ASCB 2005 Abstracts - American Society for Cell Biology

789 Pages·2005·7.52 MB·English

Abstracts
The American Society
for Cell Biology
th
45 Annual Meeting
December 10-14, 2005
The Moscone Center
San Francisco
CONTENTS
SATURDAY CoordinationofCytoskeletalNetworks.....................................207
NuclearCompartments..............................................................208
BigScience,LittleScience........................................................4 PathogensCo-optingHostCellFunctions.................................210
RegulatingIntercellularJunctions.............................................211
SUNDAY Signalingin3DEnvironments...................................................213
TraffickingProteins&Complexes............................................215
QuantitativeStudiesofCellSignalingNetworks......................5 SignalTransductionII...............................................................216
BruceAlbertsAwardPresentation............................................5 ApoptosisI.................................................................................224
ProkaryoticOriginsoftheCytoskeleton...................................5 Mitosis&MeiosisII..................................................................231
E.E.JustLecture.......................................................................6 G2-M.........................................................................................237
CargoSorting&VesicularTransport........................................7 CytokinesisI..............................................................................240
CellBiologyoftheSynapses.....................................................8 Actin-AssociatedProteinsII......................................................245
Differentiation&Cancer...........................................................10 ActinDynamics&AssemblyI..................................................250
ExtracellularMatrix&Signaling..............................................11 ConventionalMyosin.................................................................255
Formins&Arp2/3:RegulatorsofActin....................................13 Tubulin......................................................................................260
NuclearEnvelopeFunctions......................................................15 DyneinII....................................................................................262
RegulationoftheCellCycle.....................................................16 MicrotubuleDynamics&AssemblyI.......................................269
SignalingintheImmuneSystem...............................................18 Cilia&FlagellaI.......................................................................275
EBWilsonMedalPresentation&Lecture................................20 CellMotilityII...........................................................................282
GrowthFactors&Receptors.....................................................20 CytoskeletalOrganizationI.......................................................290
SignalTransductionI................................................................26 Cytoskeleton-MembraneInteractionsI......................................296
Mitosis&MeiosisI...................................................................32 CellAttachmenttotheExtracellularMatrix..............................300
G1-S/DNAReplication.............................................................39 ExtracellularMatrix&CellSignalingII...................................306
Actin..........................................................................................42 Cell-CellInteractionsII.............................................................312
Actin-AssociatedProteinsI.......................................................44 OrganizationandRegulationoftheExtracellularMatrix..........316
UnconventionalMyosins...........................................................50 MembraneReceptors.................................................................318
DyneinI.....................................................................................57 ERtoGolgiTransport...............................................................326
Microtubule-AssociatedProteins...............................................60 EndocytosisII............................................................................330
CellMotilityI............................................................................67 ProteinTargetingtotheCellSurface.........................................335
CentrosomesI............................................................................75 ProteinFolding&AssemblyintheEndoplasmic
IntracellularMovement.............................................................79 ReticulumI..............................................................................339
IntermediateFilamentsI............................................................85 MechanismsofNuclearTranscription.......................................342
FocalAdhesions........................................................................91 Chromatin&ChromosomesI....................................................347
Cell-CellAdherensJunctions....................................................96 NuclearImportandExportSignals............................................352
ExtracellularMatrix&CellSignalingI....................................100 Organogenesis...........................................................................355
Cell-CellInteractionsI..............................................................106 CellPolarityII...........................................................................358
MembraneChannels&TransportersI.......................................110 Development&Carcinogenesis................................................362
MembraneFusion......................................................................117 SynapseFormation&FunctionI...............................................364
EndocytosisI.............................................................................119 GolgiComplex..........................................................................368
ProteinTargeting.......................................................................124 EpitheliaII.................................................................................375
GeneStructureandExpression..................................................132 CancerII....................................................................................379
ChromatinRemodeling.............................................................137 MetabolicDiseases....................................................................383
NuclearMatrixandNuclearArchitecture.................................139 OtherDiseasesI.........................................................................391
GermCells&Fertilization........................................................142
CellPolarityI............................................................................148 TUESDAY
Neurotransmitters,Peptides&Receptors..................................152
Chloroplasts&Mitochondria....................................................157 ReprogrammingCellFate..........................................................399
EndoplasmicReticulum.............................................................163 CommunityBuildingtoPromoteCareersinBiomedical
EpitheliaI..................................................................................169 Science.....................................................................................399
Parasitology...............................................................................173 Host-PathogenInteractions........................................................400
CancerI.....................................................................................180 BuildingSensoryNetworks.......................................................401
ImagingTechnology..................................................................186 CoordinatingAdhesion&Signaling..........................................402
MolecularBiology.....................................................................193 CytoskeletalMolecularMotors..................................................404
BloodVessels............................................................................198 IntermediateFilaments..............................................................405
IntersectionofSignaling&Trafficking:SmallGTPases..........407
MONDAY Mitosis&Meiosis.....................................................................409
OrganelleDynamics..................................................................410
WiringtheNervousSystem.......................................................202 ProteinMisfolding&Disease....................................................412
TheNatureofLife:AnOrientationCoursethatIntroduces KeithR.PorterLecture..............................................................413
FreshmentotheDisciplinesofBiology,BuildsCommunity, Oncogenes&TumorSuppressors.............................................414
andTeachesStrategiesforSuccessinCollege........................202 CellCycleControlsI.................................................................419
AdaptingtoStress:SpotlightonOrganelles..............................203 ApoptosisII...............................................................................425
CellMigration/Motility.............................................................203 Mitosis&MeiosisIII.................................................................431
ChromatinDynamics.................................................................205 Kinetochores..............................................................................440
CONTENTS
Pre-CollegeandCollegeScienceEducation..............................447 Caveolae....................................................................................718
Actin-AssociatedProteinsIII....................................................455 TraffickinginPolarizedCells....................................................721
ActinDynamics&AssemblyII................................................461 DevelopmentalControlofGeneExpression..............................726
Muscle:Biochemistry&CellBiologyI....................................466 Ribonucleoproteins....................................................................728
KinesinI....................................................................................470 StructureofNuclearEnvelope...................................................732
MicrotubuleDynamics&AssemblyII......................................474 InvertebrateDevelopment..........................................................737
CellMotilityIII.........................................................................479 SignalTransductioninDevelopment.........................................740
Cytoskeleton-MembraneInteractionsII....................................486 StemCellsII..............................................................................747
ExtracellularMatrix&CellBehaviorI.....................................490 Endosomes&LysosomesII......................................................753
ExtracellularMatrix&Morphogenesis.....................................494 Leukocytes.................................................................................757
Cadherins...................................................................................499 CellCulture...............................................................................760
GapJunctions............................................................................504 CancerIV...................................................................................765
Structure&FunctionofMembraneProteinsI..........................511 NeuronalDiseasesII..................................................................771
GolgitoCellSurfaceTransport.................................................517 OtherDiseasesIII......................................................................778
EndocyticMachinery:Structure,Function&Regulation..........522 Bioinformatics/BiologicalComputing.......................................785
ProteinTargetingtotheEndocyticPathway.............................529 RNAiTechnology......................................................................787
ProteinFolding&AssemblyintheEndoplasmic
ReticulumII.............................................................................534
Tissue-SpecificGeneExpression..............................................538
Chromatin&ChromosomesII..................................................543
MammalianDevelopment.........................................................548
GrowthFactorsinDevelopment................................................555
StemCellsI...............................................................................558
SynapseFormation&FunctionII.............................................564
Endosomes&LysosomesI.......................................................568
EndothelialCells.......................................................................571
CancerIII..................................................................................575
NeuronalDiseasesI...................................................................581
OtherDiseasesII.......................................................................588
WEDNESDAY
CellGrowthandDivision..........................................................594
YeastGenomicsintheClassroom:UsingtheYeastDeletion
CollectiontoStudyEnvironmentalToxinsandFood
Additives.................................................................................594
CytoskeletalDynamicsinLivingCells.....................................595
EpithelialMorphogenesis&Polarity........................................596
Lipid-MediatedSignals.............................................................598
TheMembraneCytoskeleton.....................................................599
NeuronalPolarity&Axo-DendriticGrowth.............................601
ProteinFolding&QualityControl............................................603
RNASilencingMechanisms.....................................................604
StemCellNiches.......................................................................606
SignalTransductionIII..............................................................607
CellCycleControlsII................................................................614
Mitosis&MeiosisIV................................................................621
CytokinesisII............................................................................629
ActinDynamics&AssemblyIII...............................................633
Muscle:Biochemistry&CellBiologyII...................................638
KinesinII...................................................................................643
Cilia&FlagellaII......................................................................648
CellMotilityIV.........................................................................655
CytoskeletalOrganizationII......................................................662
CentrosomesII..........................................................................669
NerveCellCytoskeleton............................................................673
IntermediateFilamentsII..........................................................679
ExtracellularMatrix&CellBehaviorII....................................684
Integrins.....................................................................................689
Metalloproteases........................................................................695
TightJunctions..........................................................................699
Structure&FunctionofMembraneProteinsII.........................705
MembraneDomains..................................................................708
Exocytosis:RegulatedSecretion...............................................713
Saturday
BigScience,LittleScience(1-2)
1
UnravelingSmell
L.Buck;HowardHughesMedicalInstitute,FredHutchinsonCancerResearchCenter,Seattle,WA
Odorantsaredetectedby~1000differentodorantreceptors(ORs),whicharelocatedonolfactorysensoryneuronsinthenose.Ourstudiesshow
that ORs are used combinatorially to detect different odorants and encode their unique identities. Toexplore howthe nervous systemtranslates
odorouschemicalsintoperceptions,weaskedhowinputsderivedfromdifferent mouseORsareorganizedassignalstravel fromthenosetothe
olfactorybulbandthentheolfactorycortex.Inthenose,eachsensoryneuronexpressesasingleORgene.NeuronswiththesameORaredispersed
inthenose,buttheiraxonsconvergeinafewglomeruliattwofixedlocationsinthebulb.Theresultisastereotypedsensorymapinwhichinputs
fromdifferentORsaresegregatedindifferentglomeruliandrelayneurons.Intheolfactorycortex,inputsfromoneORaretargetedtoclustersof
neuronsatspecificsites,creatingastereotypedmapunrelatedtothatinthebulb.IncontrasttothesegregationofdifferentORinputsseeninboth
the nose and bulb, it appears that different OR inputs overlap extensively in the cortex and single neurons receive combinatorial inputs from
multipledifferentORs.Usingc-Fosasanindicatorofneuronalactivator,wefoundthatdifferentodorantselicitdifferent,butpartiallyoverlapping,
activation patterns in the cortex. The representation of each odorant is composed of a small subset of sparsely distributed neurons. Quantitative
analysisoftheodorrepresentationssuggeststhatcorticalneuronsmayfunctionascoincidencedetectorsthatareactivatedonlybycorrelatedinputs
fromdifferentORs.
2
Discovery-DrivenResearch:ANewFrontierintheBiologicalSciences
C.Fraser;TheInstituteforGenomicResearch,Rockville,MD
The application of large-scale approaches to the study of biological questions has produced a fundamental change in the way that we approach
scientific discovery. In this new era, high-throughput technologies are providing enormous amounts of new data and computational biology is
allowingustomakelinksbetweengenomesequenceandbiologicalprocessesandfunction.Theultimategoalofsuchbigscienceistoachievea
predictiveunderstandingofbiology.
4a
Sunday
QuantitativeStudies ofCell SignalingNetworks (3-4)
3
TheDeeperCorrelations:SingleCellMeasuresofKinaseSignalingforMechanisticandClinicalAnalyses
G.Nolan;Microbiology/Immunology,StanfordUniversity,Stanford,CA
Intracellularassaysofsignalingsystemshasbeenlimitedbyaninabilitytocorrelatefunctionalsubsetsofcellsincomplexpopulationsbasedon
activekinasestatesorothernodalsignalingjunctions.Suchcorrelationscouldbeimportanttodistinguishchangesinsignalingstatusthatarisein
rare cell subsets during functional activation or in disease manifestation. Simultaneous detection of activated kinases and phosphoproteins in
simultaneous pathways in subpopulations of complex cell populations by multi-parameter flow cytometric analysis allows identification of
signaling cascades for disease states by ordering of kinase activation and phosphoprotein status in signaling hierarchies. Importantly, we
demonstrate that ordering of these activations requires multiple interrogations of cells, and that thenetworks discovered are reflective of deeper
correlations.UsingBayesianNetworkanalysis(aformofmachinelearning)onecaninferpathwayconnectivityinanautomatedfashion,allowing
forhighthroughputderivationsofsignalingsystemnetworksgraphsinPRIMARYCELLS.Theapproachhaspowerfulapplicationsinmechanistic
understanding,drugscreening,andpatientstratificationforpredictionofdiseaseoutcomeincancer,autoimmunity,infection,basedonsignaling
network status. (1) Irish J.M., Hovland R., Krutzik P.O., Perez O.D., Bruserud O., Gjertsen B.T., Nolan G.P. (2004) Single Cell Profiling of
PotentiatedPhospho-Protein Networksin Cancer Cells. Cell. 118:217-228. (2)SachsK.,PerezO.,Pe'er D., Lauffenburger D.Aand NolanG.P.
2005.Causalprotein-signalingnetworksderivedfrommultiparametersingle-celldata.Science.308:523-9.
4
SystemsBiologyofCytokineSignalinginHumanCells
P. Sorger, D. Lauffenburger, K. Janes, S. Gaudet, J. Albeck, B. Schoeberl; Department of Biology and Biological Engineering, Massachusetts
InstituteofTechnology,Cambridge,MA
Cytokines and their receptors activate complex signaling cascades to regulate cell proliferation, death and differentiation. We seek to develop
quantitative, mechanistic models that describe cytokine-induced signaling with an eye to understanding cell-type variation and the differences
between healthy and diseased states. We focus on decisions controlled by pro-apoptotic cytokines such as Tumor Necrosis Factor (TNF) and
TRAIL and pro-survival cytokines such as EGF and the insulin-like growth factors (IGF). By mining a compendium comprising ~10,000
measurements of signal protein activities elicited by cytokines individually and in combination we can construct both statistical and
physicochemicalmodels.Classifier-basedregressionhasbeenparticularlyhelpfulinestablishingthatcellsrespondtoTNFdirectly,viaactivated
TNFreceptor,andindirectlyviaautocrinecircuitsinvolvingtransforminggrowthfactoralpha(TGF- α),interleukin-1α(IL-1α)andIL-1receptor
antagonist(IL1-ra).Thesecytokinesparticipateinathree-partautocrineloopthatplaysoutoveratleast24hrandaddssequentiallayersofproand
anti-apoptoticsignalingthatsetscelldeathataself-limitinglevel. Experimentalworktodatehasbeeninhumantumorcells.However,itseems
highlylikelythatTNF-triggeredautocrinecascadeswilldifferfromonecelltypetothenext.Wearecurrentlyattemptingtocomparediseasedand
normalprimarycellsfrombreastandconnectivetissue.OneinterestinginitialfindingisthatthelogicoftheTNF-TGF-IL-1α- IL-1racascadecan
bere-wired in somecell typesbyinflammatorycytokinessuch asinterferons. Thereismuch interest in theroleofintracellular crosstalkamong
signaling circuits. We propose that that time-dependent crosstalk among synergistic and antagonistic autrocrine circuits may equally important.
Morevoer, it should be easier to modulate the activity of autocrine than intracellular loops thanks to the increasing range of protein-based
therapeuticsavailabletotargetcytokinesandtheirreceptors.
BruceAlberts Award Presentation (5)
5
ColumbiaUniversity’sSummerResearchProgramforSecondarySchoolScienceTeachers
S.C.Silverstein;DeptPhysiology/CellBiophys,ColumbiaUnivCollPhys&Surg,NewYork,NY
Concerns about the quality of secondary science education stimulated me in 1990 to found Columbia’s Summer Research Program
(www.scienceteacherprogram.org).Theprogram’spurposeistoincreasestudentinterestandachievementinsciencebyimprovingthequalityof
science instruction. To this end, Columbia’s program provides secondary school science teachers with paid fellowships that support their
participationinlifeandphysicalscienceresearchlaboratoriesfortwoconsecutivesummersundertheguidanceofColumbiafaculty.Todate,202
teachers have participated in the program. At ASCB’s 2004 Education Forum, I reported that 8.4% more students in classes of participating
teacherspass a NY State Regents exam in science than students studying the same subject in classes of non-participating teachers in the same
school. This is objective evidence that teacher participation in Columbia’s program has a significant positive impact on student achievement in
science.ThepresentvalueofthisincreaseinRegentsscienceexampassrateis$11,782perteacherinschoolcostssavedannually,and$32,885per
teacher in additional tax revenues generated annually, yielding total annual economic benefits of each teacher’s participation in Columbia’s
programthatare3.4-foldgreaterthantheprogram’sannualcostperteacher.Policyimplicationsofthesefindings: Anationalinvestmentof$75
millionannuallycouldsupportsimilarprogramsat250U.S.medicalschoolsandresearchuniversities,whilereturningover$220millionannually
in school costs saved and tax revenues generated. If each of these 250 programs enrolled 10 newscience teachers annually, over 10 years they
couldprovidescienceworkexperiencesfor25,000scienceteachers,approximatelyhalfthecurrentmembershipoftheNationalScienceTeachers
Association.
ProkaryoticOrigins of theCytoskeleton (6-8)
6
BacterialtubulinhomologFtsZ
H.P.Erickson;DepartmentofCellBiology,DukeUniversityMedicalCenter,Durham,NC
5a
Sunday
FtsZisthemajorcytoskeletalproteininbacterialcytokinesis.Whenviewedbylightmicroscopyitappearsasa“Zring”inthecenterofthecell.
TheZringconstrictstodividethecell,disassemblesduringtheconstrictionandthenreassemblesinthedaughtercells.ThesubstructureoftheZ
ringhasnotbeenvisualizedbyEM,butwehaveturnedtoinvitrostudiestodeduceit.FtsZassemblesintoshort,single-strandedprotofilaments
(pfs),whicharestructuralhomologsofthetubulinprotofilamentsthatmakethemicrotubulewall.Wehavedevelopedfluorescencetechniquesto
study the kinetics of initial assembly and subunit turnover at steady state. FtsZ assembly is cooperative, showing a weak dimer nucleusand a
criticalconcentration.(Itisanenigmahowasingle-strandedpfcanassemblecooperatively.)Theassemblyisverydynamic - pfsareturningover
with a half time of 8 sec at steady state. The subunit turnover is regulated by GTP hydrolysis, and may involve a mechanism like microtubule
dynamic instability. We believe that the Z ring in vivo is constructed from these dynamic pfs. This must involve a lateral association (the
mechanismforthisisunknown)andattachmenttothemembrane.FtsZistetheredtothemembranebyFtsA,abacterialactinhomolog.Weused
FRAPtodeterminetheassemblydynamicsoftheZringinvivo.Itisturningoverwithahalftimeof8sec,justaswedeterminedinvitro.Thisis
themostrapidcytoskeletaldynamicsknown.Animportantquestioniswhatgeneratestheforceofconstriction?WehavefoundinvitrothatFtsZ
can formcurved pfs, which areequivalent to tubulin rings. Thestraight-to-curved pfconformational changeispowered byGTP hydrolysis, and
maygeneratetheforceforconstriction.
7
DynamicsofaDNA-SegregatingCytoskeletalSysteminProkaryotes
D.Mullins,C.Campbell,E.Garner;Cellular&MolecularPharmacology,UniversityofCalifornia,SanFrancisco,SanFrancisco,CA
Themechanismsthatprovideforcetosegregatebacterialchromosomesarestillmysterious.Wedo,however,understandoneimportantexampleof
bacterialDNAsegregationinmoleculardetail- segregationoftheR1andR100drug-resistanceplasmids.Theselarge(100kb),low-copyplasmids
encodegenesforantibioticandheavy-metalresistanceandhavebeenisolatedfrommanypathogens.Toensureinheritancebybothdaughtercells
during division, the R1par operon constructs a simple DNA-segregating machine from three components. One of these components, ParM, is
relatedtoeukaryoticactinsandassemblyofParMintoactin-likefilamentsappearstodriveplasmidsegregationdirectly.Wefindthatthissimple
prokaryotic cytoskeleton exhibits a remarkable collection of activities usually associated with eukaryotic cytoskeletons, including: dynamic
instability,processivecapping,insertionalpolymerization,andtheabilitytogenerateforce.WealsofindthatR1plasmidsegregationisadynamic
process in which assembly of unstable ParM filaments induces plasmids to oscillate rapidly from pole to pole of the cell producing a dynamic
rather than static bipolar distribution. Our results indicate that theassembly dynamics of prokaryotic cytoskeletal systems are important to their
cellularfunctionandthattheprokaryoticsystemsalso makeuseofmechanismssimilartothoseoftheeukaryoticcytoskeletontoestablishlong-
rangeorderandtomoveintracellularcargo.
8
TheBacterialCytoskeletonandCellShape
C. Jacobs-Wagner,1N. Ausmees,2G. Charbon,1M. Cabeen1;1Molecular, Cellular &Developmental Biology, YaleUniversity, NewHaven, CT,
2UppsalaUniversity,Uppsala,Sweden
Similarly to eukaryotic cells, prokaryotic cells come in a variety of shapes. In eukaryotes, the cytoskeleton, which is made of microtubules,
microfilamentsandintermediatefilaments,constitutesaninternalframeworkthatisessentialforthemaintenanceofcellshape.Fordecades,itwas
thought that the external cell wall was the sole determinant of cell shape in bacteria.It is now apparent that bacteria also possess an actin-like
cytoskeleton madeofMreB and that thiscytoskeleton isinvolved in determiningrod cell morphology. Our laboratoryhasrecentlydiscovered a
prokaryotic counterpart of intermediate filament (IF) proteins, termed crescentin. Crescentin is a fibrous protein with a tripartite domain
architecturesimilartothatofmetazoanIFproteins.Purifiedcrescentinself-assemblesspontaneouslyinto~10nmwidefilamentsinvitrowithout
exogenous energy sources, which is a distinct biochemical property of IF proteins. The function of crescentin is required for the characteristic
crescentshapeofCaulobactercrescentusascellslackingcrescentinlosetheircurvatureandadoptastraight-rodcellmorphology.Consistentwith
its role in cell curvature, crescentin forms a filamentous structure along the inner cell curvature of wild-type cells. Localization and proper
organizationofthecrescentincytoskeletonisdependentonthebacterialhomologofactin,MreB,indicatingthatMreBalsoplaysanactiverolein
cellcurvature.
E.E.JustLecture(9)
9
StillWatersRunDeep:InvestigationsintotheQuiescentStateinYeast
M.Werner-Washburne;DepartmentofBiology,UniversityofNewMexico,Albuquerque,NM
Mylaboratoryhasstudiedentranceinto,survivalduring,andexitfromstationaryphaseinyeastforthepast17years.Previously,yeastandother
microbeswerethoughttolackatrueG phase,becausebuddedcellswerealwayspresentinstationary-phasecultures.Wehaverecentlyisolated
0
twodistinctcellpopulationsfromyeaststationary-phaseculturesthatcontainverydifferentcelltypes.Wehaveconcludedfromouranalysisthat
onefractioncontainsquiescent(G )cellsandtheothernon-quiescentcells.Thequiescentcellsaregenerallythelastcellsformedafteryeastcells
0
exhaustglucose(thediauxicshift).Theyarerefractilebyphasecontrastmicroscopy,unbudded,thermotolerant,andsynchronousduringexitfrom
quiescence.Strangely,inthequiescentcellsonlynucleiandvacuolesarevisiblebyEM.Non-quiescentcellscontainmanymoremembrane-bound
organelles,includingER,Golgi,andmitochondriaandlipidbodiesandlackglycogen.Bothcelltypesaremetabolicallyactive,butnon-quiescent
cells show a reduced ability to form colonies. We are continuing to characterize these cell types and their formation. The identification and
characterizationofthesecelltypesprovidesthebasisforongoing,novelanalysesofthequiescentstate,asymmetriccelldivision,andtheprocesses
ofcelldifferentiationandaginginyeast.
6a
Sunday
CargoSorting&VesicularTransport(10-15)
10
AnIntramoleculart-SNAREComplexFunctionsinvivowithouttheSyntaxinN-terminalRegulatoryDomain
J.S.VanKomen,X.Bai,B.L.Scott,J.A.McNew;BiochemistryandCellBiology,RiceUniversity,Houston,TX
Membrane fusion in the secretory pathway is mediated by SNAREs (located on the vesicle membrane (v-SNARE) and the target membrane (t-
SNARE).Inallcasesexamined,t-SNAREfunctionisprovidedasathree-helixbundlecomplexcontainingthree~70aminoacid‘SNARE-motifs’.
OneSNAREmotifisprovidedbyasyntaxinfamilymember(thet-SNAREheavychain)andtheothertwohelicesarecontributedbyadditionalt-
SNARElightchains.ThesyntaxinfamilyisthemostconformationallydynamicgroupofSNAREsandappearstobethemajorfocusofSNARE
regulation. An N-terminal region of plasma membrane syntaxins has been assigned as a negative regulatory element in vitro. This region is
absolutelyrequiredforsyntaxinfunctioninvivo.WenowshowthattherequiredfunctionoftheN-terminalregulatorydomainoftheyeastplasma
membranesyntaxin, Sso1p, can becircumvented when t-SNARE complex formation is madeintramolecular. Our resultssuggest theN-terminal
regulatorydomainisrequiredforefficientt-SNAREcomplexformationanddoesnotrecruitnecessaryscaffoldingfactors.
11
Arf1p,Chs5p,andtheChAPsareRequiredforExportofSpecializedCargofromtheGolgi
M.Trautwein,C.Schindler,R.Gauss,A.Spang;Friedrich-Miescher-LaboratoriumderMax-Planck-Gesellschaft,Tübingen,Germany
InSaccharomycescerevisiae,thesynthesisofchitinistemporallyandspatiallyregulatedthroughthetransportofChs3p(ChitinsynthaseIII)tothe
plasmamembraneinthebudneckregion.TrafficofChs3pfromtheTGN/earlyendosometotheplasmamembranerequiresthefunctionofChs5p
andChs6p.Chs6pbelongstoafamilyoffourproteinsthatwehavenamedChAPsforChs5p-Arf1p-bindingProteins.Thisnovelproteinfamilyis
conservedthroughoutfungiandseemedtohavearisenbythreegeneduplicationevents.WeshowthatallChAPsphysicallyinteractnotonlywith
Chs5pbutalsowiththesmallGTPaseArf1p.AshortsequenceattheC-terminusoftheChAPsisrequiredforproteinfunctionandtheabilityto
bindtoChs5p.Disruptionoftwomembers,Δbud7 and Δbch1,phenocopiesaΔchs6or Δchs5deletionwithrespecttoChs3ptransport.Moreover,
theChAPsinteractwitheachotherandformhighermolecularweightcomplexes.Inaddition,theyareallatleastpartiallylocalizedtotheTGNin
aChs5p-dependentmanner.Mostimportantly,theChAPsinteractphysicallywithChs3p.WeproposethattheChAPsfacilitateexportofcargoout
oftheGolgi.
12
TheRoleofARF4andARF-GAPsinRhodopsinTrafficking
D. Deretic,1 L. Astuto-Gribble,1 N. Ransom,1 P. A. Randazzo2; 1Surgery/Ophthalmology, University of New Mexico, Albuquerque, NM,
2LaboratoryofCellularOncology,NationalCancerInstitute,Bethesda,MD
Thesmall GTP-bindingproteinARF4, aclassII ARF, specificallyrecognizesand bindsto theVXPX-COOHsortingmotifofthe light receptor
rhodopsin. Using a retinal cell-free system, which reconstitutes rhodopsin trafficking in vitro, we have established that the rhodopsin-ARF4
interactionregulatesthebuddingfromthetrans-Golginetwork(TGN)andtheincorporationofrhodopsinintothetransportcarriers(RTCs).RTCs
are targeted to the rod outer segment (ROS), a highlyspecialized subcellular domain of retinal photoreceptors. To test if rhodopsin controlsthe
ARF4-GTPasereaction byregulatingtheaccess of an ARF-GAP to ARF4, in thisstudywesought to identifythe ARF-GAP thatinteracts with
ARF4 in retinal photoreceptors. ARF-GAPs containing ankyrin repeats and plekstrin homology domains (AZAPs) were particularly attractive
candidatesgiventheirknownrolesintheregulationofmembranetraffickingandactincytoskeleton.Bywesternblottingwithspecificantibodies,
we determined that photoreceptors express high levels of ASAP1, an AZAP that also contains the Src homology domain 3 (SH3), and has a
preferenceforARF1,aclassIARF,andARF5,aclassIIARF.ARAP1,anAZAPthatcontainsarho-GAPdomain,wasdetectedatlowerlevels.
ByconfocalmicroscopyASAP1waslocalizedtopunctatestructuresdistributedalongthephotoreceptormicrofilamentsandinthevicinityofthe
Golgi/TGN.ARAP1wasparticularlyconcentratedattheouterlimitingmembranewheretheactincablesareanchored.Wearecurrentlytestingthe
GAP activityof ASAP1 on ARF4. SincetheBARdomain of ASAP1 participatesin membranebendingand fission, weareexploringif ARF4-
dependent recruitment of ASAP1 to the sites of RTC budding may be the underlying mechanism for the regulation of rhodopsin trafficking by
ARF4.SupportedbyNIHgrantEY12421.
13
FunctionalInvolvementofAnnexinIIincAMP-InducedAQP2ExocytosisinRenalCells
G.Tamma,A.Strafino,F.Addabbo,M.Svelto,G.Valenti;DepartmentofGeneralandEnvironmentalPhysiology,UniversityofBari,Bari,Italy
The membraneassociated protein annexin IIisknown toberequired for theapical transport in epithelial cells. Inthisstudyweinvestigated the
involvementofannexinIIincAMP-inducedAQP2translocationtotheapicalmembrane.RT-PCRperformedusingdegeneratedprimersrevealed
thepresenceofannexinIImRNAtranscriptinAQP2expressingrenalCD8cells.Interestingly,annexinIIwasfoundinAQP2-containingvesicles
immunoisolatedfromCD8cells.Consistentwiththisobservation,cellfractionationfollowedbyWesternblottinganalysisshowedthatstimulation
of CD8 cells with the cAMP-elevating agent forskolin, caused a significant increase of annexin II in the particulate fraction paralleled with a
decrease in the soluble fraction. To investigate the functional involvement of annexin II in AQP2 exocytosis the fusion process between highly
purifiedAQP2bearingvesiclesandplasmamembranewasinvitroreconstructedandmonitoredbyafluorescenceassaybasedonthedequenching
of the lipophilic fluorescent probe octadecylrhodamine B-chloride (R18). We designed a peptide reproducing the N-terminal 14 aminoacids of
annexin II and including binding site of the the calcium binding protein p11, a protein required for the formation of a complex with annexin
necessaryfortightlyanchoringtheproteintothecorticalcytoskeleton.Acontrolpeptidehaving1000timesreducedaffinityforp11wasusedasa
control.WefoundthatpreincubationofcellularfractionswithannexinIIpeptidestronglyinhibitedthefusioninducedbytheadditionofcytosol.
In contrast, the control peptide had no effect on fusion. Together these data demonstrate the association of annexin II with AQP2 containing
vesiclesandpointtoapossiblefunctionalinvolvementofannexinIIincAMP-inducedAQP2exocytosisinrenalcells.
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14
RabCouplingProtein(RCP)RegulatestheSortingofMembraneProteinsinRecyclingEndosomes
J. J. Burden,1E. Schonteich,2G. Wilson,2 R. Prekeris,2C. R. Hopkins1;1Department of Biological Sciences, Imperial College, London, United
Kingdom,2UniversityofColoradoHealthSciencesCenter,Aurora,CO
Rabcouplingprotein(RCP)isaclassImemberoftheRab11 familyofinteractingproteins(FIPs),afamilycharacterisedbytheirabilitytobind
Rab11 via a Rab11/25 binding domain (RBD), and to bind phospholipids via aC2 domain. Through its association with Rab11, RCP has been
proposed to play a role in protein recycling. Whilst many proteins, like the transferrin receptor (TfR), have been extensively characterised and
showntofaithfullyfollowthisrecyclingpathway,littleisknownaboutthemolecularmachineryinvolvedinregulatingthistraffickingroute.Using
RNA interference and a combination of biochemical techniques and electron microscopy, we have investigated the role that RCP plays in the
intracellulartraffickingofrecyclingproteins.Byimmuno-electronmicroscopywehavelocalisedRCPtoatubular-vesicularcompartment,thatcan
be loaded with endocytosed transferrin-HRP. Depletion of RCP in Hela cells, was found to significantly reduce the amount of internalised
transferrin in comparison to control cells, whilst the rate of internalisation of the TfR was unaffected. The reduction in transferrin uptake was
accompanied by a reduction in the levels of TfR, suggesting mis-sorting of the TfR away from the recycling pathway towards the degradation
pathway.Toinvestigatethisfurther,weusedRNAinterferencetodepletecellsofanotherclassIFIPmember,Rip11,andfoundthatTfRrecycling
wasnotinhibited(seeWilsonetal.poster).Interestingly,theco-down-regulationofRCPwithRip11wasabletorescuetheeffectofRCPdown-
regulationontheTfR,implicatingRip11insortingmembraneproteinstowardsthedegradativepathway.Thus,weproposethatRCPplaysarolein
theregulationofTfRtrafficking,specificallysortingthereceptorfromtheendosomalcompartmenttowardstheplasmamembraneandawayfrom
thedegradativepathway.
15
UbiquitinBindingProteinsandLysosomalSorting
R.C.Piper,S.Winistorfer,J.McDermott;PhysiologyandBiophysics,UniversityofIowa,IowaCity,IA
Oneofthesortingsignalsthatdirectsmembraneproteinstolysosomesistheirpost-translationalattachmenttoubiquitin.Ubiquitinactsasaself-
containedsortingsignal,whichactsatmayintracellularlocalestoultimatelysendmembraneproteinstothelysosome.Ubiquitinworksasasignal
for internalization, asaTGNsortingsignal and asasignal for incorporatingproteinsintolumenal vesiclesof multivesiclular bodies. Inorder to
understandhowubiquitinsortsproteinstothelysosome,wehavefocusedonidentifyingubiquitin-sortingreceptorsattheTGNandendosomesthat
bind cargo proteinsand guidethemto lysosomes. At theTGN, we find that theGGA familyof clathrin bindingproteinsbindubiquitin viatwo
equivalent motifs within their GAT domains. This binding is required to direct ubiquitinated membrane proteins from the TGN directly to
endosomes.Wealsofindthatseveralubiquitin-bindingproteinsattheendosomearerequiredforsortingintomultivesicularbodies.Amongthese
aretheVps27-Hse1complex,theESCRT-IcomplexandESCRT-IIcomplex.WeshowthatphysicalinteractionoftheVps27-Hse1complexand
ESCRT-I complextriggersahand-off mechanismwherebyubiquitinated cargo couldbetransferred fromonecomplexto thenext.Interestingly,
however,wefindthattheubiquitinbindingcapacityoftheESCRT-IandESCRT-IIcomplexesisnotrequiredforpropersortingofubiquitinated
cargo into the MVB. Thus, we propose that the ubiquitin-binding function of these complexes is used to help Vps27-Hse1 release from cargo
bindingandhelppreventtheVps27-Hse1complexfrombeingincorporatedintotheMVBlumen.
Cell Biologyof theSynapses (16-21)
16
ProN-cadherinInhibitsSynapseFormation
A.G.Reines,W.Shan,A.W.Koch,D.R.Colman;BTRC,MontrealNeurologicalInstitute,McGillUniversity,Montreal,PQ,Canada
N-cadherinparticipatesintheregulationofsynapticstrengthandstabilization.Thisproteinissynthesizedasaprecursormolecule(ProN-cadherin)
which has anti-adhesive properties. The Pro domain is thought to be cleaved off in the late Golgi by a furin protease, after which adhesively
activatedN-cadherinisdirectedtothecellmembrane.WeraisedanantibodyrecognizingtheProNsequence,andstudiedtheexpressionofProN-
cadherinindevelopinghippocampalcultures.WedemonstratedthepresenceofthisimmatureN-cadherinthroughoutneuronaldifferentiation,from
5h to 14 days in vitro. Biotinylation of the proteins on theneuronal surface revealed that a proportion of ProN-cadherin is sorted to the plasma
membraneandthatthemature/immatureN-cadherinratioonthesurfaceincreasesasneuronaldifferentiationprogresses.Interestingly, we found
thatthePropieceisreleasedintotheculturemediacoincidentwithitsdecreaseontheplasmamembrane.TostudythefunctionofProN-cadherin
whenexpressedontheneuronalsurface,weemployedaconstructinwhichtheendogenouscleavagesitewasreplacedbyafactorXacleavagesite.
ProN-cadherinoverexpressionhadalargeimpactonsynapsenumber,measuredasadecreaseinthenumberofsynaptophysinandPSD-95puncta.
Thesameeffect wasobserved whenthefunctionallabelingofpresynapticboutonswiththeFM4-64dyewasanalyzed. Thiseffect was partially
overcomewhenfactorXawasappliedtothecultures.Ourresultsdemonstratethatanti-adhesiveProN-cadherinisexpressedinneuronsandsorted
totheplasmamembranewhereitmayactasanegativeregulatorofsynapseformation.Weproposethatchangesintheratioofmature/immature
N-cadherinontheneuronalsurfacemightbeanovelmechanismbywhichneuronsregulatesynapticjunctionformation.
17
ImmaculateConnections,aKinesinMotor,isNecessaryforPresynapticDifferentiationandfortheTransportofSynapticComponents
E.Pack-Chung,D.K.Dickman,T.L.Schwarz;Children'sHospital,HarvardMedicalSchool,Boston,MA
Thebuildingblocksofsynapsesarepresentwithingrowingaxonsandarerecruitedtositesoftargetcellcontact.Thus,thetransportofsynaptic
components,includingtheproteinsofsynapticvesiclesandactivezones,isnecessaryforsynaptogenesis.Theidentityandregulatorymechanisms
of the molecular motors that move synaptic cargoes, however, remain elusive. We have identified a Drosophila gene (named immaculate
connections)thatisrequiredforsynaptogenesis.immaculateconnections(imac)encodesamemberofthekinesin3familyofmotors.Mutationsin
imacdonotpreventaxonoutgrowthbutpreventgrowthconesfromtransformingintosynapses.Attheembryonicneuromuscularjunction,growth
cones reach their target muscles but do not mature into synaptic boutons. Thus the development of nmj appears to be blocked just at
synaptogenesis.Examinationofintracellulartransportrevealedthatthetraffickingofsynapticvesicleandactivezonecomponentsareblockedin
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imac.Theseproteinswerelackinginimacaxonsandaccumulatedinthecellbodies.However,post-Golgivesicles,mitochondria,andcytoskeletal
componentswereproperlytraffickedinimacaxons.Interestingly,theabsenceofpresynapticdifferentiationinimacdidnotpreventtheassembly
ofpostsynapticcomponents;clusteringofpostsynapticreceptorsandpostsynapticdensityelementsweredetected.WethusconcludethatImacis
selectivelyrequiredforthetransportofmaterialsforsynaptogenesisandthatinitsabsence,presynapticdifferentiationfailstotranspire.Ourdata
alsoprovesthatitisdistinctfromthemotorormotorsnecessaryforaxonoutgrowth.Bybringingsynapticmaterialsintothegrowingaxon,imac
permitstherapidformationoffunctionalsynapticconnections.Thecouplinganduncouplingofthemotoranditscargoarelikelytobeimportantin
regulatingtheformationofsynapses.
18
HIP1ExpressionisRequiredforNormalNMDARFunction:ImplicationsforaRoleofHIP1inHuntington’sDisease
M.Metzler,1L.Gan,1J.Helm,1T.P.Wong,2L.Liu,2Y.Wang,2L.Liu,1Y.T.Wang,2M.R.Hayden1;1CMMT,Dept.ofMedicalGenetics,UBC,
Vancouver,BC,Canada,2TheBrainResearchCentre,UBC,Vancouver,BC,Canada
The initial identification of the endocytic protein HIP1 (huntingtin interacting protein 1) resulted from its interaction with the polyglutamine-
containing protein huntingtin that, in its polyglutamine-expanded form, causes Huntington's Disease (HD). The interaction between HIP1 and
huntingtin is significantly altered following polyQ-expansion in huntingtin suggesting that HIP1 is a possible component of the pathogenic
mechanisminHD.Inpreviousstudieswehaveshownthat AMPA-inducedAMPAreceptor(AMPAR)traffickingisblockedincorticalneurons
fromHIP1 knock-out mice(Metzler et al., 2003).Here, wedemonstrateasimilar blockin NMDA-induced AMPARendocytosis, andhencethe
reduction ofcell-surface AMPARexpression, in cultured hippocampal neuronsfromHIP1 knock-out mice. Moreover, theNMDA-induced long
term potentiation of AMPAR-mediated synaptic transmission at the CA1 synapses in hippocampal brain slices from HIP1 knock-out mice is
significantly reduced compared to wild-type littermates. Results from coimmunoprecipitation and GST-pulldown experiments revealed direct
interaction between HIP1 and the NR2 subunit of NMDARs. Furthermore, colocalization between HIP1 and NR2-cointaining NMDARs is
observedinprimaryhippocampalneurons.MostimportantforourunderstandingofHD,NMDA-inducedexcitotoxicityisblockedinneuronsfrom
HIP1knock-outmice.Together,thesedataprovidestrongevidencethatHIP1regulatesNMDARfunctionandthatthisfunctionofHIP1maybe
contributingtoenhancedexcitotoxicityinHD.
19
AssociationofanAKAPSignalingScaffoldwithCadherinAdhesionMoleculesinNeuronsandEpithelialCells
J.A.Gorski,1L.L.Gomez,1J.D.Scott,2M.L.Dell'Acqua1;1Pharmacology,UniversityofColoradoDenverHealthSciencesCenter,Aurora,CO,
2VollumInstitute,HowardHughesMedicalInstitute,OregonHealthSciencesUniversity,Portland,OR
A-kinase anchoring protein (AKAP) 79/150 organizes a scaffold of PKA, PKC and protein phosphatase 2B/calcineurin that is localized to
epithelialadherensjunctionsandthepostsynapticdensityofneuronalsynapses.TargetingoftheAKAPtothesesubcellularlocationsrequiresthree
N-terminal basic domains that bind F-actin and acidic phospholipids. Here we report a novel interaction of this targeting domain with cadherin
adhesion moleculesthat arelinked to actin through β-catenin (β-cat). MappingtheAKAP bindingsitein cadherinsidentified overlap withβ-cat
binding; however, no competition between AKAP and β-cat binding to cadherins was detected in vitro. Accordingly, AKAP79/150 exhibited
polarizedlocalization withβ-catand cadherinsin epithelialcell lateral membranes, andβ-cat waspresent inAKAP-cadherin complexesisolated
fromepithelial cells, culturedneurons, and ratbrain synapticmembranes. Inhibitionofepithelialcell cadherin adhesion induced byextracellular
calciumswitchandinhibitionofactinpolymerizationbytreatmentwithLatrunculinAredistributedintactAKAP-cadherincomplexesfromlateral
membranes to intracellular compartments. In contrast, stimulation of neuronal pathways implicated in long term depression that depolymerize
postsynaptic F-actin disrupted AKAP-cadherin interactions and resulted in loss of the AKAP, but not cadherins, from synapses. This neuronal
regulationofAKAP79/150targetingtocadherinsmaybeimportantinfunctionalandstructuralsynapticmodificationsunderlyingplasticity.
20
StructuralPlasticitywithPreservedTopologyinaPostsynapticProteinNetwork
T. A. Blanpied,1 M. D. Ehlers2; 1Physiology, University of Maryland School of Medicine, Baltimore, MD, 2Neurobiology, Cell Biology,
PharmacologyandCancerBiology,HowardHughesMedicalInstituteandDukeUniversityMedicalCenter,Durham,NC
Multiprotein complexes form structural networks to mediate diverse cellular events including adhesion, signaling, nuclear transport, and
intercellular communication. The function of such interconnected protein machines is determined by the spatial positioning and dynamic
rearrangementsofindividualcomponentswithinthecomplex.Thepostsynapticdensity(PSD)isaprominentexampleofsuchnetworks.ThePSD
positions neurotransmitter receptors across from presynaptic sites of neurotransmitter release and links postsynaptic receptors with intracellular
signaling cascades. Nearly all molecular theories of learning postulate morphological and molecular alteration of the PSD during plasticity at
excitatory synapses. However, despite recent documentation of rapid mobility of receptors near the PSD, there is little known about dynamic
behavior ofcorePSDconstituentsin thecomplex. To probethestructureoflivingPSDs, wehavemeasured theirinternaldynamicsusinghigh-
resolutionimaginganalyses.TheseexperimentsindicatethatthePSDisassembledasaflexibleyettopologicallystablematrixonwhichenduring
changes in function can be rapidly encoded. Exchange, addition, or removal of PSD elements can occur at independent matrix coordinates,
providingamolecularmapfororganizingsynapticnanoarchitecture.
21
TwoTypesofEndocyticIntermediatesatthePeriactiveZoneofaCentralSynapse
A.Sundborger,N.Tomilin,O.Shupliakov;Neuroscience,KarolinskaInstitutet,Stockholm,Sweden
Dynamin is the GTPase implicated in fission of vesicles from the plasma membrane. In synapses it is an important component of the protein
complexresponsiblefordetachingclathrinvesiclesfromthepresynapticmembrane.Ithasbeenshownininvitrostudieswithisolatedmembranes
and with synaptosomes that GTPγS blocks fission resulting in the accumulation of clathrin-coated pits with elongated necks decorated with
dynamin-containing spirals. To visualize dynamin-dependent endocytic intermediates in an intact synapse we microinjected GTPγS into living
giant axons in lamprey and studied them using electron microscopy. GTPγS did not alter morphology of synapses at rest. Stimulation of
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microinjected axons with action potentialsat 5 Hzinduced areduction in thenumber ofsynaptic vesiclesat active zonesand theappearanceof
numerousendocyticintermediatesatperiactivezones. Twodifferentclassesofintermedientswerefoundatperiactivezones:clathrin-coatedpits
withelongatednecks,andmembraneinvaginations.Coatedpitswerepresentonlyonsomeofthesemembraneinvaginations.Bothwereconnected
to the presynaptic membrane. Spiral-like structures were found at sites of these membrane connections. To investigate if these spirals contain
dynamin, axons microinjected with GTPγS we cut along the longitudinal axis and stained with anti-dynamin (DG-1) antibodies using a pre-
embedding immunogold technique. An accumulation of gold particles occurred at necks of clathrin-coated pits and at sites of connection of
membrane invaginations to the presynaptic membrane. Our results show that two endocytic intermediates, clathrin-dependent and clathrin-
independent,areformedatperiactivezonesduringsynapticactivity.Thus,inadditiontotheclathrinmechanism,bulkretrievaloflargemembrane
compartmentsmayoccurinintactsynapsesduringneurotransmitterrelease.
Differentiation &Cancer(22-27)
22
UnderstandingWntSignalinginDevelopmentandDisease
X.He1,2;1Children'sHospital,Boston,MA,2HarvardMedicalSchool,Boston,MA
Wnt signaling is essential for development and tissue homeostasis. Disruption of Wnt signal transduction causes abnormal embryogenesis and
cancers. Using a combination ofmolecular, biochemical and embryological techniques, we have focused on the mechanismof Wnt signaling in
Xenopusembryodevelopmentandhumancancer.WeareparticularlyinterestedinhowtheWntreceptorcomplextransducesWntsignalacrossthe
plasmamembrane,howtheWntreceptorcomplexspecifiesdistincttransductionpathwaystogoverndifferentaspectsofembryogenesis,andthe
molecularcompositionandlogicofthesetransductionpathways.ProteinphosphorylationispivotalforWntsignaling.Wehavecharacterizedtwo
key phosphorylation events in the canonical Wnt/beta-catenin pathway. One is phosphorylation of the Wnt coreceptor, LDL receptor related
protein 6 (LRP6). This phosphorylation leads to LRP6 activation and the initiation of Wnt signal transduction. The other is beta-catenin
phosphorylation, which results in beta-catenin degradation and is inhibited upon Wnt signaling. 1). The mechanism of phosphorylation and
activationoftheWntcoreceptorLRP6.WehaveshownthatWntinducesLRP6phosphorylationatPPPS/TPmotifs,andthisphosphorylationis
necessaryandsufficienttotriggerWntsignaling.WehavegeneratedantibodiesthatspecificallyrecognizephosphorylatedLRP6.Theseantibodies
areusefultoolsfordetectionofWntsignalingactivationinvivoandforidentificationofkinasesinvolvedinLRP6phosphorylation.Iwilldiscuss
ourrecentprogressinstudyingWnt-inducedLRP6phosphorylationandidentifyingLRP6kinases.2).Themechanismofphosphorylationand
degradationofbeta-catenin.Wehavedemonstratedthattwokinases,caseinkinase1(CK1)andglycogensynthasekinase3(GSK3),sequentially
phosphorylatebeta-catenininaproteincomplexassembledbythescaffoldingproteinAxin.IwilldiscusshowWntreceptoractivationmayleadto
inhibitionofbeta-cateninphosphorylation,andourattempttoidentifyotherkeymoleculesviainvitroreconstitution.
23
p21WAF1/Cip1MediatesNotch1-dependentSuppressionofWntExpressionandSignaling
V. Devgan,1 C. Mammucari,2S. E. Millar,3C. Brisken,4 G. P. Dotto2; 1Cutaneous Biology Research Center, Massachusetts General Hospital,
Charlestown, MA, 2Department of Biochemistry, Lausanne University, Epalinges, Switzerland, 3Department of Dermatology, University of
PennsylvaniaMedicalSchool,Philadelphia,PA,4SwissCancerResearchInstitute,Epalinges,Switzerland
Besides controlling cyclin/CDK and PCNA activities, p21WAF1/Cip1 can directly bind transcription factors and co-activators, modulating their
functions. However, thebiological significanceofthesefindingshasnotbeen established. In keratinocytes, p21isadirectdownstreamtarget of
Notch1 activation, and loss of either p21 or Notch1 expands keratinocyte stem cell populations and facilitates tumor development. The tumor
suppressorfunctionofNotch1hasbeenassociatedwithnegativeregulationofβ-cateninsignaling,throughanunknownmechanism.Weshowhere
that Notch1 activation down-regulates β-catenin signaling through suppression of Wnts gene expression, and that p21 is a key mediator of this
down-modulation. p21 suppresses Wnts expression independently of the cell cycle, while lack of p21 prevents Notch-dependent suppression of
Wntgeneexpressionandresults,bothinculturedkeratinocytesandintheintactskin invivo,inincreasedWnt4expression.Morespecifically,p21
associates with the E2F-1 transcription factor at the Wnt4 promoter and causes curtailed recruitment of c-Myc and p300, and histone
hypoacetylationatthispromoter.Thus,p21functionsasamediatorofthenegativeeffectofNotch1activationonWntsignaling,byspecificdown-
modulationofWntgeneexpressionatthetranscription-chromatinlevelandindependentlyofcellcycle.
24
Gata-3isaCriticalRegulatorofDifferentiationintheMammaryGlandandBreastCancer
H.Kouros-Mehr,Z.Werb;Anatomy,UCSF,SanFrancisco,CA
AseriesofbreastcancermicroarraystudieshaveshownthatthetranscriptionfactorGATA-3isstronglycorrelatedwithEstrogen Receptor(ER)
status, tumor grade and survival. We show here that GATA-3 plays a fundamental role in maintaining the luminal epithelial cell fate in the
mammaryglandandinbreastcancer.WeinitiallyidentifiedGATA-3throughamicroarrayscreenasthemosthighlyexpressedtranscriptionfactor
inthemammarygland.ImmunostainingrevealedthatGATA-3isexpressedexclusivelyinallluminalprogenitorsanddifferentiatedluminalcells.
TodeterminethefunctionofGATA-3,wecrossedfloxedGATA-3micewithMMTV-CreandWAP-rtTA-Crelines.HomozygousfloxedGATA-3
micecarryingMMTV-Credisplayedrunting,progressivealopecia,andahighlydefectivemammarygland.Themammaryglandsdisplayedanear
lackofepitheliumduetoaninabilitytoformterminalendbuds.TomorecloselyanalyzethefunctionofGATA-3,wecrossedthefloxedmicewith
the Tet-inducible Cre line WAP-rtTA-Cre. After short-term (72 hr) administration of doxycycline to adult mice, GATA-3 null luminal cells
displayedlossofbasalpolarityanddiedwithintheductallumenassinglecells.TodeterminehowGATA-3affectsbreastcancerprogression,we
usedthePyMTmousemodelofbreastcancer.WetransplantedGFP+hyperplasiasintosyngeneicmicetodeterminewhenmalignantconversion
occurred.WefoundthatlossofGATA-3stronglycorrelatedwithlossoftumordifferentiation,theprogressionfromadenomatoearlycarcinoma,
andtheonsetoftumordisseminationintodistantsites.Furthermore,overexpressionofGATA-3inprimaryPyMTtumorswassufficienttoinduce
elementsoftumor differentiation. Thiswork suggeststhat GATA-3 specifiesand maintainsluminal cell differentiation and suggeststhat lossof
cellfateisacriticaleventinthemalignantprogressionofbreastcancer.
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