ARMENISE-HARVARDSYMPOSIUM2014

MECHANISMSTOMOLECULES

15thBiennialSymposiumJune22-24,2014,PoianoResortHotel,LakeGardaItaly

AbouttheSymposium

L1050036ThegentlebrushstrokesofaXVIIIcenturyoilpaintingseemedtomakeupthesettingofthe15thArmenise-HarvardSymposium.Amélangeofolivetrees,cypressesandoleanderssurroundedbygreenhillsandreflectedintheshimmeringblueoftheGardaLake:inthisbucolicscenario,80scientistsmettodiscussthelatestadvancementsincellbiology.

BetweenJune22and24,thesymposiumheldintheluxuriousPoianoResortbroughttogetherresearchersworkingonthecuttingedgeofscience.“Interdisciplinary”and“heterogeneity”werethemainkeywords:thiswasalreadyprettyclearfromthesymposiumtitle,“MechanismstoMolecules”.

Fromsingleproteinsactivitytocompletecellsystems,frombasicresearchtoclinicalapplication:thetwo-and-a-half-daymeetingwasanexcitingopportunityforcross-disciplinarydiscussionsandhigh-levelscientificexchanges.

Twenty-oneresearchers,allleadersoftheirresearchgroupsinItalyandtheUnitedStates,talkedabouttheirlatestresults,inmanycasespresentingpreliminaryandunpublisheddata.Thefriendlyandfreedialogueledtosharpdebatesaboutthemostfascinatingenginesofourbody:humancells.

Theoutstandingtalks,verydifferentfromeachother,hadacommonchallenge:makinglongstoriesshort.Infacteachpresentedresearchbeganmanyyearsago,whenmostofthecellularmechanismsdiscussedduringthesymposiumwereunknown.

Today,newtechnologicaltoolsandhighlyadvancedmicroscopictechniquesaremaking“visible”whatwaspreviouslyunseen;butatthesametime,itisimportantnottolosethebiggerpicture.

ThisiswhatemergedfromtheenthrallingkeynotespeechgivenbyHarvardProfessorStephenHarrison,whodeliberatelyjuxtaposedthesymposiumname,entitlinghispresentation“MoleculetoMechanisms”.Heoutlinedvariouswaystothinkofmolecularactivitystartingfromtheparadigmaticexampleofinfluenzavirushemagglutinin(HA),whosebindsreceptorcandefeatourimmuneresponse.

Thisstartedthediscussionabouttheincrediblenumberofinteractionshappeningfrommoleculartocellmechanismlevel,andviceversa.Thefirstsymposiumsession,entitled“MechanismsRegulatingEpigeneticRegulation”,openedupthedoortoinvestigatethisintriguingcellularcomplexity.DaneshMoazedandStephenBuratowskitalkedabouttwofundamentalingredientsof

cellregulation,chromatineandnoncodingRNA.DiegoPasiniexposedtheactivitiesofpolycombgroupproteinsinproliferationanddevelopmentalcontrol,whileGiuseppeTestabroadenedthefieldbyexplaininghowcellreprogrammingrevolutionizedbiologyoverthelastyears.

Thesecondsession,“MechanisticInsightsfromSingleMoleculeAnalysis”,tooktheaudiencebacktothemolecularlevel.SamaraReck-PetersonandAndreasLeschzinertalkedaboutafundamentalaspectforcells’survival,themechanicsbehindmolecularmotors.JosephLoparogaveastimulatingtalkaboutDNA“intelligence”,showinghowourgenesstrivetoovercomeobstacles.SherefMansychallengedtheaudiencewithaverynovelfrontierofbiotechnology,theintegrationbetweenartificialandnaturalcells.

Anotherimportantaspectisthecommunicationamongstdifferentcells,andthiswasthetopicofthethirdsession,“MechanismsRegulatingTrafficking”.RobertoSitiaexplainedhowthequalitycontrolworkswithinthesignaturepathway.TomasKirchhausenshowedtheexceptionalpotentialitiesofthemodern3Dmicroscopytostudyendocytosis.DanielaCordaandAlbertoLuinigavetwointerestingtalksaboutmembranetraffickingandtransportapparatus.

Withthebackdropoftherelevanceofthisresearchtomedicalapplications,thefourthsessionfocusedon“DegradationMechanisms”.SimonaPolotalkedaboutcellmigrationundernormalandpathologicalconditions.AlfredGoldbergpresentedthesysteminvolvedinproteindegradationbyproteasomes.AndreaMusacchioshowedhislatestresultsonthereconstructionofmitoticsignaling,whileFrancescoCecconidiscussedtheimplicationofoneofthemostincrediblebehaviorofourcells,autophagy.

Thisledtothefifthandlastsession,“TransductionMechanisms”,openedbyGianniCesareni’stalkaboutthesignalingmechanismsunderlyingtheanti-tumoractivityofmetformin.StephenBlacklowandMichaelEckrespectivelypresentedthesignalingintheNotchcellandthecontroloftheepidermalgrowthreceptor.PierPaolodiFioreclosedthesessiondiscussingtheconnectionsbetweencellfatedeterminationandtumorsuppressioninmammarystemcells.

Thetaskofclosingthe15thArmenise-HarvardSymposiumfelltoTomasKirchhausen,whowrappedupthegeneraltake-homemessageofthemeeting:thatweareconstantlydealingwithanumberofcomplexinteractions,fromcellularmechanismstomolecularcommunication.Unfoldingthemysteriesoftheseinteractionsisthemainchallengeofcellbiology,fascinatingandever-growingfieldexploringthefundamentalunitsoflife.

Moleculetomechanisms—acasehistory

StephenHarrison

DepartmentofBiologicalChemistry&MolecularPharmacology,HarvardMedicalSchool,Boston,USA

Thetitleofthesymposiumwasreversedbythekeynotespeech:frommolecule–intentionallysingular–tomechanisms.Withthistalk,StephenHarrison“setthetoneforallthefollowingpresentations”,aswouldhavelaterobservedTomasKirchhausenduringthemeetingclosingremarks.

Harrisonisoneoftheworld’sleadingexpertsonviruses.JeffreyFlier,PresidentoftheArmenise-HarvardFoundationandDeanofHarvardMedicalSchool,brieflyintroducedHarrison’soutstandingscientificactivity:“Hehasmadeimportantcontributionstostructuralbiology,mostnotablybydeterminingandanalyzingstructuresofvirusesandviralproteins,andbycrystallographicanalysisofprotein-DNAcomplexes,aswellasstructuralstudiesofprotein-kinaseswitchingmechanisms.Hisworkhasbeenwidelyrecognized,andhehasmadepioneeringresearchestoaddressarangeoffundamentalproblems”.

Oneoftheseproblemsconcernstheinfluenzavirushemagglutinin(HA).TheHarrisonlabatHarvardMedicalSchoolstudieshowinfluenzaviruspenetratescellsbyfusionofviralandendosomalmembranescatalyzedbytheviralhemagglutinin.

SoHarrisonusedthisvirusasthecasehistoryofhispresentation–averyeffectivewaytodevelopthethemeofthemeetingattheoutset.

“Thehemagglutininisatrimericstructurewiththreefunctions”heexplained.“Itbindsthevirustoitsreceptor,catalyzesthemembranefusionprocess,andhasstructuresontheoutsidethatcanvarywithoutcompromisingitstwootheressentialfunctions”.

Inthiswaytheviruscanevolvetoescapeneutralizationbytheimmunesystemofitshost.

Intheinfluenza,theproteinhemagglutininsticksoffofthevirussurfacealongwithanotherprotein,whichisanenzyme,calledtheneuraminidase(NA).

Harrisonfocusedontwoparticularmechanisms:HA-mediatedfusionandHAantigenicity.

Membranefusionisthermodynamicallyfavorable,butitgenerallypresentsahighkineticbarrier.Fusionproteinslowerthisbarrier,sotheyarecatalystsforthemergeroftwobilayers;inthecaseofviralfusionproteins,theybecome“suicide”catalysts.Afterseveralexperimentssetuptomeasurefusionkinetics,Harrisonandcolleaguesshowedthatforinfluenzavirusfusionrequiresengagementwiththetargetbilayeroffusionpeptidesfrom3or4neighboringHAtrimmers.

“Thecasehistorylessonissummarizedhere”saidHarrison,“Thatthereisacontactpatchbetweenthevirusandthetargetmembrane.”

Thisintermediateisafundamentalaspectofthefusionmechanism.ButhowdoesHAantigenicityreacttothismechanism?

Antigenicityisthecapacitytostimulatetheproductionofantibodies.Duringtheimmuneresponse,theprocessbywhichBcellsproduceantibodieswithincreasedaffinityforantigeniscalledaffinitymaturation.

“Antibodystructures,fromB-celllineagesinhumanresponsestoinfluenzavirusvaccines,mapevolutionofproteininteractionsduringantibodyaffinitymaturation”explainedHarrison.

Thefuturechallenge,heconcluded,isthereforetounderstandtheaffinitymaturationmechanismswellenoughtodesignmodifiedimmunogensthatmightselectivelyelicitbroadimmuneresponses.

Frommoleculetomechanisms,then:theinspiringexampleofinfluenzahemagglutiningotstraighttotheheartofthesymposiumtheme.Showingfirstofallhowbiologycantravelbetweenthemicroandthemacrolevelofourcells.

Geneon,geneoff.Ineukaryoticcells,DNAactivationisoftenamatterofswitches.Butwhatarethebiologicaltriggerscapableofunchaininggeneexpression?ThisisoneofthecorequestionsaddressedbyDaneshMoazedatHarvardMedicalSchool.

HislaboratoryfocusesonunderstandingthemechanismsthatmediatetheformationofoneofthemosttightlypackedformsofDNA:heterochromatin,whichplaysacrucialroleingenesilencing.Forthisreason,thestudyofheterochromatin–alsocalledsilentchromatin–helpsunderstandingthemechanismsthatkeepgenesintheirrightonoroffstate.

Theformationofthesesilentdomainsisalsoresponsiblefortheso-calledepigeneticmemory,whichmaintainscellidentityduringdevelopmentanddifferentiation.

Biologistsknowthateverysinglecellofourorganismonlyturnsonthesubsetofgenesnecessarytocarryoutparticularfunctions.Thisisthereasonwhy,forexample,thegenesofnerveimpulsetransmissionremainshutoffinlivercells:theyarejustnotusefulinlivercellsandcaninterferewithliverfunction.

Moazedandcolleaguesaimatunderstandinghowcellssilenceunnecessarygenestomaintaintheirspecificidentities.

Theyapplyacombinationofapproaches:frombiochemistrytocellbiology,fromproteomicstogenomics,withthecommongoalofidentifyingthefactorsabletomediateheterochromatinassemblyandfunction.

Analyzingsilentdomainsinyeast–single-celledfungithatreproducebyfission–researchersidentifiedaspecialingredientplayingacentralrolesintheformationofrepressiveheterochromatin:noncodingRNAs.

“NowadaystherearealotofideasaboutwhatsmallRNAsarecapableofdoing,andsomemechanismsareclearerthanothers”Moazedexplained.“ThewaytheseRNAsaregeneratedisafundamentalquestion,becausetheyspecifythepartsofthegenomethatshouldbesilenced”.

Inparticular,histeamshowedthatsmallRNAmolecules(overall,about20nucleotides)workthroughtheRNAinterference(RNAi)toregulategenesilencingandexpression.Developingmanybiochemicalexperimentsinfissionyeast,theypurifiedtheRNA-InducedTranscriptionalSilencing(RITS)complex,whichdirectlylinkstheRNAipathwaytoheterochromatinassembly.

Moazed’slabfocusedontwocriticalprocessesformaintenanceofheterochromatin,histonemethylationandsiRNAamplification.Thesetwoprocesseswereshowntobemutuallydependent

andtoformself-reinforcingpositivefeedbackloopsofcrucialimportanceformaintenanceofsilentdomains.

SuchnoncodingRNA-basedmechanismsmaybeinvolvedinregulatingheterochromatinformationandgeneexpressioninotherorganisms.Andthisisexactlywhatstudiesinotherlaboratoriesarebeginningtoreveal.

“Futureapplications?UsingsmallRNAstosilencegenesandreprogramtheepigenome”hesaid.“Itmaybepossibletoturn-offgenesatthetranscriptionallevel,althoughmanytechnicalchallengeshavetobeovercomebeforethiscanbedoneinsystemsoutsidesimplermodelorganismslikeyeast”.

Thenextstep,then,istheinvestigationofotherbiologicalpathwaysthatplaymajorrolesinregulatingheterochromatin:inordertounderstandbetterandbetterthecomplex,fascinatingswitchesofDNA.

Shapingtheeukaryotictranscriptomewithchromatinandnon-codingRNA

StephenBuratowski

DepartmentofBiologicalChemistryandMolecularPharmacology,HarvardMedicalSchool,Boston,USA

Thehugedictionarybuildingupourgenomehasaverycomplexpunctuation.DNAsequencesarejustsmallstringsofletters,butunderstandingtheirrulesisabigchallenge.

AtHarvardMedicalSchool,theBuratowskiLabstudiesthemarksofgeneexpressions:inparticular,theenzymesthat“write”thesemarks,aswellastheproteinsthat“read”them.

Usingyeastasamodelsystem,researchersanalyzethemechanismofgeneexpressionineukaryotes,workingonRNApolymeraseIItranscriptioninitiationandthesubsequentprocessingofthemRNA.Severaldozenproteinsarerequiredsimplytoinitiatetranscription,andmanymoretakeactionsinotherprocesseslinkedtotranscription.Forthisreason,understandingtranscriptionmeansdecipheringthefunctionsofeverysinglefactor.

Duringthe15thArmenise-HarvardSymposiumStephenBuratowski,headofthelaboratory,presentedhisapproachtounfoldthemysteriesoftranscription’spunctuation.Hisresearchteamfocusesonthecommunicationbetweenchromatinandthetranscriptionmachinery.

Theyshowedthattheactoftranscriptioncausesmajorchangesinthenucleosomesthatpackagethegene.UsingtheyeastSaccharomycescerevisiae,theydiscoveredthatSet1(atypeofhistonemethyltransferases,orHMT)proteinlevelsarecarefullycalibratedtotheamountoftranscriptionoccurringinthecell.Set1issubjecttodegradationbytheubiquitin-proteasomesystemunlessitisstabilizedbyongoingtranscriptionandhistonemethylation.Disruptionofthisfeedbackloopcausesaberrantmethylationpatternsandgenemisregulation.

Thismechanismmayhaveclinicalrelevance,asmanyleukemiaandlymphomascontaintranslocationsinthemammalianMLL1gene,anHMTrelatedtoSet1.

Carryingoutgenome-wideexpressionscreenstoanalyzeachromatin“reader”calledSet3,Buratowskiandcolleaguesfoundthatthemajorityofgeneswhoseexpressionlevelschangedwererepressedbythisfactor.Butwhatwasmoreunexpected,theydemonstratedthattheseeffectsstronglycorrelatewithoverlappingnon-codingtranscription.Thisdoesnotmeanthatnon-codingRNAsthemselvesmediatethegeneexpressionchanges;instead,thelatterdependonhistonemethylationsplacedovergenepromotersbyoverlappingtranscription.

Andthisisexactlywhereclinicalmedicinecouldfindnewapplications:“Ithinkitiscertainlyworththinkingabouthowchangesintranscriptioncanaffectdiseaseslikecancer”Buratowskiconcluded.“Thisisbecausemanydiseasesarecausedbyproblemsineitherwritingorreadingthemarksofgeneexpressions”.

ActivitiesofPolycombGroupProteinsinProliferationandDevelopmentalControl

DiegoPasini

DepartmentofExperimentalOncology,EuropeanInstituteofOncology,Milan,Italy

Manyhumandiseasesarecausedbythelossofcellularidentity.Canceristheclearestexample:inalltumors,cellsacquirefeaturesthatleadtoabnormalgrowthanddifferentiationdefects.

Buthowhappensthatcellsstartlosingtheiridentity?Andwhatarethemechanismsregulatingcellfateduringdevelopmentanddifferentiation?

DiegoPasini’scurrentresearchattheEuropeanInstituteofOncologyrevolvesaroundthesequestions.Heaimstounderstandhowdiseaseslikecancercanform,maintainanddevelop.

DuringtheArmenise-HarvardSymposium,PasiniexplainedthathislabisinterestedinstudyingthesemechanismsbyfocusingonPolycombGroup(PcG)proteins.

PcGareafamilyofproteinsresponsibleforcellulardifferentiationduringdevelopmentviatranscriptionalrepression.

“PolycombGroupproteinsaremasterregulatorsofcelldevelopment”saidPasini.“Theyarealsoessentialforcellularproliferation,andplayanactiveroleincancerformation”.

ThefirsttodescribethepolycombgroupwasgeneticistEdwardB.Lewis,whoin1978observedinDrosophilathatPcGwasinvolvedinthesilencingofHoxgeneexpression.

Sincethen,theseproteinshavebeenthesubjectofintensestudyasitisclearthattheyarevitalformaintenanceofcell-typeidentityanddifferentiation.

“Atthebiochemicallevel,mostPcGproteinsformtwomajorpolycombrepressivecomplexes:PRC1andPRC2”explainedPasini.

PRC1andPRC2represstranscriptionrespectivelybyUbiquitylatingHistoneH2Alysine(K)119andbytri-methylating(me3)HistoneH3K27.DeregulationofbothPRC1andPRC2activitiesisacommonfeatureofhumantumors.

TostudytheroleofPcGproteinsinregulatingnormalandcancercellsproliferation,Pasiniandcolleaguescombinecellcultureandinvivostudies.TheirexperimentsdemonstratedthatPRCsindependentlyregulatecellularproliferationandtransformation.

SotheabilityofPRC1andPRC2topromoteproliferationisamainfeaturethatlinksPolycombGroupproteinsactivitytocancer.

Forthisreason,PcGinhibitionhasbeenproposedasastrategyfortumortreatment.

JoiningtheZoo:Cellreprogrammingandtheriseofhumandiseasemodels

GiuseppeTesta

DepartmentofExperimentalOncology,EuropeanInstituteofOncology,Milan,Italy

Haveyoueverthoughtofyourbodyasarovinglaboratory?Intheverylatestyears,thisisexactlywhathashappened.Atruerevolutionoccurredincellularbiology:itiscalled“cellreprogramming”,anditallowsscientiststodirectlystudydiseasesinhumantissues.

Cellreprogrammingdependsontheseminalderivationofhumaninducedpluripotentstemcells(iPSC)fromsomaticcells.Bornin2007,thistechniquehasalreadydeeplychangedtheprospectsnotonlyofregenerativemedicinebutalso,andlikelyinanevenshortertimeframe,ofourcapacitytodissectthegeneticcontributiontohumandiseases.

AttheEuropeanInstituteofOncology,GiuseppeTestaisworkingonthiscutting-edgelineofresearch.Hislaboratoryfocusesontheepigeneticsofgenomeprogrammingandreprogramming,inparticularthemechanismsenablinglineagecommitmentandtheiraberrationsincancerandneurologicaldiseases.

DuringtheArmenise-HarvardSymposium,Testagaveabrilliantoverviewofwhathecalledthe“zoojoining”:withpluripotentstemcellsonboard,themenagerieofavailabletissuesamplesbecamepotentiallyinfinite.

Thiswasunimaginableuntilthebeginningofourcentury,whenmodelorganismswerethemainresourcetointerrogatehumandiseasepathogenesis.Obtainingprimarysamplesdirectlyfrompatientswasverydifficult;andwhenithappened,mostofthetimeitwastoolate,postmortemoratnotsomeaningfulstagesofdiseasehistory.

Testaexplainedhowcellreprogrammingallowed,forthefirsttimeinthehistoryofmedicine,tomakehumangeneticvariationexperimentallytractablethroughthecreationofgeneticallymatchedcelllineages.Ontheselineagesitisnowpossibletodecipherandtargetdiseasepathogenesis,biologicalstand-insor“avatars”ofourselves.

Sowearethenewmodelstoworkon:thismeansthatone’sdiseaseisdirectlystudiedinvitroonone’sDNA,andpersonalizedtreatmentsaremorelikelytobedeveloped.

TheTestaLabharnessesthispotentialtodevelopphysiopathologicallymeaningfulmodelsofbothcancerandneurodevelopmentaldisorders.

Incancer,researchersaimatthedissectionofthegenomicversusepigenomiccomponents:sincetumorshavethemboth,cellreprogrammingallowstoseewhichcomponentispredominantineveryphaseofthedisease.

Withinneurodevelopmentaldisorders,theyfocusonauniquerangeofintellectualdisabilitysyndromes(includingautismspectrumdisorders)causedbymutationsordosagealterationsinepigeneticregulatorsandtranscriptionfactors.AfterreprogrammingandanalyzingiPSCandtheirdifferentiatedderivativeswiththesemutations,itisalsopossibletoscreendrugsonthemataverylargescale.

Theseexperimentalsettings,togetherwiththeirimportantclinicalapplications,areagreatexampleofhowcellularphenomenahavebecomeaninterfacebetweenmolecularbiologyandmedicine.

TestaquotedwhatbiologistHaroldKincaidcalledthe“placeholders”,processesforwhichwehavegoodevidence,butwhosenatureisunknown.Nowcellreprogrammingisprobablybringingthis“unknown”tolight.

SpatialRegulationofMolecularMotors

SamaraReck-Peterson

DepartmentofCellBiology,HarvardMedicalSchool,Boston,USA

Thecellsthatmakeupourbodyareconstantlybusy:theymove,divide,andcommunicatewithneighboringcells.Atthesametime,theyneedtomaintainhomeostasis,sothattheinternalcellularconditionsremainstableandrelativelyconstant.Molecularmotors,whichtransportcellularcargos,areresponsibleforallofthesefunctions.Transportoccursalongtwotypesoftracks,calledactinfilamentsandmicrotubules.

Alleukaryoticcellsusemotorsfortransportalongactinfilamentsandmicrotubules,anddamagetothesetransportmechanismscanleadtoseriousdiseases.Forexample,neurodegenerativeandneurodevelopmentaldiseasesareknowntoresultfromdefectsinmicrotubule-basedtransport.

Forthisreason,explaininghowmicrotubule-basedintracellulartransportworkscouldrepresentasignificantbreakthroughinclinicalmedicine.

TheReck-PetersonLabatHarvardMedicalSchoolismovinginthisdirection.“Wewanttounderstandhowthemotorsofthecellwork”explainedSamaraReck-Peterson,headofthelaboratory.“Inparticular,wearefocusingonthedyneinmotor”.

Cytoplasmicdyneinisthemainmotorproteindrivingmicrotubule-basedintercellulartransport,togetherwithanotherfamilyofmotorscalledkinesins.Dyneinmotorsmoveonlytowardstheminus-endsofmicrotubules(towardsthecellcenter),whilekinesinmotormovetowardstheplus-endsofmicrotubules(towardsthecellperiphery).

“Wehaveover40kinesingenesthathostdiversefunctions,butdyneinisalittlebitdifferent”explainedReck-Peterson.“Wehave15differentdyneinsinthehumangenomeand14oftheseareonlyfoundincellsthathaveciliaorflagella.Theremainingcytoplasmicdyneinmotorhashundredsofcargos,manyofwhicharestilltobeidentified;weknowthatduringinterphasedyneinhasmanyfunctionsincludingthetransportofmRNAs,RNPs,proteins,andorganelles.Virusesareanotherimportantcargo,theycanhijackdyneintogettothecenterofthecell”.

Tounderstandthemolecularmechanismsunderlyingthesefunctionsofdynein,theReck-PetersonLabusesahighlyinterdisciplinaryapproach,fromcelltosystemsbiology,frombiophysicstosyntheticbiology.

Therearefourgenesimplicatedinlocalizingdyneintotheplus-endofmicrotubule.Intheirexperiments,theypurifiedthesefourproteinsandreconstitutedinvitrothetransportofdyneintotheplus-endofthemicrotubule.

Theyfoundthattwoproteins–homologsofLis1andClip170–aresufficienttocoupledyneintoKip2,aplus-end-directedkinesin.Kip2transportsdyneintothemicrotubuleplusend,butnotasapassivepassenger:dyneinresistsitsownplus-end-directedmotionthoughitsmicrotubule-bindingdomain.

Twoothersmicrotubule-associatedproteins,homologsofClip170andEB1,actasprocessivityfactorsforKip2,helpingitovercomedynein’sintrinsicminus-end-directedmotility.

Thus,therearefourmainplayersinvolvedindyneinspatialregulation:twoproteinsthatarerequiredtocouplethedyneintokinesin,andthentwomoreproteinsthatmakethekinesinabettermotor.Thisrevealshowaminimalsystemofproteinstransportsamolecularmotortothestartofitstrack.

“Ourmaingoalnowistounderstandhowthismotorworksandisregulated,”saidReck-Peterson.“Andifweunderstandthat,weareastepclosertodiscoveringwhythemutationsinthetransportmachinerycausediseases”.

Mechanismsandregulationofcytoplasmicdynein

AndresLeschziner

Dept.ofMolecularandCellularBiology,HarvardUniversity,Boston,USA

Stepafterstep,walkingtakesprettyadvancedcoordinationabilities.Legs,muscles,nerves,brain:everysingleingredientfindsitsrole,buildingupthecomplexandharmoniousactionofmoving.

Atmicroscopiclevel,almostthesamethinghappensinyourcells,asexplainedAndresLeschzinerduringtheArmenise-HarvardSymposium.“Molecularmotorsaretheproteinmachinesthat‘walk’alongcytoskeletaltracks”hesaid.“Inparticular,cytoplasmicdyneinisresponsiblefortransportingmostcellularcargofromtheperipherytowardsthecellinterior.Itscomplexmotoractivityisessentialformanyfunctionsineukaryotes,suchaschromosomesegregationandintracellulartransport”.

Togetherwithkinesinsandmyosins,dyneinsmakeupthethreefamiliesofmolecularmotors.However,dyneinsarethelargestandmostcomplexgroup,andmanyoftheirregulatingmechanismsarestillunknown.

OneofthebiggestunsolvedquestionsconcernsLis1,aconserveddyneinregulator.Lis1isknowntokeepdyneinboundtomicrotubules,butitisnotunderstoodhowitaccomplishesthisaction.

AtHarvardMedicalSchool,theLeschzinerLabteamedupwiththeReck-PetersonLabtounfoldthemysteriesofdyneinusing3Delectronmicroscopy,single-moleculeimaging,biochemistryandinvivoassays.

Leschzinerandcolleagueswereparticularlyinterestedinunderstandinghowdyneinmovesalongmicrotubules,componentsofthecytoskeletonfoundthroughoutthecytoplasm.

A3Dstructureofthedynein-Lis1complexwasobtained.ThismodelrevealedthatbindingofLis1todynein’sAAA+ring(whichbelongstoasuperfamilyofring-shapedproteins)physicallyblocksdynein’smainmechanicalelement,the“linker”,frommakingcriticalinteractionswiththering.

“Lis1isaubiquitousdyneinco-factor,actingasaclutchtouncoupledynein’scyclesofATPhydrolysisandmicrotubulebindingandrelease”explainedLeschziner.

Sotherearetwodifferentcyclesinvolved:thefirst(acycleofforce-generatingATPhydrolysis)occursinthering-shapedAAA+motordomain;thesecond(acycleofmicrotubulebindingandrelease)occursinthemicrotubulebindingdomain,locatedattheendofdynein’slong“leg”.

Thesetwocyclesoccur25nmawayfromeachother,yettheirfunctioncriticallydependsontheircoordination.

Regulationofdyneinisthereforeoneofthecrucialaspectsforthecomplex,articulatedworldofmovementoccurringatthecellularlevel.AndapparentlyahugeresponsibilityfallsonLis1,playingacentralroleinregulatingdynein’smovements.

Overcomingobstacles:Single-moleculestudiesofDNArepair

JosephJ.Loparo

DepartmentofBiologicalChemistryandMolecularPharmacology;HarvardMedicalSchool

WecanthinkofDNAastheinstructionmanualpackedinsideourcells.IfourDNAbecomesdamaged,thenourcellsmaygetthewronginstructions,whichcanleadtodiseases.Forthisreason,whenaDNAlesionoccurs,ourcellsimmediatelyactivatetheirDNArepairmechanismstofixtheproblem.

Buttherearesomelesionsthattherepairmachineryfailstofind.InthiscasetheproteinmachinerythatcopiesourDNAcancollidewiththesedamagedDNAbases,stoppingtheDNAreplicationmachineryinitstracks.Itisthenthatourcellshaveatoughdecisiontomake:donothingandperhapsdieorutilizeanerrorproneDNAcopyingenzymethatcansynthesizethroughthedamagebutcouldintroducediseasecausingmutationsintoourDNA.

“ThisishowcellsovercomeDNAobstacles:sometimes,itisbetterforthemtotaketherisk”saidJosephLoparo,headofalaboratoryatHarvardMedicalSchoolworkingonDNAdamagetoleranceandrepair.

Gamblingasageneticstrategy,inotherwords.Loparoandcolleaguesareworkingtounravelhowbacterialcellschoosetousetheseerrorproneenzymes:“Mostbacteriahaveasinglecircularchromosomewhichtheycopywithamulti-proteinmachineknownasthereplisome,theirreplicationmachinery”heexplained.“ThereplisomeiscomposedoftworeplicativepolymerasecomplexeswhichquicklyandaccuratelycopyeachparentalstrandofDNA”.

IfDNAbecomesdamagedandtheDNArepairmachineryisunabletocorrectthelesion,thiscanbeablocktothereplisome.

“TranslesionpolymerasesarespecializedDNApolymerasescapableofsynthesizingovercertainDNAlesionsthatstallthereplicativeDNApolymerase”saidLoparo.“Mylabisinterestedinunderstandingthemechanismsofthisprocess,andhowtranslesionpolymerasesarerecruitedtothereplicationmachinery”.

Byreconstitutingtranslesionsynthesis(TLS)andobservingitoccuronsingleDNAmoleculesinrealtime,theLoparoLabshowedthattheEscherichiacolibclamp,aring-shapedmoleculethatencirclesDNAandtetherspolymerasestotheirsubstrates,cansimultaneouslybindtwokindsofpolymerases,thereplicativepolymerasePolIIIandtheerror-pronetranslesionpolymerase,PolIV.ThisenablesanexchangeofthetwopolymerasesandarapidbypassofaDNAlesionwhichisinmanywaysanalogoustohowonemaintainsasparetireinthetrunkoftheircar.

Furthermore,theyfoundthatadditionalbindingsitesbetweenPolIVandbacttolimitPolIVdependentDNAsynthesisundernormalconditions,yetfacilitatesthedisplacementofPolIIIfromtheDNAuponthedetectionofDNAdamage.

Theseresultssupportanewmodelinwhichinteractionsbetweenpolymerasesandthebclampacttobothinactivateandactivateerror-pronepolymerases.Withinthisregulatorynetwork,itislikethecellissaying:“Icantellthatthereplicationmachineryisintrouble.Itisworthutilizinganerrorpronepolymeraseasitismyonlyhopeforsurvival”.

Thismechanismisalsoveryinterestingfromahumanhealthperspective,becausemanyofthebasicmechanismsofthispolymeraseregulationlikelyoccurinhumancells.Understandinghowgeneticmutationsarisewillleadtoabetterunderstandingofdiseaseslikecancer.

“Thenextstepsforusaretolookattheseprocessesinlivebacteriacells,wherewehaveallthephysiologicalcomplexity”concludedLoparo.“Additionally,wearereconstitutingthereplicationmachineryinitsentiretyinatesttube”.

Thiscouldansweronceandforalltheodd,yetimportantquestion:howproneareourcellstowardsgeneticgambling?

Integratingartificialwithnaturalcells

SherefMansy

UniversityofTrento,ViadelleRegole,101,Mattarello(TN)Italy

Crossingtheboundarybetweenlivingandnon-living,bringingartificialsystemstolife.Sciencefiction?Probablynot.AttheCentreforIntegrativeBiology(CIBIO)oftheUniversityofTrento,biochemistSherefMansyhastakenastepforwardtowardsmakinganartificialcell“breath”.Thiswouldbeatruerevolutionfortraditionalcellularbiology,whichcouldeventuallyleadtochangethedefinitionitselfoflife.

“Achickisalivingorganism,astoneisnot.Everybodycanclearlyseethedifference.Butisthereanythinginthemiddle?”askedMansyattheArmenise-Harvardsymposiumaudience.Hethenpresentedthelatestresultsofhisresearch,showinganewwaytoaddressthechallengeofartificiallife.

Thecontrolofcellularbehaviorlargelyreliesongeneticengineering,butartificialcellscouldbedesignedtobetterregulatecellprocessesthroughchemicalcommunication.WithhisteamatCIBIO,MansydevelopedanartificialcellwhichisabletotranslateachemicalmessageintoasignalthatcanbesensedbyEscherichiacoli.Thiscouldactivateacellularresponseotherwiseimpossibletobedetected.

Withinthissystem,theartificialcellsworkaschemicaltranslators,sensingmoleculesthatE.colialonecannotsense.Asaconsequence,theartificialcellsreleaseamoleculefamiliartoE.coli,therebytranslatinganunrecognizedchemicalmessageintoawell-knownone.

Thisallowsa“dialogue”betweentheartificialandthenaturalcells,expandingthesensorycapabilitiesofE.coliwithoutalteringthegeneticcontentofthebacterium.

Mansy’sartificialcellhasacomplexstructure,bothfromengineeringandbiologicalpointofviews.Itisbuiltwithaphospholipidvesiclecontainingisopropylb-D-1-thiogalactopyranoside(IPTG),DNA,andtranscription-translationmachinery.TheDNAtemplatecodesforapreviouslyselectedriboswitch,activatingtranslationinresponsetothepresenceoftheophylline.Thetheophyllineriboswitchcontrolsthesynthesisoftheporeformingproteina-hemolysin(aHL).

colialonedoesnotrespondtotheophylline,andIPTGdoesnotcrossthevesiclemembraneoftheartificialcellintheabsenceofthepore.Andhere’sexactlywheretheartificialcellscometoaction:theyallowE.colitoreceivethechemicalmessage,thusbecomingIPTG-responsive.

“Bacteriadonaturallycommunicatetoeachother”saidMansy.“Ourgoalwastoseeifasinglebacteriumcouldstillcommunicateincaseoneofthecellsisartificial.Apparently,theanswerisyes.”

Theseresults,publishedinNatureCommunications,areapromisingbasisforpossiblemedicalapplications.“Ourartificialcellsdegradeinacoupleofhours:therearenolong-termconsequences.Thisimpliesthattheycouldbeusedinbiologicalsystems–forexample,toidentifypollutants–withoutgeneticintervention”explainedMansy.Sofarthisisthefirstartificial,cell-likesystemcapableofcreatingacommunicationpathwaybetweenartificialandlivingcells.Thenextstepwillbebroadeningthisapproach,makingartificialcellsableto“talk”withcomplexbiologicalsystems.

“Ifwehadartificialcellsthatcandetectallthesignalsofthelivingcells,theycouldalsorecognizethesignalsexpressedbyproblemcells,likecancer”concludedtheresearcher.

Thedefinitionof“livingcell”isthusbecomingmoreandmorefoggy:butthiscouldnotbeaproblem,asfaraswechangethequestionstobeaddressed.JustasAlanTuringdidabout60yearsago,whenhegavebirthtothefieldofArtificialIntelligence.

Biogenesisandqualitycontrolofoligomericproteinsintheearlysecretorypathway

RobertoSitia

UniversitàVita-SaluteSanRaffaele,DivisionofGeneticsandCellBiology,SanRaffaeleScientificInstitute,Milan,Italy

Prestoebeneraroavviene.ThistypicalItalianproverb,meaningthatitisdifficulttoworkfastandwellatthesametime,isbroadlydisregardedbyourcells.

AsRobertoSitiaexplainedduringtheArmenise-HarvardSymposium,manybiologicalprocessessuchasproteinsecretionmusthavehighfidelityandefficiency.

AtSanRaffaeleScientificInstitute,Sitiaaddressesafundamentalquestionincellbiology:howarethesizeandactivityofthedifferentcompartmentsconstantlycoordinated?

Inmulticellularorganisms,cellsmustpromptlyrespondtomultiplestimuli.Totaketherightdecision,theyneedtocontinuouslyexchangeinformationamongsteachotherandwiththeexternalworld,andtounambiguouslyintegratethecorrespondingsignals.

Thistaskbecomesparticularlydemandingduringdifferentiationorinresponsestoenvironmentalchanges.Sitiaandcolleaguesinvestigatedthemolecularmechanismsthatallowcellstointegratesignalling,proteinqualitycontrolandsortingintheearlysecretorycompartment.

“Theearlysecretorypathwayisemergingasakeyhub,performingmanydifficulttasksatthesametime”saidSitia.“Itensuresefficienthigh-qualityreleasebytheproteinfactory”.

Theendoplasmicreticulum(ER)isamultifunctionalcompartmentfoundinalleukaryoticcells.“FromtheER,secretoryproteinsbegintheirjourneytowardstheirfinaldestinations,theorganellesoftheexocyticandendocyticcompartments,theplasmamembraneortheextracellularspace”Sitiaexplained.HisresearchteamdiscoveredthatERp44,amultitaskproteinattheER-Golgiinterface,isamasterregulatorinthishub.

Fidelityofprotein-basedintracellularcommunicationisguaranteedbyqualitycontrol(QC)mechanismslocatedattheER–Golgiinterface,whichrestrictforwardtransporttonativeproteins.

AndhereiswhereERp44comestoaction:Sitiafoundthatitisakeyregulatorofproteinsecretion,Ca2+signallingandredoxregulation.

“IfthesignalsconveyedbytheERp44-centeredmolecularhubarenotworking,cellssuffer;butiftheyaretoomuch,thecell’sfunctionarealsocompromised”hesaid.

WearebeginningtounderstandwhatmakesERp44capableofsatisfyingitsmultipletasks,allowingtheproteinfactorytoperformprestoebene.

Imagingendocytosiswithhighspatiotemporalresolution

TomasKirchhausen

DepartmentofCellBiology,HarvardMedicalSchool,Boston,USA

“Seeingisbelieving.Biologyisbasedonobservation.ButwhatIwanttodoisalsomeasuring:thisquantificationcanbesize,canbelength,canbevolume,canbenumberofmolecules,canbewherearethemoleculesinagivenmoment”.

BiologistTomasKirchhausenknowswhathe’stalkingabout.HislaboratoryatHarvardMedicalSchoolisoneofthefewintheworldtocombinestandardand3Dmicroscopycapableofgivingrapid,high-precisionthree-dimensionalimagingoflivingcells.

Thiscutting-edgetechnologyisputattheserviceofunderstandingthemovementofmembraneproteinsthroughoutcells.Thesemechanismsareofkeyimportanceforthecell’ssortingmachineries,andcanbehijackedbytoxins,virusesandbacterialpathogens.

Studyinghowcellscanbeattacked,KirchhausenLabaimsatfindingtreatmentfordiseasesdependingonviralinfectionandpathogeninvasion:fromcancertoLGMD2B/Miyoshimusculardystrophies,fromAlzheimerdiseasetoALS(amyotrophiclateralsclerosis),aswellasotherneurologicaldiseases.

Inpreparationtothesestudies,hisgroupdeterminedthefirststructureatnearatomicresolutionofclathrin,aproteinplayingamajorroleinthecreationofcoatedvesicles.Clathrinhasatriskelionshapecomposedofthreeclathrinheavychainsandthreelightchainsandtheyformthecoatsurroundingthevesicleswhoselatticeoftenappearsastheseemofasoccerball.

Live-cellandsinglefluorescencemicroscopyimagingwereusedto“see”inthreedimensionsthemoleculareventsandtheintracellularcompartmentsresponsiblefortheformationofclathrin-coatedpitsandcoatedvesicles–aconserved“nano-machine”thatgeneratesintracellularvesicularcarriersinallanimalsandplants.

“Inourstudieswehavetwooppositeextremes”explainedKirchhausen.“Thefirstoneistheuseofmethodsgivingacompletepicture,likecrystallographyorNMR;thesecondextremeistheuseoffluorescencemicroscopyvisualizationmethodsgoingtoaverylowresolution,withalotofdynamics”.

“Sotheideaistocombinethesetwoextremestohaveaclearerviewofthemechanismofendocytosis”hesaid.

Endocytosis,theprocessthatcellsusetoingestmolecules(likeLDL–thebadcholesterolandcertainviruses)byengulfingthem,wasobservedwiththemicroscopyvisualizationtechniques.Theyallowedsufficienttemporalandspatialresolutiontofollowthelifeofasingleclathrincoatedpit.

“Theinitiationprocessofendocytosisishighlystochastic”continuedKirchhausen.“Weobservedthattheclathrinadaptors,proteinsthatconnectclathrinwiththemembranesurroundingtheclathrincoatarriveanddepartfromthemembranewithverylowbinding.Whenwehavetheadaptorsintherightplace,thetriskelioncanmapthisinteraction,thatstabilizesthestructureforafewsecondsandformationofthepitensues”.

ThelevelofdetailreachedbyKirchhausen’sobservationispioneerinquantitativebiology.“Nowthatwehavetherighttechniques,wecancounthowmanymoleculesofacertaintypearerequiredforthedifferentstepsofendocytosis:aquestionthathadremainedunansweredfor30years”commentedEmanueleCocucci,postdocworkinginKirchhausenLab.“Thenextstepswillbeunderstandingthetotalnumberofmoleculesinvolvedinendocytosis,includingallthereceptors.Sowehavetogoevendeeper”.

Inthenearfuture,temporalresolutionandspatialprecisionof3Dmicroscopycouldthenreachhigherdetails.ButasKirchhausenpointedout,itiscrucialnottolosethebiggerpicture.“It’slikethecartraffic”hesaid.“WecanseetheglobaldifferencesbetweenthetrafficofRomeandMilan,orwecancountthedetails,likethecoloursofthecar,thedrivers,thenumberofpassengers,andsoon.Bothtypeofquantificationsareimportant”.

Mono-ADP-ribosylationandmembranetrafficking

DanielaCorda

InstituteofProteinBiochemistry,NationalResearchCouncil,Napoli,Italy

Between20.000and25.000:thisisapproximatelythenumberofgenesinyourgenome.Butifyouthinkthisquantityishigh,youshouldprobablylookatyourproteome.Infactthenumberofproteinsmakingupthehumanproteomeisestimatedatover1million.

Overthelastdecades,scientistshavediscoveredthatsinglegenesencodemultipleproteins,andthismakestheproteomefarmorecomplexthanthegenome.

Butthereisasecretingredientwhichcanfacilitatestudyingthiscomplexity:itistheprocesscalledproteinpost-translationalmodifications(PTMs).Thisisafundamentalstepoccurringafterproteinbiosynthesis,playingakeyroleindeterminingtheregulationandfunctionofproteinsandothercellularmolecules.

Mostoftenmediatedbyenzymaticactivity,post-translationalmodificationscanoccuratanystepduringthe“lifecycle”ofaprotein,sounderstandingtheseprocessesiscrucialtounfoldthecomplexityofproteome.

AttheInstituteofProteinBiochemistryoftheItalianNationalResearchCouncil,DanielaCordaisleadingpioneeringresearchonPTMs.Amongthevariouspost-translationalmodifications,sheisfocusingonmono-ADP-ribosylation(mono-ADPR).Thisreactionhasanimportantphysiologicalroleincellularprocessessuchasmembranetraffic,immuneresponse,DNArepairandsignalling.

Cordawasoneofthefirstscientiststolookatmono-ADP-ribosylationasakeymechanismincellbiology.

“Formanyyearsithasbeenconsideredatoxicreaction”sheexplained.“Buttogetherwithothercolleagues,Ithoughtthatifatoxinmodifiesaprotein,perhapsthismodificationisinterferingwithaphysiologicalmechanism.Ithoughtthatifatoxinmodifiesaprotein,perhapsthismodificationisinterferingwithaphysiologicalmechanism.Thismeansthatisolatedpathologicalmechanisms(withouttheirphysiologicalcounterpart)donotexist”.

Thisintuitionshookuptheunderstandingoftheproteinenzymaticactivity.StudyinghowtoxinsinducetheADP-ribosylationofproteins,Cordaandothercolleaguescouldidentifyspecificcellmechanismsthatwerepreviouslyunknown.

Inparticular,theCordalaboratorystudiedbrefeldinA(BFA),afungaltoxincausingthedisassemblyoftheGolgicomplexmembranes.BFAinducestheADP-ribosylationofBARS,aproteininvolvedinthefissionofmembranesatseveraltrafficstepsofthesecretoryandendocyticpathways.

TheproteinBARSwasdiscovered20yearsagobyDanielaCordateam,andnowturnedouttobeanessentialelementofthemembranefissionmachinery.

SelectivelyanalyzingthebrefeldinAactivity,researchersidentifiedanintermediatethatcovalentlybindsBARS,calledBFA-ADP-riboseconjugate(BAC).

“BACmodifiesthisproteinonly,itisveryspecific”saidCorda.“SoifwecanmimicBAC,wecaninhibitasingleprotein,BARS.Thisiswhatweareworkingon:synthetizingBACanalogues,andperformingvirtualscreeningstoselectsothersmallmoleculeswithsimilaractivity”.

Theinhibitionbysmallmoleculesofproteinsknowntocauseadiseasemayeventuallyleadtoclinicalapplications,incancerasinotherpathologies.

“Wearetestingthesesmallmoleculesinvitrohopingtosoonfindanapplicationintumorssuchaslymphomaandbreastcancer”Cordaconcluded.

BARS,arelativelynewentryinscientificlabs,isthereforesheddinganewlightonthecellularfunctionscontrol.Andthisproteincouldsoonbecomeapharmacologicaltargetforanticancertherapies.

ControlsystemsofmembranetransportattheinterfacebetweentheendoplasmicreticulumandtheGolgi.

AlbertoLuini

IstitutodiBiochimicadelleProteine(IBP),CNRNapoli,Italy;TelethonInstituteofGeneticsandMedicine(TIGEM),Napoli,Italy.

Ifyouloseyourbalance,youwillprobablyfall.Yourcellsbehaveprettymuchthesameway:theyhaveaninternalequilibriumthatneedstoremainconstant,otherwisethey“fall”.Thiscellularbalanceiscalledhomeostasis,whichguaranteesthecells’internalstability.

Maintaininghomeostasisdespitethevariationofinternalandexternalconditionsisafundamentaltaskourcellularsystemneedstofulfill.Sounderstandingthistaskcanrevealimportantaspectsofourcellactivity.

AttheItalianNationalResearchCouncil,AlbertoLuiniandhisresearchgroupstudytheprocessofhomeostasisstartingfromthecomplexcellmembranetransportapparatus.

Withinthissystem,variationsinmembranefluxesfromtheendoplasmicreticulum(ER)totheGolgicomplexarebalancedbyoppositefluxesfromtheGolgitotheER,tomaintainhomeostasisbetweenthetwoorganelles.

AsLuiniexplainedtotheArmenise-HarvardSymposiumaudience,heandhisteamdescribedamoleculardevicethatbalancestransportfluxesbyintegratingsignaltransductioncascadeswiththetransportmachinery.

Inparticular,theyfoundthatER-to-GolgitransportactivatestheKDELreceptorattheGolgi.ThistriggersacascadeinvolvingGsandadenylylcyclaseandphosphodiesteraseisoforms,andthenPKAactivation,andresultsinthephosphorylationofproteinsinvolvedinretrogradetraffic.ThisinducesrecyclingtotheERandtendstobalancetransportfluxesbetweenERandGolgi.

Moreover,theKDELreceptoractivatesCREB1andothertranscriptionfactorsthatup-regulatetransport-relatedgenes.InthiswayaGolgi-basedcell-autonomouscontrolsystemmaintainstransporthomeostasisthroughbothsignalingandtranscriptionalnetworks.

Anotherinterestingthingisthatthismodelwasobtainedusingveryadvancedmicroscopyincludinganextremelypowerfultechnique:correlativemicroscopy.ItwasdevelopedforthefirsttimeinLuini’slaboratory,anditisusedtostudyinvivodynamicsandultrastructureofintracellularstructuresatincrediblelevelsofdetail.

Combiningcorrelativelightandelectronmicroscopy,researcherswereabletoseedynamicfunctionalassaysinlivecellsdirectlywithhighresolution3Dmorphology.

Thiswayitbecamepossibletoobservethecontrolsystemsofmembranetransportintegratinginformationondynamics,ultrastructureandmolecularcompositionassemblyofmolecularmachinery.Atthesametime,“seeing”theenginethatallowsourcellstomaintaintheirbalance.

MyosinVIBridgesUbiquitinSignalingandCellMigration

SimonaPolo

IFOMFondazioneIstitutoFIRCdiOncologiaMolecolare,20139Milan,Italy;DipartimentodiScienzedellaSalute,UniversitàdegliStudidiMilano,Milan,Italy.

Humancellscannotfreelytravelaroundthebody:theirmovementistightlyregulatedandnormallyquitelimited.Butcancercellscanlosethiscontrol,travellinginthebloodstreamthroughthewell-knowprocessofmetastasis.MyosinVIisamotor-proteinableto“travel”alongactinfilamentsandisinvolvedintumorformationandmetastasis.

AttheIFOM–FIRCInstituteofMolecularOncologyofMilan,SimonaPoloandcolleaguesarenowinvestigatingthecontrolsystemofMyosinVIfromanalternativepointofview.ThiswasthefocusofthefascinatingtalkgivenbyDr.PoloduringtheArmenise-HarvardSymposium:MyosinVIplaysanexplicitroleincellmigration,underbothpathologicalandnormalconditions.

DrPoloistheleaderofaresearchgroupinvestigatingthemechanismsofregulationmediatedbyubiquitin.Ubiquitinisaregulatoryproteinthathasbeen“ubiquitously”foundinalmostalltissues;itsaddictiontootherproteinscanaffecttheminmanyways.

“Thebest-knownfunctionofubiquitinisthedegradationviatheproteasome,butabout10yearsagoanovelfunctionofubiquitinationinsignallingwasdiscovered”sheexplained.“Wearestudyingthismechanism,andshowedthatMyosinIVharboursaparticularubiquitinbindingdomain(UBD)differentfromanypreviouslydescribedUBD”.

UBDsareacollectionofmodularproteindomainsthatnon-covalentlybindtoubiquitin;thenewUBDidentifiedbyPolo’steamwasgiventhenameofMyosinVIUbiquitinBinding(MyUb)domain.

Ubiquitincancomeindifferentflavoursandtheyallperformdifferentfunctionsintothecells,mostofwhicharestillunknown.

“Wefoundoutthat,differentlyfromthevastmajorityoftheUBDsthatshownobindingspecificity,MyUbdomainshaveaclearpreferenceforK63dimersimplicatingthatMyosinVIandubiquitininteractionisnotforproteindegradation”saidPolo.

ThesefindingspreparedthegroundtoexplaintheroleofUbandMIU-MyUbdomainsinthephysiologicalandpathologicalregulationofMyosinVI.

“IthasbeenobservedanoverexpressionofMyosinVIintumortissues”saidPolo.“Butwefoundoutthatthisisnotagenericoverexpression:itislimitedtothespecificformabletointeractwithubiquitin.Wearenowdissectingindetailstheunprecedentedroleplayedbyubiquitinincellmigration”.

Newinsightsintoproteasomemechanismsinnormalanddiseasestates

AlfredGoldberg

DeptCellBiology,HarvardMedicalSchool,Boston,USA

“Proteasomeissmarterthanyouthink!”ThisexclamationcrownedtheconclusionofthesharpandbrightpresentationbyAlfredGoldberg,duringthethirddayoftheArmenise-HarvardSymposium.

HislaboratoryatHarvardMedicalSchoolstudieshowproteinsinourbodyareconstantlybeingfabricatedandthenbrokendownintoaminoacids.Proteasomestakeprideofplaceinthisprocess:theyareproteincomplexeswhosemainfunctionistodegradeunneededordamagedproteinsbyproteolysis,achemicalreactionthatbreakspeptidebonds.

ThesmartproteasomeGoldbergreferredtoisthe26Sproteasome,themajorsiteforproteindegradationinmammaliancells.Inrecentyears,inhibitorsofits20Speptidaseactivities(e.g.bortezomib)havegreatlyadvancedthetreatmentofmultiplemyeloma.

Moreover,theprocessingofubiquitin(asmallregulatoryproteinfoundinalmostalltissuesofeukaryoticorganisms)conjugatesby26Sproteasome’s19Sregulatoryparticleinvolvesmanyenzymaticstepsthatmaybetargetsfordrugdevelopment.

Sothestudyof26Sproteasomehasimmediateapplicationalsoinclinicalmedicine,andthisisoneofthereasonswhyAlfredGoldbergtookitasthe“maincharacter”ofhisresearchandhistalk.Herethischaracterbecametheprotagonistoffourshortstories:foursurprisingmechanismscontrollingproteasomefunction.

“Thefirststoryisthattheproteasomeistightlyregulated”heexplained.“Wenowthinkweunderstoodthemechanism,theoveralllinkagebetweentheubiquitinchainandthebreakingdownoftheproteins:theregulationthatoccursinthatprocess”.

InfactGoldberdLabdiscoveredthatwhenmammalian26Sproteasomesareinhibited,theubiquitin-receptorsubunit,Rpn13,becomespolyubiquitinatedbya26S-associatedubiquitinligase.Thismodificationpreventsbindingofubiquitinatedsubstrates,andpresumablyevolvedtopreventbuild-upofconjugateswhenproteasomefunctionisstalled.

Andherewecometothesecondstory.“Thesurpriseisthatproteasometriggersunexpectedresponses”Goldbergtold.“Whentheproteasomeisinhibited,ithasamechanismsaying:‘don’tgivemeanymoreubiquinatedproteins!’It’saveryadvancedself-regulatedmechanism”.

Proteasomeregulationconcernsthethirdstoryaswell:“Wefoundthatwhentheproteasomeispartiallyinhibited,evenjustalittlebit,thesignalsofproductionofnewproteasomesevolveveryspecifically.Thisisaverysmartstructurethatknowshowtogetthecelltocompensatepossibleproblems”.

Thefourthstoryisaboutproteasomeanddisease.“Proteasomeisreallyaffectedincommonneurodegenerativediseases:thishadbeensuggestedmanytimes,butwithnoclearevidences”saidGoldberg.“Wehaveusednewtechniquestoshowinmousemodeldiseaseoffrontotemporaldementia,whichisveryclosetoAlzheimerdiseasebecausehasmutationsinTaugene”.

Inthiscasetheproteasomesaredefected:thisistightlyboundwithdiseasedevelopmentinmice.“Ourhypothesisisthatinalltmajorneurodegenerativediseasesthereisaprogressivefailuretodegradetheubiquinatedconjugates,andthisconditionisassociatedtotheaccumulationoftauandphospho-tau”concludedGoldberg.

Connectingthesefourstoriescouldgettheproteasomemechanismtoworkbetter,improvingNeurodegenerativediseasestherapies.Takingatthesametimeadvantageoftheproteasome’sunexpectedsmartness.

Towardsinvitroreconstitutionofspindlecheckpointsignaling

AndreaMusacchio

DepartmentofMechanisticCellBiology,Max-Planck-InstituteofMolecularPhysiology,Dortmund,Germany

Thedistributionoftheparentalgenometotwodaughtercellsduringmitosisandmeiosisistheessenceofgeneinheritanceandthereforeoflifeitself.Notsurprisingly,therefore,thisprocessinvolveswhatisprobablythemostcomplexensembleofcellularmolecularmachineryandprovidesanastoundingexampleoftheabilityofbiologicalmattertoself-organize.

Themitoticspindle,astructuremadeofmicrotubules,molecularmotorsandtheirregulators,hostsacruciallylargefractionofthemachineryofcelldivision.Thisbipolar,elongated,andremarkablydynamicstructuredefinesthedivisionplaneofthemothercell,anddevotesitselftothecaptureofchromosomes,totheirclusteringinthemiddleplane,andtotheirsubsequentsegregationtothedaughtercells.Thus,themitoticspindleiscrucialtoensurethatthedaughtercellsinheritexactlythesamenumberandtypeofchromosomes,thereforeguaranteeingcellularandorganismalviability.

Despitetheimportanceofthespindleforcelldivision,theexactmolecularbasisofitsfunctionremainspoorlyunderstood.TryingtounfoldthismysteryisthemaingoalofAndreaMusacchioattheMax-Planck-InstituteofMolecularPhysiologyinDortmund,Germany.Musacchioleadsaresearchteamfocusedoncomplexproteinscaffoldsknownaskinetochores.Kinetochoresarelargeproteinaceousstructuresbuiltonthecentromereregionofchromosomes.Theirprimaryfunctionistoprovideasiteofattachmentofchromosomestothemitoticspindle.

Kinetochoresplayasecond,subtlerfunctionthatishowevercrucialforaccuratecelldivision:theycontrolafeedbackmechanismknownasmitoticcheckpoint,orspindleassemblycheckpoint(SAC).Thefunctionofthischeckpointistopreventseparationoftheduplicatedchromosomes–thesisterchromatids–untiltheyhaveproperlyattachedtothespindleapparatus.DysfunctionoftheSACmayresultinincorrectpartitioningofthesisterchromatidstothedaughtercells,creatingapathologicalcellconditionknownasaneuploidy.Suchconditionstronglycorrelateswithtumorigenesis,supportingthespeculationthatcheckpointdysfunctionmaybe–amongothers–aprominentcauseoftransformation.

Overthelastyears,Musacchio’slaboratoryhasmadesignificantcontributionstothefield:“Wereconstitutedseveralkinetochoresub-complexesintheinnerandtheouterkinetochore,theregionsofthekinetochoreimplicatedincentromerebindingandmicrotubulebindingandcheckpointcontrol,respectively”,saidtheresearcher.

Indeed,biochemicalreconstitution,coupledwithmedium-orhigh-resolutionstructuralinvestigationsisplayingaleadingroleindevelopinganunderstandingofkinetochoreorganization.Forinstance,Musacchioandcolleagueswererecentlyableforthefirsttimetogainadetailedviewofacrucialkinetochoresub-complexmadeoffoursubunits,theCENP-HIKMcomplex.

Thenextchallengeisexplainingtherelationshipbetweenkinetochoresandcheckpointcontrol:thisiswhatMusacchiocalledthe“thirddecadeofcheckpointstudies”,afterthediscoveryofthemaincheckpointcomponents(1991-2000)andtheinvestigationoftheirinteractions(2001-2010).Toaddressthischallenge,hisresearchgroupisapproachingthereconstitutioninvitroofspindle

checkpointsignalling.Thiseffortfollowsatraditionalpathof“reductionist”studiesthatinterprettheemergenceofcomplexbiologicalfunctionsasaresultofspecificinteractionsofthemacromolecularbuildingblocksthatpopulatecells.

ThescaleofambitionimplicitinMusacchio’splans,however,transcendsthatofmostcurrentefforts.“Theword‘towards’isveryimportant:we’restillnotthere”pointedoutMusacchio.“Butwebelievethatitispossibletoreconstitutethecatalyticapparatusofthecheckpointonreconstitutedkinetochores”.Invitroreconstitutionofthiscomplexbiologicalstructurewillrevolutionizetheunderstandingofchromosomesdivisionduringmitosis,sheddinganewlightononeofthemostexcitingexamplesofself-organizationinlivingmatter.

Theautophagysignalingnetworkinthecoordinationofacell’sresponse

FrancescoCecconi

UnitofCellStressandSurvival,DanishCancerSocietyResearchCenter,2100Copenhagen,Denmark;DepartmentofBiology,UniversityofRomeTorVergata,Rome,Italy

Theworld“cannibalism”usuallyconjuresupimagesofprimitiveandbrutalpractices.Butdeepdown,weareallcannibals:ourcellsareconstantlyeatingthemselves,discardingoftheirredundantmoleculargarbagethroughaprocessknownasautophagy.

DerivingfromaGreektermmeaning“selfeating”,autophagyismostofthetimesasurvivingmechanism,allowingcellsto“recycle”theirbiologicalwaste.Andtomanyscientists’surprise,recentdiscoverieshaveshownthatfaultyautophagymechanismscontributetothedevelopmentofvariousdiseases.

FrancescoCecconi’slaboratoryiscommittedtounravelingtheupstreamregulationofautophagyandelucidatingtheroleitplaysinthreedifferentpathologicalconditions:neurodegeneration,autoimmunityandcancer.

Thelatterinparticularisstillobjectofdiscussion:“Incancerthereisasortofcontroversygoingonabouttheroleofautophagy”explainedCecconi.“Ifweknock-outanautophagygene,thenwegetaccumulationsoftoxiccompoundsthatautophagycan’thelpgettingridof.Forexample,damagedtissuescanoverproduceROS,whichcandamageDNAcausingchromosomeinstabilityandcancer.Inthiscase,mutationsintheautophagygenessetareresponsibleforcancerogenesis”.

Ontheotherside,autophagycanhavetheoppositefunction,beingevendetrimentalforthepatient.“Ifatumororiginatesfromacompletelydifferentsetofmutationsandwehavetheautophagysystemperfectlyworkingwithinthecells,thentheautophagyishelpingcancercellstosurvive”.

Thereisthenincanceradualfunctionofautophagy:dependingonthetumororigin,autophagyshouldeitherbepushedtoworkorblocked.

Inthiscontext,Cecconi’slabhasidentifiedanovelproteincalledAmbra1(ActivatingMoleculeinBeclin1-RegulatedAutophagy),playingmultiplerolesasascaffoldfactorinautophagycontrol.

“WearestudyingthefunctionofAmbra1incellcycleregulationanditsimplicationsintumorinsurgence”saidCecconi.

Togetherwithhisresearchteam,hedevelopedanexperimenttounderstandtheroleofAmbra1incancer.Theresultscouldshedanewlightonthelinkbetweenautophagyandtumordevelopment.

“IhopeIconvincedyouabouttheimportanceofthisprotein”concludedCecconitotheArmenise-Harvardaudience,andheprobablydid.

Thesignalingmechanismsunderlyingtheanti-tumoractivityofmetformin

GianniCesareni

DepartmentofBiology,UniversityofRomeTorVergata,RomeItaly

Fightingcancerwithawell-knownmedicine.Afewyearsago,thishypothesisdominatedmanyscientificmagazines,asanewpossiblefrontierfortumortreatment.

Thepromisingmedicinewasmetformin,themostfrequentlyprescribeddrugfortype2diabetespatients.Thesefindingsimmediatelytriggeredseveralclinicaltrials.Butthemechanismsunderlyingtheanti-tumoractivityofmetforminweren’tfullyclarified,anditsroleasacancersuppressorisstillunderexamination.

AttheUniversityofRome,TorVergata,GianniCesareniusedanewapproachtoaddressthesequestions.Togetherwithhisresearchgroup,hedecidedtolookatthebehaviorofacancercellasasystemandnotasasimplelinearcombinationofitsparts.Theserelativelynewapproachiscommonlyreferredtoassystemsbiology,asexplainedduringthe15thArmenise-HarvardSymposium.

“Welookatthecellinitsentirety,aimingatdescribinghowmetforminperturbsthephysiologicalprocessesofthecancercelltherebypreventingitsproliferation”heexplained.“Thisisanewapproachinmodernbiology,aimingtodescribethecellasasystemandnotsimplyasthesumofitsparts”.

Inordertoworkoutmetformin’sroleincancer,theCesareniLabappliedacombinationofhighcontentmulti-parametricanalysiswithlogicmodelingandsimulationtechniques.Theobjectivewastomapcellperturbationsoncomplexlogicnetworks.

Inthiswaytheyobtainedapredictivecell-specificlogicmodel,laterappliedtofunctionallycharacterizethemolecularmechanismunderlyingtheanti-canceractivityofmetformin.

“Weanalyzedhowsignalingnetworksarerewiredinbreastcancercellsuponmetformintreatment”saidCesareni.“First,wemonitoredtheactivationofabout20keysignalingproteinsincancercellstreatedwithavarietyofperturbationsbeforeandaftermetformintreatment.Next,webuilttwocellspecificnetworkmodelsofmetforminfortreatedanduntreatedcells”.

Theresultswereextremelyinteresting:theexperimentsshowedthatmetforminrewiresthesignalingnetworks,modifyingdifferentconnectionsbetweensignalingproteins.

“Wefoundthatmetforminchangesthestructureofthenetworksunderlyingsomecellpathways”explainedtheresearcher.“Sothecellsbecomemoresensitivetotopictreatmentsandlesspronetoproliferation”.

Tosumup,metformindoesn’tkillthetumorcells,butmakethemweaker.Andthisiscertainlyquiteagoodstartingpoint.

Mechanisticeventsregulatingnotchsignaltransduction

StephenC.Blacklow

HarvardMedicalSchool,Boston,MA,USA;DanaFarberCancerInstitute,Boston,USA

Thisstorybeginsin1917,whenThomasHuntMorganidentifiedallelesofageneresponsiblefornotchingofthewingsinfruitflies.Subsequentwork,ledinlargepartbySpyrosArtavanis-TsakonasofHarvardMedicalSchool,revealedthatthegeneresponsibleforthisphenotype,calledNotch,isahighlyconservedreceptorinasignaltransductionpathwaythatcontrolnumerouscellfatedecisionsinorganismsrangingfromfliestohumans.WhereasnormalNotchsignalingmakesitpossibleforadjacentcellstocommunicateeffectivelywitheachother,dysregulatedNotchsignalingoftenasaresultofmutationsintheNotchreceptorsortheirligands,contributestoavarietyofhumandiseases,includingneurodegenerationandcancer.

Thus,understandinghownormalandaberrantNotchsignalingtakesplacehasimportantimplicationsforclinicalmedicine.AtHarvardMedicalSchool,thisisonetaskoftheBlacklowLab:tounderstandthemolecularlogicofcell-surfacereceptorssuchasNotchproteins,whicharehighlyrelevanttohumanphysiologyanddisease.

“Amajorfocusofthelaboratoryistounderstandhownormalnotchsignalingtakesplace.ThisknowledgewillalsoyieldimportantinsightsintotheeffectsofaberrantNotchsignalingincancerandotherdiseases”saidStephenBlacklow,headofthelaboratory.

Hiseffortsaremainlydirectedtowardunderstandinghowactivationisinducedbyligandsandhownotchcooperateswithotherfactorstoregulatetargetgenetranscription.

“Onecentralunansweredquestioninsignalingishowthebindingsiteforligandscancommunicatewitharegulatoryswitchthatis600Angstroms(alongdistanceinmolecularterms)away,andhowligandstimulationnormallyrelievesautoinhibition”saidBlacklow.

Oneimportantcluetoansweringthisquestionisthatsignal-sendingcellsrelyonendocytosisoftheligandstodeliverthesignaltotheNotchreceptorsonthesignal-receivingcells.Twopossibilitiesmightexplainthisdependence:oneisthatligandendocytosisexertsmechanicalforcetopullontheNotchreceptorinordertoexposeametalloproteasecleavagesitetoactivatingproteolysis,andthesecondisthatligandsrelyonendocytosisforan“activating”modificationthatrendersthemcompetentforsignaling.Inotherwords,endocytosisandrecyclingofligandmayberequiredforconversionofligandsfromalatentstateintoanactiveform.

Thelaboratoryiscurrentlyworkingondistinguishingbetweenthesetwopossibilities.“Itispossibletosubstitutethenormalligand-receptorinteractionwithasyntheticsystem,whichretainsthedependenceonligandendocytosisinsendingcells,andontheactivationswitchinreceivingcells,”saidBlacklow.“Sotheworkingmodelthatcanbetestedinthesyntheticsystemisthatendocytosisisactuallysupplyingtheforcethatopenstheregulatoryswitch”.

MechanismofinhibitionoftheepidermalgrowthfactorreceptorbyMig6

MichaelJ.Eck

DepartmentofBiologicalChemistry&MolecularPharmacology,HarvardMedicalSchool;CancerBiology,Dana-FarberCancerInstitute,Boston,USA

Lungcanceristheleadingkillerintheworldamongtumors.AccordingtotheWorldHealthOrganization1,59millionofdeathsforlungcancerwereregisteredin2012.

Althoughlungcanceriscloselylinkedtosmoking,everyyearitaffectstensofthousandsofpeoplewhoneversmoked.

Inrecentyears,scientistscontinuedtounravelgeneticfactorsinvolvedincancerdevelopment.Andforlungcancerinnonsmokers,thereisanincreasedlikelihoodoffindingasomaticmutationoftheepidermalgrowthfactorreceptor(EGFR).EGFRisthecell-surfacereceptorformembersoftheepidermalgrowthfactorfamily(EGF-family)ofextracellularproteinligands.

AtHarvardMedicalSchool,MichaelEckisworkingonthestructureofsignalingcomplexesthatunderliecancer,withaparticularfocusonlungcancer-derivedmutationsintheepidermalgrowthfactorreceptor.Hislab’sstructuralapproachesarealsousedtofacilitatedevelopmentofanti-cancerdrugs.

“About15%oflungcancersarecausedbyEGFRmutations”explainedEckduringtheArmenise-HarvardSymposium.“Inparticular,somaticmutationsinEGFRareamajorcauseofnon-smallcelllungcancer”.

Hepresentedthelatestresultsofhislaboratory:thediscoveryofaquiteunexpectedmechanismofinhibitionofEGFRbyanendogenousregulator,Mig6.

Mig6(Mitogen-inducedgene6,alsocalledRALT)isafeedbackinhibitorofEGFRfamilymembersthatactsbydirectlybindingactivatedEGFR,inhibitingitscatalyticactivityanddirectingitsinternalizationanddegradation.

Mig6isprobablyatumorsuppressor:thismeansthatitslossleadstotumorformation.Inparticular,focaldeletionsspanningitschromosomallocusoccurinGBM(Glioblastomamultiforme)andlungcancer.

HisstudiesrevealedthatMig6isactuallya“mechanism-based”inhibitorofEGFR.“EGFRistrappedintheactofphosphorylatingMig6”explainedEck.“OncetheEGFRkinasephosphorylatesMig6,itiseffectivelyirreversiblyinhibited”.

ThesefindingsmayleadtonewtherapeuticstrategiesforlungandothercancerscausedbymutantEGFR.

Connectingthemachineriesofcellfatedeterminationandtumorsuppressioninmammarystemcells

PierPaoloDiFiore

FondazioneIFOM-IstitutoFIRCdiOncologiaMolecolare,MilanItaly

Itishardtoimaginethatstemcells,themost“immaculate”biologicalexistingmaterial,couldleadtocancer.Butaccordingtoarecenttheory,“stem-like”cancercellsareresponsibleforthegenerationoftumorsandforsustainingtumorgrowth.

Thismodelpredictstheexistenceofcancerstemcells(CSCs)withpropertiescharacteristicallyassociatedtonormalstemcells,suchasself-renewal,multipotencyandquiescence.

AttheInstituteofMolecularOncologyFoundation,PierPaoloDiFioreisexploringthischallengingstem-celltheoryofcancer.Heisinvestigatingthemolecularmechanismsgoverningthemaintenanceofthestemcellcompartmentinnormaltissues,andhowthesemechanismsaresubvertedincancer.

DuringtheArmenise-HarvardSymposiumclosingtalk,heexplainedhowislabisaddressingthisstudyperformingbothbasicandtranslationalcancerresearch.

“Theexistenceofcancerstemcellshasbeenprovedforanumberofcancertypes,includingbreastcancer”hesaid.“However,thebreaststemcellcompartmentremainspoorlycharacterizedduetothelackofreliabletechniquesfortheiridentificationandisolation.Wehavedevelopedanewtechniquetospecificallylabelandpurifybreaststemcellsfrommammaryglandtissue”.

Thistechniqueexploitsboththepropensityofbreaststemcellstogeneratemammospheres(3Dclustersofcells)insuspensionculture,andtherelativequiescenceofstemcellscomparedtootherbreastcelltypes,whenpropagatedinvitro.

DiFioreandcolleaguesareusingpurifiednormalandcancerstemcellstoisolatea“stemness”signaturefromwhichtheycanextractdiagnostic,prognosticandtherapeuticmarkersthatcanthenbeevaluatedfortestinginclinicaltrials.

Inthiscontext,acrucialroleisplayedbythemechanismsofasymmetriccelldivision.Inparticular,researchersareinvestigatingwhether,andhow,Numbandendocytosis-basedmechanismsareinvolvedintheregulationofasymmetriccelldivisionofhumanbreaststemcells.

“Numbisacellfatedeterminantthatbyasymmetricallypartitioningatmitosiscontrolsbinarycellfatedecisions”explainedDiFiore.“Inhumanbreastcancers,thereisfrequentlossofNumbexpression,duetoitsexaggeratedubiquitinationandensuingdegradation”.

ThisNumblosscausesalterationsintwomajordownstreampathways.Ontheonehand,lackofNumballowsforuncheckedsignalingactivityoftheNotchreceptor.Ontheother,lackofNumbcausesattenuationofthep53signalingpathway.Tumorsdisplayingloss-of-Numbexpressionareaddictedtothiseventandtoitsmolecularconsequences.

DiFioreclaimedthatthisleadstoafirstimportantconclusion,whichalsoconstitutedthe“take-homemessage”ofhistalk:“WhenyouhavehighNumb,youhavehighp53;whenyouhavelowNumb,youhavelowp53”.

ThissuggestsadoubleroleofNumb:itisprobablyatumorsuppressor,anditsactioncausesdecreasedp53activityinbreastcancers.Therefore,Numbcontrolsbothanoncogenicpathwayandatumorsuppressorpathway,andthismayleadtonewpromisingclinicalapplicationstofightcancerstemcells.

Armenise-HarvardSymposiumandyoungresearchers:theCareerDevelopmentAward

“Myfatherbelieved,andsodoI,thatsuperbresultsmostoftencomefromcollaboration.TheFoundationintendstocontinuelookingforwaystoenhanceconversationandcooperativeworkonbothsidesoftheAtlantic”.

WiththesewordstheArmenise-HarvardFoundation’sChairman,CountGiampieroAulettaArmenise,gaveatributetohislatefather,founderCountGiovanniAulettaArmenise,onthelastdayofthesymposium.HegreatlysummarizedtheFoundation’smission:establishingamultidisciplinary-basedscienceresearch.

The15thArmenise-HarvardSymposiumfullyreflectedthisvision.Intheend,everyoneagreedithadbeenanexcitingmeeting,withastronginterdisciplinaryapproach.

Alongwiththescientificsessions,over30scientistspresentedposters.Thisgavegroupleadersandyoungerresearchersthechancetotalkovertheirwork,findingnewcollaborationopportunities.

ThisisthespiritofthewholeFoundation,bestexpressedbyitsmostforward-lookinggrantprogram:theCareerDevelopmentAward(CDA).Since2001,theArmenise-HarvardFoundationhasfundedthedevelopmentoftalentedyoungscientists,establishingcollaborativerelationshipsbetweenItalianresearchersandHarvardMedicalSchool.

Inthelast14years,20scientistshavemovedtoItalyfromelsewherearoundtheworld,supportedbytheFoundation.Theysetuptheirownlabsalloverthecountry,fromPalermotoTrento.

The15thArmenise-HarvardSymposiumwitnessedthesuccessofthisfundingprogram.8CareerDevelopmentAwardeesattendedthemeeting:VincenzoCostanzo,FedericoForneris,ClaudiaLodovichi,MarieLaureBaudet,RosellaVisintin,TizianaBonaldi,StefanoGustincichandSherefMansy,whoalsowasoneofthespeakers.Allofthemaredevelopingapromisingcareerinscience.

“ThisisoneofthefewopportunitiestoindependentlyworkinItalyonaspecificresearchprogram”commentedVincenzoCostanzo,winnerofthe2013CDAgrant.HerecentlymovedtotheVertebrateGenomeStability,IFOMIstitutoFIRCdiOncologiaMolecolareinMilan,whereheisworkingontheroleofDNAdamageresponsefactorsinvertebrateDNAreplication.

“WestudyhowDNArepairsitself,andwhytumorcellsarenotcapableoffulfillingthistask”heexplained.“Cellsarelikeplanes:everysinglemechanismisrelatedtomanyothers.Ifoneofthesemechanismsisdamaged,aDNAdamageresponseisactivated.Wewanttounderstandhowtoselectivelyinterveneontumorcells,whichdefectsinthespecificgenesoftheDNAdamageresponse”.

Tothisend,VincenzoCostanzoisapplyingamultidisciplinaryapproach–thesamevisionbroadlypromotedduringthe15thArmenise-HarvardSymposium.

AsCountGiampieroAulettaArmenisestated:“Ourfoundersbelievedinlookingatproblemsfrommultipleangles,andtheFoundationwillcontinuetopursuethisgoal”.

Glossary

Actin:aglobularmulti-functionalproteinfoundthatformsmicrofilaments.Itisthemonomericsubunitoftwotypesoffilamentsincells:microfilaments,oneofthethreemajorcomponentsofthecytoskeleton,andthinfilaments,partofthecontractileapparatusinmusclecells.Actinparticipatesinmanyimportantcellularprocesses,includingmusclecontraction,cellmotility,celldivisionandcytokinesis,vesicleandorganellemovement,cellsignalling,andtheestablishmentandmaintenanceofcelljunctionsandcellshape.

Autophagy:anevolutionarilyconservedcatabolicmechanismthatinvolvescelldegradationofunnecessaryordysfunctionalcellularcomponentsthroughtheactionsoflysosomes.Althoughoriginallyclassifiedasatypeofprogrammedcelldeath,autophagyismorewidelyviewedasabasiccellsurvivalmechanismtocombatenvironmentalstressors

Chromatin:acomplexofmacromoleculesfoundincells,consistingofDNA,proteinandRNA.Itsprimaryfunctionsare:topackageDNAintoasmallervolumetofitinthecell;toreinforcetheDNAmacromoleculetoallowmitosis;topreventDNAdamage;4tocontrolgeneexpressionandDNAreplication.

Clathrin:aproteinthatplaysamajorroleintheformationofcoatedvesicles.Itformsatriskelionshapecomposedofthreeclathrinheavychainsandthreelightchains.

Dynein:amotorproteinconvertingthechemicalenergycontainedinATPintothemechanicalenergyofmovement.Ittransportsvariouscellularcargoby“walking”alongcytoskeletalmicrotubulestowardstheminus-endofthemicrotubule,whichisusuallyorientedtowardsthecellcenter.

Endocytosis:anenergy-usingprocessbywhichcellsabsorbmolecules(suchasproteins)byengulfingthem.

Epidermalgrowthfactorreceptor:thecell-surfacereceptorformembersoftheepidermalgrowthfactorfamily(EGF-family)ofextracellularproteinligands.

Heterochromatin:atightlypackedformofDNA,whichcomesindifferentvarieties.Thesevarietieslieonacontinuumbetweenthetwoextremesofconstitutiveandfacultativeheterochromatin.Bothplayaroleintheexpressionofgenes,whereconstitutiveheterochromatincanaffectthegenesnearthem(position-effectvariegation)andwherefacultativeheterochromatinistheresultofgenesthataresilencedthroughamechanismsuchashistonedeacetylationorpiRNAthroughRNAi.

Histone:ahighlyalkalineproteinfoundineukaryoticcellnucleithatpackagesandorderstheDNAintostructuralunitscallednucleosomes.Histonesarethechiefproteincomponentsofchromatin,actingasspoolsaroundwhichDNAwinds,andplayaroleingeneregulation.

Histonemethylation:aprocessbywhichmethylgroupsaretransferredtoaminoacidsofhistoneproteinsofchromosomes.Dependingonthetargetsite,methylationcanmodifyhistonessothatdifferentportionsofchromatinareactivatedorinactivated.Thisprocessiscriticalfortheregulationofgeneexpressionthatallowsdifferentcellstoexpressdifferentportionsofthegenome.

Histonemethyltransferases(HMT):histone-modifyingenzymesthatcatalyzethetransferofone,two,orthreemethylgroupstolysineandarginineresiduesofhistoneproteins.

Kinesin:aproteinbelongingtoaclassofmotorproteinsfoundineukaryoticcells.Kinesinsmovealongmicrotubulefilaments,andarepoweredbythehydrolysisofATP.Mostkinesinswalktowardstheplusendofamicrotubule,which,inmostcells,entailstransportingcargofromthecentreofthecelltowardstheperiphery.

MessengerRNA(mRNA):alargefamilyofRNAmoleculesthatconveygeneticinformationfromDNAtotheribosome,wheretheyspecifytheaminoacidsequenceoftheproteinproductsofgeneexpression.

Metformin:anoralantidiabeticdruginthebiguanideclass.Itisthefirst-linedrugofchoiceforthetreatmentoftype2diabetes,inparticular,inoverweightandobesepeopleandthosewithnormalkidneyfunction.

Microtubule:acomponentofthecytoskeleton,foundthroughoutthecytoplasm.Microtubulesareformedbythepolymerizationofadimeroftwoglobularproteins,alphaandbetatubulin;theyareinvolvedinmaintainingthestructureofthecelland,togetherwithmicrofilamentsandintermediatefilaments,theyformthecytoskeleton.

Myosin:aproteinofATP-dependentmotorfamily,bestknownforitsroleinmusclecontractionanditsinvolvementinawiderangeofothereukaryoticmotilityprocesses.Myosinsareresponsibleforactin-basedmotility.

Notch-1:ahumangeneencodingamemberoftheNotchfamily.Notchfamilymembersplayaroleinavarietyofdevelopmentalprocessesbycontrollingcellfatedecisions.

Notchproteins:afamilyoftransmembraneproteinswithrepeatedextracellularEGFdomainsandthenotch(orDSL)domains.Theseproteinsareinvolvedinlateralinhibitioninembryogenesis.

Notchsignalingpathway:ahighlyconservedcellsignalingsystempresentinmostmulticellularorganisms.Notchsignalingpromotesproliferativesignalingduringneurogenesis,anditsactivityisinhibitedbyNumbtopromoteneuraldifferentiation.

Numb:aproteinthatinhumansisencodedbytheNUMBgene.Itplaysaroleinthedeterminationofcellfatesduringdevelopment.

Phosphorylation:theadditionofaphosphate(PO43−)grouptoaproteinorotherorganicmolecule.Phosphorylationturnsmanyproteinenzymesonandoff,therebyalteringtheirfunctionandactivity.Proteinphosphorylationisonetypeofpost-translationalmodification.

Proteasomes:proteincomplexesinsidealleukaryotesandarchaea,andinsomebacteria.Ineukaryotes,theyarelocatedinthenucleusandthecytoplasm.Themainfunctionoftheproteasomeistodegradeunneededordamagedproteinsbyproteolysis,achemicalreactionthatbreakspeptidebonds.

Reactiveoxygenspecies(ROS):chemicallyreactivemoleculescontainingoxygen.ROSareformedasanaturalbyproductofthenormalmetabolismofoxygenandhaveimportantrolesincellsignalingandhomeostasis.

RNAinterference(RNAi):abiologicalprocessinwhichRNAmoleculesinhibitgeneexpression,typicallybycausingthedestructionofspecificmRNAmolecules.

RNA-inducedtranscriptionalsilencing(RITS):aformofRNAinterferencebywhichshortRNAmolecules–suchassmallinterferingRNA(siRNA)–triggerthedownregulationoftranscriptionofaparticulargeneorgenomicregion.

Yeast:single-celledfungithatreproducebybudding.Yeastsizecanvarygreatlydependingonthespecies,typicallymeasuring3–4µmindiameter,althoughsomeyeastscanreachover40µm.