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regulation of protein transport and the maintenance of
axonal identity
Christophe Leterrier, Bénédicte Dargent
To cite this version:
Christophe Leterrier, Bénédicte Dargent. No Pasaran! Role of the axon initial segment in the
regula-tion of protein transport and the maintenance of axonal identity. Seminars in Cell and Developmental
Biology, Elsevier, 2014, 27, pp.44 - 51. �10.1016/j.semcdb.2013.11.001�. �hal-01701549�
SeminarsinCell&DevelopmentalBiologyxxx (2013) xxx–xxx
ContentslistsavailableatScienceDirect
Seminars
in
Cell
&
Developmental
Biology
j ou rn a l h o m e pa g e :w w w . e l s e v i e r . c o m / l o c a t e / s e m c d b
Review
No
Pasaran!
Role
of
the
axon
initial
segment
in
the
regulation
of
protein
transport
and
the
maintenance
of
axonal
identity
Christophe
Leterrier
∗,
Bénédicte
Dargent
AixMarseilleUniversité,CNRS,CRN2MUMR7286,13344MarseilleCedex15,France
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Available online xxx Keywords: Axoninitialsegment Neuronalpolarity Neuronaltransport Proteintraffic Diffusionbarrier AnkyrinG Cytoskeleton
a
b
s
t
r
a
c
t
Thetransmissionofinformationinthebraindependsonthehighlypolarizedarchitectureofneurons. Anumberofcellulartransportprocessessupportthisorganization,includingactivetargetingof pro-teinsandpassivecorrallingbetweencompartments.Theaxoninitialsegment(AIS),whichseparatesthe somatodendriticandaxonalcompartments,hasakeyroleinneuronalphysiology,asboththe initia-tionsiteofactionpotentialsandthegatekeeperoftheaxonalarborization.Overtheyears,theAISmain componentsandtheirinteractionshavebeenprogressivelyunraveled,aswellastheirroleintheAIS assemblyandmaintenance.Twomechanismshavebeenshowntocontributetotheregulationofprotein transportattheAIS:asurfacediffusionbarrierandanintracellulartrafficfilter.However,amolecular understandingoftheseprocessesisstilllacking.IntheviewofrecentresultsontheAIScytoskeleton structure,wewilldiscusshowabetterknowledgeoftheAISarchitecturecanhelpunderstandingitsrole intheregulationofproteintransportandthemaintenanceofaxonalidentity.
© 2013 Elsevier Ltd. All rights reserved.
Contents
1. Introduction... 00
2. AISstructureandcomponents... 00
2.1. Ankyrin,spectrinandactin ... 00
2.2. Membrane-partnersofankG... 00
2.3. RegulatedcomponentinteractionsattheAIS... 00
2.4. LinkbetweenankGandmicrotubules... 00
3. AISassemblyandmaintenance... 00
3.1. AISassembly... 00
3.2. AISmaintenance... 00
4. TrafficandtargetingofAIScomponents ... 00
4.1. TrafficofintracellularAIScomponents... 00
4.2. TargetingmechanismsofAISmembraneproteins... 00
5. RoleoftheAISinthemaintenanceofpolarity... 00
6. ThediffusionbarrierattheAIS... 00
6.1. RoleofankG-bindingproteins... 00
6.2. Roleofspectrinandactin... 00
7. TheintracellularfilterintheAIS... 00
7.1. Theintracellularfilter... 00
7.2. Roleofactin... 00
7.3. Roleofmicrotubules... 00
8. AdditionalrolesformicrotubulesintheAIS ... 00
8.1. TheAISasalaunchpad ... 00
∗ Correspondingauthorat:FacultédeMédecine–SecteurNord,Aix-MarseilleUniversitéCS80011,BdPierreDramard,13344MarseilleCedex15,France. Tel.:+33491698930;fax:+33491090506.
E-mailaddress:christophe.leterrier@univ-amu.fr(C.Leterrier). 1084-9521/$–seefrontmatter © 2013 Elsevier Ltd. All rights reserved.
2 C.Leterrier,B.Dargent/SeminarsinCell&DevelopmentalBiologyxxx (2013) xxx–xxx 8.2. Moretodiscover ... 00 9. Conclusion ... 00 Acknowledgements... 00 References... 00 1. Introduction
Thedirectionalityof signaltransmissioninthe brainis sup-portedbytheexquisiteasymmetryofneurons.Neuronsaredivided intwo main compartments that differin theirmolecular com-positionand organization:the somaanddendrites that receive andintegratesynapticinputs(somatodendriticcompartment),and theaxonthatsendsthesignalstodownstreamcellsthroughan extensivearborization(axonalcompartment,Fig.1A).Anumber ofstudieshave progressivelyuncoveredhowcellularpolarityis established during neuronal development [1]. Once polarity is established,a sequential growthphase leadstothe buildingof axonalanddendriticarborizations.Matureneuronsthenfacethe dauntingtaskofmaintainingtheasymmetryofthisintricate archi-tectureforyearsinrodents,anddecadesinhumans[2].
A key player for the maintenance of neuronal polarity lies between the somatodendritic and axonal compartments: the axoninitialsegment(AIS,Fig.1A).Itslocalizationalongthefirst 20–40moftheaxonunderpinsitstwomainfunctions:the gener-ationoftheactionpotential,andthepreservationofaxonalidentity
[3].ThisreviewwillfocusonhowtheAISmaintainsneuronal polar-itybyregulatingproteinmobilityandtrafficbetweenthesomaand theaxon.WefirstdescribetheAISmaincomponents,howtheAISis assembled,andhowitscomponentsaretargetedandconcentrated. Then,wereviewhowtheAIScontrolsproteintransportinthecell
viatheformationofasurfacediffusionbarrierandan intracellu-lartrafficfilter.Wefinallydiscusshowtheexpandingrepertoireof identifiedproteinsandinteractionattheAIS,togetherwithrecent resultsontheAIScytoskeletonstructure,sparknewhypotheseson themoleculardetailsoftheseprocesses.
2. AISstructureandcomponents 2.1. Ankyrin,spectrinandactin
FirstdescribedbyKöllikerandRemakinthemiddleofthe19th century[4],theAISmorphologywascharacterizedbythepioneers ofelectronmicroscopyinthe1960s[5].Theydefined morpholog-icalcriteriaforidentifyingtheAISonbrainelectronmicroscopy images,includingfasciclesofmicrotubules,andadenselayerof materialundercoatingtheplasmamembrane.Likeintherestof theaxon,AISmicrotubulesareuniformlyorientedwithplus-ends towardthedistalaxon.TheAISmicrotubulefasciclesareclearly seenontransversesections,wheretheyappearassmallrings con-nectedbyfilaments(Fig.1B).
The plasma membrane undercoat is composed of an ankyrin–spectrin scaffolding complex that defines the iden-tity and functions of the AIS. The main component of the AIS scaffoldis ankyrinG[6](ankG,Fig.1Cand D).Themammalian ankyrinfamilyiscomposedofthreegenesencodingforankyrinR
Fig.1. Theaxoninitialsegment(AIS).(A)Theneuronintegratesinputsreceivedinthesomatodendriticcompartment(blue).TheAIS(red),locatedatthebeginningofthe axon,generatestheactionpotentialthatpropagatesuptotheterminals(lightred),andisregeneratedatnodesofRanvier(red)acrossinternodes.(B)TheAISofaPurkinje cell(transversesection,right).ThiselectronmicroscopyimagedemonstratestwomorphologicalfeaturesoftheAIS:microtubulefascicles(arrows)andthemembrane undercoat(arrowheads).AdaptedwithpermissionfromSynapseWeb(J.SpacekandK.Harris,PI,http://synapses.clm.utexas.edu).Scalebar,0.5m.(C)TheAISofacultured rathippocampalneuronlabeledforankG(red)andIV-spectrin(green).Map2(blue)isexcludedfromtheaxonanddelineatesthesomatodendriticcompartment.Scale bar,20m.(D)TheAIScomponents.ThemainAISscaffoldisankG(orange),linkedtoIV-spectrin(green)thatinturnbindstoactinfilaments(darkgray).AnkGbindsto Nav1.xchannelsandKv7.2/7.3channels(darkblue),aswellasadhesionproteinsNF-186andNrCAM(blue).EB1/3proteins(lightorange)linkankGtomicrotubules(light gray).AnkGalsobindstoSCHIP1(purple).OtherchannelspresentattheAISareKv1.xchannelslinkedtoPSD-93(pink),andCav2/3channels.Kinasesareshowninyellow (seedetailsinmaintext).
C.Leterrier,B.Dargent/SeminarsinCell&DevelopmentalBiologyxxx (2013) xxx–xxx 3 (ankR,firstidentifiedinerythrocytes),ankyrinB(ankB,isolated
from brain), and ankG (initially characterized as a component of nodes of Ranvier). Although the short isoformsof ankGare ubiquitous,thetwolongankGisoformsof 270and480kDaare uniquelyfoundinneurons,concentratedattheAISandnodesof Ranviermembrane undercoat [7–9]. Thelongest isoform, ankG 480,isalarge4400aminoacidsproteincomposedofamembrane bindingdomain,aspectrin-bindingdomain,aserine-richdomain, alongtail(2200aa),andacarboxy-terminaldomain.Thesecond longisoformofankGfoundattheAIS,ankG270,lacksthelast 1900amino-acidsofthetailcomparedtoankG480[7].Theother largeankyrinexpressedinneurons,ankB,isfoundastwo440and 220kDaisoformsthatarenotconcentratedintheAIS,butlocalize tothedistalpartofunmyelinatedaxons[6].
Similarlytotheankyrin–spectrincomplexliningthe erythro-cytemembrane,ankGisboundviaitsspectrin-bindingdomainto aspecificmemberofthespectrinfamily,identifiedasIV-spectrin in theAIS [10] (Fig. 1C and D). Classically, spectrins are found ashetero-tetrameric complexesof two ␣-spectrinsand two -spectrins,with-spectrinsboundtoactinfilaments[6].Although ␣II-spectrinisfoundalongthewholeaxon,aspecific␣-spectrinhas notyetbeencharacterizedattheAIS.ItisthusunknownifAIS spec-trinsexistasclassical␣–tetramersorasadifferentcomplex.The IV-spectrinisoformsconcentratedattheAISarethe1and6 IV-spectrins.TheIV6isoformisnecessaryforproperAIS orga-nization[11,12]althoughitlackstheactin-bindingdomainfound onotherisoforms,questioningwhetherthisAISspectrincomplex islinkedtoactin.Inlinewiththisquestion,actinenrichmentin theAIShasbeenreportedinahandfulofstudies[13–15],butonly detectedafterspecificdetergentextractionforothers[16,17]. How-ever,evenwithnoconcentration,specificactinorganizationoccurs intheAIS[15],consistentwithitsmajorroleinestablishingtheAIS mobilitybarrier(seebelow).Finally,ankGhasbeenshownto inter-actwithschwannomin-interactingproteinSCHIP1,aproteinthat concentratesintheAIS,butwhosefunctionremainsunknown[18]. 2.2. Membrane-partnersofankG
AnkGalsobinds viaitsmembrane-binding domainto mem-braneproteinsthataccumulate attheAIS(Fig.1C).AnkG binds tovoltage-gatedsodium(Nav)channels[19]viaa motiflocated intheirintracellularloopbetweentransmembranedomainsIIand
III[20,21].ItleadstotheconcentrationofNavchannelsintheAIS
comparedtothesoma,withanenrichmentestimatedbetween
3-[22]and40-fold[23,24],dependingoftheexperimentalapproach. NavchannelaccumulationunderliestheroleoftheAISinthefinal integrationofsomatodendriticinputsand thegenerationofthe actionpotentialinmostneuronaltypes(reviewedin[4,25]).The mainNavchanneltypespresentintheAISareNav1.6andNav1.2, althoughNav1.1isalsofoundatbeginningoftheAISincertain neurons(reviewedin[26]).Additionally,adevelopmentalswitch occursbetweenNav1.2andNav1.6[27],aswellasa proximodis-talcomplementarydistributionofNav1.2and Nav1.6inmature neurons[28].How differentNav channelsbearing similarankG bindingsitecanbeselectivelyenrichedalongtheAISisan inter-estingandstillopenquestion.PotassiumchannelsKv7.2andKv7.3 (KCNQ2/KCNQ3)shareabindingsitetoankGthatisanalogousto theNavchannelmotif,andaccumulateattheAISasheteromers, where theydampen excitability [29]. Ion channels that do not possessanidentifiedankyrin-bindingsitehavealsobeenlocalized totheAIS.PotassiumchannelsKv1.1/1.2localizeatthedistalAIS, bindtopost-synapticdensityproteinPSD-93[30,31],andcan mod-ulateAPinitiation,asdoCav2/3calciumchannelspresentalongthe AIS[32].
AnkGalsobindsviaitsmembrane-bindingdomaintocell adhe-sionmoleculesfromtheL1-CAMfamily[6].BindingtoankGresults
intheAISandnodalconcentrationoftwofamily members,the 186kDaisoform of neurofascin(NF-186), and theneuronalcell adhesionmolecule(NrCAM)[33].Notably,asingleankGprotein cansimultaneouslybindtwoCAMsandoneionchannel[6]. NF-186hasbeenshowntorecruitcomponentsoftheextracellular matrixsuchasbrevicantotheAISofhippocampalneurons[34],and ankG-directedconcentrationofNF-186isimportantforGABAergic innervationofthepinceauterminalaroundthePurkinjecellsAIS
[35].
2.3. RegulatedcomponentinteractionsattheAIS
Althoughitisastablecomplex,theAISis notstatic: a num-berofkinasesregulatetheinteractionbetweenAIScomponents. NF-186and NrCAMcanbephosphorylated attheircytoplasmic FIGQYmotif,inhibitingtheirinteractionwithankG[36].Protein kinaseCK2phosphorylatestheankGbindingsiteonNavchannels tostrengthentheirinteractionwithankG[37],and participates in AISformation[38].Cyclin-dependent kinasescdk2 and cdk5 phosphorylatetheKvsubunit, regulatingtheirsurface expres-sion in the distal AISKv complex [39], whereas cdk5 controls the lengthof an AIS-like compartment in Drosophila[40].The Ca2+/calmodulindependentproteinkinaseCaMKIIassociateswith
IV-spectrin andphosphorylates Navchannels[41].Finally, the glycogensynthasekinaseGSK3phosphorylates-catenininthe AIS,regulatingthemaintenanceoftheAIS[42],andcontrolsthe interactionofNavchannelswithfibroblastgrowthfactorFGF14, participatinginchannelsconcentrationintheAIS[43].
2.4. LinkbetweenankGandmicrotubules
ThelargesizeoftheankGisoformsfoundattheAISsuggests thattheycouldinteractwithintracellulartargets.Potential part-nersforankGinsidetheAISaremicrotubules,asankRandankBbind microtubulesinvitro,directlyorindirectly[44,45].Sobotziketal. studiedtheAISstructureinPurkinjecellslackingankG,thankstoa cerebellum-specificankGknockoutmouse[46].Theyshowedthat thecharacteristicmicrotubulefasciclesattheAISaredispersedin theabsenceofankG,suggestinginterplaybetweenankGand micro-tubules[47].Leterrieretal.thussearchedforpotentialpartnersin thisinteractionandidentifiedtheend-bindingproteinsmembers EB1andEB3aslinksbetweenmicrotubulesandankG[48].EB pro-teinsareusuallyassociatedwithgrowingmicrotubuleplus-ends, wheretheyorganizeacompleximplicatedininteractionswiththe cellularcortexstructures[49].EB1and EB3interactwithankG, andarestabilizedalongthemicrotubulelatticeintheAIS.Thislink betweenankGandmicrotubulescouldhaveimportantimplications forthefilteringofintracellulartransport[48](seebelow). 3. AISassemblyandmaintenance
3.1. AISassembly
HowisthelargeAISmacromolecularcomplexbuiltand main-tained?In neuronal cultures,allcomponentsassemble inquick successionbetween3and4daysinculture[34,50].Invivo stud-iesofmicelackingankGinPurkinjecells[35,46,51],togetherwith knockdownofAIScomponentsindeveloping neurons[34]have demonstratedthatankGisnecessaryfortherecruitmentofother AIScomponents:Navchannels,IV-spectrin,NF-186andNrCAM. Conversely,theankG-bindingmotifofNavchannels[20,21,52],Kv7 channels[29,53],NF-186[50]andIV-spectrin[54]isnecessaryfor theirconcentrationintheAIS.Interestingly,Navchannel knock-downcanpreventformationoftheAISinmotoneurons,suggesting thattheycouldalsoplayaroleinAISassemblyforcertainneuronal
4 C.Leterrier,B.Dargent/SeminarsinCell&DevelopmentalBiologyxxx (2013) xxx–xxx types[55].TheankG-dependent“inside–out”AISformation
con-trastswiththe“outside–in”assemblyofnodesofRanvier,where extracellularglialsignalsfirstbindtoNF-186,whichrecruitsankG, allowingNavchannelconcentration[56–58].IfankGrecruitsall othercomponentstotheAIS,howisankGisrecruitedtotheAISin thefirstplace?TheRasbandlabrecentlydemonstratedthe exist-enceofanantagonisticmechanismbetweenankGandankB[59]. Aftertheinitialspecificationoftheaxon,ankBistargetedtothe distalaxonandprogressivelyfillstheaxontowardthesoma.The lateronsetofankGexpressionresultsinthedistributionofankG alongtheremainingproximalstretchoftheaxonthatbecomesthe AIS[59].Indevelopingmotoneurons,theearlyexpressionofankG couldleadtoadifferentAISformationpattern:ankGappearsalong thewholeaxonatembryonicdayE9.5andconcentratesintheAIS afterE11.5,ankBbeingpresentalongthewholeaxon[9].
3.2. AISmaintenance
AnkGalsohasaveryimportantroleforAISmaintenance: deplet-ingankGin matureneurons resultsin AISdisassembly,with a dispersionoftheaccumulatedIV-spectrin,ionchannelsandCAMs
[60].However,onceassembled,theintegrityoftheAISalsodepends onthepresenceoftheothercomponents.Long-termabsenceof IV-spectrininknockoutmicedecreasesankGand Navchannel accumulationintheAIS[11,12,61].Invivo depletionof NF-186 inPurkinjecellsresultsinaprogressivedisappearanceoftheAIS
[62,63]. Finally, depletionof themicrotubule-ankG linkformed
byEB1orEB3resultsinapartialdisassemblyoftheAISin cul-turedneurons[48].Theseresultssuggest thatalthoughankGis theprimaryorganizer oftheAIS,itscomponentsareultimately interdependent.
4. TrafficandtargetingofAIScomponents 4.1. TrafficofintracellularAIScomponents
BeyondtheinteractionsandhierarchiesbetweenAIS compo-nents,howeachcomponentistargetedanddeliveredtotheAIS remainselusive.HowdoesankGentertheaxonbeforecompeting withankBasproposedbyGalianoetal.[59]?SeveralankGdomains, notably the serine-rich domain, seem to be important for its axonaltargeting[64].PalmitoylationoftheankGmembrane bind-ingdomain,togetherwiththelinkerbeforethespectrin-binding domain,areimplicatedinankGsubmembranelocalizationinaxons
[65,66].WeknowthatankGrecruitsitspartnerstotheAIS, but
whetheritassociateswithanyofthemandisco-transportedbefore arrivingattheAISremains anopenquestion.In particular,this includesotherAISintracellularcomponentssuchasIV-spectrin andEB1/3.
4.2. TargetingmechanismsofAISmembraneproteins
Two general mechanisms participate in the concentration of membrane proteins the AIS: selective endocytosis from the somatodendritic compartment, and capture by ankG. Using chimerasbearingvariousintracellularpartsofNavchannels,the DargentlabhasshownthattheC-terminusandtheloopbetween transmembranedomainsIIandIIItargetNavchannelstotheaxon. Interestingly,thereisnodirecttargetingtotheaxon,butrather unpolarizedexporttotheplasmamembranefollowed by selec-tiveendocytosisfromthesomatodendriticcompartment[67,68].It wouldbeinterestingtotestwhetheraxonaltargetingofKv7.2/7.3 heteromers,whichoccursviamotifsintheC-terminusdomain[69], alsodependsonaselectiveendocytosisstep.Interestingly,NF-186 alsoaccumulatesattheAISafterselectiveendocytosisfromthe
somatodendriticcompartment,inadoublecortin-dependent man-ner[70].OnceAISmembraneproteinshavereachedtheaxonal surface,bindingtoankGisthoughttodrivetheirconcentrationat theAISbycapturingproteinsthatdiffusealongtheplasma mem-brane.IthasbeenobservedthatNavchanneldiffusionisblocked
attheAIS[13,71].Brachetetal.usingNav–Kvchimerasbearing
mutantsoftheNavankyrin-bindingdomain,havedemonstrated thatchannelimmobilizationintheAISiscausedbytheirinteraction withankG[72].BindingtoankGisalsoabletoslowdownNF-186 diffusion[36],resultinginitsimmobilizationattheAIS[50],and preventsNF-186endocytosismediatedbydoublecortin[70]. 5. RoleoftheAISinthemaintenanceofpolarity
Onceassembled,theAISseparatestheaxonfromtherestof theneuron and thus helps maintainingneuronal polarity. First proposedafterthediscoveryofthediffusionbarrierbetweenthe
somaandtheaxon[71,73],thefunctionalevidenceconfirmingthis
hypothesis wasprovidedonly afew years ago.Hedstrom etal. usedlong-termdepletionofankGinculturedneurons,andshowed thatitnotonlyinducesdisassemblyoftheAIS,butalsoleadsto aprogressivelossofaxonalidentity,withappearanceof somato-dendriticmarkersintheproximalaxon[60].Theseresultswere confirmedinvivobyexaminingtheaxonalmorphologyofmice lackingankGinPurkinjecells. Theproximalaxonofthesecells exhibitsspinescontaining post-synapticassembliesthat appear graduallyduringmaturation[47].HowistheAISkeeping somato-dendriticproteinsfrominvadingtheaxon?Studieshaveshown thattheAISregulatesproteintransportbetweenthesomaandthe axonthroughtwomechanisms:asurfacediffusionbarrier,andan intracellulartrafficfilter(Fig.2AandB).
6. ThediffusionbarrierattheAIS
Lipidsandmembraneproteinscannotfreelydiffusethroughthe AIS,butaresloweddownorimmobilized,resultingintheisolation ofthesomaticandaxonalmembranecomposition(Fig.2A).This diffusionbarrierwasfirstidentifiedbyKobayashietal.,who dis-coveredthatfluorescentlipidscannotdiffusefromtheaxonbackto thesoma[73],beforebeingconfirmedatthesinglemoleculelevel byvideo-ratetrackingofmembranelipids[74].Restricteddiffusion ofaxonalproteinsattheAISwasfirstshownbyexperiments mea-suringtractabilityofL1orThy-1attachedbeads[17]andconfirmed bysingleparticletrackingofproteinslabeledwithquantumdots
[14,72].
6.1. RoleofankG-bindingproteins
Theimmobilizationoflipidsintheproximalaxonistemporally correlatedtotheAISformation[13].Furthermore,disassemblyof theAISthroughankGdepletionleadstothedisappearanceofthe diffusionbarrier[14].HowdoestheAISrestrictthediffusionof membranecomponents?Theanchored-proteins“pickets”model fromtheKusumilabproposesthatasubsetofmembraneproteins interactswiththecytoskeletalelementsunderlyingthemembrane, resultingin rows of immobilized picketsthat slow diffusionof membraneproteinsandlipids[13].IntheAIS,thepicketswouldbe themembraneproteinsimmobilizedbytheirinteractionwiththe ankG/spectrinundercoat:ionchannelsandCAMs[50,71,72]. Bra-chetetal.showedthatankG-bindingproteinswereimmobilized assoonasankGaccumulatedintheAIS,beforetheestablishment ofthediffusionbarrier[72].Thus,ankG-bindingmembrane pro-teinsarerecruitedasapreliminarystep,suggestingtheyarethe picketsoftheAISdiffusionbarrier[13].Interestingly,the phospho-regulationofchannelsandCAMsthatmodulatetheirbindingto
C.Leterrier,B.Dargent/SeminarsinCell&DevelopmentalBiologyxxx (2013) xxx–xxx 5
Fig.2. RegulationofproteinmobilityandactinstructuresintheAIS.(A)TheAISdiffusionbarrier.Lipidsandmembraneproteins(red)diffusionisimpededintheAIS,due tocorrallingfromthesubmembranescaffold(green)andtheconcentrationofAISmembraneproteins(gray).Diffusionalongdistalaxonandsomaislessrestricted,as measuredfromtrajectoriesofindividualmolecules(above,red),despitethepresenceofthedistalaxonankB/II-spectrinscaffold(orange).(B)TheAIStrafficfilter.Vesicles containingsomatodendriticproteins(blue)areexcludedfromenteringtheaxon(bluearrow),whereasvesiclestransportingaxonalproteins(orange)canproceedthrough theAIS(orangearrow).Specificrecruitmentofaxonalkinesinsishelpedbycuesonmicrotubulesuchaspost-translationalmodifications(lightorange).(C)Actinringsalong theaxon[15].Ringsofactinfilaments(gray)cappedbyadducing(red)arespacedregularlyevery∼180nmalongtheaxon.Longitudinal-spectrindimers(IV-spectrin intheAIS,green,andII-spectrininthedistalaxon,orange)jointwoadjacentrings.(D)Actinpatchessize(∼1minsize)insidetheAIS[16].Somatodendriticvesicles transportedalongmicrotubules(bluearrow)bykinesins(blue)alsobearmyosins(green),causingthemtostopattheseactinpatchesandkeepingthemfromenteringthe axon(greenarrow).Alsodepictedisthehypotheticalroleofdyneinintransportingnon-axonalcargoesbacktothesoma(orange).
ankGalsomodifytheirdiffusiveproperties[36,72],suggestingthat thestrengthofthediffusionbarriercouldbedynamicallyregulated. 6.2. Roleofspectrinandactin
Actinisnecessaryforthepresenceofthediffusionbarrier,as actinfilamentsdisruptionleadstoitsdisappearance[13,14,17]. IV-SpectrinplaysaroleinrestrictingthediffusionofL1intheAIS comparedtothedistalaxon[75].Inerythrocytes,spectrinconnects complexesofactinandankyrinwitha∼100nmhexagonalpattern thatlimitsdiffusionofmembraneproteins[6].AttheAIS,what specificorganizationofthespectrin/actincytoskeletonleadstothe immobilizationofmembranecomponents?Recently,Xuetal.used super-resolutionmicroscopytorevealthelocalizationofactinand spectrininaxonswitha∼20nmresolution.Theyshowedthatactin isformingring-likestructuresspaced∼180nmapartaroundthe circumferenceoftheaxons.Themiddlepartofspectrintetramers ispreciselylocatedbetweentheactinrings,resultingina comple-mentarybandspattern.Thissuggeststhatspectrinsliealongthe plasmamembrane,connectingsuccessiveactinrings[15](Fig.2C). Thisorganizationisstrikinglyreminiscentoftheperiodicannular structurepreviouslyobservedinelectronmicroscopyimagesofthe AISundercoat[76].
Dothesesuccessiveactin/spectrinhurdlescausetheAIS diffu-sionbarrier?Theactinringsaredetectedalongthewholeaxon, linkedbyIV-spectrinintheAISandbyII-spectrininthedistal axon[15](Fig.2C).Thus,thepresenceoftheseringscannotexplain thediffusivepropertiesoftheAIScomparedtothedistalaxon.The diffusionbarrierislikelycausedbyadditionalAISproteins associ-atedtothesestructures,suchastheionchannelsandCAMsacting aspickets.Itwouldneverthelessbeinterestingtoextendthese find-ingsinrelationtotheknownAISstructure.Isthereadifferencein thearrangementsofthetwoAISIV-spectrinisoforms,giventhat oneofthemlacksanactin-bindingdomain?IsankGalsoorganized
inring-likestructures?Howaretheactin/spectrinringsassembled duringdevelopment,giventhatankGrecruitsIV-spectrinduring AISformation[54]?Interestingly,impairmentoftheAISstructure hasbeenreportedafteractinfilamentsdisruptioninyoungneurons
[14,55],whereasAISofolderneuronsdonotseemtobeaffected
[38,77].
7. TheintracellularfilterintheAIS 7.1. Theintracellularfilter
Besidesthesurfacediffusionbarrier,theAISparticipatesinthe maintenanceofaxonalidentitybyregulatingtheintracellular traf-fic.Live-cell imaging of GFP-fused proteinsshows that vesicles transportingaxonalproteinscanenterinbothaxonanddendrites, whereasthosecontainingsomatodendriticproteinsareexcluded fromenteringtheaxon[78,79].Theintracellulardiffusionoflarge moleculesandvesiculartransporttowardtheaxonissignificantly slowedattheAIS,demonstratingtheexistenceofanAIStransport filter[14].Importantly,thisfilteractslikeasieve,astheentryof macromoleculesdependsontheirsize,andthepassageof vesi-clesdependsontheprocessivityofthemotorproteinspropelling them(Fig.2B).ThisfilterisestablishedatthesametimeastheAIS (between3and5daysinculture),anddependsonAISintegrity,as shownbyitsdisappearanceafterankGdepletion[14].
7.2. Roleofactin
Disruption ofactin filamentsimpairsthe AISfilter,allowing theentryoflargemacromoleculesandsomatodendriticvesicles
intotheaxon[14,80].Howcanactinregulate intracellular
traf-ficin the AIS? Lewiset al. have shown that myosin V binding cantargetmembrane proteinstothesomatodendritic compart-ment[80].SubsequentworkshowedthatmyosinVI,whichtravels
6 C.Leterrier,B.Dargent/SeminarsinCell&DevelopmentalBiologyxxx (2013) xxx–xxx towardtheoppositesideofactinfilaments,canconversely
partici-pateinthetargetingofaxonalprotein[81].Thisledtheauthorsto proposethattheAIScontainsactinfilaments,whichstop somato-dendriticvesiclesenteringtheaxonviamyosinV-basedinteraction andmaybringthembacktothesoma[79].Strikingly,scanning electronmicroscopyimagesofdetergent-extractedcellsshowed anaccumulation of actin into∼1m globular clusterstheAIS. Somatodendriticvesicleswereoftenstoppedattheseclusters[16], supportinga role of actin as a selective road block in theAIS
(Figure2D).Itisnotcleariftheseclusterscouldmediateselective
retrievalofvesiclestowardthesoma,andtheyareclearly differ-entfromtheringsobservedbyXuetal.[15].Observingthefine structureofactinintheAISoflivingcells,forexampleusing photo-activatedlocalizationmicroscopy(PALM)couldhelptoconciliate thesefindings.Coupledwithimagingofvesicularcargoes,PALM ofactincouldfurtherclarifytheroleofactininfilteringtransport throughtheAIS.
7.3. Roleofmicrotubules
Recentresultssuggest thatmicrotubules, besidessupporting vesiculartransport,couldplay arole intheAISsieveassembly. Acutemicrotubuledisruptionorstabilizationdoesnot disassem-bletheAISofmatureneurons[26,77,82].However,microtubules arenecessaryforblockingretrogradediffusionofthe microtubule-associated protein Tau from the axon to the soma [77]. The discoveryofaninteractionbetweenankGandmicrotubulesvia EBproteins[48] revigoratesthehypothesisthat thelargeankG isoformscouldstretchacrosstheAISbetweentheplasma mem-braneandthecytoplasm,andconnecttheundercoatcomplexto microtubules[33].TheankGtailisflexibleandunstructured,and couldstretchupto0.5minlengthiffullyextendedinthecase ofthelongestisoform[6].ThiscouldexplainhowtheAISscaffold regulatestraffictowardtheaxon:anaccumulationofankG teth-ersbetweenmembraneproteinpartnersandmicrotubuleswould formasieveandimpedeentryoflargemacromoleculesandweakly poweredvesiclesintotheaxon[14].
This physical link between membrane proteins and micro-tubulesviaankG,anditsrelevanceforfilteringtrafficthroughthe AIS,hasyettobetested.Importantly,thedetails ofan interac-tionbetweenankGand microtubulesarestill largelyunknown. AlthoughitisknownthatankGbindstothehydrophobicpocket of EBs [26], what part of ankG binds to EBs is not. The large ankG480 contains a putative SxIP EB-binding motif in its tail
[83], but it does not seem to be present in the shorter ankG 270. Besides EBs, it is likely that ankG can bind microtubules directlyorvia otheryet unidentifiedpartners. Antibodies reac-tingwithmicrotubule-associatedepitopesintheAISsuggestthat unknownmicrotubule-boundAIScomponentsremaintobe dis-covered[82,84].Interestingly,arecentstudyfoundthattheankyrin repeatsoftheDrosophilanomechanoreceptorpotentialC(NOMPC) channelareboundtomicrotubules,thismembrane-microtubule tetheringresultinginthemechanosensitivepropertiesofthe chan-nel[85].CombiningthedetailsoftheankG-microtubulelinkage andtheankGinteractionwiththespectrinringsdiscoveredbyXu etal.[15]wouldhelptestingamolecularmodelwheretheAIS traf-ficfilterisformedbyanaccumulationofankGtethersacrossthe axon.
8. AdditionalrolesformicrotubulesintheAIS 8.1. TheAISasalaunchpad
Agrowingbody ofevidencesuggeststhatmicrotubules con-tributetoaxonalcargoes steeringviaspecificmodifications and
selectiverecruitment of kinesins[2].Kinesins are microtubule-associatedmotorsthatdrivetransportoforganellesandvesicular cargoes.Mostkinesinstraveltowardthemicrotubuleplusends, resultingin anterogradetransportin axons,which contain uni-formplusend-outmicrotubules[86].ThekinesinKIF5/kinesin-1 has been shown to transport several axonal cargoes, and also a few somatodendritic ones [86]. KIF5, when expressed as a motorheadfragment,isselectivelytargetedtoaxons,anda non-processiveKIF5headrigormutantaccumulatesselectivelyalong theAISmicrotubules,suggestingthatthesemicrotubulesharbor aspecificcueforKIF5recruitment[74].Whatarethesignalson AISmicrotubules thatare recognized bythe KIF5 motor head? AISmicrotubulesareenrichedinpost-translationalmodifications (PTMs)typicalofstablemicrotubulessuchasacetylation, detyrosi-nationandpolyglutamylation [87–89].AsKIF5 preferablybinds toPTM-rich,stabilizedmicrotubules[90,91],itwasproposedthat PTMsdirecttherecruitmentofKIF5totheAIS.However,reports differonwhereasdetyrosination[87],acetylation[89,90]ora com-binationofmultiplePTMs[88]havea roleforKIF5 preferential recruitment.AlternativecandidatesformicrotubulecuesareGTP remnantsalongtheAISmicrotubules.Amicrotubuletypicallyhas tubulinmonomersboundtoGTPatthegrowingplus-end,andto GDPalongtheshaft,reflectingprogressivehydrolysisofGTP.But microtubulescanalsocontainsmallstretchesofnon-hydrolyzed GTPalongtheirlengththatarecalledGTPremnants[92].Nakata etal.showedthattheseremnantsareenrichedalongtheaxonal microtubules,andplayaroleinrecruitingKIF5towardtheaxon
[93].Moreover,astheplus-endGTPcapisalsothepreferred loca-tionofEBproteinsinteractions,onecouldspeculatethattheseGTP remnantshavearoleintheenrichmentofEBsattheAIS,allowing themtolinkthemicrotubuleshafttoankG[48].
8.2. Moretodiscover
Resultsinmodelorganismssuggestthatkeymechanismsmay bestillmissinginourunderstandingofpolarizedtargeting.Dynein is a molecularcomplex that moves along microtubules toward theirminusend,anddrivesretrogradetransportinaxons[2].In mammalianneurons,dyneinhasaroleintargetingproteinsto den-dritesviatransportalongtheminusend-outmicrotubulesfoundin proximaldendrites,wheremicrotubuleorientationismixed[94]. Furthermore,dyneincontributestothepolarizedorganizationof theGolgi apparatus[95], and organizesaxonal microtubulesin Drosophilaneurons[96].Onecouldhypothesizethatdyneinalso excludessomatodendriticcargoesfromtheaxonbybringingthem backtothesomaalongAISmicrotubules,similarlytowhatwas proposedfor myosinValong actinfilaments[80].Inthe nema-todeCaenorhabditis elegans,theankyrinhomologunc-44allows theaxonaltargetingofunc-33/CRMP(collapsinresponsemediator protein),theestablishmentofuniformmicrotubulepolarity,and thepropertargetingofunc-116/KIF5totheaxon[97].Finally,the C.elegansunc-16/SundayDriver/JIP3(JNKinteractingprotein),an adaptorthatinteractswithbothkinesinanddynein,ispresentin theproximalaxonandnecessaryforregulatingorganelleentryinto theaxon[98].Altogether,theseresultssuggestthatfurther inter-playbetweentheAISscaffold, microtubulesandmotor proteins mayexistandcouldplayacrucialroleinshapingpolarizedtraffic andaxonalidentity.
9. Conclusion
TheAISisnowrecognizedasakeycompartmentinneuronal physiology.Itisinstructivetodrawaparallelwithanotherneuronal structurethathasbeenthoroughlystudiedin thepastdecades: the neuronal synapse. Both structures are characterized by an
C.Leterrier,B.Dargent/SeminarsinCell&DevelopmentalBiologyxxx (2013) xxx–xxx 7 accumulation of channelsand adhesionproteins, controlledby
interactionswithspecializedscaffolds.However,atthelevelofthe cell,thesynapseisanendpoint,whereastheAISisagatebetween theneuron’smainpolarizeddomains.Thisunderliesitsroleinthe organizationofthecellandinthemaintenanceofpolarity.Multiple componentsmaybestillmissinginourviewoftheAISarchitecture, andhowcomponentsarearrangedislargelyunknown. Decipher-ingthe finestructure of theAISwill allowunderstandinghow theAISshapesintracellulartrafficandmaintainsaxonalidentity. Moreover,similartothesynapse,theAISmacromolecularcomplex isnotastaticassembly.Recentresultshaveshownthatitiscapable ofstructuralplasticity,allowingadaptationtophysiological and pathologicalconditions[99–101].Thisplasticitylikelyimplicates kinasesandphosphatasesregulatingtheinteractionsbetweenAIS components.Itwillbeveryinterestingtotestiftheseregulations, andthisplasticityoftheAISstructure,haveanimpactontheneuron organizationviatheregulationofproteintrafficandmobility.
Acknowledgements
TheauthorswouldliketothankKirstenHarrisfortheuseof theAISelectronmicroscopyimage,FrancisCastetsandAmapola Autillo-Touatiforreadingthemanuscript,andthemembersofthe Dargentlab for helpfuldiscussions. We apologizetothose col-leagueswhoseworkcouldnotbecitedbecauseofspaceconstraints.
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