自噬檢測指南

細胞自噬研究方法概述研究生：王穎導師：劉乃豐教授AutophagicCompartmentsAutophagicCompartmentsPhagophore(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)Induce/promoteInhibitionAutophagicmolecularmechanismsInitiation：Induction，CargorecognitionandselectivityElongation，Closure：AutophagosomeformationMaturation，Degradation:Vesiclefusionandautophagosomebreakdown

AutophagicmolecularmechanismsInductionNormalconditionsBasal-levelautophagyisverylow;Autophagyinhibitor:serine/threonineproteinkinaseTOR(targetofrapamycin)inputinformationfrommultipleupstreamsignaltransductionpathways(discussedbelow)andnegativelyregulatesanotherserine/threoninekinase,Atg1,innutrient-richconditionsStarvationconditonsorRapamycinTORinhibited;Atg1activated;Atg1bindingaffinitytoAtg13andAtg17↑;PromotestheformationofanAtg1-Atg13-Atg17scaffold;Atg1-Atg13-Atg17recruitmentofmultipleAtgproteinstothePAStoinitiateautophagosomeformation.CargorecognitionandselectivityP62/sequestosome1(SQSTM1).P62directlybindsbothpoly-ormono-ubiquitinviaitsubiquitin-associated(UBA)domainandLC3linkstheubiquitinatedcargostotheautophagymachineryforautophagicdegradation.

ClassIIIphosphatidylinositol3-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.ThefunctionofBeclin1inautophagyisregulatedbyBcl-2;Bcl-2inhibitsautophagybybindingandsequesteringBeclin1;DissociationofBeclin1fromBcl-2isrequiredforautophagyinduction.Vps34Atg14Vps15Beclin1Bcl-2Atg12isactivatedbyAtg7,transferredtoAtg10(E2conjugatingenzyme)andattachedtoaninternallysineofthesubstrateproteinAtg5covalently.TheAtg12–Atg5conjugatefurtherinteractswithacoiled-coilproteinAtg16,whichlinkstheAtg12–Atg5-Atg16complexintoatetramerbyself-oligomerizationandattachesittothephagophore.AutophagosomeformationAtg8isfirstprocessedbyacysteineprotease,Atg4,exposingaC-terminalglycineresidue.ThesameE1enzymeAtg7activatesAtg8andtransfersitAtg8isfinallyconjugatedtothetargetlipidPEviaanamidebond.Innutrient-richconditions,themajorityofAtg8iscytosolic（16Kd）;uponautophagyinduction,Atg8largelyexistsasthelipid-conjugatedform(14Kd)andislocalizedtobothsidesofthephagophore.Atg8controlsthesizeoftheautophagosome,whichmayresultfromitsability

todeterminemembranecurvature.ThelipidationofAtg8anditsmammalianhomologLC3arewidelyusedtomonitorautophagyinduction..AutophagosomeformationVesiclefusionandautophagosomebreakdownInmammaliancells,thefusioneventrequiresthelysosomalmembraneproteinLAMP-2andthesmallGTPaseRab7.Afterfusion,degradationoftheinnervesicleisdependentonaseriesoflysosomal/vacuolaracidhydrolases,includingproteinasesAandB(encodedbyPEP4andPRB1,respectively)andthelipaseAtg15inyeastandcathepsinB,D(ahomologofproteinaseA),andLinmammaliancellsTheresultingsmallmoleculesfromthedegradation,particularlyaminoacids,aretransportedbacktothecytosolforproteinsynthesisandmaintenanceofcellularfunctionsunderstarvationconditions.1.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-mediatedautophagyTransmissionelectronmicroscopy一、取材快速、低溫細胞株：胰酶消化：細胞完整性保存良好，但是對自噬有一定影響細胞刮片：最大程度維持細胞原生理狀態(tài)，細胞物理損傷大先固定，后刮?。杭毎暾浴⑸硇跃S持良好，但細胞分散，不易成團組織：優(yōu)選低溫灌注固定時間點：1~2h，8h，24hTransmissionelectronmicroscopy二、結(jié)構(gòu)特點Autophagosomes

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

：24husuallyhaveonlyonelimitingmembrane,andcontaincytoplasmicmaterialand/ororganellesatvariousstagesofdegradation

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

thanthethinnersequestrationmembranetype

(9–10nm,vs.7–8nm,respectively).

Agoodfeaturetodistinguishbetweenautophagosomesanddoubleplasmamembrane-boundstructuresisthelackofthedistendedemptyspacebetweenthetwomembranesofthephagocyticvacuoles.Engulfedapoptoticbodiesusuallyhavea

largeraveragesizethanautophagosomes.DuetothecisternalstructureoftheER,doublemembrane-likestructuressurroundingmitochondriaorotherorganellesareoftenobservedaftersectioning.EmploytomographicreconstructionsoftheTEMimagestoconfirmthattheautophagiccompartmentsaresphericalandarenotbeingconfusedwithendomembranecisternaeordamagedmitochondria.Ifthereareribosomesassociatedwiththesemembranestheycanhelpdistinguishthemfromtheribosomefreedouble-membraneofthephagophoreandautophagosome.Cautionarynotesa.Westernblottingandubiquitin-likeproteinconjugationsystems

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

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

f.Autophagicfluxdeterminationusingflowandmultispectralimagingcytometry

g.Immunohistochemistry.

Atg8/LC3detectionandquantificationWesternblottingThemammalianhomologsofAtg8LC3(microtubule-associatedprotein1lightchain3)LC3A,B,B2andCGABARAP:GABAAreceptor-associatedproteinGABARAPL1/GEC1:GABAAreceptorassociatedproteinlike1/GlandularEpithelialCell1GABARAPL2/GATE-16/GEF2:GABAAreceptor-associatedproteinlike2/Golgi-associatedATPaseenhancerof16kDa/gangliosideexpressionfactor2GABARAPL3:GABAAreceptorassociatedproteinlike3Thereisnotalwaysaclearprecursor/productrelationshipbetweenLC3-IandLC3-II,changesinLC3-IIamountsaretissue-andcellcontext-dependentMoreover,LC3-IismorelabilethanLC3-II,beingmoresensitivetofreezingthawingandtodegradationinSDSsamplebuffer,freshsamplesshouldbeheatedandassessedassoonaspossibleandshouldnotbesubjectedtorepeatedfreeze-thawcycles.PVDFmembranesmayresultinastrongerLC3-IIretentionthannitrocellulosemembranesTritonX-100maynotefficientlysolubilizeLC3-IIinsomesystemsHeatinginthepresenceof1%SDS,oranalysisofmembranefractions,mayassistinthedetectionofthisprotein.Insomecasesbeta-actinlevelsdecreasewhenautophagyisinducedCautionary

notes:WesternblottingTurnoverofLC3-II/Atg8–PE.PreventLysosomalDegradationAutophagicfluxcanbemeasuredbyinferringLC3-II/Atg8–PEturnoverbywesternblotinthepresenceandabsenceoflysosomaldegradation;TherelevantparameterinthisassayisthedifferenceintheamountofLC3-IIinthepresenceandabsenceofsaturatinglevelsofinhibitors;Iffluxisoccurring,theamountofLC3-IIwillbehigherinthepresenceoftheinhibitor.ProteaseinhibitorspepstatinAandE-64d:NeutralizethelysosomalPHbafilomycinA1（洛霉素A1）chloroquine：氯喹NH4Cl：氯化銨BlockfusionofautophagosomeswithlysosomesbafilomycinA1Knockingdownorknockingoutlysosomal-associatedmembraneprotein2(LAMP2)

PreventLysosomalDegradation注：抑制劑作用時間：1~2h；

設(shè)陽性對照組

時間點：4h/24hbafilomycinA1,NH4Clorchloroquine,alsodirectlyinhibittheendocytosis/uncoatingofvirusesandotherendocyticeventsrequiringlowpHmonitorbothturnoverofLC3-IIandanautophagosomesubstrateinparallel.1hofpre-incubationwith10mg/mlE-64dissufficientinmostcases,sincethisinhibitorismembranepermeableandrapidlyaccumulateswithinlysosomes.pepstatinAismembraneimpermeable(ethanolorpreferablyDMSOmustbeemployedasavehicle)andrequiresaprolongedincubation(.8h)andarelativelyhighconcentration(.50mg/ml)tofullyinhibitlysosomalcathepsinDGFP-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GFP單體條帶Cautionarynotes:AreductionintheintensityofthefreeGFPbandmayindicatereducedflux,butitmayalsobeduetoefficientturnover.Usingarangeofconcentrationsandtreatmenttimesofcompoundsthatinhibitautophagycanbeusefulindistinguishingbetweenthesepossibilities.GFP-Atg8/LC3fluorescencemicroscopyFluorescencemicroscopyAconstantincreaseinthenumberofcellsaccumulatingGFP-LC3punctaissuggestiveofdefectivefusionofautophagosomeswithlysosomes;Conversely,adeclineimpliesthatGFP-LC3isconsumedwithinnewlyformedautolysosomes.Fluorescencemicroscopy+lysosomalproteaseorfusioninhibitorsMonitoringchangesinthenumberofpuncta.ThepresenceoflysosomalinhibitorsshouldincreasethenumberofGFP-LC3-positivestructures,andtheabsenceofaneffectonthetotalnumberofGFP-LC3punctaoronthepercentageofcellsdisplayingnumerouspunctaisindicativeofadefect(s)inautophagicflux.

TandemmRFP-GFPfluorescencemicroscopyTheGFPsignalissensitivetotheacidicand/orproteolyticconditionsofthelysosomelumen,whereasmRFPismorestable.

Therefore,colocalizationofbothGFPandmRFPfluorescenceindicatesacompartmentthathasnotfusedwithalysosome,suchasthephagophoreoranautophagosome.Incontrast,anmRFPsignalwithoutGFPcorrespondstoanamphisomeorautolysosome.

OneofthemajoradvantagesofthetandemmRFP/mCherry-GFPreportermethodisthatitenablessimultaneousestimationofboththeinductionofautophagyandfluxthroughautophagiccompartmentsinessentiallynativeconditions,withoutrequiringanydrugtreatment.自噬體和自噬溶酶體分別成黃色和紅色標記，如果自噬潮增加，兩種顏色的點狀聚集均增加。如果自噬體向自噬溶酶體成熟受阻，黃色點狀聚集物增加，紅色不增加。Flowandmultispectralimagingcytometry在自噬誘導一開始，GFP-LC3點狀聚集顯著增多，隨后信號可能會出現(xiàn)下降，代表著自噬性降解的發(fā)生．高通量檢測ImmunohistochemistryWhenautophagosomesareabsent,thelocalizationpatternofLC3inthecellsofvarioustissuesisdiffuseandcytosolic.OneproblemwithimmunohistochemistryforLC3isthatinsometissuesthisproteincanbelocalizedinstructuresotherthanautophagosomes.Forexample,inmurinehepatocytesandcardiomyocytesunderstarvedconditions,endogenousLC3isdetectednotonlyinautophagosomesbutalsoonlipiddroplets.InneuronsinATG7-deficientmice,LC3isaccumulatedinubiquitin-andSQSTM1-positiveaggregates.p62andrelatedLC3bindingproteinturnoverassaysTheSQSTM1proteinservesasalinkbetweenLC3andubiquitinatedsubstrates.decreasedSQSTM1levelsareassociatedwithautophagyactivationThephosphorylationofSQSTM1atSer403appearstoregulateitsroleintheautophagicclearanceofubiquitinatedproteins,andanti-phospho-SQSTM1/p62antibodiescanbeusedtodetectthemodifiedformoftheprotein.WesternblotanalysisusingNP40orTritonX-100lysisinautophagicconditionstypicallyshowsareductioninSQSTM1levels.However,thisdoesnotnecessarilyindicatethatSQSTM1isdegraded,becauseSQSTM1aggregatesareinsolubleinthesedetergentlysisconditions.WhereasLC3changesmayberapid,clearanceof