自噬-检测指南课件

细胞自噬研究方法概述研究生：王颖导师：刘乃丰教授细胞自噬研究方法概述研究生：王颖1AutophagicCompartmentsAutophagicCompartments2AutophagicCompartmentsPhagophore(pre-autophagosomal)：

previouslycalledtheisolationorsequestrationmembrane吞噬泡：参与自噬体形成早期事件的膜池。也指“隔离膜isolationmembrane”或“杯状结构cup-shapedstructure”Autophagosome:自噬体：双层膜包裹胞质形成的囊泡Amphisome

：generatedbythefusionofautophagosomeswithendosomes,alsoreferredtoasanacidiclateautophagosome自噬内涵体：溶酶体和内涵体融合的中间囊泡Autolysosome

：generatedbyfusionofautophagosomesoramphisomeswithalysosome自噬溶酶体：自噬小体和溶酶体融合形成的终末结构AutophagicCompartments3Induce/promoteInhibitionAutophagicmolecularmechanismsInduce/promoteInhibitionAutop4Initiation：Induction，CargorecognitionandselectivityElongation，Closure：AutophagosomeformationMaturation，Degradation:Vesiclefusionandautophagosomebreakdown

AutophagicmolecularmechanismsInitiation：Autophagicmolecula5InductionNormalconditionsBasal-levelautophagyisverylow;Autophagyinhibitor:serine/threonineproteinkinaseTOR(targetofrapamycin)inputinformationfrommultipleupstreamsignaltransductionpathways(discussedbelow)andnegativelyregulatesanotherserine/threoninekinase,Atg1,innutrient-richconditionsStarvationconditonsorRapamycinTORinhibited;Atg1activated;Atg1bindingaffinitytoAtg13andAtg17↑;PromotestheformationofanAtg1-Atg13-Atg17scaffold;Atg1-Atg13-Atg17recruitmentofmultipleAtgproteinstothePAStoinitiateautophagosomeformation.InductionNormalconditionsSta6CargorecognitionandselectivityP62/sequestosome1(SQSTM1).P62directlybindsbothpoly-ormono-ubiquitinviaitsubiquitin-associated(UBA)domainandLC3linkstheubiquitinatedcargostotheautophagymachineryforautophagicdegradation.

Cargorecognitionandselectiv7ClassIIIphosphatidylinositol3-kinase(PtdIns3K)complex:PtdIns3KVps34(vacuolarproteinsorting34),amyristoylatedserine/threoninekinaseVps15,Atg14;

Beclin1;Autophagosomeformation

Vps34Atg14Vps15Beclin1ThePtdIns3KcomplexproducesPtdIns3P(phosphatidylinositol3-phosphate)andisinvolvedinPAStargetingofanumberofyeastAtgproteinsthatbindPtdIns3P,suchasAtg18,Atg20,Atg21,andAtg24.Inyeast,Atg20andAtg24interactwiththeAtg1-Atg13-Atg17complex,andthelattermediatesautophagyinduction;ThePtdIns3Kcomplex,recruitstwointerrelatedubiquitin-like(Ubl)conjugationsystems,Atg12–Atg5-Atg16andAtg8–PE

(phosphatidylethanolamine),tothephagophorewhichplayanessentialroleinregulatingthemembraneelongationandexpansionoftheformingautophagosome.ClassIIIphosphatidylinositol8ThefunctionofBeclin1inautophagyisregulatedbyBcl-2;Bcl-2inhibitsautophagybybindingandsequesteringBeclin1;DissociationofBeclin1fromBcl-2isrequiredforautophagyinduction.Vps34Atg14Vps15Beclin1Bcl-2Atg12isactivatedbyAtg7,transferredtoAtg10(E2conjugatingenzyme)andattachedtoaninternallysineofthesubstrateproteinAtg5covalently.TheAtg12–Atg5conjugatefurtherinteractswithacoiled-coilproteinAtg16,whichlinkstheAtg12–Atg5-Atg16complexintoatetramerbyself-oligomerizationandattachesittothephagophore.AutophagosomeformationThefunctionofBeclin1ina9Atg8isfirstprocessedbyacysteineprotease,Atg4,exposingaC-terminalglycineresidue.ThesameE1enzymeAtg7activatesAtg8andtransfersitAtg8isfinallyconjugatedtothetargetlipidPEviaanamidebond.Innutrient-richconditions,themajorityofAtg8iscytosolic（16Kd）;uponautophagyinduction,Atg8largelyexistsasthelipid-conjugatedform(14Kd)andislocalizedtobothsidesofthephagophore.Atg8controlsthesizeoftheautophagosome,whichmayresultfromitsability

todeterminemembranecurvature.ThelipidationofAtg8anditsmammalianhomologLC3arewidelyusedtomonitorautophagyinduction..AutophagosomeformationAutophagosomeformation10VesiclefusionandautophagosomebreakdownInmammaliancells,thefusioneventrequiresthelysosomalmembraneproteinLAMP-2andthesmallGTPaseRab7.Afterfusion,degradationoftheinnervesicleisdependentonaseriesoflysosomal/vacuolaracidhydrolases,includingproteinasesAandB(encodedbyPEP4andPRB1,respectively)andthelipaseAtg15inyeastandcathepsinB,D(ahomologofproteinaseA),andLinmammaliancellsTheresultingsmallmoleculesfromthedegradation,particularlyaminoacids,aretransportedbacktothecytosolforproteinsynthesisandmaintenanceofcellularfunctionsunderstarvationconditions.Vesiclefusionandautophagoso111.Transmissionelectronmicroscopy

2.Atg8/LC3detectionandquantification3.SQSTM1/p62andrelatedLC3bindingproteinturnoverassays4.MTOR,AMPKandAtg1/ULK1

5.Additionalautophagy-relatedmarkers6.Transcriptionalandtranslationalregulation

7.Autophagicproteindegradation8.Selectivetypesofautophagy

MethodsforMonitoringAutophagy9.Autophagicsequestrationassays

10.Turnoverofautophagiccompartments

11.Autophagosome-lysosomecolocalizationanddequenchingassay12.Tissuefractionation13.Analysesinvivo14.Celldeath

15.Chaperone-mediatedautophagy1.Transmissionelectronmicro12Transmissionelectronmicroscopy一、取材快速、低温细胞株：胰酶消化：细胞完整性保存良好，但是对自噬有一定影响细胞刮片：最大程度维持细胞原生理状态，细胞物理损伤大先固定，后刮取：细胞完整性、生理性维持良好，但细胞分散，不易成团组织：优选低温灌注固定时间点：1~2h，8h，24hTransmissionelectronmicrosco13Transmissionelectronmicroscopy二、结构特点Autophagosomes

：1~2h,8hdoublemembrane，visibleastwoparallelmembranebilayersseparatedbyanelectron-lucentcleftcontaincytosoland/ororganellesthatlookmorphologicallyintactAmphisomescansometimesbeidentifiedbythepresenceofsmallinternalvesiclesinsidetheautophagosome/autophagicvacuole(AV).Theseinternalvesiclesaredeliveredintothelumenbyfusionwithmultivesicularendosomes.Late/degradativeautophagicvacuolesandautolysosomes(AVd)

：24husuallyhaveonlyonelimitingmembrane,andcontaincytoplasmicmaterialand/ororganellesatvariousstagesofdegradation

Transmissionelectronmicrosco14TransmissionelectronmicroscopyTransmissionelectronmicrosco15CautionarynotesFixationofexcisedtissuesrequirescaretoavoidsamplinganonrepresentativeoruninformativesectionoftissue.Quantifyautophagosome(and/orautolysosome)profilespertotalcytoplasmicorcellularareainsections.Atleast20cellprofilespersample.Eachimagedcellprofileiscapturedandscoredatthesamemagnification.CautionarynotesFixationofex16CautionarynotesNotalldouble-membranestructuresareautophagosomesApoptoticbodiesfromneighboringcellsarereadilyphagocytosedbysurvivingcellsofthesametissue.Phagosomeshavedoublelimitingmembranes,inneroneisfromtheplasmamembraneoftheapoptoticbodyandtheouteroneisthatofthephagocytizingcell.Amajordifference,isthatthesurroundingmembranesarethethicker

thanthethinnersequestrationmembranetype

(9–10nm,vs.7–8nm,respectively).Agoodfeaturetodistinguishbetweenautophagosomesanddoubleplasmamembrane-boundstructuresisthelackofthedistendedemptyspacebetweenthetwomembranesofthephagocyticvacuoles.Engulfedapoptoticbodiesusuallyhavea

largeraveragesizethanautophagosomes.CautionarynotesNotalldouble17DuetothecisternalstructureoftheER,doublemembrane-likestructuressurroundingmitochondriaorotherorganellesareoftenobservedaftersectioning.EmploytomographicreconstructionsoftheTEMimagestoconfirmthattheautophagiccompartmentsaresphericalandarenotbeingconfusedwithendomembranecisternaeordamagedmitochondria.Ifthereareribosomesassociatedwiththesemembranestheycanhelpdistinguishthemfromtheribosomefreedouble-membraneofthephagophoreandautophagosome.CautionarynotesDuetothecisternalstructure18a.Westernblottingandubiquitin-likeproteinconjugationsystems

b.TurnoverofLC3-II/Atg8–PEc.GFP-Atg8/LC3lysosomaldeliveryandproteolysis

d.GFP-Atg8/LC3fluorescencemicroscopye.TandemmRFP/mCherry-GFPfluorescencemicroscopy

f.Autophagicfluxdeterminationusingflowandmultispectralimagingcytometry

g.Immunohistochemistry.

Atg8/LC3detectionandquantificationa.Westernblottingandubiqui19WesternblottingThemammalianhomologsofAtg8LC3(microtubule-associatedprotein1lightchain3)LC3A,B,B2andCGABARAP:GABAAreceptor-associatedproteinGABARAPL1/GEC1:GABAAreceptorassociatedproteinlike1/GlandularEpithelialCell1GABARAPL2/GATE-16/GEF2:GABAAreceptor-associatedproteinlike2/Golgi-associatedATPaseenhancerof16kDa/gangliosideexpressionfactor2GABARAPL3:GABAAreceptorassociatedproteinlike3WesternblottingThemammalian20Thereisnotalwaysaclearprecursor/productrelationshipbetweenLC3-IandLC3-II,changesinLC3-IIamountsaretissue-andcellcontext-dependentMoreover,LC3-IismorelabilethanLC3-II,beingmoresensitivetofreezingthawingandtodegradationinSDSsamplebuffer,freshsamplesshouldbeheatedandassessedassoonaspossibleandshouldnotbesubjectedtorepeatedfreeze-thawcycles.PVDFmembranesmayresultinastrongerLC3-IIretentionthannitrocellulosemembranesTritonX-100maynotefficientlysolubilizeLC3-IIinsomesystemsHeatinginthepresenceof1%SDS,oranalysisofmembranefractions,mayassistinthedetectionofthisprotein.Insomecasesbeta-actinlevelsdecreasewhenautophagyisinducedCautionary

notes:Thereisnotalwaysaclearpr21WesternblottingWesternblotting22自噬_检测指南课件23TurnoverofLC3-II/Atg8–PE.PreventLysosomalDegradationAutophagicfluxcanbemeasuredbyinferringLC3-II/Atg8–PEturnoverbywesternblotinthepresenceandabsenceoflysosomaldegradation;TherelevantparameterinthisassayisthedifferenceintheamountofLC3-IIinthepresenceandabsenceofsaturatinglevelsofinhibitors;Iffluxisoccurring,theamountofLC3-IIwillbehigherinthepresenceoftheinhibitor.TurnoverofLC3-II/Atg8–PE.Pre24ProteaseinhibitorspepstatinAandE-64d:NeutralizethelysosomalPHbafilomycinA1（洛霉素A1）chloroquine：氯喹NH4Cl：氯化铵BlockfusionofautophagosomeswithlysosomesbafilomycinA1Knockingdownorknockingoutlysosomal-associatedmembraneprotein2(LAMP2)PreventLysosomalDegradation注：抑制剂作用时间：1~2h；

设阳性对照组

时间点：4h/24hbafilomycinA1,NH4Clorchloroquine,alsodirectlyinhibittheendocytosis/uncoatingofvirusesandotherendocyticeventsrequiringlowpHmonitorbothturnoverofLC3-IIandanautophagosomesubstrateinparallel.ProteaseinhibitorsPreventLy251hofpre-incubationwith10mg/mlE-64dissufficientinmostcases,sincethisinhibitorismembranepermeableandrapidlyaccumulateswithinlysosomes.pepstatinAismembraneimpermeable(ethanolorpreferablyDMSOmustbeemployedasavehicle)andrequiresaprolongedincubation(.8h)andarelativelyhighconcentration(.50mg/ml)tofullyinhibitlysosomalcathepsinD1hofpre-incubationwith1026GFP-Atg8/LC3lysosomaldeliveryandproteolysisWersternblotGFP-LC3在自噬溶酶体的酸性环境中被降解GFP单体释放到胞质中，蛋白印迹检测GFP单体条带Cautionarynotes:AreductionintheintensityofthefreeGFPbandmayindicatereducedflux,butitmayalsobeduetoefficientturnover.Usingarangeofconcentrationsandtreatmenttimesofcompoundsthatinhibitautophagycanbeusefulindistinguishingbetweenthesepossibilities.GFP-Atg8/LC3lysosomaldeliver27GFP-Atg8/LC3fluorescencemicroscopyFluorescencemicroscopyAconstantincreaseinthenumberofcellsaccumulatingGFP-LC3punctaissuggestiveofdefectivefusionofautophagosomeswithlysosomes;Conversely,adeclineimpliesthatGFP-LC3isconsumedwithinnewlyformedautolysosomes.Fluorescencemicroscopy+lysosomalproteaseorfusioninhibitorsMonitoringchangesinthenumberofpuncta.ThepresenceoflysosomalinhibitorsshouldincreasethenumberofGFP-LC3-positivestructures,andtheabsenceofaneffectonthetotalnumberofGFP-LC3punctaoronthepercentageofcellsdisplayingnumerouspunctaisindicativeofadefect(s)inautophagicflux.GFP-Atg8/LC3fluorescencemicr28

TandemmRFP-GFPfluorescencemicroscopyTheGFPsignalissensitivetotheacidicand/orproteolyticconditionsofthelysosomelumen,whereasmRFPismorestable.

Therefore,colocalizationofbothGFPandmRFPfluorescenceindicatesacompartmentthathasnotfusedwithalysosome,suchasthephagophoreoranautophagosome.Incontrast,anmRFPsignalwithoutGFPcorrespondstoanamphisomeorautolysosome.

OneofthemajoradvantagesofthetandemmRFP/mCherry-GFPreportermethodisthatitenablessimultaneousestimationofboththeinductionofautophagyandfluxthroughautophagiccompartmentsinessentiallynativeconditions,withoutrequiringanydrugtreatment.TandemmRFP-GFPfluorescence29自噬体和自噬溶酶体分别成黄色和红色标记，如果自噬潮增加，两种颜色的点状聚集均增加。如果自噬体向自噬溶酶体成熟受阻，黄色点状聚集物增加，红色不增加。自噬体和自噬溶酶体分别成黄色和红色标记，如果自噬潮增加，两种30Flowandmultispectralimagingcytometry在自噬诱导一开始，GFP-LC3点状聚集显著增多，随后信号可能会出现下降，代表着自噬性降解的发生．高通量检测Flowandmultispectralimaging31ImmunohistochemistryWhenautophagosomesareabsent,thelocalizationpatternofLC3inthecellsofvarioustissuesisdiffuseandcytosolic.OneproblemwithimmunohistochemistryforLC3isthatinsometissuesthisproteincanbelocalizedinstructuresotherthanautophagosomes.Forexample,inmurinehepatocytesandcardiomyocytesunderstarvedconditions,endogenousLC3isdetectednotonlyinautophagosomesbutalsoonlipiddroplets.InneuronsinATG7-deficientmice,LC3isaccumulatedinubiquitin-andSQSTM1-positiveaggregates.ImmunohistochemistryWhenautop32p62andrelatedLC3bindingproteinturnoverassaysTheSQSTM1proteinservesasalinkbetweenLC3andubiquitinatedsubstrates.decreasedSQSTM1levelsareassociatedwithautophagyactivationThephosphorylationofSQSTM1atSer403appearstoregulateitsroleintheautophagicclearanceofubiquitinatedproteins,andanti-phospho-SQSTM1/p62antibodiescanbeusedtodetectthemodifiedformoftheprotein.p62andrelatedLC3bindingpr33WesternblotanalysisusingNP40orTritonX-100lysisinautophagicconditionstypicallyshowsareductioninSQSTM1levels.However,thisdoesnotnecessarilyindicatethatSQSTM1isdegraded,becauseSQSTM1aggregatesareinsolubleinthesedetergentlysisconditions.WhereasLC3changesmayberapid,clearanceofautophagysubstratesmayrequirealongertime.Therefore,ifLC3changesareassessedat6hor24hafteradrugtreatment,SQSTM1levelscanbetestednotonlyatthesametimepoints,butalsoatlatertimepoints(24hor48h)fordeterminingthemaximalimpactonsubstrateclearance.CautionarynotesWesternblotanalysisusingNP34TOR,AMPKandAtg1/ULK1TORC1

isanautophagy-suppressiveregulatorthatintegratesgrowthfactor,nutrientandenergysignals.inhibitionofmTORleadstoinductionofautophagyTheenzymeactivityofAMPKisabsolutelydependentonphosphorylationofthea-subunitonThr172,andcanmonitoredbywesternblottingwithaphosphospecificantibodyagainstthissite.Activation/assemblyULK1complexinmammals(ULK1-RB1CC1-ATG13-C12orf44/ATG101)isoneofthefirststepsofautophagyinduction.，activationofthiscomplexcanbeassessedtomonitorautophagyinduction.thephosphorylationstatusofULK1attheactivatingsites(Ser317,467,555,637,777,orThr574)

ordephosphorylationatinactivatingsites(Ser638,757)canbedeterminedusingphospho-specificantibodies,orbywesternblotting.TOR,AMPKandAtg1/ULK1TORC135根据自噬机制主要负责降解长寿命蛋白的特性，先让细胞在含有同位素标记氨基酸(如14C-或3H-缬氨酸或亮氨酸)的培养基中生长一段时间(数小时至数天)细胞在此期间合成的蛋白质都将被同位素标记，然后换成不含同位素的培养基，让一些被标记的短寿命蛋白通过蛋白酶体途径降解．在自噬诱导后，通过检测培养上清中释放的自噬性降解产物的放射性活度即可反映细胞自噬性降解的能力．同时加入自噬抑制剂作为对比，更能特异性地反映自噬引起的蛋白质降解.长寿命蛋白降解检测根据自噬机制主要负责降解长寿命蛋白的特性，先让细胞在含有同位36自噬的实验性调控

二、基因干预通过RNAi干扰Atg3、Atg5、Atg7及Beclin1等自噬相关基因的表达后，细胞表现为自噬功能缺失．与工具药相比，通过基因沉默或敲除技术来抑制自噬具有相对强的特异性．一、药物干预自噬的实验性调控

二、基因干预一、药物干预37谢谢聆听！谢谢聆听！38谢谢观看！2020

谢谢观看！39细胞自噬研究方法概述研究生：王颖导师：刘乃丰教授细胞自噬研究方法概述研究生：王颖40AutophagicCompartmentsAutophagicCompartments41AutophagicCompartmentsPhagophore(pre-autophagosomal)：

previouslycalledtheisolationorsequestrationmembrane吞噬泡：参与自噬体形成早期事件的膜池。也指“隔离膜isolationmembrane”或“杯状结构cup-shapedstructure”Autophagosome:自噬体：双层膜包裹胞质形成的囊泡Amphisome

：generatedbythefusionofautophagosomeswithendosomes,alsoreferredtoasanacidiclateautophagosome自噬内涵体：溶酶体和内涵体融合的中间囊泡Autolysosome

：generatedbyfusionofautophagosomesoramphisomeswithalysosome自噬溶酶体：自噬小体和溶酶体融合形成的终末结构AutophagicCompartments42Induce/promoteInhibitionAutophagicmolecularmechanismsInduce/promoteInhibitionAutop43Initiation：Induction，CargorecognitionandselectivityElongation，Closure：AutophagosomeformationMaturation，Degradation:Vesiclefusionandautophagosomebreakdown

AutophagicmolecularmechanismsInitiation：Autophagicmolecula44InductionNormalconditionsBasal-levelautophagyisverylow;Autophagyinhibitor:serine/threonineproteinkinaseTOR(targetofrapamycin)inputinformationfrommultipleupstreamsignaltransductionpathways(discussedbelow)andnegativelyregulatesanotherserine/threoninekinase,Atg1,innutrient-richconditionsStarvationconditonsorRapamycinTORinhibited;Atg1activated;Atg1bindingaffinitytoAtg13andAtg17↑;PromotestheformationofanAtg1-Atg13-Atg17scaffold;Atg1-Atg13-Atg17recruitmentofmultipleAtgproteinstothePAStoinitiateautophagosomeformation.InductionNormalconditionsSta45CargorecognitionandselectivityP62/sequestosome1(SQSTM1).P62directlybindsbothpoly-ormono-ubiquitinviaitsubiquitin-associated(UBA)domainandLC3linkstheubiquitinatedcargostotheautophagymachineryforautophagicdegradation.

Cargorecognitionandselectiv46ClassIIIphosphatidylinositol3-kinase(PtdIns3K)complex:PtdIns3KVps34(vacuolarproteinsorting34),amyristoylatedserine/threoninekinaseVps15,Atg14;

Beclin1;Autophagosomeformation

Vps34Atg14Vps15Beclin1ThePtdIns3KcomplexproducesPtdIns3P(phosphatidylinositol3-phosphate)andisinvolvedinPAStargetingofanumberofyeastAtgproteinsthatbindPtdIns3P,suchasAtg18,Atg20,Atg21,andAtg24.Inyeast,Atg20andAtg24interactwiththeAtg1-Atg13-Atg17complex,andthelattermediatesautophagyinduction;ThePtdIns3Kcomplex,recruitstwointerrelatedubiquitin-like(Ubl)conjugationsystems,Atg12–Atg5-Atg16andAtg8–PE

(phosphatidylethanolamine),tothephagophorewhichplayanessentialroleinregulatingthemembraneelongationandexpansionoftheformingautophagosome.ClassIIIphosphatidylinositol47ThefunctionofBeclin1inautophagyisregulatedbyBcl-2;Bcl-2inhibitsautophagybybindingandsequesteringBeclin1;DissociationofBeclin1fromBcl-2isrequiredforautophagyinduction.Vps34Atg14Vps15Beclin1Bcl-2Atg12isactivatedbyAtg7,transferredtoAtg10(E2conjugatingenzyme)andattachedtoaninternallysineofthesubstrateproteinAtg5covalently.TheAtg12–Atg5conjugatefurtherinteractswithacoiled-coilproteinAtg16,whichlinkstheAtg12–Atg5-Atg16complexintoatetramerbyself-oligomerizationandattachesittothephagophore.AutophagosomeformationThefunctionofBeclin1ina48Atg8isfirstprocessedbyacysteineprotease,Atg4,exposingaC-terminalglycineresidue.ThesameE1enzymeAtg7activatesAtg8andtransfersitAtg8isfinallyconjugatedtothetargetlipidPEviaanamidebond.Innutrient-richconditions,themajorityofAtg8iscytosolic（16Kd）;uponautophagyinduction,Atg8largelyexistsasthelipid-conjugatedform(14Kd)andislocalizedtobothsidesofthephagophore.Atg8controlsthesizeoftheautophagosome,whichmayresultfromitsability

todeterminemembranecurvature.ThelipidationofAtg8anditsmammalianhomologLC3arewidelyusedtomonitorautophagyinduction..AutophagosomeformationAutophagosomeformation49VesiclefusionandautophagosomebreakdownInmammaliancells,thefusioneventrequiresthelysosomalmembraneproteinLAMP-2andthesmallGTPaseRab7.Afterfusion,degradationoftheinnervesicleisdependentonaseriesoflysosomal/vacuolaracidhydrolases,includingproteinasesAandB(encodedbyPEP4andPRB1,respectively)andthelipaseAtg15inyeastandcathepsinB,D(ahomologofproteinaseA),andLinmammaliancellsTheresultingsmallmoleculesfromthedegradation,particularlyaminoacids,aretransportedbacktothecytosolforproteinsynthesisandmaintenanceofcellularfunctionsunderstarvationconditions.Vesiclefusionandautophagoso501.Transmissionelectronmicroscopy

2.Atg8/LC3detectionandquantification3.SQSTM1/p62andrelatedLC3bindingproteinturnoverassays4.MTOR,AMPKandAtg1/ULK1

5.Additionalautophagy-relatedmarkers6.Transcriptionalandtranslationalregulation

7.Autophagicproteindegradation8.Selectivetypesofautophagy

MethodsforMonitoringAutophagy9.Autophagicsequestrationassays

10.Turnoverofautophagiccompartments

11.Autophagosome-lysosomecolocalizationanddequenchingassay12.Tissuefractionation13.Analysesinvivo14.Celldeath

15.Chaperone-mediatedautophagy1.Transmissionelectronmicro51Transmissionelectronmicroscopy一、取材快速、低温细胞株：胰酶消化：细胞完整性保存良好，但是对自噬有一定影响细胞刮片：最大程度维持细胞原生理状态，细胞物理损伤大先固定，后刮取：细胞完整性、生理性维持良好，但细胞分散，不易成团组织：优选低温灌注固定时间点：1~2h，8h，24hTransmissionelectronmicrosco52Transmissionelectronmicroscopy二、结构特点Autophagosomes

：1~2h,8hdoublemembrane，visibleastwoparallelmembranebilayersseparatedbyanelectron-lucentcleftcontaincytosoland/ororganellesthatlookmorphologicallyintactAmphisomescansometimesbeidentifiedbythepresenceofsmallinternalvesiclesinsidetheautophagosome/autophagicvacuole(AV).Theseinternalvesiclesaredeliveredintothelumenbyfusionwithmultivesicularendosomes.Late/degradativeautophagicvacuolesandautolysosomes(AVd)

：24husuallyhaveonlyonelimitingmembrane,andcontaincytoplasmicmaterialand/ororganellesatvariousstagesofdegradation

Transmissionelectronmicrosco53TransmissionelectronmicroscopyTransmissionelectronmicrosco54CautionarynotesFixationofexcisedtissuesrequirescaretoavoidsamplinganonrepresentativeoruninformativesectionoftissue.Quantifyautophagosome(and/orautolysosome)profilespertotalcytoplasmicorcellularareainsections.Atleast20cellprofilespersample.Eachimagedcellprofileiscapturedandscoredatthesamemagnification.CautionarynotesFixationofex55CautionarynotesNotalldouble-membranestructuresareautophagosomesApoptoticbodiesfromneighboringcellsarereadilyphagocytosedbysurvivingcellsofthesametissue.Phagosomeshavedoublelimitingmembranes,inneroneisfromtheplasmamembraneoftheapoptoticbodyandtheouteroneisthatofthephagocytizingcell.Amajordifference,isthatthesurroundingmembranesarethethicker

thanthethinnersequestrationmembranetype

(9–10nm,vs.7–8nm,respectively).Agoodfeaturetodistinguishbetweenautophagosomesanddoubleplasmamembrane-boundstructuresisthelackofthedistendedemptyspacebetweenthetwomembranesofthephagocyticvacuoles.Engulfedapoptoticbodiesusuallyhavea

largeraveragesizethanautophagosomes.CautionarynotesNotalldouble56DuetothecisternalstructureoftheER,doublemembrane-likestructuressurroundingmitochondriaorotherorganellesareoftenobservedaftersectioning.EmploytomographicreconstructionsoftheTEMimagestoconfirmthattheautophagiccompartmentsaresphericalandarenotbeingconfusedwithendomembranecisternaeordamagedmitochondria.Ifthereareribosomesassociatedwiththesemembranestheycanhelpdistinguishthemfromtheribosomefreedouble-membraneofthephagophoreandautophagosome.CautionarynotesDuetothecisternalstructure57a.Westernblottingandubiquitin-likeproteinconjugationsystems

b.TurnoverofLC3-II/Atg8–PEc.GFP-Atg8/LC3lysosomaldeliveryandproteolysis

d.GFP-Atg8/LC3fluorescencemicroscopye.TandemmRFP/mCherry-GFPfluorescencemicroscopy

f.Autophagicfluxdeterminationusingflowandmultispectralimagingcytometry

g.Immunohistochemistry.

Atg8/LC3detectionandquantificationa.Westernblottingandubiqui58WesternblottingThemammalianhomologsofAtg8LC3(microtubule-associatedprotein1lightchain3)LC3A,B,B2andCGABARAP:GABAAreceptor-associatedproteinGABARAPL1/GEC1:GABAAreceptorassociatedproteinlike1/GlandularEpithelialCell1GABARAPL2/GATE-16/GEF2:GABAAreceptor-associatedproteinlike2/Golgi-associatedATPaseenhancerof16kDa/gangliosideexpressionfactor2GABARAPL3:GABAAreceptorassociatedproteinlike3WesternblottingThemammalian59Thereisnotalwaysaclearprecursor/productrelationshipbetweenLC3-IandLC3-II,changesinLC3-IIamountsaretissue-andcellcontext-dependentMoreover,LC3-IismorelabilethanLC3-II,beingmoresensitivetofreezingthawingandtodegradationinSDSsamplebuffer,freshsamplesshouldbeheatedandassessedassoonaspossibleandshouldnotbesubjectedtorepeatedfreeze-thawcycles.PVDFmembranesmayresultinastrongerLC3-IIretentionthannitrocellulosemembranesTritonX-100maynotefficientlysolubilizeLC3-IIinsomesystemsHeatinginthepresenceof1%SDS,oranalysisofmembranefractions,mayassistinthedetectionofthisprotein.Insomecasesbeta-actinlevelsdecreasewhenautophagyisinducedCautionary

notes:Thereisnotalwaysaclearpr60WesternblottingWesternblotting61自噬_检测指南课件62TurnoverofLC3-II/Atg8–PE.PreventLysosomalDegradationAutophagicfluxcanbemeasuredbyinferringLC3-II/Atg8–PEturnoverbywesternblotinthepresenceandabsenceoflysosomaldegradation;TherelevantparameterinthisassayisthedifferenceintheamountofLC3-IIinthepresenceandabsenceofsaturatinglevelsofinhibitors;Iffluxisoccurring,theamountofLC3-IIwillbehigherinthepresenceoftheinhibitor.TurnoverofLC3-II/Atg8–PE.Pre63ProteaseinhibitorspepstatinAandE-64d:Neutralizethely