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1、CELL POLARITY: FUNCTIONS AND REGULATIONPlant Cell and Developmental BiologyOrganizers:Tetsuya Higashiyama,Nagoya University, JapanBo Liu, University ofCaliformia, Davis, USAHong Ma, FudanUniversity/Pen StateUniversity, China/USAZhenbiao Yang, University ofCalifornia Riverside, USA Keynote Speakers:J
2、iayang Li, ChinaNatasha Raikhel, USAMajor Topics:1.Membrane, organelle remodeling, and cytoskeleton2.Cell division cycle: meiosis and mitosis for plant growth regulation3.The regulation of cell polarity in plants4.Intracellular and intercellular signaling5.Sexual plant reproduction: from gametogenes
3、is to embryogenesis6.Morphogenesis: from cells to tissues7.Technologies for plant cell and developmental biology More detailed information is available at www.csh-asia.orgJune 17-21, 2013Suzhou, ChinaCell Polarity: the asymmetric property of a cellFUNCTIONS OF CELL POLARITYExamples of axialized and
4、polarized cells in plants. (a) The concepts of axiality and polarity. (b) Sperm entry and axis fixation in algae. (c) Asymmetric division of Arabidopsis zygote and hypophyseal cell. (d) Axialized and polarized cells in the multicellular context of the Arabidopsis root. Note polarity at the organ lev
5、el associated with distal auxin accumulation. (e) Pollen tube growth. (f) Trichome branch initiation. (g)Cytoskeletal components guiding the asymmetric subsidiary mother cell division (smc) in maize. gmc, guard mother cell. Black or shaded circles represent the localization of the proteins indicated
6、 next to these symbols.(Glebe et al. Curr Opin Plant Biol 2001 Dec;4(6):520-6)Cell Polarity: Critical for Plant Development Different levels of cell polarity expressionPolar distribution of moleculesCytoplasmic polarity and asymmetric cell divisionPolar cell expansionPolarized zygotes divide asymmet
7、ricallyApical PIN1 localization & auxin gradientsPIN1Polar cell growthLeaf epidermis zygoteRootCell MorphogenesisPolar Growth is critical for cell morphogenesis and sexual reproduction PollinationPollen tube guidanceEmbryogenesisDifferentiation of guard cellsPolar Distribution of the Cytoplasmnucleu
8、snucleusAsymmetric Cell Division (ACD): Cell Fate DiversificationMother CellDaughterDaughterAMother CellDaughter Daughter Symmetric Cell DivisionAsymmetric Cell Division （ （common property of stem cells) AAAAABequal cell fatesdifferent cell fatesSignificance of stem cell asymmetric division in multi
9、cellular organism developmentAsymmetric cell divisionDifferentiationSelf-renewalSignificance of stem cell asymmetric division in multicellular organism developmentNormal neuroblasts ACDAbnormal neuroblasts ACD-Proliferating cells (tumor)Typical asymmetric cell divisions in plantsArabidopsis embryo d
10、evelopmentpollen developmentTypical asymmetric cell divisions in plantsRoot DevelopmentLateral Root InitiationStomata Patterning and DevelopmentThree steps of asymmetric cell division (ACD)1.Specification of a subset of cells for ACD2.Numerous cellular eventspolar localization of proteinsestablishme
11、nt of gradientscorrect positioning of the nucleuspolar accumulation of cytoplasmformation of PPB band and cell plate3.Asymmetrically inherited cell fate determinants or asymmetrically located external signals decide the cell identity and future differentiation and development Three steps of asymmetr
12、ic cell division (ACD)step 1step 2step 3Most extensively studied three developmental contexts for mechanisms of ACD in plantsEmbryo developmentStomata Patterning and DevelopmentRoot DevelopmentLife starts with asymmetry: Division of the zygoteEmbryo developmentThe first division of a new life!ACDThe
13、 external cues specify division asymmetry?Embryo developmentZygotic ACDExternal Cues?-Not identified yet.Embryo developmentMAPK pathway executes the first ACD YDA: MAPKKKSSP: membrane RLKFailure of zygote expansion Defective zygotic ACDLoss of basal cell fateEmbryo developmentSSP(RAK)External signal
14、?Nuclear EnvelopeZygote PMYDA (MAPKKK)YDAMAPK3, 6gene expressioncytoplasmic activityZygote successful ACD Intrinsic determinants: WOX homeobox gene familyEmbryo developmentWOX2WOX8WOX2WOX8Differential expression of WOX2 and WOX8 (transcriptional factors) in the apical and basal cell, respectivelywtw
15、ox8;9ACDs to generate root layersRoot Stem Cell Niche cortexendodermisQCEpidermisLateral root capcolumellaPositive feedback loop between SHR and SCRRoot Stem Cell Niche transcription factor movement and sequestration SHR mRNA made in stele cellsSHR protein diffused in stele neighbor cellsSHR protein
16、 initiates SCR expressionSCR binds to SHR for more expression of SCRpositive feedback loop between SHR and SCR limits both in endodermal cellsACDs in dispersed population: the stomata lineageStomatal Patterning and DevelopmentMonocots (Maize)Dicots (Arabidopsis) MMMeristemoidGMCGMCGuard Mother CellG
17、CsGCsGCGuard CellSLGCSLGCSLGCStomatal Lineage Ground CellPavement (PC)Stomatal Development and Cell TypesStomatal Patterning and DevelopmentStem cell behavior of the stomata lineageStomatal DevelopmentStomatal DevelopmentStomatal PatterningStomatal PatterningStomatal Patterning and DevelopmentIntrin
18、sic and extrinsic regulation in the stomata lineageCurrently one of the best understood modelsIntrinsic factors: Intrinsic factors: transcription factor (SPCH), unknown (BASL), etc. Extrinsic factors: Extrinsic factors: Ligands (EPF family), RLP/RLKs (TMM, ER family), etc.Stomatal Patterning and Dev
19、elopmentMaster transcription factor: SPCHLESS (SPCH, bHLH)Absence of the stomatal lineage in spch-1 mutantStomatal Patterning and DevelopmentEntryTransitionDifferentiationMMCGMCSLGCMGCsPCSeries of transcription factors for stomatal developmentStomatal Patterning and DevelopmentMaster transcription f
20、actor: SPCHLESS (SPCH, bHLH)EntryTransitionDifferentiationMMCGMCSLGCMGCsPCStomatal Patterning and DevelopmentEntryTransitionDifferentiationMMCGMCSLGCMGCsPCIntrinsic regulatory factor: beyond the master SPCHStomatal Patterning and DevelopmentThe Conserved Model for Animal ACDNeuroblast (NB)Polarity P
21、roteins (Par-aPKC complex)NBNeuronsCell Fate Determinants ACD(Example: Neuroblasts ACD)Stomatal Patterning and DevelopmentBASL: Breaking of Asymmetry in the Stomatal LineageStomatal Patterning and Developmentbasl phenotypeBASL: Breaking of Asymmetry in the Stomatal LineageWTbasl?Stomatal Patterning
22、and DevelopmentBASL: Breaking of Asymmetry in the Stomatal LineageMUTE- Meristemoid Cell Fate MarkerWTbaslStomatal Patterning and DevelopmentBASL localization in the stomatal lineageStomatal Patterning and DevelopmentBASL localization in the stomatal lineageStomatal Patterning and DevelopmentStomata
23、l Patterning and DevelopmentBASL functions at the cell periphery region, polarizedNICBASLBASL-IC; baslPeriphery & PolarizationFunctionalNucleusBASL-N; baslNot Sufficient Stomatal Patterning and DevelopmentBASL Polarization, Cell Outgrowth & Stomatal ACDBASLStomatal Patterning and DevelopmentPlasma M
24、embraneNuclear EnvelopeYDA (MAPKKK)MAPKK 4,5MAPK 3,6Stomatal Patterning and DevelopmentEPF1 EPF2 STOMAGENTMMERfSPCHExtrinsic factors and mediatorsIntrinsic factorROLES OF POLAR CELL EXPANSIONAsymmetric cell divisionCell morphogenesisPollen tube elongation and guidanceCell MorphogenesisPolar Growth i
25、s critical for cell morphogenesis and sexual reproduction PollinationPollen tube guidanceMechanisms of Cell PolarizationApical-basal Polarity(single cell)Planar Cell Polarity(Cell-Cell Interaction)Budding yeastWormembryoPollentubeWinghairEmbryoConvergentextensionLeaf epidermisPIN1-GFP in SAMEstablis
26、hing polaritySelecting a polar siteMaintaining polarity/polar growthCytoskeleton polarization/ vesicular traffickingPolarity FormationPolarity cueRho GTPaseSignaling paradigm for cell polarity formation in single cellsThe CytoskeletonA network of interconnected fibrous protein polymers that run thro
27、ugh the cell within the cytoplasmThe cytoskeleton has four general functionsA) Provide structural stability to the cytoplasm, anchoring proteins, macromolecules, and supporting organelles B) Provide mechanisms for cell movement and molecular and organellar traffickingThe cytoskeleton has four genera
28、l functions:C) Participates in processing cellular information, particularly spatial information. D) Provides directional cues within the cell due to its polarityApical-basal Polarity(single cell)Planar Cell Polarity(Cell-Cell Interaction)Budding yeastWormembryoPollentubeWinghairEmbryoConvergentexte
29、nsionLeaf epidermisPIN1-GFP in SAMPollen Tube Growth and Guidance: An intricate growth control in time and spaceSpatial control:Germination sitePolar cell expansion (tip growth)Guided growthDevelopmental and temporal control:Signals from stigma, style, and ovuleVarying growth rates in different spec
30、iesAnalogy to Neuron guidanceStimgaOvulePollen tubePollen grainSimilar to other tip growing systems, e.g., root hairs, fungal hyphae and animal neuronsUniformly shaped “single cells”Robust transient expressionHaploid genomeVarious forms of polarity expressionSimilarity to hyphae and neuronsIn Vitro
31、Pollen Tubes: A Model for Tip Growth. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Actin CablesMicrotubulesVacuoles1NSperm/Veg. nucleusTip GrowthDomain1.What is the molecular mechanism that defines the tip growth domain 2.How does the tip growth domain signal to the growth machinery?3.
32、How is the tip growth domain maintained and regenerated during rapid oscillatory growth?4.How does the intracellular molecular network coordinate cell wall mechanics in tip growthWhat is the molecular mechanism that defines the tip growth domain?ROPGTPaseCdc42Rho2ScRhoAHsRhoCHsRhoBHsRho1ScCdc42pHsCd
33、c42bHsCdc42ScRac1 HsRac2 HsRop4Rop3Rop5Rop2Rop7Rop6Rop1Rop8Rop9Rop10ROPRacRhoRop11Fungi & animalsArabidopsisZheng & Yang. 2000. Plant Mol Biol. 44:1-9 Rop: the sole and unique subfamily of Rho small GTPases from plantsROP: Rho-like GTPase from plantsSmall GTPase: A Binary Switch That IntegratesMulti
34、ple Inputs, Coordinates Multiple Outputs, and Involves Feedback RegulationMultipleEffectors MembraneOffGTPGDPPiGAPOnGDP-RhoRho-GTPGEFCytoskeletal organizationGene expressionH2O2productionCell wall constructionVesicular traffickingGDP-RhoGDIOutInUpstreamSignalsGDI: guanine nucleotide dissociation inh
35、ibitor GEF: guanine nucleotide exchange factorGAP: GTPase-activating proteinPollen-specific transgenic expression of CA-rop1 and DN-rop1 alters pollen tube growthEffectorsUpstreamSignals MembraneOffGTPGDPPiGAPOnGDP-RhoRho-GTPGEFDN (Dominant Negative) mutantCA (Constitutively Active) mutantOutInRo p
36、4Rop 3R op5Rop2Ro p7Rop 6Rop1Ro p 8Rop9Rop10Rop11Pollen-specificPollen-specific transgenic expression of CA-rop1 and DN-rop1 alters pollen tube growthWTDN-ROP1CA-ROP1WTA localized cue activates ROP1Active ROP1 promotes growthModel: A spatially regulated ROP1-dependent pathway controls polar tip grow
37、th in pollen tubes DN-rop1Inhibits ROP1 activationCA-rop1or ROP1 OXCauses depolarized localization of the ROP1 pathwayTip-localized ROP1 activitySmall GTPase: A Binary Switch That IntegratesMultiple Inputs, Coordinates Multiple Outputs, and Involves Feedback RegulationMultipleEffectors MembraneOffGT
38、PGDPPiGAPOnGDP-RhoRho-GTPGEFCytoskeletal organizationGene expressionH2O2productionCell wall constructionVesicular traffickingGDP-RhoGDIOutInUpstreamSignalsGDI: guanine nucleotide dissociation inhibitor GEF: guanine nucleotide exchange factorGAP: GTPase-activating proteinRICsGFP-RIC4 localization is
39、a marker for ROP1 activity and forms an apical cap during pollen tube growthROP1GDPGTPRIC4RIC4 specifically binds GTP-ROP1GFP-RIC4ROP 1 activity distribution is correlated with tube widthHwang et al. 2010.J. Cell Sci.A rapid burst of GFP-RIC4 localization to the tip leads growth in a spatiotemporal
40、fasionGFP-RIC4 localization to the apical region of the PM (the RIC4 cap) reports ROP1 activity at the tipThe apical cap of active ROP1 defines the tip growth domain and leads growth burst TimeThe ROP1 activity spatially predicts the direction of pollen tube growthThe apical cap of ROP1 activity def
41、ines the tip growth domain and directs pollen tube growthGFP-RIC4How does active ROP1 regulate tip growth?Tip localized active ROP1 regulates apical actin dynamicsWTGAP1OXROP1OXActin MFsActive ROP LocalizationTip-localized activation of ROP1Tip actin dynamics polarized tube growthUnknown Localized C
42、ue(Fu et al. 2001. J Cell Biol 152:1019)Tip localized active ROP1 regulates apical actin dynamicsWTGAP1OXROP1OXActin MFsActive ROP LocalizationTip-localized activation of ROP1Tip actin dynamics polarized tube growthUnknown Localized Cue(Fu et al. 2001. J Cell Biol 152:1019)Check and balance between
43、the RIC3 and RIC4 pathways is critical for polarized tip growth RIC3 affects the tip-focused gradient of intracellular Ca2+ Control RIC3 OX RIC4 OX Actin DynamicsPolymerization(assembly)Depolymerization(disassembly)+-RIC4Tip actin assemblyRIC3Ca2+Tip actin disassemblyROP1Actin DynamicsGu et al. 2005
44、. J Cell Biol. 169:127-139.A model for the ROP1 signaling networkROP1-GDPGrowth cueROP1-GTPCa2+Pollen tube tip growthRIC3RIC4F-actinassemblyActindynamicsRopGAP1REN1Actin disassemblyRIP1How does actin dynamics control polarized tip growth?Tip growth requires tip-targeted exocytosis. . . . . . . . . .
45、 . . . . . . . . . . . . . . . . . . . . .Actin CablesMicrotubulesVacuoles1NSperm/Veg. nucleusTip GrowthDomainLocalized cueGTPGDPGAP1Ca2+Polarized pollen tube growthRIC3RIC4F-actinassemblyProfilinActindynamicsActin disassembly?Lee et al. 2008.J. Cell Bio.YFP-RabA4d marked exocytic vesicles oscillate
46、 at the tip Lee et al. 2008.J. Cell Bio.Targeting of exocytic vesicles to the tip is mediated by RIC4-dependent actin and is suppressed by calcium RLK-GFP FRAP at the apical PM is a novel means for measuring vesicle fusion ROP1 controls exocytic fusionRIC4 inhibits vesicle fusion by promoting actin
47、assembly Localized cueGTPGDPGAP1Ca2+Polarized pollen tube growthRIC3RIC4F-actinassemblyProfilinActindynamicsActin disassemblyVesicle targetingVesicledockingVesicledockingVesiclefusionThis model may also be applicable to other tip growth systemsWhat is the mechanism underlying the formation of the ap
48、ical cap of ROP1 activity?Oscillation of the apical ROP1 cap indicates a self-organizing mechanism for ROP1 activity regulation: Interlinked positive and negative feedbacksGFP-RIC4 localization to the apical region of the PM (the RIC4 cap) reports ROP1 activity at the tipPositive feedback Hypothesis
49、: ROP1 is activated locally at the tip and is regulated by a positive feedback loop that amplifies laterallyGTPGDPLocalized cueGTP?0 sec30 secGFP-RIC4 time-lapse imagingLocalActivationLateral amplification via position feedback loopRopGEF: A Novel Family of GEFs for Rop GTPasePlant specificContains
50、a conserved domain, DUF315Variable N- and C-terminal regions14 RopGEFs in ArabidopsisGu et al. 2006. Plant CellRopGEF1-induced depolarization is ROP1 activation-dependent, suggesting the involvement of RopGEF1 in the feed-forward loop Both ROP1 activator (RopGEF1) and ROP1-dependent exocytosis are i
51、nvolved in the positive feedback loopsROP1-GDPGrowth cueROP1-GTPRIP1 RIP1ROP1-GTPRIP1RopGEF1ExocytosisRop1 activatorsSuppression of the positive feedback loop is required for cell polarity maintenanceCA-rop1How is the tip growth domain maintained and regenerated during rapid oscillatory growth?Two p
52、ossible mechanisms to restrict ROP1 activity to the tipLateral Inhibition_Global Inhibition_Pollen tubes of ren1 (ROP1 OX enhancer) knockout mutants are severely depolarized ren1-1ren1-2ren1-1,ROP1 OXROP1 OXWTWTHwang et al. 2009. Cur. Bio.PHGAPNCoiled-coilCREN1 encodes a new RopGAP essential for pol
53、arity GTPGDPPiRopGAPGDP-RopRop-GTPREN1CRIBREN1RopGAPRopGAP (GTPase activating protein) acts as negative regulator in ROP signaling by promoting GTP hydrolysis on active ROPsROP localization WTren1-1Does REN1 act laterally or globally?Lateral Inhibition_Global Inhibition_REN1Active ROP1?GFPGFP-REN1+R
54、OP1Similar to active ROP1 and YFP-RabA4b (exocytic vesicles)Apical REN1 localization supports its global actionMathematical modeling is consistent with REN1 mediated global inhibitionIs the REN1 RhoGAP regulated by a feedback mechanism?ROP1-GDPGrowth cueROP1-GTPCa2+Pollen tip growthRIC3RIC4F-actinas
55、semblyActindynamicsREN1GAPActin disassemblyGEFFu et al. 2001. J. Cell Biol 152: 1092-32Gu et al. 2005. J. Cell Biol. 169:127-39Lee et al. 2008. J. Cell Biol. 181:1155-68Polar exocytosisVesicle accumulation to the tipVesicle docking/fusionICR1/RIP1SEC3ExocystLi et al. Mol Plant. 2008. In press Low le
56、vels of LatB and BFA disrupts the inverted cone localization of GFP-REN1 and compromise the function of GFP-REN1 Apical localization and function of REN1 requires F-actin andpolarized exocytosisREN1 is colocalized with and functionally interacts with RabA4d (marker for exocytic vesicles)A model for
57、the generation of the ROP1 capROP1-GDPGrowth cueROP1-GTPPollen tip growthRIC4F-actinassemblyREN1GEFPolar exocytosisVesicle TargetingCa2+RIC3Actin disassemblyVesicle docking/fusionPIPKEndocytosisPIP2 PRKIntegrated Partial Differential Equation Model for the Spatiotemporal Regulation of Tip GrowthAn a
58、nd Liu, unpublishedSimulation of ren1 mutants and model validationSimulating and testing the effect of exocytosis: when exocytosis is strongly inhibited Simulating and testing the effect of exocytosis: when exocytosis is weakly inhibited Integrated Partial Differential Equation Model for the Spatiotemporal Regulation of Tip Growth
