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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.