl, %25 (v/v) gliserol, 0.2 mM EDTA, proteaz/fosfataz inhibitörleri =) ve 10 kez solüsyon içeren temizleyicide (5 üzerinde 10 s s Çekil, % 30 amp.) buz üzerinde</li>
	<li>Santrifüj 18.000 x g 4 ° C'de 10 dk de</li>
	<li>Yeni bir microcentrifuge tüp süpernatant aktarın. Nükleer kesir bu.</li>
	<li>Miktarını BCA tahlil kullanarak protein miktarı</li></ol>
 3. ham mitokondrial kesir <ol><li>buz gibi PBS 10 mL ile her 10 cm çanak hücrelerde yıkama, 5 mL buz gibi PBS ekleyin ve bir hücre kazıyıcı kullanarak hücreleri ayırmak. 
	Not: Western Blot mitokondri, iyi bir verim almak için üç 10 cm yemekleri hücre kullanın.</li>
	<li>Hücre süspansiyon 15 mL konik tüp aktarmak ve her üç 10 cm yemeklerini bir 15 ml konik tüp içine birleştirmek.</li>
	<li>Hücreleri, 10 min 600 x g 4'te santrifüj kapasitesi ° C.</li>
	<li>PBS Aspire edin ve Pelet 1 mL buz gibi mitokondri yalıtım arabellek yeniden askıya alma (10 mM Tris-BEZLERİ, 1 mM EGTA/Tris, 200 mM Sükroz, ayarlamak için pH 7.4 =).</li>
	<li>Buz üzerinde politetrafloroetilen kaplamalı havaneli ile 25 vuruş 2 mL homogenizer hücrelerle homojenize 
	Not: Bu mitokondri bütünlüğünü korumak ve mitokondrial kesir verimi en üst düzeye çıkarmak için kritik bir adımdır. Vuruş sayısı her hücre türü için optimize edilmelidir. Yordamına başlamadan önce homogenizer precool.</li>
	<li>Homogenate bir microcentrifuge tüp aktarmak ve vasıl 600 x g 4 ° C'de çekirdeği ve kesilen hücreleri kaldırmak için 10 dk santrifüj kapasitesi.</li>
	<li>Süpernatant toplamak, yeni bir microcentrifuge tüp aktarmak ve 7000 x g 10 min için de 4'te santrifüj kapasitesi ° C. 
	Not: Pelet gevşemiş, süpernatant özenle toplamak ve Pelet bozmamaya çalışın.</li>
	<li>Süpernatant kaldırmak, pelet 200 μL buz gibi mitokondri yalıtım arabelleği ile yeniden askıya alma ve çözüm için yeni bir microcentrifuge tüp aktarın. 7000 x g 10 dk de tüp 4'te santrifüj kapasitesi ° C.</li>
	<li>Pelet yıkamak için yineleme adım 3.8. Bu adımda yeni bir microcentrifuge tüp süpernatant aktarmak gerekli değildir.</li>
	<li>Süpernatant yıkanmış Pelet kaldır ve mitokondri ile 50 μL RIPA arabelleği içeren Pelet yeniden askıya alma.</li>
	<li>Incubate 10 dakika süreyle buzda süspansiyon</li>
	<li>Süspansiyon 16.000 x g 15dk için de 4'te santrifüj kapasitesi ° C.</li>
	<li>Yeni bir microcentrifuge tüp süpernatant aktarın. Mitokondrial kesir bu.</li>
	<li>BCA tahlil kullanarak protein miktarı ölçmek.</li>
</ol> 4. Western Blot fosfo-spesifik proteinler için <ol><li>bir SDS-polyacrylamide Jel Elektroforez (8-%16 erken jel) gerçekleştirmek ve protein bir PVDF membran 4 ' e transfer.</li>
	<li>Kuluçkaya membran TBST %5 BSA ile (% 0,1 Polysorbate 20) arka plan belirsiz bağlayıcı siteleri engellemek için oda sıcaklığında 30 dk için. 
	Not: fosforile protein tespiti için membran BSA değil süt ile engelleyin. Süt içeren kazein, yüksek nonspesifik sinyal sonuçları bir bol miktarda Fosoprotein.</li>
	<li>Membran 4 ° C'de birincil antikoru (anti-fosfo-ERK1/2) ile bir gecede kuluçkaya.</li>
	<li>Ertesi gün, membran TBST ile yıkayın (% 0,1 polysorbate 20) üç kere oda sıcaklığında 5 min için.</li>
	<li>Membran ile ikincil antikoru (Anti-tavşan HRP) oda sıcaklığında 1 h için kuluçkaya.</li>
	<li>Membran TBST ile yıkayın (% 0,1 polysorbate 20) üç kere oda sıcaklığında 5 min için.</li>
	<li>Membran ECL çözüm ile kuluçkaya ve Image analyzer membran görüntü.</li>
	<li>Şerit arabellek 15 dakika oda sıcaklığında ve yıkama membran ile TBST üç kez 5 dakika süreyle sıyırma ile membran</li>
	<li>4.2-4.7 kullanarak anti-toplam ERK1/2 antikor arasındaki adımları yineleyin.</li>
	<li>Her kesir saflığı kontrol etmek için SDS-sayfa çalıştırmalarına ve işaretleri her bölmesi (örneğin, Okan B1 çekirdeği, sitozol için GAPDH ve TOM20 mitokondri için) için tanıma antikorları kullanarak immunoblots.</li>
</ol>

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Bir hissedar ve Yen CohBar, Inc. bir danışman olarak hizmet vermiştir CohBar, Inc Kelvin danışmanlığını Pinchas Cohen olduğunu

Burada, biz iki farklı hücre tiplerinde humanin peptid-aracılı ERK1/2 Etkinleştirme oluşuyor ve aktif ERK1/2 hücre altı lokalizasyonu (örneğin, peptid, zaman nokta ve hücre doz koşullara bağlı olarak farklı olabilir gösterdi yazın). O humanin göstermiştir-sinyal içinde iki hücre hatları gibi farklı dozda kullanımının humanin gereksinimini farklılıkları açıklayabilir iki farklı reseptörleri22,23, sinyalleri. Bu fizyolojik ...

Ortaya çıkan çalışmalar mitokondrial kaynaklı peptidler (MDP'ler) cytoprotection ve metabolizma1,2,3' te önemli rol oynarlar göstermektedir. Sinyal iletim yolu MDP'ler huzurunda anlama bize hangi çeşitli işlevleri MDP'ler modüle mekanizması görmemizi sağlar. İlk tanımlanan MDP, humanin, hücre dışı sinyal düzenlenir kinaz 1/2 (ERK1/2) artırmak için gösterilmiştir fosforilasyon, reseptör4,5bağlama aracılığıyla. Ancak, ERK1/2 aktivasyon aşağı akım etkisi hala underexplored.
ERK1/2 basamaklı hücresel süreçler yayılması, hücre göç, hücresel metabolizma, hayatta kalma ve Apoptozis6,7,8de dahil olmak üzere çeşitli önemli bir aracı olarak hizmet vermektedir. Tüm alacak için ayrı bu hücresel süreçler, aktivite ve ERK1/2 hücre altı yerelleştirme sıkıca fosfatazlar ve İskele proteinler9,10tarafından düzenlenmektedir. Translasyon modifikasyon ek olarak, dinamik ERK1/2 / mekik onun sinyal işlevi, aktivite ve özgüllük11,12düzenlemektedir. ERK1/2 öncelikle sitozol13' te lokalize edilmiştir. Ankraj ve İskele proteinler bir dizi yardımcı organelleri, yüzey veya dağınık sitoplazma13' te hücre iskeleti elemanları ERK1/2 korumak. Stimülasyon, üzerine ERK1/2 fosforile ve onun ankraj proteinler ERK1/2 translocation çekirdeği, mitokondri, Golgi ve organellerin14, de dahil olmak üzere diğer hücre altı bölmeleri için sağlayan ayrışmış olur 15 , 16.
Humanin ERK1/2 yolu sinyal etkinleştirmek için bilinen rağmen ERK1/2 aktivasyonu sadece toplam hücresini lysates görülmektedir. ERK1/2 hücre altı yerelleştirme aşağı akım etkisi, hem hücre altı yerelleştirme analizi ve toplam düzeyde önemli bir rol oynar bu yana daha önce açıklandığı gibi fosforile ERK1/2 kapsamlı bir anlayış sağlamak için gerekli humanin kaynaklı ERK1/2 harekete geçirmek ve aşağı akım hedefleri aktivasyonu.
Aktif ERK1/2 hedef organelleri anlamak için hücre altı ayırma fosforile ERK1/2 için western Blot tarafından takip gerçekleştirildi. Standart laboratuar ekipmanları ve Kimyasalları kullanır gibi bu yöntem kolayca uygulanabilir. İzole hücre altı bölmeleri delikanlı yorumlanması için sonuçlar sağlayan yüksek saflık vardır. Immunostaining ERK1/2 benzer sonuçlara neden olabilir. Ancak, belirli hücre altı bölmeleri nispeten zor görselleştirmek ve özel fiksasyon ve permeabilization yöntemleri gerektirir. Hücre altı bölmeleri ERK1/2 düzeyleri farklılık ve bu değişim tüm cep telefonu lysates bakıldığında yanlış pozitif ve yanlış olumsuz sinyaller neden olabilir. Bu nedenle, bir immunoblot izole hücre altı bölmeleri kullanarak bize ERK1/2 yerelleştirme daha iyi bir anlayış verir.
Yöntem çok yönlülük değişiklikler diğer MDP'ler veya translocation ile STAT3 başka sinyal moleküllerin de dahil olmak üzere diğer uyarıcı etkilerini araştırmak için iletişim kuralı sağlar. Son zamanlarda keşfedilen küçük humanin benzeri peptidler (yelken) nerede humanin kodlanır ve onlar-si olmak benzer ancak farklı özellikleri HN17' ye göre 16S rRNA bölgesinden kodlanır. Örneğin, 5 dk. ERK1/2 İnceleme yanıt-e doğru farklı peptidler hücre altı yerelleştirme içinde ERK1/2 humanin etkinleştirir, ancak 8 h bize Bu peptidler Biyoloji daha iyi bir anlayış verir sonra SHLP2 ve SHLP3 ERK1/2 etkinleştirin. Kanıt ortaya çıkan sinyal molekülleri, hücre altı yerelleştirme aşağı akım etkileri önemli bir rol oynar gösterdi. Örneğin, STAT3 geleneksel olarak öncelikle resting hücrelerdeki sitozol yerelleştirilecek bilinmektedir ve sonra gen ekspresyonu sitokinler18yanıt olarak etkinleştirmek için çekirdek içine translocates. STAT3 Ayrıca mitokondri için translocates ve TCA döngüsü ve ATP üretimi19düzenlemektedir. Autophagy Yönetmeliği ile ilgili çeşitli yolları20autophagy STAT3 farklı hücre altı yerelleştirme modüle. Örneğin, nükleer STAT3 transcriptionally autophagy ile ilgili genleri düzenler ve bir autophagy modülatör davranır. Sitoplazmik STAT3 moleküller sinyal autophagy ile etkileşerek autophagy yapısal engeller. Mitokondrial STAT3 engeller ve oksidatif stres indüklenen autophagy baskılayarak mitophagy engeller. Bu nedenle, bu hücre altı yuvası ayırma yöntemi diğer sinyal molekülleri yanı sıra ERK1/2 rolünün anlaşılması için önemlidir.

<table><tbody><tr><td>p44/42 MAPK (ERK1/2)</td><td>Cell signaling</td><td>9102</td><td>Dilution 1:1,000</td></tr><tr><td>phospho-p44/42 MAPK (ERK1/2)(Thr202/Tyr204)</td><td>Cell signaling</td><td>4370</td><td>Dilution 1:1,000</td></tr><tr><td>Lamin B1</td><td>Cell signaling</td><td>12586</td><td>Nuclear Marker, Dilution 1:1,000</td></tr><tr><td>GAPDH</td><td>Cell signaling</td><td>5174</td><td>Cytoplasmic marker, Dilution 1:2,000</td></tr><tr><td>Tom20</td><td>Santa cruz</td><td>SC-17764</td><td>Mitochondria marker, Dilution 1:2,000</td></tr><tr><td>anti-Rabbit-HRP conjugated</td><td>Cell signaling</td><td>7074</td><td>Dilution 1:30,000</td></tr><tr><td>RIPA Lysis and Extraction Buffer</td><td>ThermoFisher SCIENTIFIC</td><td>89900</td><td></td></tr><tr><td>100 mm Culture Dish</td><td>ThermoFisher SCIENTIFIC</td><td>12556002</td><td></td></tr><tr><td>HNG peptide</td><td>Genescript</td><td></td><td></td></tr><tr><td>25mm sylinge filter</td><td>ThermoFisher SCIENTIFIC</td><td>09-719A</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>MgCl2</td><td>Sigma</td><td>M8266</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G9012</td><td></td></tr><tr><td>Triton X-100</td><td>ThermoFisher SCIENTIFIC</td><td>BP151-100</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>3609</td><td></td></tr><tr><td>MOPS</td><td>Sigma</td><td>M1254</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E3889</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S7903</td><td></td></tr><tr><td>Tris-base</td><td>ThermoFisher SCIENTIFIC</td><td>BP152-1</td><td></td></tr><tr><td>HCL</td><td>Sigma</td><td>H1758</td><td></td></tr><tr><td>PBS</td><td>Lonza</td><td>17-512F</td><td></td></tr><tr><td>Cell Scraper</td><td>FALCON</td><td>353085</td><td></td></tr><tr><td>Halt&amp;trade; Protease and Phosphatase Inhibitor Cocktail (100X)</td><td>ThermoFisher SCIENTIFIC</td><td>78440</td><td></td></tr><tr><td>Thomas Pestle Tissue Grinder Assemblies with Smooth Pestles</td><td>Thomas Scientific</td><td>3432S90</td><td></td></tr><tr><td>Tween-20</td><td>ThermoFisher SCIENTIFIC</td><td>BP337-500</td><td></td></tr><tr><td>BSA</td><td>ThermoFisher SCIENTIFIC</td><td>BP1600-100</td><td></td></tr><tr><td>8-16% Mini-PROTEAN TGX Precast Protein Gels</td><td>BIO RAD</td><td>4561104</td><td></td></tr><tr><td>Mini Trans-Blot Module</td><td>BIO RAD</td><td>1658030</td><td></td></tr><tr><td>Trans-Blot Turbo Transfer System</td><td>BIO RAD</td><td>1704150</td><td></td></tr><tr><td>Trans-Blot Turbo RTA Mini PVDF Transfer Kit</td><td>BIO RAD</td><td>1704272</td><td></td></tr><tr><td>Clarity Western ECL Blotting Substrates</td><td>BIO RAD</td><td>1705060</td><td></td></tr><tr><td>Restore Western blot stripping buffer</td><td>ThermoFisher SCIENTIFIC</td><td>21059</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>ThermoFisher SCIENTIFIC</td><td>11965-092</td><td></td></tr><tr><td>Sonicator, Medel: FB120</td><td>ThermoFisher SCIENTIFIC</td><td>695320-07-12</td><td></td></tr></tbody></table>

subcellular fractionation for erk activation upon mitochondrial-derived peptide treatment

Mitokondrial kaynaklı peptidler (MDP'ler) küçük açık okuma çerçevesi mitokondri genom kopyası bilinen diğer genler içinde tarafından kodlanmış peptidler yeni bir sınıf vardır. MDP'ler çok çeşitli nöronların apoptosis korumak, metabolik işaretçileri artırma ve kemoterapi hücrelerin korunması gibi biyolojik etkileri var. Humanin ilk MDP keşfedilmeyi ve en çok çalışılan peptid MDP aile arasında. Membran reseptörleri ve humanin aşağı akım sinyal yollar dikkatle karakterize. MOTS-c ve SHLP1-6 gibi ek MDP'ler daha Geçenlerde bulunduktan ve sinyal mekanizmaları henüz aydınlatılmamıştır zorunda. Burada Bu peptidler işlevini belirlemek için bir hücre kültür dayalı yöntem açıklanmaktadır. Özellikle, hücre ayırma teknikleri, western Blot birlikte harekete geçirmek kantitatif tayin ve translocation önemli sinyal molekülünün için izin verir. Hücre ayırma başka yöntemler olmakla birlikte, burada açıklanan bir kolay ve basit yöntemdir. Bu yöntemler Bu peptidler ve diğer terapötik ajanlar etki mekanizması daha fazla aydınlatmak için kullanılabilir.

Burada sunulan işlemi uygulayarak, biz 1 mikron ve 100 mikron HEK293 ve SH-SY5Y hücrelerle tedavi S14G-humanin, bir güçlü humanin analog21, sırasıyla, içinde tamamlamak medya belirtilen süreler için (Resim 1A ve şekil 1 B). sonra ERK1/2 Toplam ve fosforile şeklinde Thr202/Tyr204 toplam proteini özleri itibariyle incelenmiştir. S14G-huma...

Watch this Scientific Journal Video about Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment at JoVE.com

Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

Bu iletişim kuralı, mitokondrial kaynaklı peptidler içeren hücreleri uyarmak ve sinyal cascade ve yerelleştirme fosfo-proteinlerin değerlendirmek açıklar.

Mitokondrial kaynaklı peptid tedavi üzerine ERK harekete geçirmek için hücre altı ayırma

Mitochondrial-derived peptides (MDPs) are a new class of peptides that are encoded by small open reading frames within other known genes of the ...

subcellular-fractionation-for-erk-activation-upon-mitochondrial

Research

JoVE Journal

Biology

7.8K Görüntüleme.  University of Southern California. Bu iletişim kuralı, mitokondrial kaynaklı peptidler içeren hücreleri uyarmak ve sinyal cascade ve yerelleştirme fosfo-proteinlerin değerlendirmek açıklar.

Video: Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

Assaying Protein Kinase Activity with Radiolabeled ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [&#947;-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([&#947;-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

Protein Kinaz Aktivitesini Radyoaktif Etiketli ATP ile Tahlil

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

Biyokimya

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

İnsan Embriyonik Kök Hücreleri Mitokondrial Kompleksi Aktivite araştırmayı

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

Biyoloji

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

Aktif ERK Mekansal ve Zamansal Analizi<em&gt; C. elegans</em&gt; Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Gelişim Biyolojisi

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. This method utilizes hypotonic buffers, digitonin, mechanical lysis and differential centrifugation to isolate the cytoplasm, mitochondria and plasma membrane. The process can be scaled to accommodate the needs of researchers, is inexpensive and straightforward. This method will allow researchers to determine protein localization in cells without specialized centrifuges and without the use of commercial kits, both of which can be prohibitively expensive. We have successfully used this method to separate cytosolic, plasma membrane and mitochondrial proteins in the human monocyte cell line U937.

Here, we present a protocol to isolate the plasma membrane, cytoplasm and mitochondria of U937 cells without the use of high-speed centrifugation. This technique can be used to purify subcellular fractions for subsequent examination of protein localization via immunoblotting.

Yüksek hızlı Santrifüjü yokluğunda U937 hücrelerinin hücre ayırma

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. ...

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances, many yeast mitochondrial proteins still remain with their functions completely unknown. This protocol provides a simple and reliable method to determine the submitochondrial localization of proteins, which has been fundamental for the elucidation of their molecular functions.

Tomurcuklanan Maya Sakkaromices cerevisiae'de Submitochondrial Protein Lokalizasyonunun Değerlendirilmesi

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. Despite a broad genetic heterogeneity, germline gain-of-function mutations in key regulators of the RAS-MAPK pathway underlie the majority of the cases, and, thanks to advanced sequencing techniques, potentially pathogenic variants affecting the RAS-MAPK pathway continue to be identified. Functional validation of the pathogenicity of these variants, essential for accurate diagnosis, requires fast and reliable protocols, preferably in vivo. Given the scarcity of effective treatments in early childhood, such protocols, especially if scalable in cost-effective animal models, can be instrumental in offering a preclinical ground for drug repositioning/repurposing. 
Here we describe step-by-step the protocol for rapid generation of transient RASopathy models in zebrafish embryos and direct inspection of live disease-associated ERK activity changes occurring already during gastrulation through real-time multispectral F&#246;rster resonance energy transfer (FRET) imaging. The protocol uses a transgenic ERK reporter recently established and integrated with the hardware of commercial microscopes. We provide an example application for Noonan syndrome (NS) zebrafish models obtained by expression of the Shp2D61G. We describe a straightforward method that enables registration of ERK signal change in the NS fish model before and after pharmacological signal modulation by available low-dose MEK inhibitors. We detail how to generate, retrieve, and assess ratiometric FRET signals from multispectral acquisitions before and after treatment and how to cross-validate the results via classical immunofluorescence on whole embryos at early stages. We then describe how, via examining standard morphometric parameters, to query late changes in embryo shape, indicative of a resulting impairment of gastrulation, in the same embryos whose ERK activity is assessed by live FRET at 6 h post fertilization.

RASopathies are multisystem genetic syndromes caused by RAS-MAPK pathway hyperactivation. Potentially pathogenic variants awaiting validation emerge continuously while poor preclinical evidence limits therapy. Here, we describe our in vivo protocol to test and cross-validate RASopathy-associated ERK activation levels and its pharmacological modulation during embryogenesis by live FRET imaging in Teen-reporter zebrafish.

Canlı FRET mikroskobu ve immünofloresan ile erken zebra balığı Noonan sendromu modellerinde ERK aktivite modülasyonunu değerlendirmek için etkili bir protokol

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

Böbrek Hücreleri aşırı eksprese eden protein kinaz C izozimlerinin Mitokondriyal Fonksiyonlar ve Hücre Canlılık Değerlendirilmesi

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

Gap Kavşak Sınavı Fonksiyonel Testi; İndiyum-Kalay Oksit Yapışık hücrelerin elektroporasyonu

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

Deneysel Yaklaşımlar Mitokondriyal Yerelleşme ve Nükleer Hücre Döngüsü Kinaz, Cdk1 Fonksiyonu Eğitim için

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

Sinirbilimde Mitokondriyal Ağları Analiz Etmek için MATLAB Optimizasyonu

Optimized Automated Analysis of Live Neuronal Mitochondria Homeostasis Modulation by Isoform-Specific Retinoic Acid Receptors

The complex mitochondrial network makes it very challenging to segment, follow, and analyze live cells. MATLAB tools allow the analysis of mitochondria in timelapse files, considerably simplifying and speeding up the process of image processing. Nonetheless, existing tools produce a large output volume, requiring individual manual attention, and basic experimental setups have an output of thousands of files, each requiring extensive and time-consuming handling. 
To address these issues, a routine optimization was developed, in both MATLAB code and live-script forms, allowing for swift file analysis and significantly reducing document reading and data processing. With a speed of 100 files/min, the optimization allows an overall rapid analysis. The optimization achieves the results output by averaging frame-specific data for individual mitochondria throughout time frames, analyzing data in a defined manner, consistent with those output from existing tools. Live confocal imaging was performed using the dye tetramethylrhodamine methyl ester, and the routine optimization was validated by treating neuronal cells with retinoic acid receptor (RAR) agonists, whose effects on neuronal mitochondria are established in the literature. The results were consistent with the literature and allowed further characterization of mitochondrial network behavior in response to isoform-specific RAR modulation. 
This new methodology allowed rapid and validated characterization of whole-neuron mitochondria network, but it also allows for differentiation between axon and cell body mitochondria, an essential feature to apply in the neuroscience field. Moreover, this protocol can be applied to experiments using fast-acting treatments, allowing the imaging of the same cells before and after treatments, transcending the field of neuroscience.

Yazar Spotlight: Nöronal Mitokondride Retinoik Asit Reseptörlerini Araştırmak İçin Optimize Edilmiş Otomatik Bir Yöntem

The mitochondrial network is extremely complex, making it very challenging to analyze. A novel MATLAB tool analyzes live confocal imaged mitochondria in timelapse images but results in a large output volume requiring individual manual attention. To address this issue, a routine optimization was developed, allowing for speedy file analysis.

İzoforma Özgü Retinoik Asit Reseptörleri ile Canlı Nöronal Mitokondri Homeostaz Modülasyonunun Optimize Edilmiş Otomatik Analizi

The complex mitochondrial network makes it very challenging to segment, follow, and analyze live cells. MATLAB tools allow the analysis of mitochondria in ...