Guidelines-for-the-use-and-interpretation-of-assay

GuidelinesfortheuseandinterpretationofassaysformonitoringautophagyReporter:LongtaoHuangfuTHETERM“AUTOPHAGY”Thepastdecadehaswitnessedanexplosionofresearchonafundamentalcellbiologypathwaycalledautophagy(Greekfor“self-eating”).自噬是细胞内的一种“自食（Self-eating）”的现象，凋亡是“自杀（Self-killing）”的现象，二者共用相同的刺激因素和调节蛋白，但是诱发阈值和门槛不同，如何转换和协调目前还不清楚THEMAINDEGRATIONPATHWAYS1、蛋白酶体途径：降解细胞内短寿(short-lived)、多聚泛素化(ubiquitination)的蛋白质。2、内吞途径：将跨膜蛋白运送到溶酶体降解。原核生物：通过19S的蛋白酶体能识别靶蛋白的特定氨基酸序列并将其降解。真核生物：则是通过26S的蛋白酶体降解蛋白质。3、细胞自噬途径：而长寿蛋白(long-livedprotein)、蛋白聚集物及膜包被的细胞器是通过细胞自噬的方式在溶酶体降解。THREETYPESOFAUTOPHAGYTHREETYPESOFAUTOPHAGY1、巨型细胞自噬(macroautophagy)，又称为大自噬，是指细胞内新生的杯状脂质双层包裹胞浆蛋白和细胞器，并运送到溶酶体降解的自噬行为。2、微型细胞自噬(microautophagy)，又称为小自噬，是指通过溶酶体膜的内陷、突起和/或分隔，直接吞入细胞浆的自噬行为。3、分子伴侣介导的细胞自噬(chaperonemediatedautophagy，CMA)，是指由分子伴侣将靶蛋白转送至溶酶体内的自噬行为。这只见于哺乳类动物细胞。THREETYPESOFAUTOPHAGYBLUKANDSELECTIVEAUTOPHAGYAUTOPHAGYINCELLULARGROWTHCONTROLAUTOPHAGYINCELLULARGROWTHCONTROLAUTOPHAGYDURINGVERTEBRATEDEVELOPMENTAUTOPHAGYREGULATESVERTEBRATEDISEASESAUTOPHAGYPROCESSTHEAMPK-MTORC1-ULK1/2NETWORKTHEAMPK-MTORC1-ULK1/2NETWORKTHEBECLIN-1NETWORKBeclin-1isaproteinthatinhumansisencodedbytheBECN1gene.Beclin-1isalsoknownasautophagy-relatedgene(Atg)6.Beclin-1anditsbindingpartnerclassIIIphosphoinositide3-kinase(PI3K),alsonamedVps34,arerequiredfortheinitiationoftheformationoftheautophagasomeinautophagy.Beclin1isalsoanovelBcl-2-homology(BH)-3domainonlyprotein.NuclearExportSignalATG6=Beclin-1Vps38=UVRAGThereisonlyoneknownclassIIIPI3-kinase,Vps34,whichisalsotheonlyPI3-kinaseexpressedinalleukaryoticcells.InhumansitisencodedbythePIK3C3gene.InhumancellsVps34associateswitharegulatorysubunit,p150.THEFUNCTIONOFPI3KC3THEFUNCTIONOFPI3KC3DFCP1:doubleFYVE-containingprotein1WIPI:WD-repeatdomainphosphoinositideinteractingMajorKeyPlayersintheMaturationandDegradationMICRORNA-MODULATEDAUTOPHAGY导致大多数神经性疾病的元凶REGULATIONOFMITOPHAGYREGULATIONOFMITOPHAGY•CCCP和FCCP都可以引起强烈的短期线粒体去极化，常用作线粒体自噬诱导剂•CyclosporinA（CsA，环孢菌素A）和ALC左旋肉毒碱可以抑制线粒体自噬，稳定电位miR-137抑制NixREGULATIONOFMITOPHAGYCROSSTALKBETWEENAPOPTOSISANDAUTOPHAGYCROSSTALKBETWEENAPOPTOSISANDAUTOPHAGYCROSSTALKBETWEENAPOPTOSISANDAUTOPHAGYCROSSTALKBETWEENAPOPTOSISANDAUTOPHAGY钙蛋白酶EffectsofDrugsontheAutophagicPathwayEffectsofDrugsontheAutophagicPathwayMethodsinMammalianAutophagyResearchMethodsinMammalianAutophagyResearchMethodsinMammalianAutophagyResearchMethodsinMammalianAutophagyResearchMethodsinMammalianAutophagyResearchChangesinthedetectionandlocalizationofGFP-LC3upontheinductionofautophagy.U87cellsstablyexpressingGFP-LC3weretreatedwithPBS,rapamycin(200nM),orrapamycinincombinationwith3-MA(2mM)for24h.RepresentativefluorescenceimagesofcellscounterstainedwithDAPI(nuclei)areshown.Scalebar,10mm.MethodsinMammalianAutophagyResearchMovementofactivatedpDendra2-hp62(orange)fromthenucleus(middle)totheaggregateinARPE-19cells,revealedbyconfocalmicroscopy.ThecellswerethenexposedtoaUVpulse(theUV-inducedareaisshownbyredlinesthatareinsideofthenucleus)thatconvertsDendra2fromgreentored,andthetimeshownafterthepulseisindicated.MethodsinMammalianAutophagyResearchMethodsinMammalianAutophagyResearchMethodsinMammalianAutophagyResearchLAMP1/2在溶酶体中的作用尚无定论MethodsinMammalianAutophagyResearchSaponin皂苷处理细胞膜后通透性改变，LC3-puncta无法出细胞MethodsinMammalianAutophagyResearchWortmanin:渥曼青霉素，PI3K非特异性抑制剂（已经被3-MA取代）MethodsinMammalianAutophagyResearch磷脂酰乙醇胺MethodsinMammalianAutophagyResearchE-64dandPepstainA:溶酶体蛋白酶抑制剂12%gelMethodsinMammalianAutophagyResearch•Krebs-RingermediumSolution:Ringer’s溶液(KRPD)•Hank'sBalancedSaltSolution:Hanks平衡盐溶液(HBSS)•Earle’sBalancedSaltSolution:Earle's平衡盐溶液(EBSS)MethodsinMammalianAutophagyResearchRapamycin短时间作用（4h）MethodsinMammalianAutophagyResearchMethodsinMammalianAutophagyResearchAtg5-/-MEFsengineeredtoexpressAtg5underthecontroloftheTet-offpromoterweregrowninthepresenceofdoxycyline(10ng/ml)foroneweektosuppressautophagy.MethodsinMammalianAutophagyResearchMethodsinMammalianAutophagyResearchMethodsinMammalianAutophagyResearchREFERENCES1.Alers,S.,etal.,RoleofAMPK-mTOR-Ulk1/2intheregulationofautophagy:crosstalk,shortcuts,andfeedbacks.MolCellBiol,2012.32(1):p.2-11.2.Behrends,C.,etal.,Networkorganizationofthehumanautophagysystem.Nature,2010.466(7302):p.68-76.3.Chen,N.andV.Karantza,Autophagyasatherapeutictargetincancer.CancerBiolTher,2011.11(2):p.157-68.4.Turei,D.,etal.,AutophagyRegulatoryNetwork-asystems-levelbioinformaticsresourceforstudyingthemechanismandregulationofautophagy.Autophagy,2015.11(1):p.155-65.5.5.Kang,R.,etal.,TheBeclin1networkregulatesautophagyandapoptosis.CellDeathDiffer,2011.18(4):p.571-80.6.Klionsky,D.J.,etal.,Guidelinesfortheuseandinterpretationofassaysformonitoringautophagy(3rdedition).Autophagy,2016.12(1):p.1-222.7.Mizushima,N.,T.Yoshimori,andB.Levine,Methodsinmammalianautophagyresearch.Cell,2010.140(3):p.313-26.8.Rosenfeldt,M.T.andK.M.Ryan,Theroleofautophagyintumourdevelopmentandcancertherapy.ExpertRevMolMed,2009.11:p.e36.9.Rubinsztein,D.C.,P.Codogno,andB.Levine,Autophagymodulationasapotentialtherapeutictargetfordiversediseases.NatRevDrugDiscov,2012.11(9):p.709-30.10.Xu,J.andY.H.Li