植物生理与分子生物学：Lecture 1 Basics and apical polarity

CELLPOLARITY:FUNCTIONSANDREGULATIONPlantCelland

DevelopmentalBiologyOrganizers:TetsuyaHigashiyama,NagoyaUniversity,JapanBoLiu,UniversityofCaliformia,Davis,USAHongMa,FudanUniversity/PenStateUniversity,China/USAZhenbiaoYang,UniversityofCaliforniaRiverside,USA

KeynoteSpeakers:JiayangLi,ChinaNatashaRaikhel,USAMajorTopics:Membrane,organelleremodeling,andcytoskeletonCelldivisioncycle:meiosisandmitosisforplantgrowthregulationTheregulationofcellpolarityinplantsIntracellularandintercellularsignalingSexualplantreproduction:fromgametogenesistoembryogenesisMorphogenesis:fromcellstotissuesTechnologiesforplantcellanddevelopmentalbiology

MoredetailedinformationisavailableatJune17-21,2013

Suzhou,ChinaCellPolarity:theasymmetricpropertyofacellFUNCTIONSOFCELLPOLARITYExamplesofaxializedandpolarizedcellsinplants.(a)Theconceptsofaxialityandpolarity.(b)Spermentryandaxisfixationinalgae.(c)AsymmetricdivisionofArabidopsiszygoteandhypophysealcell.(d)AxializedandpolarizedcellsinthemulticellularcontextoftheArabidopsisroot.Notepolarityattheorganlevelassociatedwithdistalauxinaccumulation.(e)Pollentubegrowth.(f)Trichomebranchinitiation.(g)Cytoskeletalcomponentsguidingtheasymmetricsubsidiarymothercelldivision(smc)inmaize.gmc,guardmothercell.Blackorshadedcirclesrepresentthelocalizationoftheproteinsindicatednexttothesesymbols.(Glebeetal.CurrOpinPlantBiol2001Dec;4(6):520-6)CellPolarity:CriticalforPlantDevelopment

DifferentlevelsofcellpolarityexpressionPolardistributionofmoleculesCytoplasmicpolarityandasymmetriccelldivisionPolarcellexpansionPolarizedzygotesdivideasymmetricallyApicalPIN1localization&auxingradientsPIN1PolarcellgrowthLeafepidermis

zygoteRootCellMorphogenesisPolarGrowthiscriticalforcellmorphogenesisandsexualreproductionPollinationPollentubeguidanceEmbryogenesisDifferentiationofguardcellsPolarDistributionoftheCytoplasmnucleusnucleusAsymmetricCellDivision(ACD):CellFateDiversification

MotherCellDaughterDaughterAMotherCellDaughterDaughterSymmetricCellDivisionAsymmetricCellDivision（commonpropertyofstemcells)AAAAABequalcellfatesdifferentcellfatesSignificanceofstemcellasymmetricdivisioninmulticellularorganismdevelopmentAsymmetriccelldivisionDifferentiationSelf-renewalSignificanceofstemcellasymmetricdivisioninmulticellularorganismdevelopmentNormalneuroblastsACDAbnormalneuroblastsACD---Proliferatingcells (tumor)TypicalasymmetriccelldivisionsinplantsArabidopsisembryodevelopmentpollendevelopmentTypicalasymmetriccelldivisionsinplantsRootDevelopmentLateralRootInitiationStomataPatterningandDevelopmentThreestepsofasymmetriccelldivision(ACD)SpecificationofasubsetofcellsforACDNumerouscellularevents polarlocalizationofproteinsestablishmentofgradientscorrectpositioningofthenucleuspolaraccumulationofcytoplasmformationofPPBbandandcellplateAsymmetricallyinheritedcellfatedeterminantsorasymmetricallylocatedexternalsignalsdecidethecellidentityandfuturedifferentiationanddevelopmentThreestepsofasymmetriccelldivision(ACD)step1step2step3MostextensivelystudiedthreedevelopmentalcontextsformechanismsofACDinplantsEmbryodevelopmentStomataPatterningandDevelopmentRootDevelopmentLifestartswithasymmetry:DivisionofthezygoteEmbryodevelopmentThefirstdivisionofanewlife!ACDTheexternalcuesspecifydivisionasymmetry?EmbryodevelopmentZygoticACDExternalCues?---------Notidentifiedyet.EmbryodevelopmentMAPKpathwayexecutesthefirstACDYDA:MAPKKKSSP:membraneRLKFailureofzygoteexpansionDefectivezygoticACDLossofbasalcellfateEmbryodevelopmentSSP(RAK)Externalsignal?NuclearEnvelopeZygotePMYDA(MAPKKK)YDAMAPK3,6geneexpressioncytoplasmicactivityZygotesuccessfulACDIntrinsicdeterminants:WOXhomeoboxgenefamilyEmbryodevelopmentWOX2WOX8WOX2WOX8DifferentialexpressionofWOX2andWOX8(transcriptionalfactors)intheapicalandbasalcell,respectivelywtwox8;9ACDstogeneraterootlayersRootStemCellNichecortexendodermisQCEpidermisLateralrootcapcolumellaPositivefeedbackloopbetweenSHRandSCRRootStemCellNichetranscriptionfactormovementandsequestrationSHRmRNAmadeinstelecellsSHRproteindiffusedinsteleneighborcellsSHRproteininitiatesSCRexpressionSCRbindstoSHRformoreexpressionofSCRpositivefeedbackloopbetweenSHRandSCRlimitsbothinendodermalcellsACDsindispersedpopulation:thestomatalineageStomatalPatterningandDevelopmentMonocots(Maize)ACDACDDicots(Arabidopsis)ACDACDACDMM MeristemoidGMCGMC GuardMotherCellGCsGCsGC GuardCellSLGCSLGCSLGC StomatalLineage GroundCell Pavement(PC)StomatalDevelopmentandCellTypesStomatalPatterningandDevelopmentStemcellbehaviorofthestomatalineageACDACDACDStomatalDevelopmentStomatalPatterningStomatalPatterningandDevelopmentIntrinsicandextrinsicregulationinthestomatalineageCurrentlyoneofthebestunderstoodmodels

Intrinsicfactors:transcriptionfactor(SPCH),unknown(BASL),etc.Extrinsicfactors:Ligands(EPFfamily),RLP/RLKs(TMM,ERfamily),etc.StomatalPatterningandDevelopmentMastertranscriptionfactor:SPCHLESS(SPCH,bHLH)Absenceofthestomatallineageinspch-1mutantStomatalPatterningandDevelopmentEntryTransitionDifferentiationMMCGMCSLGCMGCsPCSeriesoftranscriptionfactorsforstomataldevelopmentSPCHMUTEFAMAbHLHStomatalPatterningandDevelopmentMastertranscriptionfactor:SPCHLESS(SPCH,bHLH)EntryTransitionDifferentiationMMCGMCSLGCMGCsPCSPCHMUTEFAMAStomatalPatterningandDevelopmentEntryTransitionDifferentiationMMCGMCSLGCMGCsPCIntrinsicregulatoryfactor:beyondthemasterSPCHSPCHHOW?StomatalPatterningandDevelopmentTheConservedModelforAnimalACD

Neuroblast(NB)PolarityProteins(Par-aPKCcomplex)NBNeuronsCellFateDeterminantsACD(Example:NeuroblastsACD)StomatalPatterningandDevelopmentBASL:BreakingofAsymmetryintheStomatalLineageStomatalPatterningandDevelopmentbaslphenotypeBASL:BreakingofAsymmetryintheStomatalLineageWTbasl??????StomatalPatterningandDevelopmentBASL:BreakingofAsymmetryintheStomatalLineageMUTE---MeristemoidCellFateMarkerWTbaslStomatalPatterningandDevelopmentBASLlocalizationinthestomatallineageStomatalPatterningandDevelopmentBASLlocalizationinthestomatallineageStomatalPatterningandDevelopmentStomatalPatterningandDevelopmentBASLfunctionsatthecellperipheryregion,polarizedNICBASLBASL-IC;baslPeriphery&PolarizationFunctionalNucleusBASL-N;baslNotSufficientStomatalPatterningandDevelopmentBASLPolarization,CellOutgrowth&StomatalACD

BASLStomatalPatterningandDevelopmentPlasmaMembraneNuclearEnvelopeYDA(MAPKKK)MAPKK4,5MAPK3,6StomatalPatterningandDevelopmentEPF1EPF2STOMAGENTMMERfSPCHExtrinsicfactorsandmediatorsIntrinsicfactorROLESOFPOLARCELLEXPANSIONAsymmetriccelldivisionCellmorphogenesisPollentubeelongationandguidanceCellMorphogenesisPolarGrowthiscriticalforcellmorphogenesisandsexualreproductionPollinationPollentubeguidanceMechanismsofCellPolarizationApical-basalPolarity(singlecell)PlanarCellPolarity(Cell-CellInteraction)BuddingyeastWormembryoPollentubeWinghairEmbryoConvergentextensionLeafepidermisPIN1-GFPinSAMEstablishingpolaritySelectingapolarsiteMaintainingpolarity/polargrowthCytoskeletonpolarization/vesiculartraffickingPolarityFormationPolaritycueRhoGTPaseSignalingparadigmforcellpolarityformationinsinglecellsTheCytoskeletonAnetworkofinterconnectedfibrousproteinpolymersthatrunthroughthecellwithinthecytoplasmThecytoskeletonhasfourgeneralfunctionsA)Providestructuralstabilitytothecytoplasm,anchoringproteins,macromolecules,andsupportingorganellesB)ProvidemechanismsforcellmovementandmolecularandorganellartraffickingThecytoskeletonhasfourgeneralfunctions:C)Participatesinprocessingcellularinformation,particularlyspatialinformation.D)ProvidesdirectionalcueswithinthecellduetoitspolarityApical-basalPolarity(singlecell)PlanarCellPolarity(Cell-CellInteraction)BuddingyeastWormembryoPollentubeWinghairEmbryoConvergentextensionLeafepidermisPIN1-GFPinSAMPollenTubeGrowthandGuidance:AnintricategrowthcontrolintimeandspaceSpatialcontrol:GerminationsitePolarcellexpansion(tipgrowth)GuidedgrowthDevelopmentalandtemporalcontrol:Signalsfromstigma,style,andovuleVaryinggrowthratesindifferentspeciesAnalogytoNeuronguidanceStimgaOvulePollentubePollengrainSimilartoothertipgrowingsystems,e.g.,roothairs,fungalhyphaeandanimalneuronsUniformlyshaped“singlecells”RobusttransientexpressionHaploidgenomeVariousformsofpolarityexpressionSimilaritytohyphaeandneuronsInVitroPollenTubes:AModelforTipGrowth............................................ActinCablesMicrotubulesVacuoles1NSperm/Veg.nucleusTipGrowthDomain

WhatisthemolecularmechanismthatdefinesthetipgrowthdomainHowdoesthetipgrowthdomainsignaltothegrowthmachinery?Howisthetipgrowthdomainmaintainedandregeneratedduringrapidoscillatorygrowth?HowdoestheintracellularmolecularnetworkcoordinatecellwallmechanicsintipgrowthWhatisthemolecularmechanismthatdefinesthetipgrowthdomain?ROPGTPaseCdc42Rho2ScRhoAHsRhoCHsRhoBHsRho1ScCdc42pHsCdc42bHsCdc42ScRac1HsRac2HsRop4Rop3Rop5Rop2Rop7Rop6Rop1Rop8Rop9Rop10ROPRacRhoRop11Fungi&animalsArabidopsisZheng&Yang.2000.PlantMolBiol.44:1-9Rop:thesoleanduniquesubfamilyofRhosmallGTPasesfromplantsROP:Rho-likeGTPasefromplantsSmallGTPase:ABinarySwitchThatIntegratesMultipleInputs,CoordinatesMultipleOutputs,andInvolvesFeedbackRegulationMultipleEffectors

MembraneOffGTPGDPPiGAPOnGDP-RhoRho-GTPGEFCytoskeletalorganizationGeneexpressionH2O2productionCellwallconstructionVesiculartraffickingGDP-RhoGDIOutInUpstreamSignalsGDI:guaninenucleotidedissociationinhibitorGEF:guaninenucleotideexchangefactorGAP:GTPase-activatingproteinPollen-specifictransgenicexpressionofCA-rop1andDN-rop1alterspollentubegrowthEffectorsUpstreamSignals

MembraneOffGTPGDPPiGAPOnGDP-RhoRho-GTPGEFDN(DominantNegative)mutantCA(ConstitutivelyActive)mutantOutInRop4Rop3Rop5Rop2Rop7Rop6Rop1Rop8Rop9Rop10Rop11Pollen-specificPollen-specifictransgenicexpressionofCA-rop1andDN-rop1alterspollentubegrowthWT DN-ROP1 CA-ROP1WTAlocalizedcueactivatesROP1ActiveROP1promotesgrowthModel:AspatiallyregulatedROP1-dependentpathwaycontrolspolartipgrowthinpollentubesDN-rop1InhibitsROP1activationCA-rop1orROP1OXCausesdepolarizedlocalizationoftheROP1pathwayTip-localizedROP1activitySmallGTPase:ABinarySwitchThatIntegratesMultipleInputs,CoordinatesMultipleOutputs,andInvolvesFeedbackRegulationMultipleEffectors

MembraneOffGTPGDPPiGAPOnGDP-RhoRho-GTPGEFCytoskeletalorganizationGeneexpressionH2O2productionCellwallconstructionVesiculartraffickingGDP-RhoGDIOutInUpstreamSignalsGDI:guaninenucleotidedissociationinhibitorGEF:guaninenucleotideexchangefactorGAP:GTPase-activatingproteinRICsGFP-RIC4localizationisamarkerforROP1activityandformsanapicalcapduringpollentubegrowthROP1GDPGTPRIC4RIC4specificallybindsGTP-ROP1GFP-RIC4ROP1activitydistributioniscorrelatedwithtubewidthHwangetal.2010.J.CellSci.ArapidburstofGFP-RIC4localizationtothetipleadsgrowthinaspatiotemporalfasionGFP-RIC4localizationtotheapicalregionofthePM(theRIC4cap)reportsROP1activityatthetipTheapicalcapofactiveROP1definesthetipgrowthdomainandleadsgrowthburstTimeTheROP1activityspatiallypredictsthedirectionofpollentubegrowthTheapicalcapofROP1activitydefinesthetipgrowthdomainanddirectspollentubegrowthGFP-RIC4HowdoesactiveROP1regulate

tipgrowth?TiplocalizedactiveROP1regulatesapicalactindynamicsWTGAP1OXROP1OXActinMFsActiveROPLocalizationTip-localizedactivationofROP1Tipactindynamics

polarizedtubegrowthUnknownLocalizedCue(Fuetal.2001.JCellBiol152:1019)TiplocalizedactiveROP1regulatesapicalactindynamicsWTGAP1OXROP1OXActinMFsActiveROPLocalizationTip-localizedactivationofROP1Tipactindynamics

polarizedtubegrowthUnknownLocalizedCue(Fuetal.2001.JCellBiol152:1019)CheckandbalancebetweentheRIC3andRIC4pathwaysiscriticalforpolarizedtipgrowthRIC3affectsthetip-focusedgradientofintracellularCa2+

ControlRIC3OXRIC4OXActinDynamicsPolymerization(assembly)Depolymerization(disassembly)+-RIC4TipactinassemblyRIC3Ca2+TipactindisassemblyROP1ActinDynamicsGuetal.2005.JCellBiol.169:127-139.AmodelfortheROP1signalingnetworkROP1-GDPGrowthcueROP1-GTPCa2+PollentubetipgrowthRIC3RIC4F-actinassemblyActindynamicsRopGAP1REN1ActindisassemblyRIP1Howdoesactindynamicscontrolpolarizedtipgrowth?Tipgrowthrequirestip-targetedexocytosis............................................ActinCablesMicrotubulesVacuoles1NSperm/Veg.nucleusTipGrowthDomainLocalizedcueGTPGDPGAP1Ca2+PolarizedpollentubegrowthRIC3RIC4F-actinassemblyProfilinActindynamicsActindisassembly?Leeetal.2008.J.CellBio.YFP-RabA4dmarkedexocyticvesiclesoscillateatthetipLeeetal.2008.J.CellBio.TargetingofexocyticvesiclestothetipismediatedbyRIC4-dependentactinandissuppressedbycalciumRLK-GFPFRAPattheapicalPMisanovelmeansformeasuringvesiclefusionROP1controlsexocyticfusionRIC4inhibitsvesiclefusionbypromotingactinassemblyLocalizedcueGTPGDPGAP1Ca2+PolarizedpollentubegrowthRIC3RIC4F-actinassemblyProfilinActindynamicsActindisassemblyVesicletargetingVesicledockingVesicledockingVesiclefusionThismodelmayalsobeapplicabletoothertipgrowthsystemsWhatisthemechanismunderlyingtheformationoftheapicalcapofROP1activity?OscillationoftheapicalROP1capindicatesaself-organizingmechanismforROP1activityregulation:InterlinkedpositiveandnegativefeedbacksGFP-RIC4localizationtotheapicalregionofthePM(theRIC4cap)reportsROP1activityatthetipPositivefeedbackHypothesis:ROP1isactivatedlocallyatthetipandisregulatedbyapositivefeedbackloopthatamplifieslaterallyGTPGDPLocalizedcueGTP?0sec30secGFP-RIC4time-lapseimagingLocalActivationLateralamplificationviapositionfeedbackloopRopGEF:ANovelFamilyofGEFsforRopGTPasePlantspecificContainsaconserveddomain,DUF315VariableN-andC-terminalregions14RopGEFsinArabidopsisGuetal.2006.PlantCellRopGEF1-induceddepolarizationisROP1activation-dependent,suggestingtheinvolvementofRopGEF1inthefeed-forwardloopBothROP1activator(RopGEF1)andROP1-dependentexocytosisareinvolvedinthepositivefeedbackloopsROP1-GDPGrowthcueROP1-GTPRIP1RIP1ROP1-GTPRIP1RopGEF1ExocytosisRop1activatorsSuppressionofthepositivefeedbackloopisrequiredforcellpolaritymaintenanceCA-rop1Howisthetipgrowthdomainmaintainedandregeneratedduringrapidoscillatorygrowth?TwopossiblemechanismstorestrictROP1activitytothetipLateralInhibition______GlobalInhibition______Pollentubesofren1(ROP1OXenhancer)knockoutmutantsareseverelydepolarizedren1-1ren1-2ren1-1,ROP1OXROP1OXWTWTHwangetal.2009.Cur.Bio.PHGAPNCoiled-coilCREN1encodesanewRopGAPessentialforpolarity

GTPGDPPiRopGAPGDP-RopRop-GTPREN1CRIBREN1RopGAPR