ARMENISE-HARVARDSYMPOSIUM2014
MECHANISMSTOMOLECULES
15thBiennialSymposium
June22-24,2014,PoianoResortHotel,LakeGardaItaly
AbouttheSymposium
L1050036ThegentlebrushstrokesofaXVIIIcenturyoilpaintingseemedtomakeupthesettingof
the15thArmenise-HarvardSymposium.Amélangeofolivetrees,cypressesandoleanders
surroundedbygreenhillsandreflectedintheshimmeringblueoftheGardaLake:inthisbucolic
scenario,80scientistsmettodiscussthelatestadvancementsincellbiology.
BetweenJune22and24,thesymposiumheldintheluxuriousPoianoResortbroughttogether
researchersworkingonthecuttingedgeofscience.“Interdisciplinary”and“heterogeneity”were
themainkeywords:thiswasalreadyprettyclearfromthesymposiumtitle,“Mechanismsto
Molecules”.
Fromsingleproteinsactivitytocompletecellsystems,frombasicresearchtoclinicalapplication:
thetwo-and-a-half-daymeetingwasanexcitingopportunityforcross-disciplinarydiscussionsand
high-levelscientificexchanges.
Twenty-oneresearchers,allleadersoftheirresearchgroupsinItalyandtheUnitedStates,talked
abouttheirlatestresults,inmanycasespresentingpreliminaryandunpublisheddata.Thefriendly
andfreedialogueledtosharpdebatesaboutthemostfascinatingenginesofourbody:humancells.
Theoutstandingtalks,verydifferentfromeachother,hadacommonchallenge:makinglongstories
short.Infacteachpresentedresearchbeganmanyyearsago,whenmostofthecellularmechanisms
discussedduringthesymposiumwereunknown.
Today,newtechnologicaltoolsandhighlyadvancedmicroscopictechniquesaremaking“visible”
whatwaspreviouslyunseen;butatthesametime,itisimportantnottolosethebiggerpicture.
ThisiswhatemergedfromtheenthrallingkeynotespeechgivenbyHarvardProfessorStephen
Harrison,whodeliberatelyjuxtaposedthesymposiumname,entitlinghispresentation“Moleculeto
Mechanisms”.Heoutlinedvariouswaystothinkofmolecularactivitystartingfromthe
paradigmaticexampleofinfluenzavirushemagglutinin(HA),whosebindsreceptorcandefeatour
immuneresponse.
Thisstartedthediscussionabouttheincrediblenumberofinteractionshappeningfrommolecular
tocellmechanismlevel,andviceversa.Thefirstsymposiumsession,entitled“Mechanisms
RegulatingEpigeneticRegulation”,openedupthedoortoinvestigatethisintriguingcellular
complexity.DaneshMoazedandStephenBuratowskitalkedabouttwofundamentalingredientsof
cellregulation,chromatineandnoncodingRNA.DiegoPasiniexposedtheactivitiesofpolycomb
groupproteinsinproliferationanddevelopmentalcontrol,whileGiuseppeTestabroadenedthe
fieldbyexplaininghowcellreprogrammingrevolutionizedbiologyoverthelastyears.
Thesecondsession,“MechanisticInsightsfromSingleMoleculeAnalysis”,tooktheaudiencebackto
themolecularlevel.SamaraReck-PetersonandAndreasLeschzinertalkedaboutafundamental
aspectforcells’survival,themechanicsbehindmolecularmotors.JosephLoparogaveastimulating
talkaboutDNA“intelligence”,showinghowourgenesstrivetoovercomeobstacles.SherefMansy
challengedtheaudiencewithaverynovelfrontierofbiotechnology,theintegrationbetween
artificialandnaturalcells.
Anotherimportantaspectisthecommunicationamongstdifferentcells,andthiswasthetopicof
thethirdsession,“MechanismsRegulatingTrafficking”.RobertoSitiaexplainedhowthequality
controlworkswithinthesignaturepathway.TomasKirchhausenshowedtheexceptional
potentialitiesofthemodern3Dmicroscopytostudyendocytosis.DanielaCordaandAlbertoLuini
gavetwointerestingtalksaboutmembranetraffickingandtransportapparatus.
Withthebackdropoftherelevanceofthisresearchtomedicalapplications,thefourthsession
focusedon“DegradationMechanisms”.SimonaPolotalkedaboutcellmigrationundernormaland
pathologicalconditions.AlfredGoldbergpresentedthesysteminvolvedinproteindegradationby
proteasomes.AndreaMusacchioshowedhislatestresultsonthereconstructionofmitotic
signaling,whileFrancescoCecconidiscussedtheimplicationofoneofthemostincrediblebehavior
ofourcells,autophagy.
Thisledtothefifthandlastsession,“TransductionMechanisms”,openedbyGianniCesareni’stalk
aboutthesignalingmechanismsunderlyingtheanti-tumoractivityofmetformin.StephenBlacklow
andMichaelEckrespectivelypresentedthesignalingintheNotchcellandthecontrolofthe
epidermalgrowthreceptor.PierPaolodiFioreclosedthesessiondiscussingtheconnections
betweencellfatedeterminationandtumorsuppressioninmammarystemcells.
Thetaskofclosingthe15thArmenise-HarvardSymposiumfelltoTomasKirchhausen,who
wrappedupthegeneraltake-homemessageofthemeeting:thatweareconstantlydealingwitha
numberofcomplexinteractions,fromcellularmechanismstomolecularcommunication.Unfolding
themysteriesoftheseinteractionsisthemainchallengeofcellbiology,fascinatingandevergrowingfieldexploringthefundamentalunitsoflife.
Moleculetomechanisms—acasehistory
StephenHarrison
DepartmentofBiologicalChemistry&MolecularPharmacology,HarvardMedicalSchool,Boston,USA
Thetitleofthesymposiumwasreversedbythekeynotespeech:frommolecule–intentionally
singular–tomechanisms.Withthistalk,StephenHarrison“setthetoneforallthefollowing
presentations”,aswouldhavelaterobservedTomasKirchhausenduringthemeetingclosing
remarks.
Harrisonisoneoftheworld’sleadingexpertsonviruses.JeffreyFlier,PresidentoftheArmeniseHarvardFoundationandDeanofHarvardMedicalSchool,brieflyintroducedHarrison’soutstanding
scientificactivity:“Hehasmadeimportantcontributionstostructuralbiology,mostnotablyby
determiningandanalyzingstructuresofvirusesandviralproteins,andbycrystallographicanalysis
ofprotein-DNAcomplexes,aswellasstructuralstudiesofprotein-kinaseswitchingmechanisms.
Hisworkhasbeenwidelyrecognized,andhehasmadepioneeringresearchestoaddressarangeof
fundamentalproblems”.
Oneoftheseproblemsconcernstheinfluenzavirushemagglutinin(HA).TheHarrisonlabat
HarvardMedicalSchoolstudieshowinfluenzaviruspenetratescellsbyfusionofviraland
endosomalmembranescatalyzedbytheviralhemagglutinin.
SoHarrisonusedthisvirusasthecasehistoryofhispresentation–averyeffectivewaytodevelop
thethemeofthemeetingattheoutset.
“Thehemagglutininisatrimericstructurewiththreefunctions”heexplained.“Itbindsthevirusto
itsreceptor,catalyzesthemembranefusionprocess,andhasstructuresontheoutsidethatcanvary
withoutcompromisingitstwootheressentialfunctions”.
Inthiswaytheviruscanevolvetoescapeneutralizationbytheimmunesystemofitshost.
Intheinfluenza,theproteinhemagglutininsticksoffofthevirussurfacealongwithanotherprotein,
whichisanenzyme,calledtheneuraminidase(NA).
Harrisonfocusedontwoparticularmechanisms:HA-mediatedfusionandHAantigenicity.
Membranefusionisthermodynamicallyfavorable,butitgenerallypresentsahighkineticbarrier.
Fusionproteinslowerthisbarrier,sotheyarecatalystsforthemergeroftwobilayers;inthecaseof
viralfusionproteins,theybecome“suicide”catalysts.Afterseveralexperimentssetuptomeasure
fusionkinetics,Harrisonandcolleaguesshowedthatforinfluenzavirusfusionrequires
engagementwiththetargetbilayeroffusionpeptidesfrom3or4neighboringHAtrimmers.
“Thecasehistorylessonissummarizedhere”saidHarrison,“Thatthereisacontactpatchbetween
thevirusandthetargetmembrane.”
Thisintermediateisafundamentalaspectofthefusionmechanism.ButhowdoesHAantigenicity
reacttothismechanism?
Antigenicityisthecapacitytostimulatetheproductionofantibodies.Duringtheimmuneresponse,
theprocessbywhichBcellsproduceantibodieswithincreasedaffinityforantigeniscalledaffinity
maturation.
“Antibodystructures,fromB-celllineagesinhumanresponsestoinfluenzavirusvaccines,map
evolutionofproteininteractionsduringantibodyaffinitymaturation”explainedHarrison.
Thefuturechallenge,heconcluded,isthereforetounderstandtheaffinitymaturationmechanisms
wellenoughtodesignmodifiedimmunogensthatmightselectivelyelicitbroadimmuneresponses.
Frommoleculetomechanisms,then:theinspiringexampleofinfluenzahemagglutiningotstraight
totheheartofthesymposiumtheme.Showingfirstofallhowbiologycantravelbetweenthemicro
andthemacrolevelofourcells.
Geneon,geneoff.Ineukaryoticcells,DNAactivationisoftenamatterofswitches.Butwhatarethe
biologicaltriggerscapableofunchaininggeneexpression?Thisisoneofthecorequestions
addressedbyDaneshMoazedatHarvardMedicalSchool.
Hislaboratoryfocusesonunderstandingthemechanismsthatmediatetheformationofoneofthe
mosttightlypackedformsofDNA:heterochromatin,whichplaysacrucialroleingenesilencing.
Forthisreason,thestudyofheterochromatin–alsocalledsilentchromatin–helpsunderstanding
themechanismsthatkeepgenesintheirrightonoroffstate.
Theformationofthesesilentdomainsisalsoresponsiblefortheso-calledepigeneticmemory,
whichmaintainscellidentityduringdevelopmentanddifferentiation.
Biologistsknowthateverysinglecellofourorganismonlyturnsonthesubsetofgenesnecessary
tocarryoutparticularfunctions.Thisisthereasonwhy,forexample,thegenesofnerveimpulse
transmissionremainshutoffinlivercells:theyarejustnotusefulinlivercellsandcaninterfere
withliverfunction.
Moazedandcolleaguesaimatunderstandinghowcellssilenceunnecessarygenestomaintaintheir
specificidentities.
Theyapplyacombinationofapproaches:frombiochemistrytocellbiology,fromproteomicsto
genomics,withthecommongoalofidentifyingthefactorsabletomediateheterochromatin
assemblyandfunction.
Analyzingsilentdomainsinyeast–single-celledfungithatreproducebyfission–researchers
identifiedaspecialingredientplayingacentralrolesintheformationofrepressive
heterochromatin:noncodingRNAs.
“NowadaystherearealotofideasaboutwhatsmallRNAsarecapableofdoing,andsome
mechanismsareclearerthanothers”Moazedexplained.“ThewaytheseRNAsaregeneratedisa
fundamentalquestion,becausetheyspecifythepartsofthegenomethatshouldbesilenced”.
Inparticular,histeamshowedthatsmallRNAmolecules(overall,about20nucleotides)work
throughtheRNAinterference(RNAi)toregulategenesilencingandexpression.Developingmany
biochemicalexperimentsinfissionyeast,theypurifiedtheRNA-InducedTranscriptionalSilencing
(RITS)complex,whichdirectlylinkstheRNAipathwaytoheterochromatinassembly.
Moazed’slabfocusedontwocriticalprocessesformaintenanceofheterochromatin,histone
methylationandsiRNAamplification.Thesetwoprocesseswereshowntobemutuallydependent
andtoformself-reinforcingpositivefeedbackloopsofcrucialimportanceformaintenanceofsilent
domains.
SuchnoncodingRNA-basedmechanismsmaybeinvolvedinregulatingheterochromatinformation
andgeneexpressioninotherorganisms.Andthisisexactlywhatstudiesinotherlaboratoriesare
beginningtoreveal.
“Futureapplications?UsingsmallRNAstosilencegenesandreprogramtheepigenome”hesaid.“It
maybepossibletoturn-offgenesatthetranscriptionallevel,althoughmanytechnicalchallenges
havetobeovercomebeforethiscanbedoneinsystemsoutsidesimplermodelorganismslike
yeast”.
Thenextstep,then,istheinvestigationofotherbiologicalpathwaysthatplaymajorrolesin
regulatingheterochromatin:inordertounderstandbetterandbetterthecomplex,fascinating
switchesofDNA.
Shapingtheeukaryotictranscriptomewithchromatinandnon-codingRNA
StephenBuratowski
DepartmentofBiologicalChemistryandMolecularPharmacology,HarvardMedicalSchool,Boston,
USA
Thehugedictionarybuildingupourgenomehasaverycomplexpunctuation.DNAsequencesare
justsmallstringsofletters,butunderstandingtheirrulesisabigchallenge.
AtHarvardMedicalSchool,theBuratowskiLabstudiesthemarksofgeneexpressions:inparticular,
theenzymesthat“write”thesemarks,aswellastheproteinsthat“read”them.
Usingyeastasamodelsystem,researchersanalyzethemechanismofgeneexpressionin
eukaryotes,workingonRNApolymeraseIItranscriptioninitiationandthesubsequentprocessing
ofthemRNA.Severaldozenproteinsarerequiredsimplytoinitiatetranscription,andmanymore
takeactionsinotherprocesseslinkedtotranscription.Forthisreason,understandingtranscription
meansdecipheringthefunctionsofeverysinglefactor.
Duringthe15thArmenise-HarvardSymposiumStephenBuratowski,headofthelaboratory,
presentedhisapproachtounfoldthemysteriesoftranscription’spunctuation.Hisresearchteam
focusesonthecommunicationbetweenchromatinandthetranscriptionmachinery.
Theyshowedthattheactoftranscriptioncausesmajorchangesinthenucleosomesthatpackage
thegene.UsingtheyeastSaccharomycescerevisiae,theydiscoveredthatSet1(atypeofhistone
methyltransferases,orHMT)proteinlevelsarecarefullycalibratedtotheamountoftranscription
occurringinthecell.Set1issubjecttodegradationbytheubiquitin-proteasomesystemunlessitis
stabilizedbyongoingtranscriptionandhistonemethylation.Disruptionofthisfeedbackloop
causesaberrantmethylationpatternsandgenemisregulation.
Thismechanismmayhaveclinicalrelevance,asmanyleukemiaandlymphomascontain
translocationsinthemammalianMLL1gene,anHMTrelatedtoSet1.
Carryingoutgenome-wideexpressionscreenstoanalyzeachromatin“reader”calledSet3,
Buratowskiandcolleaguesfoundthatthemajorityofgeneswhoseexpressionlevelschangedwere
repressedbythisfactor.Butwhatwasmoreunexpected,theydemonstratedthattheseeffects
stronglycorrelatewithoverlappingnon-codingtranscription.Thisdoesnotmeanthatnon-coding
RNAsthemselvesmediatethegeneexpressionchanges;instead,thelatterdependonhistone
methylationsplacedovergenepromotersbyoverlappingtranscription.
Andthisisexactlywhereclinicalmedicinecouldfindnewapplications:“Ithinkitiscertainlyworth
thinkingabouthowchangesintranscriptioncanaffectdiseaseslikecancer”Buratowskiconcluded.
“Thisisbecausemanydiseasesarecausedbyproblemsineitherwritingorreadingthemarksof
geneexpressions”.
ActivitiesofPolycombGroupProteinsinProliferationandDevelopmentalControl
DiegoPasini
DepartmentofExperimentalOncology,EuropeanInstituteofOncology,Milan,Italy
Manyhumandiseasesarecausedbythelossofcellularidentity.Canceristheclearestexample:in
alltumors,cellsacquirefeaturesthatleadtoabnormalgrowthanddifferentiationdefects.
Buthowhappensthatcellsstartlosingtheiridentity?Andwhatarethemechanismsregulatingcell
fateduringdevelopmentanddifferentiation?
DiegoPasini’scurrentresearchattheEuropeanInstituteofOncologyrevolvesaroundthese
questions.Heaimstounderstandhowdiseaseslikecancercanform,maintainanddevelop.
DuringtheArmenise-HarvardSymposium,Pasiniexplainedthathislabisinterestedinstudying
thesemechanismsbyfocusingonPolycombGroup(PcG)proteins.
PcGareafamilyofproteinsresponsibleforcellulardifferentiationduringdevelopmentvia
transcriptionalrepression.
“PolycombGroupproteinsaremasterregulatorsofcelldevelopment”saidPasini.“Theyarealso
essentialforcellularproliferation,andplayanactiveroleincancerformation”.
ThefirsttodescribethepolycombgroupwasgeneticistEdwardB.Lewis,whoin1978observed
inDrosophilathatPcGwasinvolvedinthesilencingofHoxgeneexpression.
Sincethen,theseproteinshavebeenthesubjectofintensestudyasitisclearthattheyarevitalfor
maintenanceofcell-typeidentityanddifferentiation.
“Atthebiochemicallevel,mostPcGproteinsformtwomajorpolycombrepressivecomplexes:PRC1
andPRC2”explainedPasini.
PRC1andPRC2represstranscriptionrespectivelybyUbiquitylatingHistoneH2Alysine(K)119
andbytri-methylating(me3)HistoneH3K27.DeregulationofbothPRC1andPRC2activitiesisa
commonfeatureofhumantumors.
TostudytheroleofPcGproteinsinregulatingnormalandcancercellsproliferation,Pasiniand
colleaguescombinecellcultureandinvivostudies.TheirexperimentsdemonstratedthatPRCs
independentlyregulatecellularproliferationandtransformation.
SotheabilityofPRC1andPRC2topromoteproliferationisamainfeaturethatlinksPolycomb
Groupproteinsactivitytocancer.
Forthisreason,PcGinhibitionhasbeenproposedasastrategyfortumortreatment.
JoiningtheZoo:Cellreprogrammingandtheriseofhumandiseasemodels
GiuseppeTesta
DepartmentofExperimentalOncology,EuropeanInstituteofOncology,Milan,Italy
Haveyoueverthoughtofyourbodyasarovinglaboratory?Intheverylatestyears,thisisexactly
whathashappened.Atruerevolutionoccurredincellularbiology:itiscalled“cellreprogramming”,
anditallowsscientiststodirectlystudydiseasesinhumantissues.
Cellreprogrammingdependsontheseminalderivationofhumaninducedpluripotentstemcells
(iPSC)fromsomaticcells.Bornin2007,thistechniquehasalreadydeeplychangedtheprospects
notonlyofregenerativemedicinebutalso,andlikelyinanevenshortertimeframe,ofourcapacity
todissectthegeneticcontributiontohumandiseases.
AttheEuropeanInstituteofOncology,GiuseppeTestaisworkingonthiscutting-edgelineof
research.Hislaboratoryfocusesontheepigeneticsofgenomeprogrammingandreprogramming,in
particularthemechanismsenablinglineagecommitmentandtheiraberrationsincancerand
neurologicaldiseases.
DuringtheArmenise-HarvardSymposium,Testagaveabrilliantoverviewofwhathecalledthe
“zoojoining”:withpluripotentstemcellsonboard,themenagerieofavailabletissuesamples
becamepotentiallyinfinite.
Thiswasunimaginableuntilthebeginningofourcentury,whenmodelorganismswerethemain
resourcetointerrogatehumandiseasepathogenesis.Obtainingprimarysamplesdirectlyfrom
patientswasverydifficult;andwhenithappened,mostofthetimeitwastoolate,postmortemor
atnotsomeaningfulstagesofdiseasehistory.
Testaexplainedhowcellreprogrammingallowed,forthefirsttimeinthehistoryofmedicine,to
makehumangeneticvariationexperimentallytractablethroughthecreationofgeneticallymatched
celllineages.Ontheselineagesitisnowpossibletodecipherandtargetdiseasepathogenesis,
biologicalstand-insor“avatars”ofourselves.
Sowearethenewmodelstoworkon:thismeansthatone’sdiseaseisdirectlystudiedinvitroon
one’sDNA,andpersonalizedtreatmentsaremorelikelytobedeveloped.
TheTestaLabharnessesthispotentialtodevelopphysiopathologicallymeaningfulmodelsofboth
cancerandneurodevelopmentaldisorders.
Incancer,researchersaimatthedissectionofthegenomicversusepigenomiccomponents:since
tumorshavethemboth,cellreprogrammingallowstoseewhichcomponentispredominantin
everyphaseofthedisease.
Withinneurodevelopmentaldisorders,theyfocusonauniquerangeofintellectualdisability
syndromes(includingautismspectrumdisorders)causedbymutationsordosagealterationsin
epigeneticregulatorsandtranscriptionfactors.AfterreprogrammingandanalyzingiPSCandtheir
differentiatedderivativeswiththesemutations,itisalsopossibletoscreendrugsonthematavery
largescale.
Theseexperimentalsettings,togetherwiththeirimportantclinicalapplications,areagreat
exampleofhowcellularphenomenahavebecomeaninterfacebetweenmolecularbiologyand
medicine.
TestaquotedwhatbiologistHaroldKincaidcalledthe“placeholders”,processesforwhichwehave
goodevidence,butwhosenatureisunknown.Nowcellreprogrammingisprobablybringingthis
“unknown”tolight.
SpatialRegulationofMolecularMotors
SamaraReck-Peterson
DepartmentofCellBiology,HarvardMedicalSchool,Boston,USA
Thecellsthatmakeupourbodyareconstantlybusy:theymove,divide,andcommunicatewith
neighboringcells.Atthesametime,theyneedtomaintainhomeostasis,sothattheinternalcellular
conditionsremainstableandrelativelyconstant.Molecularmotors,whichtransportcellularcargos,
areresponsibleforallofthesefunctions.Transportoccursalongtwotypesoftracks,calledactin
filamentsandmicrotubules.
Alleukaryoticcellsusemotorsfortransportalongactinfilamentsandmicrotubules,anddamageto
thesetransportmechanismscanleadtoseriousdiseases.Forexample,neurodegenerativeand
neurodevelopmentaldiseasesareknowntoresultfromdefectsinmicrotubule-basedtransport.
Forthisreason,explaininghowmicrotubule-basedintracellulartransportworkscouldrepresenta
significantbreakthroughinclinicalmedicine.
TheReck-PetersonLabatHarvardMedicalSchoolismovinginthisdirection.“Wewantto
understandhowthemotorsofthecellwork”explainedSamaraReck-Peterson,headofthe
laboratory.“Inparticular,wearefocusingonthedyneinmotor”.
Cytoplasmicdyneinisthemainmotorproteindrivingmicrotubule-basedintercellulartransport,
togetherwithanotherfamilyofmotorscalledkinesins.Dyneinmotorsmoveonlytowardsthe
minus-endsofmicrotubules(towardsthecellcenter),whilekinesinmotormovetowardstheplusendsofmicrotubules(towardsthecellperiphery).
“Wehaveover40kinesingenesthathostdiversefunctions,butdyneinisalittlebitdifferent”
explainedReck-Peterson.“Wehave15differentdyneinsinthehumangenomeand14oftheseare
onlyfoundincellsthathaveciliaorflagella.Theremainingcytoplasmicdyneinmotorhashundreds
ofcargos,manyofwhicharestilltobeidentified;weknowthatduringinterphasedyneinhasmany
functionsincludingthetransportofmRNAs,RNPs,proteins,andorganelles.Virusesareanother
importantcargo,theycanhijackdyneintogettothecenterofthecell”.
Tounderstandthemolecularmechanismsunderlyingthesefunctionsofdynein,theReck-Peterson
Labusesahighlyinterdisciplinaryapproach,fromcelltosystemsbiology,frombiophysicsto
syntheticbiology.
Therearefourgenesimplicatedinlocalizingdyneintotheplus-endofmicrotubule.Intheir
experiments,theypurifiedthesefourproteinsandreconstitutedinvitrothetransportofdyneinto
theplus-endofthemicrotubule.
Theyfoundthattwoproteins–homologsofLis1andClip170–aresufficienttocoupledyneinto
Kip2,aplus-end-directedkinesin.Kip2transportsdyneintothemicrotubuleplusend,butnotasa
passivepassenger:dyneinresistsitsownplus-end-directedmotionthoughitsmicrotubule-binding
domain.
Twoothersmicrotubule-associatedproteins,homologsofClip170andEB1,actasprocessivity
factorsforKip2,helpingitovercomedynein’sintrinsicminus-end-directedmotility.
Thus,therearefourmainplayersinvolvedindyneinspatialregulation:twoproteinsthatare
requiredtocouplethedyneintokinesin,andthentwomoreproteinsthatmakethekinesinabetter
motor.Thisrevealshowaminimalsystemofproteinstransportsamolecularmotortothestartof
itstrack.
“Ourmaingoalnowistounderstandhowthismotorworksandisregulated,”saidReck-Peterson.
“Andifweunderstandthat,weareastepclosertodiscoveringwhythemutationsinthetransport
machinerycausediseases”.
Mechanismsandregulationofcytoplasmicdynein
AndresLeschziner
Dept.ofMolecularandCellularBiology,HarvardUniversity,Boston,USA
Stepafterstep,walkingtakesprettyadvancedcoordinationabilities.Legs,muscles,nerves,brain:
everysingleingredientfindsitsrole,buildingupthecomplexandharmoniousactionofmoving.
Atmicroscopiclevel,almostthesamethinghappensinyourcells,asexplainedAndresLeschziner
duringtheArmenise-HarvardSymposium.“Molecularmotorsaretheproteinmachinesthat‘walk’
alongcytoskeletaltracks”hesaid.“Inparticular,cytoplasmicdyneinisresponsiblefortransporting
mostcellularcargofromtheperipherytowardsthecellinterior.Itscomplexmotoractivityis
essentialformanyfunctionsineukaryotes,suchaschromosomesegregationandintracellular
transport”.
Togetherwithkinesinsandmyosins,dyneinsmakeupthethreefamiliesofmolecularmotors.
However,dyneinsarethelargestandmostcomplexgroup,andmanyoftheirregulating
mechanismsarestillunknown.
OneofthebiggestunsolvedquestionsconcernsLis1,aconserveddyneinregulator.Lis1isknownto
keepdyneinboundtomicrotubules,butitisnotunderstoodhowitaccomplishesthisaction.
AtHarvardMedicalSchool,theLeschzinerLabteamedupwiththeReck-PetersonLabtounfoldthe
mysteriesofdyneinusing3Delectronmicroscopy,single-moleculeimaging,biochemistryandin
vivoassays.
Leschzinerandcolleagueswereparticularlyinterestedinunderstandinghowdyneinmovesalong
microtubules,componentsofthecytoskeletonfoundthroughoutthecytoplasm.
A3Dstructureofthedynein-Lis1complexwasobtained.ThismodelrevealedthatbindingofLis1
todynein’sAAA+ring(whichbelongstoasuperfamilyofring-shapedproteins)physicallyblocks
dynein’smainmechanicalelement,the“linker”,frommakingcriticalinteractionswiththering.
“Lis1isaubiquitousdyneinco-factor,actingasaclutchtouncoupledynein’scyclesofATP
hydrolysisandmicrotubulebindingandrelease”explainedLeschziner.
Sotherearetwodifferentcyclesinvolved:thefirst(acycleofforce-generatingATPhydrolysis)
occursinthering-shapedAAA+motordomain;thesecond(acycleofmicrotubulebindingand
release)occursinthemicrotubulebindingdomain,locatedattheendofdynein’slong“leg”.
Thesetwocyclesoccur25nmawayfromeachother,yettheirfunctioncriticallydependsontheir
coordination.
Regulationofdyneinisthereforeoneofthecrucialaspectsforthecomplex,articulatedworldof
movementoccurringatthecellularlevel.AndapparentlyahugeresponsibilityfallsonLis1,playing
acentralroleinregulatingdynein’smovements.
Overcomingobstacles:Single-moleculestudiesofDNArepair
JosephJ.Loparo
DepartmentofBiologicalChemistryandMolecularPharmacology;HarvardMedicalSchool
WecanthinkofDNAastheinstructionmanualpackedinsideourcells.IfourDNAbecomes
damaged,thenourcellsmaygetthewronginstructions,whichcanleadtodiseases.Forthisreason,
whenaDNAlesionoccurs,ourcellsimmediatelyactivatetheirDNArepairmechanismstofixthe
problem.
Buttherearesomelesionsthattherepairmachineryfailstofind.Inthiscasetheproteinmachinery
thatcopiesourDNAcancollidewiththesedamagedDNAbases,stoppingtheDNAreplication
machineryinitstracks.Itisthenthatourcellshaveatoughdecisiontomake:donothingand
perhapsdieorutilizeanerrorproneDNAcopyingenzymethatcansynthesizethroughthedamage
butcouldintroducediseasecausingmutationsintoourDNA.
“ThisishowcellsovercomeDNAobstacles:sometimes,itisbetterforthemtotaketherisk”said
JosephLoparo,headofalaboratoryatHarvardMedicalSchoolworkingonDNAdamagetolerance
andrepair.
Gamblingasageneticstrategy,inotherwords.Loparoandcolleaguesareworkingtounravelhow
bacterialcellschoosetousetheseerrorproneenzymes:“Mostbacteriahaveasinglecircular
chromosomewhichtheycopywithamulti-proteinmachineknownasthereplisome,their
replicationmachinery”heexplained.“Thereplisomeiscomposedoftworeplicativepolymerase
complexeswhichquicklyandaccuratelycopyeachparentalstrandofDNA”.
IfDNAbecomesdamagedandtheDNArepairmachineryisunabletocorrectthelesion,thiscanbe
ablocktothereplisome.
“TranslesionpolymerasesarespecializedDNApolymerasescapableofsynthesizingovercertain
DNAlesionsthatstallthereplicativeDNApolymerase”saidLoparo.“Mylabisinterestedin
understandingthemechanismsofthisprocess,andhowtranslesionpolymerasesarerecruitedto
thereplicationmachinery”.
Byreconstitutingtranslesionsynthesis(TLS)andobservingitoccuronsingleDNAmoleculesin
realtime,theLoparoLabshowedthattheEscherichiacolibclamp,aring-shapedmoleculethat
encirclesDNAandtetherspolymerasestotheirsubstrates,cansimultaneouslybindtwokindsof
polymerases,thereplicativepolymerasePolIIIandtheerror-pronetranslesionpolymerase,PolIV.
ThisenablesanexchangeofthetwopolymerasesandarapidbypassofaDNAlesionwhichisin
manywaysanalogoustohowonemaintainsasparetireinthetrunkoftheircar.
Furthermore,theyfoundthatadditionalbindingsitesbetweenPolIVandbacttolimitPolIV
dependentDNAsynthesisundernormalconditions,yetfacilitatesthedisplacementofPolIIIfrom
theDNAuponthedetectionofDNAdamage.
Theseresultssupportanewmodelinwhichinteractionsbetweenpolymerasesandthebclampact
tobothinactivateandactivateerror-pronepolymerases.Withinthisregulatorynetwork,itislike
thecellissaying:“Icantellthatthereplicationmachineryisintrouble.Itisworthutilizinganerror
pronepolymeraseasitismyonlyhopeforsurvival”.
Thismechanismisalsoveryinterestingfromahumanhealthperspective,becausemanyofthe
basicmechanismsofthispolymeraseregulationlikelyoccurinhumancells.Understandinghow
geneticmutationsarisewillleadtoabetterunderstandingofdiseaseslikecancer.
“Thenextstepsforusaretolookattheseprocessesinlivebacteriacells,wherewehaveallthe
physiologicalcomplexity”concludedLoparo.“Additionally,wearereconstitutingthereplication
machineryinitsentiretyinatesttube”.
Thiscouldansweronceandforalltheodd,yetimportantquestion:howproneareourcells
towardsgeneticgambling?
Integratingartificialwithnaturalcells
SherefMansy
UniversityofTrento,ViadelleRegole,101,Mattarello(TN)Italy
Crossingtheboundarybetweenlivingandnon-living,bringingartificialsystemstolife.Science
fiction?Probablynot.AttheCentreforIntegrativeBiology(CIBIO)oftheUniversityofTrento,
biochemistSherefMansyhastakenastepforwardtowardsmakinganartificialcell“breath”.This
wouldbeatruerevolutionfortraditionalcellularbiology,whichcouldeventuallyleadtochange
thedefinitionitselfoflife.
“Achickisalivingorganism,astoneisnot.Everybodycanclearlyseethedifference.Butisthere
anythinginthemiddle?”askedMansyattheArmenise-Harvardsymposiumaudience.Hethen
presentedthelatestresultsofhisresearch,showinganewwaytoaddressthechallengeofartificial
life.
Thecontrolofcellularbehaviorlargelyreliesongeneticengineering,butartificialcellscouldbe
designedtobetterregulatecellprocessesthroughchemicalcommunication.WithhisteamatCIBIO,
Mansydevelopedanartificialcellwhichisabletotranslateachemicalmessageintoasignalthat
canbesensedbyEscherichiacoli.Thiscouldactivateacellularresponseotherwiseimpossibletobe
detected.
Withinthissystem,theartificialcellsworkaschemicaltranslators,sensingmoleculesthatE.
colialonecannotsense.Asaconsequence,theartificialcellsreleaseamoleculefamiliartoE.coli,
therebytranslatinganunrecognizedchemicalmessageintoawell-knownone.
Thisallowsa“dialogue”betweentheartificialandthenaturalcells,expandingthesensory
capabilitiesofE.coliwithoutalteringthegeneticcontentofthebacterium.
Mansy’sartificialcellhasacomplexstructure,bothfromengineeringandbiologicalpointofviews.
Itisbuiltwithaphospholipidvesiclecontainingisopropylb-D-1-thiogalactopyranoside(IPTG),
DNA,andtranscription-translationmachinery.TheDNAtemplatecodesforapreviouslyselected
riboswitch,activatingtranslationinresponsetothepresenceoftheophylline.Thetheophylline
riboswitchcontrolsthesynthesisoftheporeformingproteina-hemolysin(aHL).
colialonedoesnotrespondtotheophylline,andIPTGdoesnotcrossthevesiclemembraneofthe
artificialcellintheabsenceofthepore.Andhere’sexactlywheretheartificialcellscometoaction:
theyallowE.colitoreceivethechemicalmessage,thusbecomingIPTG-responsive.
“Bacteriadonaturallycommunicatetoeachother”saidMansy.“Ourgoalwastoseeifasingle
bacteriumcouldstillcommunicateincaseoneofthecellsisartificial.Apparently,theansweris
yes.”
Theseresults,publishedinNatureCommunications,areapromisingbasisforpossiblemedical
applications.“Ourartificialcellsdegradeinacoupleofhours:therearenolong-termconsequences.
Thisimpliesthattheycouldbeusedinbiologicalsystems–forexample,toidentifypollutants–
withoutgeneticintervention”explainedMansy.Sofarthisisthefirstartificial,cell-likesystem
capableofcreatingacommunicationpathwaybetweenartificialandlivingcells.Thenextstepwill
bebroadeningthisapproach,makingartificialcellsableto“talk”withcomplexbiologicalsystems.
“Ifwehadartificialcellsthatcandetectallthesignalsofthelivingcells,theycouldalsorecognize
thesignalsexpressedbyproblemcells,likecancer”concludedtheresearcher.
Thedefinitionof“livingcell”isthusbecomingmoreandmorefoggy:butthiscouldnotbea
problem,asfaraswechangethequestionstobeaddressed.JustasAlanTuringdidabout60years
ago,whenhegavebirthtothefieldofArtificialIntelligence.
Biogenesisandqualitycontrolofoligomericproteinsintheearlysecretorypathway
RobertoSitia
UniversitàVita-SaluteSanRaffaele,DivisionofGeneticsandCellBiology,SanRaffaeleScientific
Institute,Milan,Italy
Prestoebeneraroavviene.ThistypicalItalianproverb,meaningthatitisdifficulttoworkfastand
wellatthesametime,isbroadlydisregardedbyourcells.
AsRobertoSitiaexplainedduringtheArmenise-HarvardSymposium,manybiologicalprocesses
suchasproteinsecretionmusthavehighfidelityandefficiency.
AtSanRaffaeleScientificInstitute,Sitiaaddressesafundamentalquestionincellbiology:howare
thesizeandactivityofthedifferentcompartmentsconstantlycoordinated?
Inmulticellularorganisms,cellsmustpromptlyrespondtomultiplestimuli.Totaketheright
decision,theyneedtocontinuouslyexchangeinformationamongsteachotherandwiththeexternal
world,andtounambiguouslyintegratethecorrespondingsignals.
Thistaskbecomesparticularlydemandingduringdifferentiationorinresponsestoenvironmental
changes.Sitiaandcolleaguesinvestigatedthemolecularmechanismsthatallowcellstointegrate
signalling,proteinqualitycontrolandsortingintheearlysecretorycompartment.
“Theearlysecretorypathwayisemergingasakeyhub,performingmanydifficulttasksatthesame
time”saidSitia.“Itensuresefficienthigh-qualityreleasebytheproteinfactory”.
Theendoplasmicreticulum(ER)isamultifunctionalcompartmentfoundinalleukaryoticcells.
“FromtheER,secretoryproteinsbegintheirjourneytowardstheirfinaldestinations,theorganelles
oftheexocyticandendocyticcompartments,theplasmamembraneortheextracellularspace”Sitia
explained.HisresearchteamdiscoveredthatERp44,amultitaskproteinattheER-Golgiinterface,
isamasterregulatorinthishub.
Fidelityofprotein-basedintracellularcommunicationisguaranteedbyqualitycontrol(QC)
mechanismslocatedattheER–Golgiinterface,whichrestrictforwardtransporttonativeproteins.
AndhereiswhereERp44comestoaction:Sitiafoundthatitisakeyregulatorofproteinsecretion,
Ca2+signallingandredoxregulation.
“IfthesignalsconveyedbytheERp44-centeredmolecularhubarenotworking,cellssuffer;butif
theyaretoomuch,thecell’sfunctionarealsocompromised”hesaid.
WearebeginningtounderstandwhatmakesERp44capableofsatisfyingitsmultipletasks,
allowingtheproteinfactorytoperformprestoebene.
Imagingendocytosiswithhighspatiotemporalresolution
TomasKirchhausen
DepartmentofCellBiology,HarvardMedicalSchool,Boston,USA
“Seeingisbelieving.Biologyisbasedonobservation.ButwhatIwanttodoisalsomeasuring:this
quantificationcanbesize,canbelength,canbevolume,canbenumberofmolecules,canbewhere
arethemoleculesinagivenmoment”.
BiologistTomasKirchhausenknowswhathe’stalkingabout.HislaboratoryatHarvardMedical
Schoolisoneofthefewintheworldtocombinestandardand3Dmicroscopycapableofgiving
rapid,high-precisionthree-dimensionalimagingoflivingcells.
Thiscutting-edgetechnologyisputattheserviceofunderstandingthemovementofmembrane
proteinsthroughoutcells.Thesemechanismsareofkeyimportanceforthecell’ssorting
machineries,andcanbehijackedbytoxins,virusesandbacterialpathogens.
Studyinghowcellscanbeattacked,KirchhausenLabaimsatfindingtreatmentfordiseases
dependingonviralinfectionandpathogeninvasion:fromcancertoLGMD2B/Miyoshimuscular
dystrophies,fromAlzheimerdiseasetoALS(amyotrophiclateralsclerosis),aswellasother
neurologicaldiseases.
Inpreparationtothesestudies,hisgroupdeterminedthefirststructureatnearatomicresolution
ofclathrin,aproteinplayingamajorroleinthecreationofcoatedvesicles.Clathrinhasatriskelion
shapecomposedofthreeclathrinheavychainsandthreelightchainsandtheyformthecoat
surroundingthevesicleswhoselatticeoftenappearsastheseemofasoccerball.
Live-cellandsinglefluorescencemicroscopyimagingwereusedto“see”inthreedimensionsthe
moleculareventsandtheintracellularcompartmentsresponsiblefortheformationofclathrincoatedpitsandcoatedvesicles–aconserved“nano-machine”thatgeneratesintracellularvesicular
carriersinallanimalsandplants.
“Inourstudieswehavetwooppositeextremes”explainedKirchhausen.“Thefirstoneistheuseof
methodsgivingacompletepicture,likecrystallographyorNMR;thesecondextremeistheuseof
fluorescencemicroscopyvisualizationmethodsgoingtoaverylowresolution,withalotof
dynamics”.
“Sotheideaistocombinethesetwoextremestohaveaclearerviewofthemechanismof
endocytosis”hesaid.
Endocytosis,theprocessthatcellsusetoingestmolecules(likeLDL–thebadcholesteroland
certainviruses)byengulfingthem,wasobservedwiththemicroscopyvisualizationtechniques.
Theyallowedsufficienttemporalandspatialresolutiontofollowthelifeofasingleclathrincoated
pit.
“Theinitiationprocessofendocytosisishighlystochastic”continuedKirchhausen.“Weobserved
thattheclathrinadaptors,proteinsthatconnectclathrinwiththemembranesurroundingthe
clathrincoatarriveanddepartfromthemembranewithverylowbinding.Whenwehavethe
adaptorsintherightplace,thetriskelioncanmapthisinteraction,thatstabilizesthestructurefora
fewsecondsandformationofthepitensues”.
ThelevelofdetailreachedbyKirchhausen’sobservationispioneerinquantitativebiology.“Now
thatwehavetherighttechniques,wecancounthowmanymoleculesofacertaintypearerequired
forthedifferentstepsofendocytosis:aquestionthathadremainedunansweredfor30years”
commentedEmanueleCocucci,postdocworkinginKirchhausenLab.“Thenextstepswillbe
understandingthetotalnumberofmoleculesinvolvedinendocytosis,includingallthereceptors.So
wehavetogoevendeeper”.
Inthenearfuture,temporalresolutionandspatialprecisionof3Dmicroscopycouldthenreach
higherdetails.ButasKirchhausenpointedout,itiscrucialnottolosethebiggerpicture.“It’slike
thecartraffic”hesaid.“WecanseetheglobaldifferencesbetweenthetrafficofRomeandMilan,or
wecancountthedetails,likethecoloursofthecar,thedrivers,thenumberofpassengers,andso
on.Bothtypeofquantificationsareimportant”.
Mono-ADP-ribosylationandmembranetrafficking
DanielaCorda
InstituteofProteinBiochemistry,NationalResearchCouncil,Napoli,Italy
Between20.000and25.000:thisisapproximatelythenumberofgenesinyourgenome.Butifyou
thinkthisquantityishigh,youshouldprobablylookatyourproteome.Infactthenumberof
proteinsmakingupthehumanproteomeisestimatedatover1million.
Overthelastdecades,scientistshavediscoveredthatsinglegenesencodemultipleproteins,and
thismakestheproteomefarmorecomplexthanthegenome.
Butthereisasecretingredientwhichcanfacilitatestudyingthiscomplexity:itistheprocesscalled
proteinpost-translationalmodifications(PTMs).Thisisafundamentalstepoccurringafterprotein
biosynthesis,playingakeyroleindeterminingtheregulationandfunctionofproteinsandother
cellularmolecules.
Mostoftenmediatedbyenzymaticactivity,post-translationalmodificationscanoccuratanystep
duringthe“lifecycle”ofaprotein,sounderstandingtheseprocessesiscrucialtounfoldthe
complexityofproteome.
AttheInstituteofProteinBiochemistryoftheItalianNationalResearchCouncil,DanielaCordais
leadingpioneeringresearchonPTMs.Amongthevariouspost-translationalmodifications,sheis
focusingonmono-ADP-ribosylation(mono-ADPR).Thisreactionhasanimportantphysiological
roleincellularprocessessuchasmembranetraffic,immuneresponse,DNArepairandsignalling.
Cordawasoneofthefirstscientiststolookatmono-ADP-ribosylationasakeymechanismincell
biology.
“Formanyyearsithasbeenconsideredatoxicreaction”sheexplained.“Buttogetherwithother
colleagues,Ithoughtthatifatoxinmodifiesaprotein,perhapsthismodificationisinterferingwith
aphysiologicalmechanism.Ithoughtthatifatoxinmodifiesaprotein,perhapsthismodificationis
interferingwithaphysiologicalmechanism.Thismeansthatisolatedpathologicalmechanisms
(withouttheirphysiologicalcounterpart)donotexist”.
Thisintuitionshookuptheunderstandingoftheproteinenzymaticactivity.Studyinghowtoxins
inducetheADP-ribosylationofproteins,Cordaandothercolleaguescouldidentifyspecificcell
mechanismsthatwerepreviouslyunknown.
Inparticular,theCordalaboratorystudiedbrefeldinA(BFA),afungaltoxincausingthedisassembly
oftheGolgicomplexmembranes.BFAinducestheADP-ribosylationofBARS,aproteininvolvedin
thefissionofmembranesatseveraltrafficstepsofthesecretoryandendocyticpathways.
TheproteinBARSwasdiscovered20yearsagobyDanielaCordateam,andnowturnedouttobean
essentialelementofthemembranefissionmachinery.
SelectivelyanalyzingthebrefeldinAactivity,researchersidentifiedanintermediatethatcovalently
bindsBARS,calledBFA-ADP-riboseconjugate(BAC).
“BACmodifiesthisproteinonly,itisveryspecific”saidCorda.“SoifwecanmimicBAC,wecan
inhibitasingleprotein,BARS.Thisiswhatweareworkingon:synthetizingBACanalogues,and
performingvirtualscreeningstoselectsothersmallmoleculeswithsimilaractivity”.
Theinhibitionbysmallmoleculesofproteinsknowntocauseadiseasemayeventuallyleadto
clinicalapplications,incancerasinotherpathologies.
“Wearetestingthesesmallmoleculesinvitrohopingtosoonfindanapplicationintumorssuchas
lymphomaandbreastcancer”Cordaconcluded.
BARS,arelativelynewentryinscientificlabs,isthereforesheddinganewlightonthecellular
functionscontrol.Andthisproteincouldsoonbecomeapharmacologicaltargetforanticancer
therapies.
Controlsystemsofmembranetransportattheinterfacebetweentheendoplasmic
reticulumandtheGolgi.
AlbertoLuini
IstitutodiBiochimicadelleProteine(IBP),CNRNapoli,Italy;TelethonInstituteofGeneticsand
Medicine(TIGEM),Napoli,Italy.
Ifyouloseyourbalance,youwillprobablyfall.Yourcellsbehaveprettymuchthesameway:they
haveaninternalequilibriumthatneedstoremainconstant,otherwisethey“fall”.Thiscellular
balanceiscalledhomeostasis,whichguaranteesthecells’internalstability.
Maintaininghomeostasisdespitethevariationofinternalandexternalconditionsisafundamental
taskourcellularsystemneedstofulfill.Sounderstandingthistaskcanrevealimportantaspectsof
ourcellactivity.
AttheItalianNationalResearchCouncil,AlbertoLuiniandhisresearchgroupstudytheprocessof
homeostasisstartingfromthecomplexcellmembranetransportapparatus.
Withinthissystem,variationsinmembranefluxesfromtheendoplasmicreticulum(ER)tothe
GolgicomplexarebalancedbyoppositefluxesfromtheGolgitotheER,tomaintainhomeostasis
betweenthetwoorganelles.
AsLuiniexplainedtotheArmenise-HarvardSymposiumaudience,heandhisteamdescribeda
moleculardevicethatbalancestransportfluxesbyintegratingsignaltransductioncascadeswith
thetransportmachinery.
Inparticular,theyfoundthatER-to-GolgitransportactivatestheKDELreceptorattheGolgi.This
triggersacascadeinvolvingGsandadenylylcyclaseandphosphodiesteraseisoforms,andthenPKA
activation,andresultsinthephosphorylationofproteinsinvolvedinretrogradetraffic.This
inducesrecyclingtotheERandtendstobalancetransportfluxesbetweenERandGolgi.
Moreover,theKDELreceptoractivatesCREB1andothertranscriptionfactorsthatup-regulate
transport-relatedgenes.InthiswayaGolgi-basedcell-autonomouscontrolsystemmaintains
transporthomeostasisthroughbothsignalingandtranscriptionalnetworks.
Anotherinterestingthingisthatthismodelwasobtainedusingveryadvancedmicroscopy
includinganextremelypowerfultechnique:correlativemicroscopy.Itwasdevelopedforthefirst
timeinLuini’slaboratory,anditisusedtostudyinvivodynamicsandultrastructureof
intracellularstructuresatincrediblelevelsofdetail.
Combiningcorrelativelightandelectronmicroscopy,researcherswereabletoseedynamic
functionalassaysinlivecellsdirectlywithhighresolution3Dmorphology.
Thiswayitbecamepossibletoobservethecontrolsystemsofmembranetransportintegrating
informationondynamics,ultrastructureandmolecularcompositionassemblyofmolecular
machinery.Atthesametime,“seeing”theenginethatallowsourcellstomaintaintheirbalance.
MyosinVIBridgesUbiquitinSignalingandCellMigration
SimonaPolo
IFOMFondazioneIstitutoFIRCdiOncologiaMolecolare,20139Milan,Italy;DipartimentodiScienze
dellaSalute,UniversitàdegliStudidiMilano,Milan,Italy.
Humancellscannotfreelytravelaroundthebody:theirmovementistightlyregulatedandnormally
quitelimited.Butcancercellscanlosethiscontrol,travellinginthebloodstreamthroughthewellknowprocessofmetastasis.MyosinVIisamotor-proteinableto“travel”alongactinfilamentsand
isinvolvedintumorformationandmetastasis.
AttheIFOM–FIRCInstituteofMolecularOncologyofMilan,SimonaPoloandcolleaguesarenow
investigatingthecontrolsystemofMyosinVIfromanalternativepointofview.Thiswasthefocus
ofthefascinatingtalkgivenbyDr.PoloduringtheArmenise-HarvardSymposium:MyosinVIplays
anexplicitroleincellmigration,underbothpathologicalandnormalconditions.
DrPoloistheleaderofaresearchgroupinvestigatingthemechanismsofregulationmediatedby
ubiquitin.Ubiquitinisaregulatoryproteinthathasbeen“ubiquitously”foundinalmostalltissues;
itsaddictiontootherproteinscanaffecttheminmanyways.
“Thebest-knownfunctionofubiquitinisthedegradationviatheproteasome,butabout10years
agoanovelfunctionofubiquitinationinsignallingwasdiscovered”sheexplained.“Wearestudying
thismechanism,andshowedthatMyosinIVharboursaparticularubiquitinbindingdomain(UBD)
differentfromanypreviouslydescribedUBD”.
UBDsareacollectionofmodularproteindomainsthatnon-covalentlybindtoubiquitin;thenew
UBDidentifiedbyPolo’steamwasgiventhenameofMyosinVIUbiquitinBinding(MyUb)domain.
Ubiquitincancomeindifferentflavoursandtheyallperformdifferentfunctionsintothecells,most
ofwhicharestillunknown.
“Wefoundoutthat,differentlyfromthevastmajorityoftheUBDsthatshownobindingspecificity,
MyUbdomainshaveaclearpreferenceforK63dimersimplicatingthatMyosinVIandubiquitin
interactionisnotforproteindegradation”saidPolo.
ThesefindingspreparedthegroundtoexplaintheroleofUbandMIU-MyUbdomainsinthe
physiologicalandpathologicalregulationofMyosinVI.
“IthasbeenobservedanoverexpressionofMyosinVIintumortissues”saidPolo.“Butwefoundout
thatthisisnotagenericoverexpression:itislimitedtothespecificformabletointeractwith
ubiquitin.Wearenowdissectingindetailstheunprecedentedroleplayedbyubiquitinincell
migration”.
Newinsightsintoproteasomemechanismsinnormalanddiseasestates
AlfredGoldberg
DeptCellBiology,HarvardMedicalSchool,Boston,USA
“Proteasomeissmarterthanyouthink!”Thisexclamationcrownedtheconclusionofthesharpand
brightpresentationbyAlfredGoldberg,duringthethirddayoftheArmenise-HarvardSymposium.
HislaboratoryatHarvardMedicalSchoolstudieshowproteinsinourbodyareconstantlybeing
fabricatedandthenbrokendownintoaminoacids.Proteasomestakeprideofplaceinthisprocess:
theyareproteincomplexeswhosemainfunctionistodegradeunneededordamagedproteinsby
proteolysis,achemicalreactionthatbreakspeptidebonds.
ThesmartproteasomeGoldbergreferredtoisthe26Sproteasome,themajorsiteforprotein
degradationinmammaliancells.Inrecentyears,inhibitorsofits20Speptidaseactivities(e.g.
bortezomib)havegreatlyadvancedthetreatmentofmultiplemyeloma.
Moreover,theprocessingofubiquitin(asmallregulatoryproteinfoundinalmostalltissuesof
eukaryoticorganisms)conjugatesby26Sproteasome’s19Sregulatoryparticleinvolvesmany
enzymaticstepsthatmaybetargetsfordrugdevelopment.
Sothestudyof26Sproteasomehasimmediateapplicationalsoinclinicalmedicine,andthisisone
ofthereasonswhyAlfredGoldbergtookitasthe“maincharacter”ofhisresearchandhistalk.Here
thischaracterbecametheprotagonistoffourshortstories:foursurprisingmechanismscontrolling
proteasomefunction.
“Thefirststoryisthattheproteasomeistightlyregulated”heexplained.“Wenowthinkwe
understoodthemechanism,theoveralllinkagebetweentheubiquitinchainandthebreakingdown
oftheproteins:theregulationthatoccursinthatprocess”.
InfactGoldberdLabdiscoveredthatwhenmammalian26Sproteasomesareinhibited,the
ubiquitin-receptorsubunit,Rpn13,becomespolyubiquitinatedbya26S-associatedubiquitinligase.
Thismodificationpreventsbindingofubiquitinatedsubstrates,andpresumablyevolvedtoprevent
build-upofconjugateswhenproteasomefunctionisstalled.
Andherewecometothesecondstory.“Thesurpriseisthatproteasometriggersunexpected
responses”Goldbergtold.“Whentheproteasomeisinhibited,ithasamechanismsaying:‘don’tgive
meanymoreubiquinatedproteins!’It’saveryadvancedself-regulatedmechanism”.
Proteasomeregulationconcernsthethirdstoryaswell:“Wefoundthatwhentheproteasomeis
partiallyinhibited,evenjustalittlebit,thesignalsofproductionofnewproteasomesevolvevery
specifically.Thisisaverysmartstructurethatknowshowtogetthecelltocompensatepossible
problems”.
Thefourthstoryisaboutproteasomeanddisease.“Proteasomeisreallyaffectedincommon
neurodegenerativediseases:thishadbeensuggestedmanytimes,butwithnoclearevidences”said
Goldberg.“Wehaveusednewtechniquestoshowinmousemodeldiseaseoffrontotemporal
dementia,whichisveryclosetoAlzheimerdiseasebecausehasmutationsinTaugene”.
Inthiscasetheproteasomesaredefected:thisistightlyboundwithdiseasedevelopmentinmice.
“Ourhypothesisisthatinalltmajorneurodegenerativediseasesthereisaprogressivefailureto
degradetheubiquinatedconjugates,andthisconditionisassociatedtotheaccumulationoftauand
phospho-tau”concludedGoldberg.
Connectingthesefourstoriescouldgettheproteasomemechanismtoworkbetter,improving
Neurodegenerativediseasestherapies.Takingatthesametimeadvantageoftheproteasome’s
unexpectedsmartness.
Towardsinvitroreconstitutionofspindlecheckpointsignaling
AndreaMusacchio
DepartmentofMechanisticCellBiology,Max-Planck-InstituteofMolecularPhysiology,Dortmund,
Germany
Thedistributionoftheparentalgenometotwodaughtercellsduringmitosisandmeiosisisthe
essenceofgeneinheritanceandthereforeoflifeitself.Notsurprisingly,therefore,thisprocess
involveswhatisprobablythemostcomplexensembleofcellularmolecularmachineryand
providesanastoundingexampleoftheabilityofbiologicalmattertoself-organize.
Themitoticspindle,astructuremadeofmicrotubules,molecularmotorsandtheirregulators,hosts
acruciallylargefractionofthemachineryofcelldivision.Thisbipolar,elongated,andremarkably
dynamicstructuredefinesthedivisionplaneofthemothercell,anddevotesitselftothecaptureof
chromosomes,totheirclusteringinthemiddleplane,andtotheirsubsequentsegregationtothe
daughtercells.Thus,themitoticspindleiscrucialtoensurethatthedaughtercellsinheritexactly
thesamenumberandtypeofchromosomes,thereforeguaranteeingcellularandorganismal
viability.
Despitetheimportanceofthespindleforcelldivision,theexactmolecularbasisofitsfunction
remainspoorlyunderstood.TryingtounfoldthismysteryisthemaingoalofAndreaMusacchioat
theMax-Planck-InstituteofMolecularPhysiologyinDortmund,Germany.Musacchioleadsa
researchteamfocusedoncomplexproteinscaffoldsknownaskinetochores.Kinetochoresarelarge
proteinaceousstructuresbuiltonthecentromereregionofchromosomes.Theirprimaryfunctionis
toprovideasiteofattachmentofchromosomestothemitoticspindle.
Kinetochoresplayasecond,subtlerfunctionthatishowevercrucialforaccuratecelldivision:they
controlafeedbackmechanismknownasmitoticcheckpoint,orspindleassemblycheckpoint(SAC).
Thefunctionofthischeckpointistopreventseparationoftheduplicatedchromosomes–thesister
chromatids–untiltheyhaveproperlyattachedtothespindleapparatus.DysfunctionoftheSAC
mayresultinincorrectpartitioningofthesisterchromatidstothedaughtercells,creatinga
pathologicalcellconditionknownasaneuploidy.Suchconditionstronglycorrelateswith
tumorigenesis,supportingthespeculationthatcheckpointdysfunctionmaybe–amongothers–a
prominentcauseoftransformation.
Overthelastyears,Musacchio’slaboratoryhasmadesignificantcontributionstothefield:“We
reconstitutedseveralkinetochoresub-complexesintheinnerandtheouterkinetochore,the
regionsofthekinetochoreimplicatedincentromerebindingandmicrotubulebindingand
checkpointcontrol,respectively”,saidtheresearcher.
Indeed,biochemicalreconstitution,coupledwithmedium-orhigh-resolutionstructural
investigationsisplayingaleadingroleindevelopinganunderstandingofkinetochoreorganization.
Forinstance,Musacchioandcolleagueswererecentlyableforthefirsttimetogainadetailedview
ofacrucialkinetochoresub-complexmadeoffoursubunits,theCENP-HIKMcomplex.
Thenextchallengeisexplainingtherelationshipbetweenkinetochoresandcheckpointcontrol:this
iswhatMusacchiocalledthe“thirddecadeofcheckpointstudies”,afterthediscoveryofthemain
checkpointcomponents(1991-2000)andtheinvestigationoftheirinteractions(2001-2010).To
addressthischallenge,hisresearchgroupisapproachingthereconstitutioninvitroofspindle
checkpointsignalling.Thiseffortfollowsatraditionalpathof“reductionist”studiesthatinterpret
theemergenceofcomplexbiologicalfunctionsasaresultofspecificinteractionsofthe
macromolecularbuildingblocksthatpopulatecells.
ThescaleofambitionimplicitinMusacchio’splans,however,transcendsthatofmostcurrent
efforts.“Theword‘towards’isveryimportant:we’restillnotthere”pointedoutMusacchio.“Butwe
believethatitispossibletoreconstitutethecatalyticapparatusofthecheckpointonreconstituted
kinetochores”.Invitroreconstitutionofthiscomplexbiologicalstructurewillrevolutionizethe
understandingofchromosomesdivisionduringmitosis,sheddinganewlightononeofthemost
excitingexamplesofself-organizationinlivingmatter.
Theautophagysignalingnetworkinthecoordinationofacell’sresponse
FrancescoCecconi
UnitofCellStressandSurvival,DanishCancerSocietyResearchCenter,2100Copenhagen,Denmark;
DepartmentofBiology,UniversityofRomeTorVergata,Rome,Italy
Theworld“cannibalism”usuallyconjuresupimagesofprimitiveandbrutalpractices.Butdeep
down,weareallcannibals:ourcellsareconstantlyeatingthemselves,discardingoftheirredundant
moleculargarbagethroughaprocessknownasautophagy.
DerivingfromaGreektermmeaning“selfeating”,autophagyismostofthetimesasurviving
mechanism,allowingcellsto“recycle”theirbiologicalwaste.Andtomanyscientists’surprise,
recentdiscoverieshaveshownthatfaultyautophagymechanismscontributetothedevelopmentof
variousdiseases.
FrancescoCecconi’slaboratoryiscommittedtounravelingtheupstreamregulationofautophagy
andelucidatingtheroleitplaysinthreedifferentpathologicalconditions:neurodegeneration,
autoimmunityandcancer.
Thelatterinparticularisstillobjectofdiscussion:“Incancerthereisasortofcontroversygoingon
abouttheroleofautophagy”explainedCecconi.“Ifweknock-outanautophagygene,thenweget
accumulationsoftoxiccompoundsthatautophagycan’thelpgettingridof.Forexample,damaged
tissuescanoverproduceROS,whichcandamageDNAcausingchromosomeinstabilityandcancer.
Inthiscase,mutationsintheautophagygenessetareresponsibleforcancerogenesis”.
Ontheotherside,autophagycanhavetheoppositefunction,beingevendetrimentalforthepatient.
“Ifatumororiginatesfromacompletelydifferentsetofmutationsandwehavetheautophagy
systemperfectlyworkingwithinthecells,thentheautophagyishelpingcancercellstosurvive”.
Thereisthenincanceradualfunctionofautophagy:dependingonthetumororigin,autophagy
shouldeitherbepushedtoworkorblocked.
Inthiscontext,Cecconi’slabhasidentifiedanovelproteincalledAmbra1(ActivatingMoleculein
Beclin1-RegulatedAutophagy),playingmultiplerolesasascaffoldfactorinautophagycontrol.
“WearestudyingthefunctionofAmbra1incellcycleregulationanditsimplicationsintumor
insurgence”saidCecconi.
Togetherwithhisresearchteam,hedevelopedanexperimenttounderstandtheroleofAmbra1in
cancer.Theresultscouldshedanewlightonthelinkbetweenautophagyandtumordevelopment.
“IhopeIconvincedyouabouttheimportanceofthisprotein”concludedCecconitotheArmeniseHarvardaudience,andheprobablydid.
Thesignalingmechanismsunderlyingtheanti-tumoractivityofmetformin
GianniCesareni
DepartmentofBiology,UniversityofRomeTorVergata,RomeItaly
Fightingcancerwithawell-knownmedicine.Afewyearsago,thishypothesisdominatedmany
scientificmagazines,asanewpossiblefrontierfortumortreatment.
Thepromisingmedicinewasmetformin,themostfrequentlyprescribeddrugfortype2diabetes
patients.Thesefindingsimmediatelytriggeredseveralclinicaltrials.Butthemechanisms
underlyingtheanti-tumoractivityofmetforminweren’tfullyclarified,anditsroleasacancer
suppressorisstillunderexamination.
AttheUniversityofRome,TorVergata,GianniCesareniusedanewapproachtoaddressthese
questions.Togetherwithhisresearchgroup,hedecidedtolookatthebehaviorofacancercellasa
systemandnotasasimplelinearcombinationofitsparts.Theserelativelynewapproachis
commonlyreferredtoassystemsbiology,asexplainedduringthe15thArmenise-Harvard
Symposium.
“Welookatthecellinitsentirety,aimingatdescribinghowmetforminperturbsthephysiological
processesofthecancercelltherebypreventingitsproliferation”heexplained.“Thisisanew
approachinmodernbiology,aimingtodescribethecellasasystemandnotsimplyasthesumofits
parts”.
Inordertoworkoutmetformin’sroleincancer,theCesareniLabappliedacombinationofhigh
contentmulti-parametricanalysiswithlogicmodelingandsimulationtechniques.Theobjective
wastomapcellperturbationsoncomplexlogicnetworks.
Inthiswaytheyobtainedapredictivecell-specificlogicmodel,laterappliedtofunctionally
characterizethemolecularmechanismunderlyingtheanti-canceractivityofmetformin.
“Weanalyzedhowsignalingnetworksarerewiredinbreastcancercellsuponmetformin
treatment”saidCesareni.“First,wemonitoredtheactivationofabout20keysignalingproteinsin
cancercellstreatedwithavarietyofperturbationsbeforeandaftermetformintreatment.Next,we
builttwocellspecificnetworkmodelsofmetforminfortreatedanduntreatedcells”.
Theresultswereextremelyinteresting:theexperimentsshowedthatmetforminrewiresthe
signalingnetworks,modifyingdifferentconnectionsbetweensignalingproteins.
“Wefoundthatmetforminchangesthestructureofthenetworksunderlyingsomecellpathways”
explainedtheresearcher.“Sothecellsbecomemoresensitivetotopictreatmentsandlessproneto
proliferation”.
Tosumup,metformindoesn’tkillthetumorcells,butmakethemweaker.Andthisiscertainly
quiteagoodstartingpoint.
Mechanisticeventsregulatingnotchsignaltransduction
StephenC.Blacklow
HarvardMedicalSchool,Boston,MA,USA;DanaFarberCancerInstitute,Boston,USA
Thisstorybeginsin1917,whenThomasHuntMorganidentifiedallelesofageneresponsiblefor
notchingofthewingsinfruitflies.Subsequentwork,ledinlargepartbySpyrosArtavanis-Tsakonas
ofHarvardMedicalSchool,revealedthatthegeneresponsibleforthisphenotype,calledNotch,isa
highlyconservedreceptorinasignaltransductionpathwaythatcontrolnumerouscellfate
decisionsinorganismsrangingfromfliestohumans.WhereasnormalNotchsignalingmakesit
possibleforadjacentcellstocommunicateeffectivelywitheachother,dysregulatedNotchsignaling
oftenasaresultofmutationsintheNotchreceptorsortheirligands,contributestoavarietyof
humandiseases,includingneurodegenerationandcancer.
Thus,understandinghownormalandaberrantNotchsignalingtakesplacehasimportant
implicationsforclinicalmedicine.AtHarvardMedicalSchool,thisisonetaskoftheBlacklowLab:
tounderstandthemolecularlogicofcell-surfacereceptorssuchasNotchproteins,whicharehighly
relevanttohumanphysiologyanddisease.
“Amajorfocusofthelaboratoryistounderstandhownormalnotchsignalingtakesplace.This
knowledgewillalsoyieldimportantinsightsintotheeffectsofaberrantNotchsignalingincancer
andotherdiseases”saidStephenBlacklow,headofthelaboratory.
Hiseffortsaremainlydirectedtowardunderstandinghowactivationisinducedbyligandsandhow
notchcooperateswithotherfactorstoregulatetargetgenetranscription.
“Onecentralunansweredquestioninsignalingishowthebindingsiteforligandscancommunicate
witharegulatoryswitchthatis600Angstroms(alongdistanceinmolecularterms)away,andhow
ligandstimulationnormallyrelievesautoinhibition”saidBlacklow.
Oneimportantcluetoansweringthisquestionisthatsignal-sendingcellsrelyonendocytosisofthe
ligandstodeliverthesignaltotheNotchreceptorsonthesignal-receivingcells.Twopossibilities
mightexplainthisdependence:oneisthatligandendocytosisexertsmechanicalforcetopullonthe
Notchreceptorinordertoexposeametalloproteasecleavagesitetoactivatingproteolysis,andthe
secondisthatligandsrelyonendocytosisforan“activating”modificationthatrendersthem
competentforsignaling.Inotherwords,endocytosisandrecyclingofligandmayberequiredfor
conversionofligandsfromalatentstateintoanactiveform.
Thelaboratoryiscurrentlyworkingondistinguishingbetweenthesetwopossibilities.“Itis
possibletosubstitutethenormalligand-receptorinteractionwithasyntheticsystem,whichretains
thedependenceonligandendocytosisinsendingcells,andontheactivationswitchinreceiving
cells,”saidBlacklow.“Sotheworkingmodelthatcanbetestedinthesyntheticsystemisthat
endocytosisisactuallysupplyingtheforcethatopenstheregulatoryswitch”.
MechanismofinhibitionoftheepidermalgrowthfactorreceptorbyMig6
MichaelJ.Eck
DepartmentofBiologicalChemistry&MolecularPharmacology,HarvardMedicalSchool;Cancer
Biology,Dana-FarberCancerInstitute,Boston,USA
Lungcanceristheleadingkillerintheworldamongtumors.AccordingtotheWorldHealth
Organization1,59millionofdeathsforlungcancerwereregisteredin2012.
Althoughlungcanceriscloselylinkedtosmoking,everyyearitaffectstensofthousandsofpeople
whoneversmoked.
Inrecentyears,scientistscontinuedtounravelgeneticfactorsinvolvedincancerdevelopment.And
forlungcancerinnonsmokers,thereisanincreasedlikelihoodoffindingasomaticmutationofthe
epidermalgrowthfactorreceptor(EGFR).EGFRisthecell-surfacereceptorformembersofthe
epidermalgrowthfactorfamily(EGF-family)ofextracellularproteinligands.
AtHarvardMedicalSchool,MichaelEckisworkingonthestructureofsignalingcomplexesthat
underliecancer,withaparticularfocusonlungcancer-derivedmutationsintheepidermalgrowth
factorreceptor.Hislab’sstructuralapproachesarealsousedtofacilitatedevelopmentofanticancerdrugs.
“About15%oflungcancersarecausedbyEGFRmutations”explainedEckduringtheArmeniseHarvardSymposium.“Inparticular,somaticmutationsinEGFRareamajorcauseofnon-smallcell
lungcancer”.
Hepresentedthelatestresultsofhislaboratory:thediscoveryofaquiteunexpectedmechanismof
inhibitionofEGFRbyanendogenousregulator,Mig6.
Mig6(Mitogen-inducedgene6,alsocalledRALT)isafeedbackinhibitorofEGFRfamilymembers
thatactsbydirectlybindingactivatedEGFR,inhibitingitscatalyticactivityanddirectingits
internalizationanddegradation.
Mig6isprobablyatumorsuppressor:thismeansthatitslossleadstotumorformation.In
particular,focaldeletionsspanningitschromosomallocusoccurinGBM(Glioblastomamultiforme)
andlungcancer.
HisstudiesrevealedthatMig6isactuallya“mechanism-based”inhibitorofEGFR.“EGFRistrapped
intheactofphosphorylatingMig6”explainedEck.“OncetheEGFRkinasephosphorylatesMig6,itis
effectivelyirreversiblyinhibited”.
Thesefindingsmayleadtonewtherapeuticstrategiesforlungandothercancerscausedbymutant
EGFR.
Connectingthemachineriesofcellfatedeterminationandtumorsuppressionin
mammarystemcells
PierPaoloDiFiore
FondazioneIFOM-IstitutoFIRCdiOncologiaMolecolare,MilanItaly
Itishardtoimaginethatstemcells,themost“immaculate”biologicalexistingmaterial,couldlead
tocancer.Butaccordingtoarecenttheory,“stem-like”cancercellsareresponsibleforthe
generationoftumorsandforsustainingtumorgrowth.
Thismodelpredictstheexistenceofcancerstemcells(CSCs)withpropertiescharacteristically
associatedtonormalstemcells,suchasself-renewal,multipotencyandquiescence.
AttheInstituteofMolecularOncologyFoundation,PierPaoloDiFioreisexploringthischallenging
stem-celltheoryofcancer.Heisinvestigatingthemolecularmechanismsgoverningthe
maintenanceofthestemcellcompartmentinnormaltissues,andhowthesemechanismsare
subvertedincancer.
DuringtheArmenise-HarvardSymposiumclosingtalk,heexplainedhowislabisaddressingthis
studyperformingbothbasicandtranslationalcancerresearch.
“Theexistenceofcancerstemcellshasbeenprovedforanumberofcancertypes,includingbreast
cancer”hesaid.“However,thebreaststemcellcompartmentremainspoorlycharacterizeddueto
thelackofreliabletechniquesfortheiridentificationandisolation.Wehavedevelopedanew
techniquetospecificallylabelandpurifybreaststemcellsfrommammaryglandtissue”.
Thistechniqueexploitsboththepropensityofbreaststemcellstogeneratemammospheres(3D
clustersofcells)insuspensionculture,andtherelativequiescenceofstemcellscomparedtoother
breastcelltypes,whenpropagatedinvitro.
DiFioreandcolleaguesareusingpurifiednormalandcancerstemcellstoisolatea“stemness”
signaturefromwhichtheycanextractdiagnostic,prognosticandtherapeuticmarkersthatcanthen
beevaluatedfortestinginclinicaltrials.
Inthiscontext,acrucialroleisplayedbythemechanismsofasymmetriccelldivision.Inparticular,
researchersareinvestigatingwhether,andhow,Numbandendocytosis-basedmechanismsare
involvedintheregulationofasymmetriccelldivisionofhumanbreaststemcells.
“Numbisacellfatedeterminantthatbyasymmetricallypartitioningatmitosiscontrolsbinarycell
fatedecisions”explainedDiFiore.“Inhumanbreastcancers,thereisfrequentlossofNumb
expression,duetoitsexaggeratedubiquitinationandensuingdegradation”.
ThisNumblosscausesalterationsintwomajordownstreampathways.Ontheonehand,lackof
NumballowsforuncheckedsignalingactivityoftheNotchreceptor.Ontheother,lackofNumb
causesattenuationofthep53signalingpathway.Tumorsdisplayingloss-of-Numbexpressionare
addictedtothiseventandtoitsmolecularconsequences.
DiFioreclaimedthatthisleadstoafirstimportantconclusion,whichalsoconstitutedthe“takehomemessage”ofhistalk:“WhenyouhavehighNumb,youhavehighp53;whenyouhavelow
Numb,youhavelowp53”.
ThissuggestsadoubleroleofNumb:itisprobablyatumorsuppressor,anditsactioncauses
decreasedp53activityinbreastcancers.Therefore,Numbcontrolsbothanoncogenicpathwayand
atumorsuppressorpathway,andthismayleadtonewpromisingclinicalapplicationstofight
cancerstemcells.
Armenise-HarvardSymposiumandyoungresearchers:theCareerDevelopment
Award
“Myfatherbelieved,andsodoI,thatsuperbresultsmostoftencomefromcollaboration.The
Foundationintendstocontinuelookingforwaystoenhanceconversationandcooperativeworkon
bothsidesoftheAtlantic”.
WiththesewordstheArmenise-HarvardFoundation’sChairman,CountGiampieroAuletta
Armenise,gaveatributetohislatefather,founderCountGiovanniAulettaArmenise,onthelastday
ofthesymposium.HegreatlysummarizedtheFoundation’smission:establishinga
multidisciplinary-basedscienceresearch.
The15thArmenise-HarvardSymposiumfullyreflectedthisvision.Intheend,everyoneagreedit
hadbeenanexcitingmeeting,withastronginterdisciplinaryapproach.
Alongwiththescientificsessions,over30scientistspresentedposters.Thisgavegroupleadersand
youngerresearchersthechancetotalkovertheirwork,findingnewcollaborationopportunities.
ThisisthespiritofthewholeFoundation,bestexpressedbyitsmostforward-lookinggrant
program:theCareerDevelopmentAward(CDA).Since2001,theArmenise-HarvardFoundationhas
fundedthedevelopmentoftalentedyoungscientists,establishingcollaborativerelationships
betweenItalianresearchersandHarvardMedicalSchool.
Inthelast14years,20scientistshavemovedtoItalyfromelsewherearoundtheworld,supported
bytheFoundation.Theysetuptheirownlabsalloverthecountry,fromPalermotoTrento.
The15thArmenise-HarvardSymposiumwitnessedthesuccessofthisfundingprogram.8Career
DevelopmentAwardeesattendedthemeeting:VincenzoCostanzo,FedericoForneris,Claudia
Lodovichi,MarieLaureBaudet,RosellaVisintin,TizianaBonaldi,StefanoGustincichandSheref
Mansy,whoalsowasoneofthespeakers.Allofthemaredevelopingapromisingcareerinscience.
“ThisisoneofthefewopportunitiestoindependentlyworkinItalyonaspecificresearchprogram”
commentedVincenzoCostanzo,winnerofthe2013CDAgrant.HerecentlymovedtotheVertebrate
GenomeStability,IFOMIstitutoFIRCdiOncologiaMolecolareinMilan,whereheisworkingonthe
roleofDNAdamageresponsefactorsinvertebrateDNAreplication.
“WestudyhowDNArepairsitself,andwhytumorcellsarenotcapableoffulfillingthistask”he
explained.“Cellsarelikeplanes:everysinglemechanismisrelatedtomanyothers.Ifoneofthese
mechanismsisdamaged,aDNAdamageresponseisactivated.Wewanttounderstandhowto
selectivelyinterveneontumorcells,whichdefectsinthespecificgenesoftheDNAdamage
response”.
Tothisend,VincenzoCostanzoisapplyingamultidisciplinaryapproach–thesamevisionbroadly
promotedduringthe15thArmenise-HarvardSymposium.
AsCountGiampieroAulettaArmenisestated:“Ourfoundersbelievedinlookingatproblemsfrom
multipleangles,andtheFoundationwillcontinuetopursuethisgoal”.
Glossary
Actin:aglobularmulti-functionalproteinfoundthatformsmicrofilaments.Itisthemonomeric
subunitoftwotypesoffilamentsincells:microfilaments,oneofthethreemajorcomponentsofthe
cytoskeleton,andthinfilaments,partofthecontractileapparatusinmusclecells.Actinparticipates
inmanyimportantcellularprocesses,includingmusclecontraction,cellmotility,celldivisionand
cytokinesis,vesicleandorganellemovement,cellsignalling,andtheestablishmentand
maintenanceofcelljunctionsandcellshape.
Autophagy:anevolutionarilyconservedcatabolicmechanismthatinvolvescelldegradationof
unnecessaryordysfunctionalcellularcomponentsthroughtheactionsoflysosomes.Although
originallyclassifiedasatypeofprogrammedcelldeath,autophagyismorewidelyviewedasabasic
cellsurvivalmechanismtocombatenvironmentalstressors
Chromatin:acomplexofmacromoleculesfoundincells,consistingofDNA,proteinandRNA.Its
primaryfunctionsare:topackageDNAintoasmallervolumetofitinthecell;toreinforcetheDNA
macromoleculetoallowmitosis;topreventDNAdamage;4tocontrolgeneexpressionandDNA
replication.
Clathrin:aproteinthatplaysamajorroleintheformationofcoatedvesicles.Itformsatriskelion
shapecomposedofthreeclathrinheavychainsandthreelightchains.
Dynein:amotorproteinconvertingthechemicalenergycontainedinATPintothemechanical
energyofmovement.Ittransportsvariouscellularcargoby“walking”alongcytoskeletal
microtubulestowardstheminus-endofthemicrotubule,whichisusuallyorientedtowardsthecell
center.
Endocytosis:anenergy-usingprocessbywhichcellsabsorbmolecules(suchasproteins)by
engulfingthem.
Epidermalgrowthfactorreceptor:thecell-surfacereceptorformembersoftheepidermal
growthfactorfamily(EGF-family)ofextracellularproteinligands.
Heterochromatin:atightlypackedformofDNA,whichcomesindifferentvarieties.These
varietieslieonacontinuumbetweenthetwoextremesofconstitutiveandfacultative
heterochromatin.Bothplayaroleintheexpressionofgenes,whereconstitutiveheterochromatin
canaffectthegenesnearthem(position-effectvariegation)andwherefacultativeheterochromatin
istheresultofgenesthataresilencedthroughamechanismsuchashistonedeacetylationorpiRNA
throughRNAi.
Histone:ahighlyalkalineproteinfoundineukaryoticcellnucleithatpackagesandorderstheDNA
intostructuralunitscallednucleosomes.Histonesarethechiefproteincomponentsofchromatin,
actingasspoolsaroundwhichDNAwinds,andplayaroleingeneregulation.
Histonemethylation:aprocessbywhichmethylgroupsaretransferredtoaminoacidsofhistone
proteinsofchromosomes.Dependingonthetargetsite,methylationcanmodifyhistonessothat
differentportionsofchromatinareactivatedorinactivated.Thisprocessiscriticalforthe
regulationofgeneexpressionthatallowsdifferentcellstoexpressdifferentportionsofthegenome.
Histonemethyltransferases(HMT):histone-modifyingenzymesthatcatalyzethetransferofone,
two,orthreemethylgroupstolysineandarginineresiduesofhistoneproteins.
Kinesin:aproteinbelongingtoaclassofmotorproteinsfoundineukaryoticcells.Kinesinsmove
alongmicrotubulefilaments,andarepoweredbythehydrolysisofATP.Mostkinesinswalk
towardstheplusendofamicrotubule,which,inmostcells,entailstransportingcargofromthe
centreofthecelltowardstheperiphery.
MessengerRNA(mRNA):alargefamilyofRNAmoleculesthatconveygeneticinformationfrom
DNAtotheribosome,wheretheyspecifytheaminoacidsequenceoftheproteinproductsofgene
expression.
Metformin:anoralantidiabeticdruginthebiguanideclass.Itisthefirst-linedrugofchoiceforthe
treatmentoftype2diabetes,inparticular,inoverweightandobesepeopleandthosewithnormal
kidneyfunction.
Microtubule:acomponentofthecytoskeleton,foundthroughoutthecytoplasm.Microtubulesare
formedbythepolymerizationofadimeroftwoglobularproteins,alphaandbetatubulin;theyare
involvedinmaintainingthestructureofthecelland,togetherwithmicrofilamentsand
intermediatefilaments,theyformthecytoskeleton.
Myosin:aproteinofATP-dependentmotorfamily,bestknownforitsroleinmusclecontraction
anditsinvolvementinawiderangeofothereukaryoticmotilityprocesses.Myosinsareresponsible
foractin-basedmotility.
Notch-1:ahumangeneencodingamemberoftheNotchfamily.Notchfamilymembersplayarole
inavarietyofdevelopmentalprocessesbycontrollingcellfatedecisions.
Notchproteins:afamilyoftransmembraneproteinswithrepeatedextracellularEGFdomainsand
thenotch(orDSL)domains.Theseproteinsareinvolvedinlateralinhibitioninembryogenesis.
Notchsignalingpathway:ahighlyconservedcellsignalingsystempresentinmostmulticellular
organisms.Notchsignalingpromotesproliferativesignalingduringneurogenesis,anditsactivityis
inhibitedbyNumbtopromoteneuraldifferentiation.
Numb:aproteinthatinhumansisencodedbytheNUMBgene.Itplaysaroleinthedetermination
ofcellfatesduringdevelopment.
Phosphorylation:theadditionofaphosphate(PO43−)grouptoaproteinorotherorganic
molecule.Phosphorylationturnsmanyproteinenzymesonandoff,therebyalteringtheirfunction
andactivity.Proteinphosphorylationisonetypeofpost-translationalmodification.
Proteasomes:proteincomplexesinsidealleukaryotesandarchaea,andinsomebacteria.In
eukaryotes,theyarelocatedinthenucleusandthecytoplasm.Themainfunctionoftheproteasome
istodegradeunneededordamagedproteinsbyproteolysis,achemicalreactionthatbreakspeptide
bonds.
Reactiveoxygenspecies(ROS):chemicallyreactivemoleculescontainingoxygen.ROSareformed
asanaturalbyproductofthenormalmetabolismofoxygenandhaveimportantrolesincell
signalingandhomeostasis.
RNAinterference(RNAi):abiologicalprocessinwhichRNAmoleculesinhibitgeneexpression,
typicallybycausingthedestructionofspecificmRNAmolecules.
RNA-inducedtranscriptionalsilencing(RITS):aformofRNAinterferencebywhichshortRNA
molecules–suchassmallinterferingRNA(siRNA)–triggerthedownregulationoftranscriptionof
aparticulargeneorgenomicregion.
Yeast:single-celledfungithatreproducebybudding.Yeastsizecanvarygreatlydependingonthe
species,typicallymeasuring3–4µmindiameter,althoughsomeyeastscanreachover40µm.