Name: characterization of mlkl-mediated plasma membrane rupture in necroptosis
Uploaded: 2018-08-07T15:00:00
Description: Watch this Scientific Journal Video about Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis at JoVE.com

1. klonlama ve hücre satırı üretimi
<ol><li>PCR yükseltmek NBB bölge çerçeve standart Restriksiyon enzimi tabanlı Oligomerizasyonda etki alanı 2 x FK506 bağlayıcı protein (2xFKBP veya 2xFV) ve Venüs floresan ile klonlama olarak amino asit kalıntıları eklenmesi 1-140 (NBB140), insan MLKL cDNA sayfasından için karşılık gelen protein doksisiklin (Dox) - indüklenebilir retroviral vektör pRetroX-NBB140elde etmek için TRE3G - 2xFV-Venüs (Tablo 1, rakamlar 1A-B)...

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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RIPK1+blocks+early+postnatal+lethality+mediated+by+caspase-8+and+RIPK3.">RIPK1 blocks early postnatal lethality mediated by caspase-8 and RIPK3.</a> Cell. 157 (5), 1189-1202 (2014).</li><li>Rickard, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RIPK1+regulates+RIPK3-MLKL-driven+systemic+inflammation+and+emergency+hematopoiesis.">RIPK1 regulates RIPK3-MLKL-driven systemic inflammation and emergency hematopoiesis.</a> Cell. 157 (5), 1175-1188 (2014).</li><li>Kaiser, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+3-mediated+necrosis+via+TRIF,+RIP3,+and+MLKL.">Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL.</a> Journal of Biological Chemistry. 288 (43), 31268-31279 (2013).</li><li>Upton, J. W., Kaiser, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DAI+Another+Way:+Necroptotic+Control+of+Viral+Infection.">DAI Another Way: Necroptotic Control of Viral Infection.</a> Cell Host &amp; Microbe. 21 (3), 290-293 (2017).</li><li>Tanzer, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necroptosis+signalling+is+tuned+by+phosphorylation+of+MLKL+residues+outside+the+pseudokinase+domain+activation+loop.">Necroptosis signalling is tuned by phosphorylation of MLKL residues outside the pseudokinase domain activation loop.</a> Biochemical Journal. 471 (2), 255-265 (2015).</li><li>Quarato, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+Engagement+of+Distinct+MLKL+Phosphatidylinositol-Binding+Sites+Executes+Necroptosis.">Sequential Engagement of Distinct MLKL Phosphatidylinositol-Binding Sites Executes Necroptosis.</a> Molecular Cell. 61 (4), 589-601 (2016).</li><li>Davies, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+brace+helices+of+MLKL+mediate+interdomain+communication+and+oligomerisation+to+regulate+cell+death+by+necroptosis.">The brace helices of MLKL mediate interdomain communication and oligomerisation to regulate cell death by necroptosis.</a> Cell Death &amp; Differentiation. , (2018).</li><li>Su, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+plug+release+mechanism+for+membrane+permeation+by+MLKL.">A plug release mechanism for membrane permeation by MLKL.</a> Structure. 22 (10), 1489-1500 (2014).</li><li>Dondelinger, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MLKL+compromises+plasma+membrane+integrity+by+binding+to+phosphatidylinositol+phosphates.">MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates.</a> Cell Reports. 7 (4), 971-981 (2014).</li><li>Llambi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BOK+Is+a+Non-canonical+BCL-2+Family+Effector+of+Apoptosis+Regulated+by+ER-Associated+Degradation.">BOK Is a Non-canonical BCL-2 Family Effector of Apoptosis Regulated by ER-Associated Degradation.</a> Cell. 165 (2), 421-433 (2016).</li><li>Malkani, N., Schmid, J. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+divergence+of+the+necroptosis+effector+MLKL.">Evolutionary divergence of the necroptosis effector MLKL.</a> Cell Death &amp; Differentiation. 23 (7), 1185-1197 (2016).</li></ol>

MLKL plazma zarı yırtılması24sözde cellat karıştığı için kombine teknikleri için iletişim kuralları sağlar. Necroptosis MLKL-aracılı düzenleyen yasal ağ deşifre ek olarak, bu tekniklerin uygun diğer biyolojik sistemleri tanımlamak için bağımsız olarak kullanılabilir. Pratik olarak konuşursak, bu teknikleri orta-düşük verimi keşif araçları vardır.
Biz düzenli olarak canlı hücre görüntüleme NBB140kullandık-2xFV-Venüs-a...

Necroptosis genetik bileşenleri tanımlayan necroptosis dolaylı fizyolojisi ve hastalığı1,2,3,4,5' te test etmek için hayvan modellerinin kullanımı kolaylaştırdı. RIPK3 veya MLKL nakavt farelerde en az dolaylı geliştirme ve yetişkin homeostazı o necroptosis için hayat3,6şart değil ima vardı. Ayrıca, bazı türler ya RIPK3 ya da MLKL genler, necroptosis olmayan temel rolü hayvanların7,8' destek içermez. Öte yandan, çeşitli patolojileri laboratuvarda indüklenen ile nakavt hayvan modelleri zorlu necroptosis inflamasyon, doğuştan gelen bağışıklık ve viral enfeksiyon9,10, önemli bir rol ortaya koymuştur 11 , 12.
Necroptosis etkinleştirilmesi birkaç şekilde farklı doğuştan gelen bağışıklık sensörler sinyal tarafından tüm belgili tanımlık harekete geçirmek RIPK31,13,14hangi sonucu. Active RIPK3 sırayla phosphorylates ve MLKL3,4,5,6,7,8,9,10 etkinleştirir ,11,12,13,14,15,16,17,18. En iyi okudu ve belki de RIPK3 aktivasyonu için önde gelen en karmaşık yolu da apoptozis ya da necroptosis1ikna etmek için sinyal kompleksleri aşağı akım kompozisyona göre hangi bifurcates ölüm reseptör ligasyonu, geçiyor. RIPK1 sinyal tercih edilir ve nişan RIPK319,20sonuç Necroptosis ensues. Bu sonuç kolayca farmakolojik inhibisyon veya caspase 8, bir sözde endojen inhibitörü olan necroptosis koyunda tutar necroptosis genetik silinmesi üzerine tercih edilir. RIPK1 bağlar ve RIPK3'ı etkinleştirir. Necroptosis etkinleştirmek için başka bir yol TLR3/TLR4 Toll benzeri reseptörler sinyal aracılığıyla yürütmektedir ve RIPK3 tır etki alanını içeren bağdaştırıcı indükleyici interferon-β (TRIF)21aktive olduğu. Henüz doğrudan yürütmektedir ve RIPK322etkinleştirir DNA sensör DAI, etkinleştirme tarafından necroptosis tarafından ölmek için başka bir yol olduğunu.
MLKL bir N-terminal helix paket (NB) ve bir düzenleyici küme ayracı bölge3ile bağlantılı bir C-terminal pseudokinase etki alanı (psKD) oluşan sitozolik bir proteindir. Normal hücrelerde, MLKL nereye RIPK314ile etkin olmayan bir karmaşık olduğu düşünülen sitozol bulunur. Harekete geçirmek-in necroptosis MLKL RIPK3 fosforilasyon harekete geçirmek ilmik psKD ve NF ve kaşlı ayraç3,15,23yılında potansiyel olarak ek siteler içinde tetikler. Fosforilasyon ayrılma RIPK314sonuç MLKL konformasyon bir değişiklik neden olmaktadır. Kötü anlaşılır konformasyon değişiklikleri küme ayracı psKD24yayın. 2 sarmal içeren küme ayracı, MLKL Oligomerizasyonda sözde trimer ile C-terminal helix25içine aracılık eder. Brace N-terminal helix membran permeabilization24,26için gerekli olan NB etki alanı engeller. Tecrit NB etki alanı plazma zarı permeabilization ve necroptosis16,24,27ikna etmek yeterlidir. NB pro-necroptotic etkinliği içinde fare embriyonik fibroblastlar MLKL (mlkl- / - MEFs) eksik öldürüldüğüne tanıklık etmişlerdir. NB tercihen fosfolipid phosphatidylinositol 4,5 nükleotittir (PIP2) yürütmektedir bir lipid bağlayıcı etki alanıdır. Biz MLKL, onda MLKL alımı için plazma zarı NB zayıf etkileşimler PIP2 kutup baş grup24ile üzerinden küme ayracı Oligomerizasyonda kolaylaştırır aktivasyon kademeli bir mekanizma önerdi. Membran NB PIP2etkin olmayan MLKL brace tarafından maskeli için bir ek yüksek afinite bağlama sitesinin düzenlenmiş pozlama uğrar. Genel olarak, her ne kadar bu olayların moleküler mekanizması-değil var aydınlatılmamıştır NB birden çok etkileşimler PIP2 ile onun kopmalara önde gelen plazma zarı istikrarsızlaştırmak.
Burada MLKL işlevini necroptosis24cellat tanımlamak için kullanılan belirli yöntemleri gösterilmektedir. Özellikle, biz odaklanmak en az etki alanı MLKL, NB ve küme ayracı (NBB), hangi küme ayracı inhibisyon tarafından düzenlenir ve plazma membran rüptürü ve necroptosis ikna etmek için zorunlu dimerization ile aktif hale getirebilirsiniz. Biz zorunlu uyuşturucu kaynaklı FKBP-aracılı dimerization canlı hücre görüntüleme ve necroptosis geçiren hücrelerin elektron mikroskobu ile birlikte bizim indüklenebilir ifade sistemi açıklanmaktadır. Ayrıca, bizim vitro NMR Analizi NBB etkileşimleri ile phosphatidylinositols (pip) göstermektedir.

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			<td height="21" style="height:21px;width:429px;">Cloning and cell line generation</td>
			<td style="width:229px;"></td>
			<td style="width:323px;"></td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pRetroX-TRE3G</td>
			<td>Clontech</td>
			<td>631188</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tet-On transactivator plasmid</td>
			<td></td>
			<td></td>
			<td>Llambi et al., 2016</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Embryonic Fibroblasts (MEFs) mlkl-/-</td>
			<td></td>
			<td></td>
			<td>Dillon et al., 2014</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blasticidin S Hydrochloride</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP2647100 CAS#3513-03-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell death quantification and live-cell microscopy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Doxycycline</td>
			<td>Clontech</td>
			<td>631311 CAS# 24390-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B/B Homodimerizer AP20187</td>
			<td>Takara</td>
			<td>635059 CAS# 195514-80-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYTOX Green</td>
			<td>Thermo Fisher Scientific</td>
			<td>S7020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syto16</td>
			<td>Thermo Fisher Scientific</td>
			<td>S7578</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NMR</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">15N Ammonium Chloride</td>
			<td>Cambridge Isotope Laboratories</td>
			<td>NLM-467-10 CAS# 12125-02-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deuterated DTT</td>
			<td>Cambridge Isotope Laboratories</td>
			<td>DLM-2622-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deuterium Oxide</td>
			<td>Sigma Aldrich</td>
			<td>617385-1 CAS# 7789-20-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">n-Dodecyl-&#946;-D-Maltopyranoside</td>
			<td>Anatrace</td>
			<td>D310 CAS# 69227-93-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-&#945;-phosphatidylinositol-4,5-bisphosphate (Brain, Porcine) (ammonium salt)</td>
			<td>Avanti Polar Lipids</td>
			<td>840046X CAS# 383907-42-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1,2-distearoyl-sn-glycero-3-phosphoinositol (ammonium salt) (18:0 PI)</td>
			<td>Avanti Polar Lipids</td>
			<td>850143 CAS# 849412-67-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1,2-dioleoyl-sn-glycero-3-phospho-(1&#39;-myo-inositol) (ammonium salt) (18:1)</td>
			<td>Avanti Polar Lipids</td>
			<td>850149 CAS# 799268-53-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Specialized Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IncuCyte FLR or ZOOM</td>
			<td>Essen BioScience, Inc.</td>
			<td></td>
			<td>Live-cell microscopy imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Helios NanoLab 660 DualBeam&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Electron microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IncuCyte 2011A Rev2 v20111.3.4288 (FLR)</td>
			<td>Essen BioScience, Inc.</td>
			<td>http://www.essenbioscience.com</td>
			<td style="width:193px;">Imaging analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FEI MAPS</td>
			<td>Thermo Fisher Scientific</td>
			<td>https://www.fei.com/software/maps/</td>
			<td style="width:193px;">EM analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TopSpin v3.2</td>
			<td>Bruker BioSpin</td>
			<td>http://www.bruker.com</td>
			<td style="width:193px;">NMR data collection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CARA v1.9.1.7</td>
			<td></td>
			<td>http://cara.nmr.ch/&#160;</td>
			<td style="width:193px;">NMR data analysis</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:429px;">Slidebook</td>
			<td style="width:229px;">3i (Intelligent Imaging Innovations)</td>
			<td style="width:323px;">https://www.intelligent-imaging.com/slidebook</td>
			<td style="width:193px;">Confocal microscopy</td>
		</tr>
	</tbody>
</table>

characterization of mlkl-mediated plasma membrane rupture in necroptosis

Necroptosis phosphorylates ve karışık soy kinaz benzeri etki alanı pseudokinase, MLKL rüptürü veya plazma zarı permeabilize, aktive protein kinaz 3 (RIPK3), etkileşim reseptör etkinleştirme tarafından tetiklenen bir programlı hücre ölümü yoludur . Necroptosis otoimmünite, bulaşıcı ve kalp-damar hastalıkları, felç, ateş ve kanser de dahil olmak üzere birden çok patolojileri ile ilişkili inflamatuvar bir yoludur. Burada, MLKL necroptosis plazma zarı kopma cellat olarak karakterize etmek için kullanılan iletişim kurallarını açıklar. Biz necroptosis confocal ve konvansiyonel floresans mikroskobu ile canlı hücre Imaging'i kullanma hücreleri ve sabit hücrelerin elektron mikroskobu, birlikte plazma için sitozol üzerinden MLKL dağıtılması ortaya kullanarak işlem görselleştirmek membran plazma zarı büyük delikler indüksiyon öncesinde. Biz MLKL-aracılı necroptosis sözde modülatörler tanımlamak için lipidler kullanarak vitro nükleer manyetik rezonans (NMR) analiz mevcut. Bu yöntem üzerinde bağlı olarak, biz nicel lipid-bağlama tercihleri ve phosphatidyl inositol fosfatlar (pip) MLKL, plazma zarı hedefleme ve necroptosis permeabilization için gerekli olan kritik bağlayıcı olarak tespit edilmiştir.

Düzenlenmiş necroptosis yürütme canlı hücrelerdeki görselleştirme en az bir kesilmiş yapı, NBB140MLKL indüklenebilir ifade yoluyla mümkün olmuştur-2xFV-Venüs. Bu yapıyı plazma zarı permeabilization ikna yeteneği korur ve Dim kaynaklı Oligomerizasyonda FKBP kaset (2xFV) etkinleştirilir. Biz gözlemlemek ve necroptosis tarafından Imaging, kinetically (her 5 dk) bir hücre geçirimsiz yeşil floresans DNA bağlama boya (Şekil 6<...

Watch this Scientific Journal Video about Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis at JoVE.com

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Biz yöntemleri konvansiyonel ve confocal live-cep mikroskobu görüntüleme, dahil olmak üzere necroptosis MLKL-aracılı plazma zarı kopma karakterizasyonu için scanning elektron mikroskobu ve lipid NMR tabanlı bağlama raporu.

Necroptosis MLKL-aracılı plazma zarı kopma karakterizasyonu

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and ...

Research

JoVE Journal

Biology

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

Astrositler Plazma Membran ve Küresel Hücre içi kalsiyum Dynamics civarında Ölçüm

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Tersinir biyotinilasyon Plazma Membran Protein endositik Oranları Ölçüm

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

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

Akış Sitometri Plazma Membran GLUT4 Translokasyon Kantitatif Ölçüm

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

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Çoklu Hedef İzleme (MTT) tarafından Plazma Membran Moleküler Difüzyon Haritalama

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Lipidik Mesophases Membran Proteinleri Yüksek throughput Kristalizasyon için bir robot kullanımı

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Plazma Retinol Bağlayıcı Protein Membran Reseptör tarafından Retinol Ulaştırma Gerçek zamanlı Analizleri

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

Imaging Plasma Membrane Deformations With pTIRFM

PTIRFM ile görüntüleme Plazma Membran deformasyonlar

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Na fonksiyonel karakterizasyonu<sup&gt; +</sup&gt; / H<sup&gt; +</sup&gt; Seçim Proton-öldürme Kullanma hücre içi Bölmeler ve Eşanjörleri Plazma Membran Them Express

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Şekilde, mikro Tekniği Sıçan mezenter mikrodamar geçirgenliği Yönetmelik Araştırma

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Memeli hücrelerinde devlet tükenmesi süper kararlılık düşsel yer

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...