Download Environmental Sensing and the Cellular Response

ARMENISE-HARVARDSYMPOSIUM2003
ENVIRONMENTALSENSINGANDTHECELLULAR
RESPONSE
7thAnnualSymposium
June21-23,2003,Trieste,Italy
AbouttheSymposium
TriesteisanancientcityontheAdriaticSeawhereItalian,Germanic,andSlavicculturesintersect.
BeforeitbecamepartofaunitedItaly50yearsago,thecitywasatvarioustimesundertheswayof
Rome,Venice,Austria,Austria-Hungary,Italy,andCommunistYugoslavia.Nowonderitspeopleare
adeptatcommunicatinginmanylanguages,enjoyingbothstrudelandgnocchi,andthrivingina
complexandrapidlychangingenvironment.Accordingtospeakersatthe7thAnnualSymposiumof
theGiovanniArmenise-HarvardFoundation,livingcellsandbiologicsystemsareeverybitas
adaptableasthehostcity’speople.</p>
<palign=""left"">“EnvironmentalSensingandtheCellularResponse”wasthethemeoftheevent,
heldinTriesteJune21-23.MarcW.KirschnerandCarlaJ.Shatz,whorespectivelyheadThe
ArmeniseCenterforIntegrativeBiologyandPhysiologyandTheArmeniseCenterfor
NeurosciencesatHarvardMedicalSchoolorganizedtheSymposium.Manyofthe15invited
lecturesand18posterpresentationsofferedmolecularinsightsintothesenses–includingvision,
hearing,andtaste–thatorganismsusetoexploreandexperiencetheoutsideworld.Other
speakersdelvedintotheinnerworldofcells,exploringhowtheycommunicatewiththeirownkind
andwithothercelltypesusingbiochemical,mechanicalorothersignals.</p>
<palign=""left"">Nearly60scientistsacceptedtheFoundation’sinvitationtothisyear’s
Symposium,halffrombasicsciencedepartmentsatHMSandhalfrepresentingItalianuniversities
andresearchinstitutionsinMilano,Padova,Pisa,Roma,Torino,andthehostcityofTrieste.This
mixreflectstheFoundation’scommitmenttostimulatingknowledgeexchangeandcollaboration
betweenAmericanandItalianscientists,amissionthatechoesthehistoryandgoalsofthe
InternationalCentreforTheoreticalPhysics,whichhostedtheSymposium.TheICTPwasfounded
in1964byAbdusSalam,aPakistani-bornphysicistwhospentmostofhiscareerinItalyandwho
sharedthe1979NobelPrizeinPhysicswithtwoHarvardUniversityprofessors.</p>
<palign=""left"">“Scientificthoughtisourcommonheritage,”Salamoftensaid,andhismotto
servesequallywellfortheArmenise-HarvardFoundation.Inhisclosingremarks,co-convenerMarc
KirschnerpaidtributetoCountGiovanniAuletta-ArmeniseandformerHMSDeanDanielTosteson
forcreatingapowerfulcatalystforcollaborationsamongItalianandAmericanscientists.</p>
<palign=""left"">Kirschnersummarizedthescientificsessionsas“aperfectbalanceofinformation,
inanenvironmentwhereitcanbeappreciated,withtherightnumberofparticipants.”Atthe
closingsession,heannouncedthisyear’srecipientsofawardsandgrantssponsoredbythe
Foundation.CareerDevelopmentawardsareintendedtohelpyoungItalianresearchers“jump
start”laboratoriesintheirhomecountry,andthisyear’swinnersareSabrinaSabatiniandStefano
Gustincich.SabatinihasbeenstudyinginUtrecht,Holland,andwillpursueherresearchongrowth
hormonesinplantsattheUniversityofRome.Gustincich,whorecentlymovedfromHarvardtothe
InternationalSchoolforAdvancedStudies(SISSA)inTrieste,workedwithindividualretinal
neuronstodevisemethodsusefulforstudyingotherneuralnetworksaswell.</p>
<palign=""left"">Asecondinitiative,theFoundation’sHMSJuniorFacultyGrants,helpsunderwrite
researchbypromisingyounginvestigatorsatHarvardMedicalSchool.Thisyear’sgrantswentto
SeanP.J.WhelanandBernardoSabatini,whodescribedhisresearchonsynapseformationina
symposiumpresentation.Whelanworksonreversegeneticsofarenaviruses,microbesthatcause
animalandhumandiseasesrangingfrommild,flu-likeillnessestolethalhemorrhagicfevers.
Sweet,BitterandUmami:TheBiologyofMammalianTaste
CharlesZuker
ProfessorofNeuroscienceandBiology,UniversityofCalifornia–SanDiego
“Howdoesthebrainknowwhatwejustate?”ThisisthecomplexquestionthatDr.Zukerposed
morethanfouryearsago,whenhesetouttoanalyzehowtasteinformationtravelsfromthetongue
tothebrainandelicitsresponsesrangingfromdelighttorepulsion.Althoughhowthebrainknows
remainsmysterious,hislabhasidentifiedtwofamiliesofcellsurfacereceptorsinthetonguethat
encodethreebasicmammaliantastes.</p>
<palign=""left"">Sweetandbitter,alongwithsourandsalty,areinstantlyrecognizableasthefour
classiccategoriesforthevastarrayofgustatorysensationsexperiencedbypeopleandanimals.
Umami,aless-familiartastemodalitystudiedintheZukerlaboratoryatUCSanDiego,hasbeen
recognizedbyscientistsonlysincethemid-1980s.“Thisistheyummy,delicioustastethatweoften
associatewithChinesefoodandheadaches,”Dr.Zukersaid.Umamiistheofficialnameforthe
uniqueflavorimpartedbymonosodiumL-glutamateandcertainnucleotides,suchasinosinateand
guanylate.</p>
<palign=""left"">Inaseriesofreportssince1999,Dr.Zukerandhisteamhavereportedontwo
familiesoftastereceptors:onedetectssweetandumami,flavorsthatattractmammalstohighcaloriefoodsources,andasecond,largergroupperceivesbitter,anaversivetastelinkedwithtoxic
substances.Thesereceptorfamiliesoccuronlyincellsthatconstitutetastebuds,andmolecular
analysisoftheirexpressionrevealsthatold-fashioned“tastemaps,”showingthatsweetandbitter
arepickedupbymutuallyexclusiveregionsofthetongue,are“utternonsense,”Dr.Zukersaid.No
matterwhereatastebudis,itscellsareoutfittedwithatleastsomereceptorsforeachofthefive
basictastes.Receptorexpressionvariesgreatlyfromcelltocell,however,andthisbalance
determineswhichtasteanareaofthetongueprimarilydetects.</p>
<palign=""left"">WhenDr.Zuker’sgroupfirstidentifiedtheT1RfamilyofGprotein-coupled
receptorstheydidn’tknowwhich–ifany–specifictastesthesestructuresencoded.Tofindout,
theydevisedascreeningassayinwhichcellsexpressingfamilymembersT1R1,T1R2,andT1R3
canbechallengedwithvarioussweetandumamitasteswhiletheircalciumresponseswere
measured.Cellsthatco-expressT1R2andT1R3respondedtoeverynaturalsweetener,artificial
sweetener,andDaminoacidtheywereexposedto,butdidnotreacttoanynon-sweetsubstances.
Incontrast,cellsbearingonlyoneofthesereceptorswereindifferenttosweetness.</p>
<palign=""left"">Otherresearchershadshownthatumami-flavoredaminoacidswereattractiveto
mice,andDr.Zukersuspectedthattherewouldbespecificmammalianreceptorsforthismodality
aswell.Inhislaboratory’sscreeningassay,cellsco-expressingT1R1andT1R3respondedtoMSG
andtoall20naturalLaminoacidsbutnottoanysweetsubstances.Resultsfromthecell-based
screeningassayswereconfirmedbyaseriesofexperimentswithknockoutmice.Inatypical
experiment,forexample,micethathadavidlyconsumedsugarwaterwhileshunningplainwater
losttheabilitytodiscriminatewhentheT1R2genewasknockedout.Parallelresultswereobtained
forumamireceptors.</p>
<palign=""left"">Turningtonoxioustastes,Dr.Zukerandhiscolleagueshaveidentifiedafamilyof
some30Gprotein-coupledreceptorswithahighaffinityforbitterflavors.Inevolutionaryterms,it
makessensetohavealargerepertoireofhighlysensitivereceptorsforharmfulsubstances,
because“ifyoueatit,youdie,”Dr.Zukersaid.Inmouseandhumantastebuds,theseT2Rreceptors
appeartobeexpressedineverycell.Acutesensitivitytobittertastesmakesnormalmiceadeptat
avoidingpoisonssuchascycloheximide,butwhentheresearchersknockedoutthereceptorforthis
bittersubstance,alteredmicedranktaintedwateranddied.</p>
<palign=""left"">Additionalexperimentsinmicerevealedthatreceptorsforsweet,umami,and
bitterrarelyoccurinthesamecells,althoughcellsbearingvariousreceptorsarebundledtogether
intastebuds.Althoughthethreemodalitiesarefunctionallysegregated,downstreamtheT1Rand
T2Rreceptorfamiliesshareatleasttwosignalingmolecules.Knockingouteitheroneofthese
yieldsmicethatcan’ttastesweet,umami,orbitter–yetdetectsaltyandsourstimuliwith
ease.</p>
<palign=""left"">Atthisstageoftheresearch,Dr.Zukersaidthathehasa“goodfeel”forhowthe
tongueknowswhatwehavejusteaten.“Ifweareluckyoverthenextfewyears,wewillbeableto
drawaconnectivitymapbetweenthetongueandthebrain,”heconcluded.</p>
<hrnoshade=""noshade""size=""1""width=""400""/>
<palign=""left""><strong>OtherPresentations</strong></p>
<blockquote>
<palign=""left""><em>ImmuneGenesandBrainWavesinBrainWiring</em>
<strong>CarlaJ.Shatz</strong>,ProfessorandChair
DepartmentofNeurobiology,HarvardMedicalSchool
Email:<ahref=""mailto:[email protected]"">[email protected]</a></p>
</blockquote>
<palign=""left"">Noonewoulddreamofworkingonacomputerwiththepowerturnedon.Yetthis
isexactlyhowconnectionsareformedinthedevelopingbrain.Thisbiologicalprocessis“much
moremarvelousthanacomputer,becausethewiringisdonewhiletheswitchisonandtheactual
functioningofthebrainisrequiredforthewiring,”Dr.Shatzsaid,introducingherlab’smostrecent
discoveriesaboutthegeneticcontrolofvisualsystemdevelopment.</p>
<palign=""left"">Dr.Shatzwasthefirsttodescribehowbrainactivitysortsamessytangleof
connectionsintothepreciseandorderlyarrangementfoundintheadultvisualsystem.Longbefore
rodsandconesarepresent,ganglioncellsfirespontaneouslyandsynchronously,generating
""waves""ofactivitythatsweepacrossdomainsoftheretina.Thegrowingtipsofthesecellsuse
molecularcuesfromtheenvironmenttogrowintothelateralgeniculatenucleus(LGN),where
signalsfromleftandrighteyesareentwinedinwhatresemblesatangleofwires.</p>
<palign=""left"">Afterbirth,arushofelectricalimpulsesstimulatedbylighttriggerssynaptic
remodelingthatprunesandreshufflestheseconnections,sothataxonalconnectionsfromganglion
cellsintheleftandrighteyesaresortedintotwomutuallyexclusivelayersintheLGN.Earlier
experimentsinherlabestablishedthatblockingsignalsfromgangliainterferedwithactivitydependentlayering.Theruleofthumbis:“Cellsthatfiretogether,wiretogether.Outofsync,lose
yourlink,”Dr.Shatzsaid,summarizinghowfrequencyofusetranslatesintoenduringstructural
change.</p>
<palign=""left"">Thenextstepwastosearchforgenesinvolvedinsynapticremodeling,whichthe
researchersthoughtwouldbeexpressedwhenthepowerswitchwasturnedon.ScreensformRNA
ofgenesactiveduringLGNdevelopmentconfirmedthattheyareexpressedwhenendogenous
activityisnormal,butnotifitisblocked.ThemostsurprisingfindwasafamilyofgenesforclassI
majorhistocompatibilitycomplex(MHCI),akeyplayerinthemammalianimmunesystemthathad
notpreviouslybeendetectedinthebrain.NotonlyisMHCIpresent,butDr.Shatz’steamshowed
thatthesegenesareexpressedbyCNSneuronsattimesandregionsofactivity-dependentaxonal
rearrangements.AseriesofexperimentsincatsandrodentssuggeststhatMHCIgenesplayanovel
roleinCNSdevelopmentandplasticity.</p>
<palign=""left"">Next,theinvestigatorsaskedwhetherMHCIisessentialforsynapticremodeling.
Tofindout,theyusedknock-outmicemissingclassIMHCorCD3zeta,arequiredsignaling
componentformanyreceptorsthatrecognizeclassMHCI.Inthesemice,activity-dependent
refinementoftheretinogeniculateprojectiondidnotdevelopintotheneatandfocalbundleof
conductivityseeninnormalmice,butinsteadwaslargeandfuzzyinappearance.MHCIisalso
expressedandregulatedbyactivityinthehippocampus;inadultmicelackingeithercellsurface
MHCIorCD3zeta,longtermpotentiationissupranormalandlongtermdepressionisabsent.
Together,theseresultssuggestthatnormalactivity-dependentsynapticremodelingcannottake
place,eitherdevelopmentallyoracutely,unlessclassIMHCandCD3zeta-containingreceptorsare
present.FutureexperimentsintheShatzlabwillfocusonmicethatoverexpresstheMHCI
gene.</p>
<palign=""left"">Sofar,theseinvestigationshavedemonstratedthatnervecellfunctionisessential
forspecificactivity-dependentgeneexpressionandfortheinitialstructuralremodelingthatwires
theadultvisualsystem.SincespontaneousneuralactivityiscommoninthedevelopingCNS,similar
activity-dependentmechanismsandmoleculesareprobablyusedelsewheretoorganizeearly
connectionsintopreciseadultpatterns,Dr.Shatzsaid.</p>
<hrnoshade=""noshade""size=""1""width=""400""/>
<blockquote>
<palign=""left""><em>TransductionandAdaptationinAuditoryHairCells</em>
<strong>DavidP.Corey</strong>
DepartmentofNeurobiology,HarvardMedicalSchool
Email:<ahref=""mailto:[email protected]"">[email protected]</a></p>
</blockquote>
<palign=""left"">Dr.Corey’slabwantstoknowhowthebrainmakessenseofthesoundsaround
us?Althoughthiscomplicatedprocessisn’tfullyunderstood,animportantfirststepiscarriedout
bybundlesofstereociliaonhaircellsintheear.These“hairbundles”looklikesheavesofgrain,
swayingwhentheyarebuffetedbywavesofsound.Intheauditoryandvestibularsystems,they
convertthesemovementsintoneuralsignalsbymeansofmechanically-gatedtransduction
channelslocatedinthetipsofthestereocilia,whichopeninresponsetotensioninfinetiplinks
connectingadjacentstereocilia.Atrest,thehaircellsmaintaintensiononthesechannelswithina
fewtenthsofapiconewton(pN),tokeepthemintheirmostsensitiverange.Atthesametime,this
tensioncanchangequicklyinresponsetomaintainedbundledeflection,animportanttypeof
adaptationthatisprobablycontrolledbyseveralindependentmechanisms.</p>
<palign=""left"">Becausethestereociliahavecoresofactinfilaments,researchershavelong
thoughtthatsometypeofmyosinisprobablythemotorthatregulatestensionatthehaircelltips.
Atleasttendifferentmyosinshavebeenidentifiedinthesensoryepitheliumoftheinnerear,and
mutationsinfiveofthesemyosingeneshavebeenlinkedwithhereditarydeafness.Dr.Corey’steam
foundthatonlyoneofthesemyosins,knownasmyosin-1c,isenrichedatthetipsofthestereocilia.
Immunogoldelectronmicroscopystudiesfounditconcentratedwithin~150nmoftiplink
insertions,wherethechannelsaremostlikelylocated.</p>
<palign=""left"">Todeterminethefunctionalsignificanceofthisobservation,Dr.Corey’slaband
collaboratorsatOregonHealthSciencesUniversitymutatedtyrosine-61ofmyosin-1ctoglycine,
whichenlargedtheADPbindingpocketsothatitcouldbeinhibitedbyoversizedN6-modifiedADP
analogs.Theinvestigatorscreatedtransgenicmicethatexpressedthemutantmyosin-1cin
utricularhaircells,deliveredanADPanalogthroughawhole-cellrecordingpipette,andfoundthat
theanalograpidlyblockedadaptationintransgenicbutnotinwild-typehaircells.Movementofthe
wild-typemyosin-1cwasapparentlyblockedaswell.“It’slikeyouhaveateamofhorsesandhalfof
themdecidetoliedown–theothersaren’tgoinganywhere,”Dr.Coreysaid.Adaptationisinhibited
andthesystemlocksdown,stronglysuggestingthatmyosin-1cindeedregulatestensionon
transductionchannels.</p>
<palign=""left"">Otherteamsofinvestigatorssuggestedthatasecondmechanismisalsoinvolved.
Accordingtothisalternativehypothesis,Ca2+entersopentransductionchannels,thenshutsthem
bybindingtoanintracellularsite.Dr.Corey’slabfolloweduponthisideabyapplyingforcestohair
bundleswithlasertweezersandmeasuringthemovementsevokedbyCa2+entry.Theresults
supportedboththemyosinandcalciummodels.Thusmechanicalforcesopenchannelsin
stereociliabundles,andtheinfluxofcalciumbothclosesthemdirectlyandallowsthemyosincontrolledtensiontorelaxfaster.Forbothmechanisms,additionalforcecanreopenchannels.Dr.
Corey’sgroupwasabletoshowthatonceCa2+closeschannelsbybinding,anadditional0.7pNof
forceissufficienttoprythemopen.</p>
<hrnoshade=""noshade""size=""1""width=""400""/>
<blockquote>
<palign=""left""><em>HowtheLeechInteractsWiththeExternalWorld</em>
<strong>VincentTorre</strong>
InternationalSchoolforAdvancedStudies,Trieste
Email:<ahref=""mailto:[email protected]"">[email protected]</a></p>
</blockquote>
<palign=""left"">Thedesiresoftheleecharenotsodifferentfromourown:thesesimple
invertebratesaredrawntofoodandsex,theywanttoescapepainandinjury,andsometimesthey
simplywanttolookaround.Theleechreliesonswimmingandcrawlingtomoveawayfrom
noxiousstimuliandtowardsfoodandmatingpartners,mechanismsthathavebeendescribedin
detailbynumerousresearchers.Theleech’sreflexesforescapingdanger,suchaswhole-body
shorteningandlocalbending,havealsobeenwellstudied.Lessisknownabouttheinnerworkings
ofexploratorymovements,inwhichtheleechattachesitselftoasurfaceusingitstailsucker,and
extendsandturnsitsheadandbody.</p>
<palign=""left"">Althoughtheleech’sexploratorymovementsappearmorecomplexandrandom
thanitsapproachorescapebehaviors,allthreeareelicitedandregulatedbyenvironmentalcues,
mediatedbycentralmechanosensoryneurons,andexecutedbymotorneurons.</p>
<palign=""left"">Dr.Torresetouttounderstandhowexternalsignalsaretranslatedinto
exploratorybehaviorbyquantifyingtheleech’sbehaviorandtheelectricalactivityunderlyingit.
Histeamusedsupergluetoattachtinyred,green,andbluebeadstotherostral,mid-body,and
caudalpositionsontheuppersurfaceoftheanimals.Acomputerizedvisualanalysissystemtracked
theirbehaviorforaslongas24hoursatastretch,usingthebeads’movementstodeterminewhat
theleechwasdoingatanytime.Someleechesrearedupintheexploratoryposturemoreoftenthan
others,butoverlongperiodsindividualdifferencesaveragedout.Theinvestigatorsobservedthat
well-fedleechesignoredenvironmentalirritantssuchasflashinglightsorchemicals,andflinched
onlywhentheywerephysicallyprodded.</p>
<palign=""left"">Additionalstudiesofescapereflexesinresponsetotouchusedahemisectionofa
leechbodywallflattenedandpinnedonarecordingchamber,withthecentralsegmentkept
innervatedbyitsganglion.Whentheleechskinwasprodded,Dr.Torre’sgroupusedmultielectroderecordingstoobservespiketrainsofmechanosensoryandmotorneurons,while
simultaneouslytakingpicturesofskincontractions.</p>
<palign=""left"">Thisapproachuncovereddifferencesbetweenspiketrainsinmechanosensory
neurons,whichwerehighlyreproducible,andthoseinmotoneurons,whichvariedgreatly.Despite
thisvariability,theinvestigatorsfoundthatmotoroutputwasreproducibleandreliable.They
identifiedtwodistinctbiophysicalmechanismsthattranslatevariablespiketrainsinto
reproduciblemotoroutputs.First,leechmotorneuronscontractslowlyenoughtosmoothoutthe
“jitter”ofmotoneuronspiketrains,whichwouldhaveaquiteadifferentimpactonrapidly
contractingmuscles.Second,themotoroutputisadistributedprocess,reflectingthesimultaneous
activationofmanymotoneurons,andthepopulation’soverallfiringaveragesoutdifferencesinhow
individualmotoneuronsreact.</p>
<palign=""left"">Theseexperimentsshowthatdependablemotorreactionscanarisefrom
unstablespiketrains,afindingthatmakessenseinevolutionaryterms.Survivalhingesonbeing
abletoescapefromdangerousassaults,Dr.Torreobserved.Nowonderthenervoussystemisa
abletoturnunreliablecomponentsintoreliableactions.</p>
<hrnoshade=""noshade""size=""1""width=""400""/>
<blockquote>
<palign=""left""><em>FunctionandMolecularDiversityoftheHypervariableReceptor
DSCAM</em>
<strong>DietmarSchmucker</strong>
DepartmentofNeurobiology,HarvardMedicalSchool&amp;DepartmentofCancerBiology,DanaFarberCancerInstitute
Email:<a
href=""mailto:[email protected]"">[email protected]</a>
</p>
</blockquote>
<palign=""left"">Alternativesplicingisahottopicinbiologythesedays,becausethismechanism
explainshowarelativelysmallnumberofgenesencodesthemyriadproteinsneededforlife.Dr.
Schmucker’sstudiesoftheDscamgenearenoteworthybecausehehasuncoveredanextreme
exampleofalternativesplicing:onegenewiththepotentialtogeneratemorethan38,000protein
isoforms.</p>
<palign=""left"">Dr.Schmucker’songoinginvestigationsfocusonhowneuronsfindtheircorrect
pathsinthedevelopingbrain,sothatmillionsofcellsrepresentingdiversecellstypesmake
preciselytherightconnections.InthecourseofstudyingthisprobleminDrosophilia,hezeroedin
onDscam,amemberoftheimmunoglobulinsuperfamilythatisexpressedinmostpost-mitotic
neuronsandlocalizedinthetipsofgrowingaxonsintheembryoniccentralnervoussystem.Dscam
takesitsnamefromthehumanproteinDownsyndromecelladhesionmolecule(DSCAM),first
foundonchromosome21ofDownsyndromepatients.(TheclinicalconnectionbetweenDSCAM
andDown’ssyndromeisunclear.)</p>
<palign=""left"">Inflies,lossoffunctionmutationsintheDscamgeneresultinearlylethalityand
disrupttheformationofmanynerveconnections.TheDrosophilaDscamgeneislarge(about270
kiloDaltons)andhighlycomplex,with115exonsincluding95thatarevariable.Dr.Schmucker
foundthatalternativesplicingofconstantandvariableexonscouldgener-ateatleast38,000
receptorisoformswithaconservedarchitecturecontainingthreevariableIgandtwoalternative
transmembranedomains.</p>
<palign=""left"">HisteamisnowexploringthefunctionalsignificanceofDscam’shypervariability
andhowitsproteannaturehelpsformhighlyspecificconnectionsincomplexneuronalcircuits.Dr.
Schmucker’shypothesisisthatdifferentnervecellsexpressdifferentDscamisoforms,providinga
specificmolecularrecog-nitioncodethatguidesindividualneuronstoconnectwiththerighttarget
neuronsinthedevelopingbrain.</p>
<palign=""left"">HesetouttotestthisinDrosophila,whereeverybristleonthefly’sbodyislinked
toaspecificsomatosensoryneuron,sothatresearcherscantracksignalsdownaspecificpath.So
far,earlyfindingsindicatethatwhenDscamisweakenedbyaninducedmutation,axonprojections
frommechanosensoryneuronsfindtheirwaytotherightareaintheCNS,butdon’tactually
communicatewithtargetcellsbecausetheylackthebranchesthatshouldmakethiscontact.
Experimentsareunderwaytolearnmoreabouthowthisaffectsspecificityandbrain
development.</p>
<hrnoshade=""noshade""size=""1""width=""400""/>
<blockquote>
<palign=""left""><em>PresynapticCalciumStoresModulateAfferentReleaseinVestibularHair
Cells</em>
<strong>FabioMammano</strong>
IstitutoVenetodiMedicinaMolecolare,IstitutoNazionalediFisicadellaMateria,andDipartimento
diFisica“G.Galilei”,UniversityofPadova
Email:<ahref=""mailto:[email protected]"">[email protected]</a></p>
</blockquote>
<palign=""left"">Justassomebundlesofhaircellstranslatethemovementofsoundwavesinto
auditoryinformation,otherstranslatethelurchofataxiintothesensationofmovement.Evena
tinyaccelerationoftheheadcausesvestibularfluidtodeflecthaircellbundles,whichthebrain
perceivesasmotion.Likeacoustichaircells,thesetinymotiondetectorsaremechanoreceptors–
presynaptictoprimaryafferentneuronsoftheeighthnerve–whichexciteneuralactivitybythe
releaseofglutamate.</p>
<palign=""left"">Dr.MammanohasbeeninvestigatingtheroleofintracellularCa2+storesin
afferenttransmissioninseveralways.Workingwithanexcisedpatchofvestibularepithelium,he
studiedpresynapticchangesbymonitoringintracellularCa2+concentration([Ca2+]i)inhaircells;
postsynapticchangeswereassessedbyrecordingfromsingleposteriorcanalafferentfibers.When
theresearchersadded1-10mMcaffeinetothehaircells,Ca2+responsesevokedbydepolarization
atselectedCa2+“hotspots”shotupbriefly,accompaniedbyanincreaseincellmembrane
capacitance(DCm).ThissignaledaburstofCa2+exocytosis,whichiswhatthepresynapticendofa
stimulatedhaircellshoulddo.Thegreatestresponsetocaffeinewasobservedinaregionlocated
about10mmfromthebaseofthehaircell.</p>
<palign=""left"">WhenDr.Mammanousedelectricitytodepolarizethehaircells,localized[Ca2+]i
increaseswereobservedfollowingdepolarizationslastingfor500milliseconds,butnotwith50ms
bursts,suggestingcalcium-inducedcalciumrelease(CICR)fromintracellularstores.BothCa2+and
DCmresponseswereinhibitedincellsthathadbeenincubatedwith40mMofryanodinefor8-10
minutes.Consistentwiththeseresults,afferenttransmissionwaspotentiatedbycaffeineand
inhibitedbyryanodinebothatthelevelofactionpotentials(APs)andofminiatureexcitatory
postsynapticpotentials(mEPSPs).Neithercaffeinenorryanodineaffectedtheshapeandamplitude
ofmEPSPs,indicatingthatbothdrugsactedatthepresynapticlevel.</p>
<palign=""left"">Dr.MammanoandcollaboratorsatUniversitadiPaviaandUniversitadiFerrara
alsousedafrogmodeltostudytheimpactofCa2+andryanodineonsinglefiberafferentnerve
activitywithandwithoutexternalstimulation.Responsestocaffieneandryanodinewereas
predicted,andinadditiontheyfoundthatcaffeine’sabilitytoincreasefiringofafferentnerves
increased3to4-foldwhentheanimalwasspunonaturntable.Theseresultsstronglysuggestthat
endogenousmodulatorsoftheCICRprocesswillaffectafferentactivityelicitedbymechanical
stimuliinthephysiologicalfrequencyrange.</p>
<hrnoshade=""noshade""size=""1""width=""400""/>
<blockquote>
<palign=""left""><em>RegulatedExocytosisotherthanRegulatedSecretion:Rolesof
Enlargeosome,aLittleBrandNewOrganelle</em>
<strong>JacopoMeldolesi</strong>
Vita-SaluteSanRaffaeleUniversity,Milan
Email:<ahref=""mailto:[email protected]"">[email protected]</a></p>
</blockquote>
<palign=""left"">Agreatdealofresearchhasbeendoneonregulatedsecretorypathwaysthat
culminateinexocytosis.Neurotransmitterreleaseanduptakeisoneofthecrucialphysiologic
processesthatdependsonregulatedexocytosis,inwhichsmallamountsofmembranedeliver
proteinsofgreatimportance,suchasglutamatereceptors,todiscretesitesonthecellsurface.For
thepastseveralyears,Dr.MeldolesiandcollaboratorsatHarvardMedicalSchoolhavebeen
exploringadifferenttypeofexocytosisthatiscrucialforprocessessuchasmitosis,phagocytosis,
andwoundhealing.</p>
<palign=""left"">Thissecondtypeofexocytosisrapidlyenlargestheplasmamembraneinresponse
tosignalsfromspecificintracellularmessengerstriggeredbyoutsidestimuli.InDr.Meldolesi’s
laboratory,patchclampcapacitanceassaysshowthatinlessthanonesecond,thesurfaceofvarious
celltypesenlargesby15-20%inresponseto[Ca2+]iincrease.Exocytoticfusionofwhatappearto
bemanysmallorganelles,eachlessthan0.1mmindiameter,seemstoaccountforthis
enlargement.Thesearchforamarkerthatcouldbeusedtotrackthisprocessledtheresearchersto
ahighmolecularweight,non-transmembraneproteincalledAhnak/Desmoyokin(dA)that
concentratesinasubcellularcompartmentandtoalesserextentinplasmamembrane.Thisprotein
appearsonthecellsurfaceonlyafteraninfluxof[Ca2+]I,whichsuggestedtoDr.Meldolesithatit
wouldbeusefulforstudyingcalcium-dependentexocytosis.</p>
<palign=""left"">TheteamdevelopedamonoclonalantibodyagainstdA,whichtheyusedtotrace
exocytosiscarriedoutbythismysteryorganelle,whichappearstohavemultiplefunctions.During
differentiation,itrushestothecellsurfacebutisnotco-locatedwithsecretoryvesicles.Whenthe
cellmembraneisinjured,theorganellerushestothesiteandfacilitatesformationofnew
membranetorepairthebreach.Althoughtheconventionalwisdomisthatlysosomesaloneare
responsibleforsuchrepairs,Dr.Meldolesi’sexperimentsshowthattheneworganelleisalso
involved.</p>
<palign=""left"">Nomatterwhatcallsthesespecialvesiclesintoaction,theirarrivalatthecell
surfacecausesrapidmembraneenlargement–anobservationthatledDr.Meldolesitocallthem
“enlargeosomes.”Monoclonalantibodystudiesindicatethatenlargeosomesrestinthecytoplasmic
layeradjacenttotheplasmalemma;whenstimulated,theyrushtothecellsurfaceandremainthere
forprolongedperiods,apparentlyanchoredtoaspecificbindingprotein.Duallabelingexperiments
demonstratedthatenlargeosomesaredistinctfromotherorganelles,includingtheER,GC,TGN
endo-andlysosomes,Glut4vesicles,andothervesiclesofconstitutivesecretion.Dr.Meldolesiand
hiscolleaguesarecontinuingtoexploretheenlargeosome’sroleincelldifferentiationandwound
healing.</p>
<hrnoshade=""noshade""size=""1""width=""400""/>
<blockquote>
<palign=""left""><em>HowdoMammalianCellsSenseDirectionality?</em>
<strong>LewisC.Cantley</strong>
DepartmentofCellBiology,HarvardMedicalSchoolandDivisionofSignalTransduction,BethIsrael
DeaconessMedicalCenter
Email:<ahref=""mailto:[email protected]"">[email protected]</a></p>
</blockquote>
<palign=""left"">Amongmammaliancells,thespeedchampionofchemotaxsisistheneutrophil:
unlikecellsthattakehourstomovetowardorawayfromadiffusiblechemical,neutrophilscanturn
onadimeoncetheydetectachemotacticsignal.MoviesmadeinDr.Cantley’slabshowneutrophils
zigzagginginpursuitofpipettetaintedwithbacterialpeptidelikehockeyplayerschasinga
puck.</p>
<palign=""left"">Thissortofdirectionalmotility,aswellasdirectionalgrowthofeukaryoticcells,
involvescomplextemporalandspatialsignaling.Shallowspatialgradientsofstimulantsinthe
externalenvironmentareamplifiedinsidethecelltoproducesharpinternalgradients.These
reorientthecytoskelton,whichcommitsthecelltogrowthormovementinaspecificdirection.The
centralcoordinatorofthissortofrapiddecision-makingistheenzymephosphoinositide3-kinase
(PI3K),whichisactivatedbyreceptorsthatdetectbacterialchemokinesorendogenousgrowth
factors.</p>
<palign=""left"">PI3Kfloatsinthecytosol,thenbindstotheplasmamembranewhereitplaysa
criticalroleindirectionalitybygeneratingalipid,phosphatidylinositol-3,4,5-trisphosphate(PIP3),
attheplasmamembraneinresponsetogrowthfactorsandchemokines.PIP3phosphorylatesa
hostofdifferentproteinsinvolvedinchemotacticresponseandcellmigration,ultimatelycausing
actintoconcentrateattheleadingedgeofthemovingcell.Giventhatreceptorsforchemotaxsis
signalsarestuddedalloverthecell,howdoesthecellknowwhichwaytoturn?</p>
<palign=""left"">Dr.Cantley’sgroupisexploringthisquestionbyexaminingwhathappensinside
neutrophilschasingamovingbacterialpeptidesignal.Theresearchersareusingbiochemical,cell
biological,andgeneticmethodstodefinethesignalingpathwaysthatpositivelyandnegatively
regulatePI3K.Theyhavefoundthatthecellsareexquisitelysensitivetostimulantgradientsinthe
environment,andautomaticallyconcentratePIP3manufactureontheedgeofthecellnearestthe
stimulus.Whenthepipettetipismoved,PIP3manufactureimmediatelyshiftstothenewleading
edge,whileproductionofPTEN,whichdegradesPIP3,breaksdownPIP3atwhatwaspreviously
thecell’sfrontend.ByconfiningPIP3productiontotheleadingedgeofmigratingcells,this
combinationoflocalpositivefeedbackloopsandglobalnegativefeedbackloopsenables
neutrophilstomakesuddenturns.</p>
<palign=""left"">Morerecently,Dr.Cantley’slabhasbeenexaminingtheroleofthePI3Kpathway
infibroblastresponsetogrowthfactors,withtheaimoflearningmoreabouthowwoundsheal.
Thisprocessisneitherasfastnorasdirectionalaswhathappensinneutrophils,butknock-out
experimentssuggestthatthePI3Kpathwayisnecessaryforproperwoundrepair.Additional
investigationsareunderway.</p>
<hrnoshade=""noshade""size=""1""width=""400""/>
<blockquote>
<palign=""left""><em>SignalingMechanismsthatRegulateAxonGuidanceinDrosophila</em>
<strong>DavidVanVactor</strong>
DepartmentofCellBiologyandPrograminNeuroscience,HarvardMedicalSchool
Email:<ahref=""mailto:[email protected]"">[email protected]</a></p>
</blockquote>
<palign=""left"">ThegreatanatomistRamonyCajaldescribedtherelationshipbetweenaneuron
anditstargetasagreatlovestorythatbeginswithaquest,Dr.VanVactorsaidatthebeginningof
hispresentation.Hisinvestigationsfocusonakeyplayerinthisquest–thegrowthconeatthetip
oftheaxon.Heviewsthisstructureas“anexquisitelysensitivemolecularcompass”thattranslates
environmentalinformationintodirectionaldecisionsthatarecrucialtonormaldevelopment.</p>
<palign=""left"">Growthconebehaviorsareregulatedbyphosphorylation-dependentevents
controlledbyproteintyrosinekinases(PTKs)andproteintyrosinephosphatases(PTPs).In
additiontoreceptor-classproteinsthatdirectlylinkPTKorPTPcatalysistoconservedextracellular
domains,severalintracellularenzymeshavebeenimplicatedinaxonguidancedecisionsas
signalingpartnersofseparatereceptorproteins.Dr.VanVactorhasbeenexploringthiscomplex
processbystudyingaxonguidancedecisionsaroundthedevelopmentalmidlineofthefly.Here,the
AbelsonPTK(Abl)andtheSlitprotein(encodedbythegenecalledRobo)teamuptopushaxons
awayfromthemidlinedivideandintoparallelpathsoneitherside.Lethallossoffunction
mutationsinSlitorAblcauseaxonstomakeincorrectdecisionsandgrowacrossthemidline,
creatingabnormalcrossoversbetweenthetwosides.</p>
<palign=""left"">ThenextstepinDr.VanVactor’squestwastoidentifynewproteinsthatinteract
withAblandmightinfluenceinteractionsbetweendifferentcytoskeletalnetworks.Heandhisteam
identifiedcandidategenesbyanalyzingmutationsthatcausedobviousabnormalitiesinthe
complexeyeofthefly,reasoningthatproductsofsuchgeneswouldprobablybeactorsinkinase
signalingpathways.Severalcandidatesfromthisscreencontainproteinmotifssuggestingthey
mightplayaroleintheAblpathway,andalsoshowexpressioninthedevelopingnervoussystem.
FutureworkintheVanVactorlabwillbefocusedontestingwhetherthesenewgenesarevital
playersinthenavigationalmachinerythatcollaborateswithAbltocontrolkeyaspectsofneuronal
morphogenesis.</p>
<hrnoshade=""noshade""size=""1""width=""400""/>
<blockquote>
<palign=""left""><em>NewMechanismsforHGFReceptorActivationandInactivation</em>
<strong>SylviaGiordano</strong>
InstituteforCancerResearchandTreatment,Candiolo,Torino,Italy
Email:<ahref=""mailto:[email protected]"">[email protected]</a></p>
</blockquote>
<palign=""left"">Invasivegrowthisamultistepmorphogenicprocessinwhichthecellsdissociate
fromtheirneighbors,leavetheiroriginalenvironmentandmigrateintothesurroundingterritories.
Oncetheysettleintotheirnewlocation,theyincreaseinnumberandeventuallyundergoterminal
differentiation.Scatterfactorreceptors,afamilyoftyrosinekinasesincludingMet(thereceptorfor
hepatocytegrowthfactor)andRon(themacrophagestimulatingproteinreceptor),arekey
moleculesthatorchestrateintercellularsignalsneededtocarryoutthisprocess.Tightlyregulated
scatterfactoractivityisessentialfornormaldevelopment.</p>
<palign=""left"">Scatterfactorreceptorsbecomeproblematic,however,whentheyescapeclose
controlofactivationorinactivation–asituationthatcanleadtomalignanttransformationand
acquisitionofmetastaticproperties.Met,forexample,isoverexpressedingastrointestinaltumors
andthoseofmysenchymalorigin,andamplifiedinmetastasis.</p>
<palign=""left"">Dr.Giordano’slaboratoryinvestigatesmolecularmechanismsthatdownregulate
Met.Shehasfoundthatthisprocessisnormallycontrolledbyacomplexthatinvolvesendophilins
(whichinitiateendocytosis),aubiquitin-likeproteincalledCbl,andaproteinCblinteractswith
calledCIN85.Inhealthycells,CblmarksMetfordestruction,whichleadstoitsendocytosis–
diminishingthereceptorpopulationandloweringHGFlevel.Ifformationofthe
endophilin/CIN85/Cblcomplexisinhibited,HGFlevelsremainhigh,prolongingsignaltransduction
thatincreasescellmobilityandcanleadtotransformation.</p>
<palign=""left"">Morerecently,theresearchershaveshownthatscatterfactorreceptorscanbe
activatedbymechanismsotherthanligandbinding,specificallybyinteractionwithothercell
surfacereceptors.HGFreceptorsphysicallyassociatewithplexins,afamilyoftransmembrane
receptorsforproteinscalledsemaphorins,andthecross-talkbetweenthesemoleculesmediates
thewholeinvasiveprogramofcells,Dr.Giordanoreported.WhenthePlexinB1receptorassociated
withMetisstimulatedbysemaphorin4D,bothreceptorsareactivated.Similarly,blockingMetalso
impedessemaphorin4Dresponses.MetandPlexinB1arefoundtogetherinhumantumorcellsthat
exhibitinvasivetraitstypicallytriggeredbyscatterfactors:comparedwithnormalcellstheyare
moremobileandlikelytomigrate,morelikelytobranch,andmorelikelytoproliferatewithout
anchors.</p>
<palign=""left"">Whilemostexamplesofcrosstalkcomefrompathwaysinthecytoplasm,these
experimentsclearlyshowthatasimilarconversation–orperhapsconspiracy–canstartatthe
upstreamendofthesignalingpath.Morerecentexperimentshaveturnedupadditional
interactionsbetweenMetandotherreceptors.Thetake-homemessageisthatfarfrombeing
loners,someofthemanyreceptorsonthecellsurfacejoinforcestomanipulatespecifickindsof
cellularactivity,Dr.Giordanosaid.</p>
<hrnoshade=""noshade""size=""1""width=""400""/>
<blockquote>
<palign=""left""><em>EnvironmentalSignalsforCellScattering:OxygenSensing</em>
<strong>PaoloMichieli</strong>
DivisionofMolecularOncology
InstituteforCancerResearchandTreatment,Candiolo,Torino
Email:<ahref=""mailto:[email protected]"">[email protected]</a></p>
</blockquote>
<palign=""left"">Ifsomemalevolentauthoritybeganpackingpeopleintoatinyroom,sodensely
thattheairsupplywouldsoonbeexhausted,anysanepersonwouldrushfortheexit.Andthisis
exactlywhatmanytumorcellsdoiftheirenvironmentbecomeshypoxic,Dr.Michielisaid.No
wondertheriskofinvasivegrowthandmetastasisishigher,andtheprognosisworse,forpatients
whosesolidtumorsareriddledwithhypoxicregions.</p>
<palign=""left"">Better-knownresponsestolowoxygenincludetheabilityofcellstoadjusttheir
metabolicprocessesandtoinducethegrowthofnew,oxygen-carryingbloodvessels.Hypoxiainducedangiogenesishasbeenahottopicinrecentyears,generatingmanypublicationsand
spawningaracetodevelopdrugsthatcanblockthisprocess.Lessattentionhasbeengiventothe
mechanismthatenablescellsto“leavetheroom”andinvadesurroundingtissueswhereoxygenand
nutrientsabound.ThisisthephenomenonthatDr.Michielihasbeenexploring.</p>
<palign=""left"">HefocusesonthesamepathwaythatDr.Giordanodiscussedinthepreceding
lecture,whichisactivatedwhenhepatocytegrowthfactor(HGF)bindstothereceptorencodedby
thec-metproto-oncogene.Dr.Michieli’sexperimentshavedemonstratedthathypoxiainduces
expressionoftheMetreceptorbothinvitroandinvivo,clearlyimplicatingitinthecellular
responsetooxygendeprivation.Whenhisteamgrewvariouslinesoftumorcellundernormal
(21%oxygen)andhypoxic(3%oxygen)conditions,theyfoundthatMetproteinandmRNAlevels
increasedbyaboutthreefoldafterseveralhoursoflowoxygen.</p>
<palign=""left"">Inexperimentaltumors,Metproteinlevelsarehighlyincreasedincoincidence
withHIF-1-positive,hypoxicareas,forminganexpressiongradientthatisinverselyproportionalto
bloodvesselproximity.WhenDr.Michieliandhiscolleaguesanalyzedthehumanmetpromoter,
theyshowedthatthisinductionistranscriptionalandismediatedbytwoHIF-1bindingsites
(HBSs)andanAP-1site.Hypoxia-inducedMetoverexpressioncausescellstobecomehighly
sensitivetoHGF,andalongwithlowoxygentensionthispushescancercellstobecomemore
aggressive.Finally,usingagenetransferapproachandRNAinterferencetechnology,the
researchersshowedthathypoxia-drivenMetoverexpressionisnecessaryandsufficienttoactivate
theinvasivegrowthprogram.HighlevelsofMethavepreviouslybeenobservedincancercells,and
theseexperimentsprovideamolecularexplanationforitspurpose.</p>
<palign=""left"">Thisfindinghasimportantimplicationsfortumorbiologyandforclinicalcare,Dr.
Michielisaid.Thedemonstrationofanangiogenesis-independent,hypoxia-induced“invasive
switch”providesalinkbetweentumorhypoxiatoincreasedmalignancy.Butclinicalcaremay
becomemorecomplexasaresult:anti-angiogenictherapiesmayincreasethemetastaticpotential
ofsolidtumorsbysuffocatingthem,drivingmalignantcellstomigrateinsearchoffreshair.</p>
<hrnoshade=""noshade""size=""1""width=""400""/>
<blockquote>
<palign=""left""><em>AstrocytesasCentralMediatorsofNeurovascularCoupling</em>
<strong>GiorgioCarmignoto</strong>
IstitutoCNRdiNeuroscienzeandDipartimentodiScienzeBiomedicheSperimentali,Universitàdi
Padova
Email:<ahref=""mailto:[email protected]"">[email protected]</a></p>
</blockquote>
<palign=""left"">“Thereisnoevidencethatgliaaredirectlyinvolvedinelectricalsignaling.
Signalingisthefunctionofneurons,”assertstheauthorofaclassicneurologytext.Butifadditional
researchcontinuestobolsterdiscoveriesmadeinDr.Carmignoto’slab,thispassagemayneedtobe
revisedbeforethenexteditionispublished.</p>
<palign=""left"">Thereisgrowingevidencefortheexistenceofadistinctsignalingsystemthat
enablesastrocytes(themostnumerousglialcelltype)tocommunicatewithneurons.Indeed,
astrocytesrespondtothesynapticreleaseoftheneurotransmitterglutamatewithrepetitive
elevationsintheirintracellularcalciumconcentration([Ca2+]i).Parallelsbetweenthese[Ca2+]i
oscillationsandfluctuationsinglutamatereleaseledDr.Carmignototocharacterizeastrocytesas
“accuratedetectorsofsynapticactivitywhichencodeinformationonthestatusofneuronalactivity
into[Ca2+]ioscillationsofdistinctfrequencies.”Furthermore,whenastroglialcellsareclosely
associatedwithbothsynapsesandcerebralvessels,theymaytransfersignalsfromneuronsto
bloodvesselsviathese[Ca2+]ioscillations.Ifso,thenastrocytesarekeyplayersinneuronalcontrol
ofmicrocirculation.</p>
<palign=""left"">Inaseriesofinvitroandinvivoexperiments,Dr.Carmignoto’slabobtained
resultsthatsupportthishypothesis.Whenneuronswerestimulatedinratcorticalslices,
glutamate-mediated[Ca2+]ioscillationswereseeninastrocyteprocessestouchingcerebral
microvessels,whichdilatedinresponse.Whenaglutamatereceptorantagonistwasintroduced,
Ca2+responseswereinhibitedandneuronalactivity-dependentvasodilationwasimpaired,while
theselectiveactivationbyapatchpipetteofsingleastrocytesincontactwitharteriolestriggered
theirrelaxation.When[Ca2+]ioscillationswerestimulatedwiththemetabotropicglutamate
receptor(mGluR)agonistt-ACPD,arterioledilationresulted.Theresearchersalsosawevidence
thatastrocyte-mediatedcontrolofarteriolesreliesmainlyonacyclooxygenaseproduct.Incultured
astrocytes,mGluR-mediated[Ca2+]ioscillationstriggerapulsatilereleaseofprostaglandins,
presumablythevasodilatorprostaglandinE2.Workinginamousemodel,Dr.Carmignotoandhis
colleaguesfoundthatblockingglutamate-mediated[Ca2+]ielevationsinastrocytesreducedblood
flowincreaseinthesomatosensorycortexduringcontralateralforepawstimulation.</p>
<palign=""left"">“Byrevealingthedirectparticipationofastrocytesinthecontrolofcerebral
microcirculation,ourdataprovideamechanisticbackgroundforadistinctroleofneuron-toastrocytesignalinginthephenomenonoffunctionalhyperemia,”Dr.Carmignotoconcluded.These
findingsmayprovidenewopportunitiesfortreatingdegenerativediseasessuchasAlzheimer’s,in
whichcerebrovascularbloodflowdiminishesasdiseaseprogresses.</p>
<hrnoshade=""noshade""size=""1""width=""400""/>
<blockquote>
<palign=""left""><em>RoleofNMDA-TypeGlutamateReceptorsinFormationandMaintenanceof
DendriticSpines</em>
<strong>BernardoSabatini</strong>
DepartmentofNeurobiology,HarvardMedicalSchool
Email:<a
href=""mailto:[email protected]"">[email protected]</a></
p>
</blockquote>
<palign=""left"">NewapproachesdevelopedinDr.Sabatini’slabareenablinghisteamtochallenge
theconventionalwisdomabouthowneuralconnectionsandsynapticfunctionareregulated.His
labisespeciallyinterestedindendriticspines,whicharetargetsformanyexcitatorysynapsesin
themammalianbrain.Thegrowthofspinesishighlyregulatedduringdevelopment,withthe
majorityappearingduringperiodsofmassivesynaptogenesis.Oncesynapsesaremade,spine
growthandremodelingispromotedbyactivitypatternsthattriggerlong-termpotentiation.</p>
<palign=""left"">Neitherthefunctioningofdendriticspinesnorthesignalingcascadesthatlead
fromsynapticactivitytospinegrowthiswellunderstood,Dr.Sababinisaid.Inhispresentation,he
describedaseriesofexperimentsthatcastdoubtonearlierclaimsthatspineformationis
independentofNMDA-typeglutamatereceptor(NMDA-R)activation.Thisinvestigationwas
triggeredbyobservationsofspinegrowthandretractioninthebrainsofyoungrats,whichcaused
Dr.Sabatinitosuspectthatfarfrombeingirrelevant,NMDA-Rmightbeessential.</p>
<palign=""left"">Incontrasttoearlierwork,whichreliedonpharmacologicalmanipulationsto
studyspinegrowth,Dr.Sabatini’steamusedRNA-inactivation(RNA-I)toknockoutNR1,an
obligatorysubunitoftheNMDA-R,inrathippocampalslices.Alow-efficiencygeneguntransfected
onlyabout10pyramidalneuronsineachslice,markingthesewithafluorescentproteinand
enablingtheresearcherstoperformpatch-clampexperimentsonlabeledcells.Therestofthecells
intheslicewerenormal,andthewholesamplecouldbemaintainedandobservedforweeks.</p>
<palign=""left"">WhencellswithnormalNMDA-Rwereelectricallystimulated,theydepolarized
andshowedlongsignaldecay.Thisresponsewaslost,however,inRNAinactivatedcells.Inthese
cells,ImmunostainingconfirmedthatRNA-IdirectedagainstNR1dramaticallyreducedNR1
proteinlevelsandeliminatedfunctionalsynapticNMDA-Rs.DeprivedofsynapticNMDA-Rs,the
numberofdendiriticspinesdeclinedgradually.Eightdaysintotheexperimenttherewasno
significantdifferencebetweenknockoutandnormalcells;after18days,therewerevirtuallyno
spinesinthealteredcellswhilecontrolsretainedfulldendritictrees,Dr.Sabatinisaid.</p>
<palign=""left"">EvidencethatNMDA-Rsarenecessaryforbothspineformationandmaintenance
motivatedDr.Sabatini’steamtoconstructa“duallaserscanningmicroscope”forlearningmore
aboutspinedynamics.Futureexperimentswillalsoinvestigategeneticcontrolofsynapticplasticity
inhumandiseasessuchasAlzheimer’s.</p>
<hrnoshade=""noshade""size=""1""width=""400""/>
<blockquote>
<palign=""left""><em>MolecularMechanismsofPlasticityintheVisualCortex</em>
<strong>LambertoMaffei</strong>
InstitutodineuroscienzedelCNReScuolaNormaleSuperiore,Pisa
Email:<ahref=""mailto:[email protected]"">[email protected]</a></p>
</blockquote>
<palign=""left"">Dr.Maffei’steamhastakenimportantstepstowardrestoringeyesighttothe
blind,atleastintermsofrestoringlostplasticitytovisualcortexcells.Decadesago,NobelPrizewinningworkbyHarvard’sDavidHubelandTorstenWieselprovedthatcoveringoneeyeduringa
criticalperiodindevelopment,evenforonlyafewdays,couldcausepermanentvisualimpairment.
Thedurationofthiscriticalperiodisproportionaltothelifespanofaspecies,rangingfromafew
weeksinmicetoseveralyearsinhumans.Duringthistime,theplasticityofthevisualcortex
dependsonelectricalactivityandmolecularsignalsinvolvedinstabilizationandremodellingof
neuralcircuits.Forexample,hisgrouphasdemonstratedthatERGpathwayactivationisnecessary
forexperience-dependentplasticity(monoculardeprivation)andforlong-termpotentiationof
corticalsynaptictransmission.</p>
<palign=""left"">Afterthecriticalperiodends,plasticitydisappearsanddeprivingoneeyeof
stimulationnolongercausesashiftinoculardominance.RecentworkinDr.Maffei’slabhassought
toidentifyfactorsthatclosethecriticalperiodandlimitadultplasticity.Oneseriesofexperiments
usedatransgenicmousethatover-expressesbrain-derivedneurotrophicfactor(BDNF),which
mediatesneurotransmittersincludinginhibitorygammaaminobutyricacid(GABA).Inthismouse,
whichhaselevatedGABAlevels,developmentofvisualacuitywasacceleratedandthecritical
periodclosedearly.TheresearchersaskedwhetherplasticitycouldberestoredbyblockingGABA
synthesisanduptakeinadultrats,thencoveringoneeye.Thesurprisinganswerwasyes:therewas
ashiftinoculardominancetypicallyfoundonlyduringthecriticalperiod,indicatingthatthe
“windowofplasticityhadbeenreopenedinadultratbrain,”Dr.Maffeisaid.</p>
<palign=""left"">Asecondseriesofexperimentsexploredtheroleofextracellularmatrixfactorsin
visualcortexplasticity.Theyfocusedonchondroitinsulphateproteoglycans(CSPGs),whichinhibit
axonsproutingandmaturejustasthecriticalperiodends.Atthistime,CSPGsundergoadramatic
reorganizationintoperineuralnets.Inanimalsrearedinthedark,however,CSPGmaturationis
delayedandthesenetsdonottakeshape.TheresearchersusedinjectionsofchondroitinaseABCto
degradeCSPGsintheadultvisualcortex,andfoundthatoculardominanceplasticitycouldbe
restoredwithoutharmingreceptivefieldsizeorvisualresponseproperties,Dr.Maffeireported.
Investigationsintotherelationshipbetweenextracellularmatrixcomponentsandvisualcortex
plasticitycontinueinhislaboratory.</p>
<hrnoshade=""noshade""size=""1""width=""400""/>
<blockquote>
<palign=""left""><em>WhatistheRelationshipBetweenPhysiologicandEvolutionary
Adaptation?</em>
<strong>MarcKirschner</strong>,professorandchair
DepartmentofCellBiology(CQ)
Email:<ahref=""mailto:[email protected]"">[email protected]</a></p>
</blockquote>
<palign=""left"">Ontheheelsofaseriesofreportsreportsdelvingintothedetailsofcellular
sensingandresponse,Dr.Kirschnersteppedbacktoexaminethesestudiesinrelationtoamuch
broaderuniverse.Helinkedbasicsciencefindingsbothtophysiologicadaptationsattheindividual
levelandtospecies-wideadaptationstotheenvironment,drawingonconceptsfeaturesinGenes,
Embryos,andEvolution.Thisbook,writtenbyDr.KirschnerandJohnGerhartin1997,callson
molecularbiologyandclassicDarwiniantheorytoshowhowfactorssuchasgeneticvariabilityand
conservationshapebothindividualsandtheenduringtraitsofaspecies.Evenaphysiologic
adaptationthatmanifestsonlyundercertainconditions,suchashemoglobin’scapacitytobind
oxygenmoretightlyathighaltitudes,canpromotelong-termhereditarychange,Dr.Kirschnersaid.
Ifindividualswithatraitsurvivethosewithoutit,aoncemarginalcharacteristicbecomes
mainstream.</p>
<palign=""left"">Biologicvariabilityoccursatmanylevels,rangingfromphenotypichallmarksthat
distinguishhorsesfromzebrastothehard-to-detectcellularanddevelopmentalvariations
describedduringthisSymposium,Dr.Kirschnersaid.Manyofthesevariationscanbeexplainedby
principlesofwhathecalleda“neo-Darwiniansynthesis”:Evolutiondependsonheritable
variability;externalenvironmentdoesnotgenerateheritablevariants(childrenofskilledpianists
don’tnecessarilyplaywell);largephenotypicchangesoccurgraduallyandaccumulate;evolutionis
descentbymodification.The“molecularaddendum”tothesetenetsisthatchangeoccursvia
randommutations,andthatresultingalterationsinproteinsorregulationaffectphenotype.What
scientistscan’tdoquiteyet,ofcourse,isexaminegeneticsequencedataandpredictitsresults,he
said.</p>
<palign=""left"">Dr.Kirschnerclosedbydescribingtheparadoxicalnatureof“explanatory
processes”suchasmicrotubules,whicharehighlyconservedyetversatileenoughtoprovide
rigiditytotheskeletonorenableleukocytestochangedirectioninaninstant.“Diversityitselfisa
selectedpropertyinbiology,”hesaid,and“wewouldn’tbehereifitwasn’tforthefactorsthat
generatediversity.Ratherthanthinkingofconservationastheendresultofwhythingscan’t
change,Ithinkweshouldlookatconservationasbeingpreservedundercontinualselection
becauseitprovidesthecapacitytochangeinbothevolutionandphysiology.”</p>";"Symposium
2003:Trieste,Italy";"";publish;open;open;"";symposium-2003-trieste-italy;"";"";2012-09-14
12:59:16;2012-09-14
12:59:16;"";0;http://armeniseharvard.sypdevelopment.com/?p=256;0;post;"";0