自噬-檢測(cè)指南課件

細(xì)胞自噬研究方法概述研究生：王穎導(dǎo)師：劉乃豐教授細(xì)胞自噬研究方法概述研究生：王穎1AutophagicCompartmentsAutophagicCompartments2AutophagicCompartmentsPhagophore(pre-autophagosomal)：

previouslycalledtheisolationorsequestrationmembrane吞噬泡：參與自噬體形成早期事件的膜池。也指“隔離膜isolationmembrane”或“杯狀結(jié)構(gòu)cup-shapedstructure”Autophagosome:自噬體：雙層膜包裹胞質(zhì)形成的囊泡Amphisome

：generatedbythefusionofautophagosomeswithendosomes,alsoreferredtoasanacidiclateautophagosome自噬內(nèi)涵體：溶酶體和內(nèi)涵體融合的中間囊泡Autolysosome

：generatedbyfusionofautophagosomesoramphisomeswithalysosome自噬溶酶體：自噬小體和溶酶體融合形成的終末結(jié)構(gòu)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、低溫細(xì)胞株：胰酶消化：細(xì)胞完整性保存良好，但是對(duì)自噬有一定影響細(xì)胞刮片：最大程度維持細(xì)胞原生理狀態(tài)，細(xì)胞物理?yè)p傷大先固定，后刮?。杭?xì)胞完整性、生理性維持良好，但細(xì)胞分散，不易成團(tuán)組織：優(yōu)選低溫灌注固定時(shí)間點(diǎn)：1~2h，8h，24hTransmissionelectronmicrosco13Transmissionelectronmicroscopy二、結(jié)構(gòu)特點(diǎn)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_檢測(cè)指南課件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：氯化銨BlockfusionofautophagosomeswithlysosomesbafilomycinA1Knockingdownorknockingoutlysosomal-associatedmembraneprotein2(LAMP2)PreventLysosomalDegradation注：抑制劑作用時(shí)間：1~2h；

設(shè)陽(yáng)性對(duì)照組

時(shí)間點(diǎn)：4h/24hbafilomycinA1,NH4Clorchloroquine,alsodirectlyinhibittheendocytosis/uncoatingofvirusesandotherendocyticeventsrequiringlowpHmonitorbothturnoverofLC3-IIandanautophagosomesubstrateinparallel.ProteaseinhibitorsPreventLy251hofpre-incubationwith10mg/mlE-64dissufficientinmostcases,sincethisinhibitorismembranepermeableandrapidlyaccumulateswithinlysosomes.pepstatinAismembraneimpermeable(ethanolorpreferablyDMSOmustbeemployedasavehicle)andrequiresaprolongedincubation(.8h)andarelativelyhighconcentration(.50mg/ml)tofullyinhibitlysosomalcathepsinD1hofpre-incubationwith1026GFP-Atg8/LC3lysosomaldeliveryandproteolysisWersternblotGFP-LC3在自噬溶酶體的酸性環(huán)境中被降解GFP單體釋放到胞質(zhì)中，蛋白印跡檢測(cè)GFP單體條帶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自噬體和自噬溶酶體分別成黃色和紅色標(biāo)記，如果自噬潮增加，兩種顏色的點(diǎn)狀聚集均增加。如果自噬體向自噬溶酶體成熟受阻，黃色點(diǎn)狀聚集物增加，紅色不增加。自噬體和自噬溶酶體分別成黃色和紅色標(biāo)記，如果自噬潮增加，兩種30Flowandmultispectralimagingcytometry在自噬誘導(dǎo)一開(kāi)始，GFP-LC3點(diǎn)狀聚集顯著增多，隨后信號(hào)可能會(huì)出現(xiàn)下降，代表著自噬性降解的發(fā)生．高通量檢測(cè)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根據(jù)自噬機(jī)制主要負(fù)責(zé)降解長(zhǎng)壽命蛋白的特性，先讓細(xì)胞在含有同位素標(biāo)記氨基酸(如14C-或3H-纈氨酸或亮氨酸)的培養(yǎng)基中生長(zhǎng)一段時(shí)間(數(shù)小時(shí)至數(shù)天)細(xì)胞在此期間合成的蛋白質(zhì)都將被同位素標(biāo)記，然后換成不含同位素的培養(yǎng)基，讓一些被標(biāo)記的短壽命蛋白通過(guò)蛋白酶體途徑降解．在自噬誘導(dǎo)后，通過(guò)檢測(cè)培養(yǎng)上清中釋放的自噬性降解產(chǎn)物的放射性活度即可反映細(xì)胞自噬性降解的能力．同時(shí)加入自噬抑制劑作為對(duì)比，更能特異性地反映自噬引起的蛋白質(zhì)降解.長(zhǎng)壽命蛋白降解檢測(cè)根據(jù)自噬機(jī)制主要負(fù)責(zé)降解長(zhǎng)壽命蛋白的特性，先讓細(xì)胞在含有同位36自噬的實(shí)驗(yàn)性調(diào)控

二、基因干預(yù)通過(guò)RNAi干擾Atg3、Atg5、Atg7及Beclin1等自噬相關(guān)基因的表達(dá)后，細(xì)胞表現(xiàn)為自噬功能缺失．與工具藥相比，通過(guò)基因沉默或敲除技術(shù)來(lái)抑制自噬具有相對(duì)強(qiáng)的特異性．一、藥物干預(yù)自噬的實(shí)驗(yàn)性調(diào)控

二、基因干預(yù)一、藥物干預(yù)37謝謝聆聽(tīng)！謝謝聆聽(tīng)！38謝謝觀看！2020

謝謝觀看！39細(xì)胞自噬研究方法概述研究生：王穎導(dǎo)師：劉乃豐教授細(xì)胞自噬研究方法概述研究生：王穎40AutophagicCompartmentsAutophagicCompartments41AutophagicCompartmentsPhagophore(pre-autophagosomal)：

previouslycalledtheisolationorsequestrationmembrane吞噬泡：參與自噬體形成早期事件的膜池。也指“隔離膜isolationmembrane”或“杯狀結(jié)構(gòu)cup-shapedstructure”Autophagosome:自噬體：雙層膜包裹胞質(zhì)形成的囊泡Amphisome

：generatedbythefusionofautophagosomeswithendosomes,alsoreferredtoasanacidiclateautophagosome自噬內(nèi)涵體：溶酶體和內(nèi)涵體融合的中間囊泡Autolysosome

：generatedbyfusionofautophagosomesoramphisomeswithalysosome自噬溶酶體：自噬小體和溶酶體融合形成的終末結(jié)構(gòu)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、低溫細(xì)胞株：胰酶消化：細(xì)胞完整性保存良好，但是對(duì)自噬有一定影響細(xì)胞刮片：最大程度維持細(xì)胞原生理狀態(tài)，細(xì)胞物理?yè)p傷大先固定，后刮?。杭?xì)胞完整性、生理性維持良好，但細(xì)胞分散，不易成團(tuán)組織：優(yōu)選低溫灌注固定時(shí)間點(diǎn)：1~2h，8h，24hTransmissionelectronmicrosco52Transmissionelectronmicroscopy二、結(jié)構(gòu)特點(diǎn)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_檢測(cè)指南課件62TurnoverofLC3-II/Atg8–PE.PreventLysosomalDegradationAutophagicfluxcanbemeasuredbyinferringLC3-II/Atg8–PEturnoverbywesternblotinthepresenceandabsenceoflysosomaldegradation;TherelevantparameterinthisassayisthedifferenceintheamountofLC3-IIinthepresenceandabsenceofsaturatinglevelsofinhibitors;Iffluxisoccurring,theamountofLC3-IIwillbehigherinthepresenceoftheinhibitor.TurnoverofLC3-II/Atg8–PE.Pre63ProteaseinhibitorspepstatinAandE-64d:Neutralizethely