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SeriesEditors
LeslieWilson
DepartmentofMolecular,CellularandDevelopmentalBiology
UniversityofCalifornia
SantaBarbara,California
PhongTran
UniversityofPennsylvania
Philadelphia,USA&
InstitutCurie,Paris,France
Contributors
StefanieAlber
DepartmentofBiologicalChemistry,WeizmannInstituteofScience, Rehovot,Israel
C.J.Alexander
CellBiologyandPhysiologyCenter,NationalHeart,LungBloodInstitute,National InstitutesofHealth,MD,USA
AdamW.Avery
DepartmentofGenetics,CellBiology,andDevelopment,UniversityofMinnesota, Minneapolis,MN,USA
PeterW.Baas
DepartmentofNeurobiologyandAnatomy,DrexelUniversityCollegeofMedicine, Philadelphia,PA,USA
AlexandreD.Baffet
DepartmentofPathologyandCellBiology,ColumbiaUniversity,NewYork, NY,USA
LisaBaker
MarineBiologicalLaboratory,WoodsHole,MA,USA
GaryBanker
JungersCenterforNeurosciencesResearch,OregonHealthandScience University,Portland,OR,USA
MarvinBentley
JungersCenterforNeurosciencesResearch,OregonHealthandScience University,Portland,OR,USA
MarkM.Black
DepartmentofAnatomyandCellBiology,TempleUniversitySchoolofMedicine, Philadelphia,PA,USA
KievR.Blasier
DepartmentofCellBiology,UniversityofVirginia,Charlottesville,VA,USA
ScottT.Brady
MarineBiologicalLaboratory,WoodsHole,MA,USA;DepartmentofAnatomy andCellBiology,UniversityofIllinoisatChicago,Chicago,IL,USA
AnthonyBrown
DepartmentofNeuroscience,TheOhioStateUniversity,Columbus,OH,USA
KerstinM.Janisch
DepartmentofCellBiology,UniversityofVirginiaSchoolofMedicine, Charlottesville,VA,USA
MinsuKang
MarineBiologicalLaboratory,WoodsHole,MA,USA;DepartmentofAnatomy andCellBiology,UniversityofIllinoisatChicago,Chicago,IL,USA
LukasC.Kapitein
CellBiology,DepartmentofBiology,FacultyofScience,UtrechtUniversity, Utrecht,TheNetherlands
EugeneA.Katrukha
CellBiology,DepartmentofBiology,FacultyofScience,UtrechtUniversity, Utrecht,TheNetherlands
NoopurV.Khobrekar
DepartmentofPathologyandCellBiology,ColumbiaUniversity,NewYork, NY,USA
EvaKlinman
DepartmentofPhysiology,UniversityofPennsylvaniaPerelmanSchoolof Medicine,Philadelphia,PA,USA;NeuroscienceGraduateGroup,Universityof PennsylvaniaPerelmanSchoolofMedicine,Philadelphia,PA,USA
KelseyLadt
DepartmentofNeurosciences,UniversityofCalifornia,SanDiego,LaJolla, CA,USA
ZofiaM.Lasiecka
Children’sNationalMedicalCenter,Washington,DC,USA
SeungJoonLee
DepartmentofBiologicalSciences,UniversityofSouthCarolina,Columbia, SC,USA
LanfrancoLeo
DepartmentofNeurobiologyandAnatomy,DrexelUniversityCollegeofMedicine, Philadelphia,PA,USA
Min-gangLi
DepartmentofGenetics,CellBiology,andDevelopment,UniversityofMinnesota, Minneapolis,MN,USA
JudyS.Liu
CenterforNeuroscienceResearch,Children’sNationalHealthSystem, Washington,DC,USA
RuiYang
JungersCenterforNeurosciencesResearch,OregonHealthandScience University,Portland,OR,USA
JulieYi
DepartmentofPathologyandCellBiology,ColumbiaUniversity,NewYork, NY,USA
WenqianYu
DepartmentofNeurobiologyandAnatomy,DrexelUniversityCollegeofMedicine, Philadelphia,PA,USA
JieZhou
DepartmentofPathologyandCellBiology,ColumbiaUniversity,NewYork, NY,USA
CHAPTER
Axonaltransport:The orderlymotionofaxonal structures 1
MarkM.Black
DepartmentofAnatomyandCellBiology, TempleUniversitySchoolofMedicine,Philadelphia,PA,USA E-mail:mark.black@temple.edu
CHAPTEROUTLINE
1.Pulse-LabelingStudiesofAxonalTransport.............................................................2
2.Live-CellImagingofAxonalTransport......................................................................7
2.1FCandtheMovementofVesicularCargoes................................................7
2.2SlowAxonalTransportandtheMovementofCytoskeletalPolymers.............8
2.3NeurofilamentsareTransportedinAxons...................................................8
2.4MicrotubulesandSlowAxonalTransport.................................................10
2.5SCbandtheMovementofSolubleProteinsofAxoplasm...........................12
3.Summary.............................................................................................................15 References...............................................................................................................15
Abstract
Axonaltransportisaconstitutiveprocessthatsuppliestheaxonandaxonterminalwith materialsrequiredtomaintaintheirstructureandfunction.Mostmaterialsaresupplied viathreeratecomponentstermedthefastcomponent,slowcomponenta,andslow componentb.Eachofthesedeliversadistinctsetofmaterialswithdistincttransport kinetics.Understandingthebasisforhowmaterialssortamongtheseratecomponents andthemechanismsthatgeneratetheirdistinctivetransportkineticshavebeenlongstandinggoalsinthefield.Anearlyviewemphasizedtherelationshipsbetweenaxonally transportedcargoesandcytologicalstructuresoftheaxon.Inthisarticle,Idiscusskey observationsthatledtothisviewandcontemporarystudiesthathavedemonstratedits validityandtherebyadvancedthecurrentunderstandingofthedynamicsofaxonal structure.
MethodsinCellBiology,Volume131,ISSN0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.06.001 © 2016ElsevierInc.Allrightsreserved.
FIGURE1
Axonaltransportofproteinsinhypoglossalandretinalganglioncellaxonsofguineapigs. (Thesedataarereprintedwithpermissionfrom Tytelletal.(1981).) Panel(A).The distributionofradioactiveproteinsinthehypoglossalnervesofguineapigs3h(uppergraph) or15days(lowergraph)afterinjectingradioactiveaminoacidsintothehypoglossalnucleus
newapproachesbasedonlive-cellimaginghavebeendevelopedthatprovidedirect visualizationofthecargoesastheyundergotransportinlivingaxons.Thesenew methodshaveprovidedcompellingsupportforthestructuralhypothesisofaxonal transport.
2. LIVE-CELL IMAGINGOFAXONALTRANSPORT
2.1 FCANDTHEMOVEMENTOFVESICULARCARGOES
Earlystudiesusingtime-lapseopticalimagingoflivingaxonsrevealedthemovementofmitochondriaandheterogeneouspopulationsofroughlysphericalobjects neartheresolutionlimitofthelightmicroscope(Forman,Padjen,&Siggins, 1977;Kirkpatrick,Bray,&Palmer,1972).Theratesofmovementaswellastheir sensitivitytometabolicinhibitorssuggestedthatthesewerefasttransportcargoes. Theintroductionofvideo-enhancedcontrastdifferentialinterferencecontrastmicroscopyrevealeddramaticallymoremovementthanpreviouslyobtainedbecause ofitsabilitytodetectstructuresassmallas30nm.Earlystudiesonaxoplasm extrudedfromthesquidgiantaxonrevealedalargevarietyofstructuresmoving atratescorrespondingtoFC(Brady,Lasek,&Allen,1982).Subsequentstudies usingcorrelativeelectronmicroscopyidentifiedmanyofthespecificcargoesasa varietyofmembrane-boundstructures,therebyconfirmingtheviewderivedfrom pulse-chasestudies(Miller&Lasek,1985;Schnapp,Vale,Sheetz,&Reese, 1985).Theyalsoestablishedthatanterogradecargoesdifferedfromthosemoving retrogradely,withtheformerincludingGolgi-derivedvesiclesandthelatter includingendocyticvesiclesandprelysosomalstructures.Thesquidaxoplasmsystemalsoledtothediscoveryofkinesin,amicrotubulemotorthatpowersfastanterogradetransport(Brady,1985;Vale,Reese,&Sheetz,1985)aswellastheexistence ofadistinctmotorthatpoweredfastretrogradetransport(Vale,Schnaapp,etal., 1985),whichwaslateridentifiedascytoplasmicdynein.Thereaderisreferredto numerousreviewsonfastaxonaltransportandthemotorsthatpowerthismotility thathaveappearedintheinterveningyears.
TwopointsregardingFCwillbehighlighted.First,itsanterogradeandretrograde cargoestypicallymovepersistentlyandunidirectionally,pausinginfrequentlyduringtheirtransitintheaxon.Second,whilemoving,theirratesapproximateboth themaximumratesreportedforFCusingpulse-chasemethodsandthemaximum ratesreportedforkinesinanddyneinmotorsinvitro.Thus,fastaxonaltransportrepresentsasystemforefficientlymovingvesicularstructuresbetweenthecellbody andaxontip.Whilemuchremainstobelearnedaboutregulatorymechanisms thatcontrolfasttransport,theinteractionsofFCcargoeswiththetransportmotors arerelativelystableandthemotorsinteractprocessivelywiththemicrotubuletracks uponwhichtransportoccurs.
Mitochondria,membrane-boundstructuresabundantinaxons,exhibitvery differenttransportbehaviorfromtypicalFCcargoes.Mitochondriahavemuch
ofneurofilamentpolymertransportseenbyliveimagingofculturedneurons.Over theyears,ithasbeensuggestedthatneurofilamentproteinsmayalsoundergotransportinaformotherthanasneurofilaments.Whilethisremainsaformalpossibility, theavailabledataindicatethatneurofilamentsconstitutetheprincipletransportform ofneurofilamentproteins.
2.4 MICROTUBULESANDSLOWAXONALTRANSPORT
Severalstudieshavedemonstratedthatmicrotubulescanredistributewithingrowing neuronsfromthecellbodyintotheaxonandfromtheaxonintothegrowthcone (Ahmad&Baas,1995;Slaughter,Wang,&Black,1997;Yu,Schwei,&Baas, 1996).Thoughnotdirectlyobserved,itwasinferredthatactivetransportaccounted fortheredistribution.Withthedevelopmentofmethodstorevealneurofilamenttransport,itwasnaturaltoapplythemtotheissueofmicrotubuletransport.Themethods clearlyrevealedtubulinmovinginlivingaxons,andlikeneurofilaments,thetubulincontainingcargomovedrapidlybutintermittentlywithanaveragerateintherange reportedfortubulintransportasseeninthepulse-chasestudies(Hasaka,Myers,& Baas,2004;He,Francis,Myers,Black,&Baas,2005;Wang&Brown,2002).The movementwasbidirectional,thoughmostlyanterograde,andduringboutsofmovementtheratewastypicalofthatseenwithfastmotors, z1 2 mm/s,butwasinterruptedbypauses.Strikingly,themovingstructureswereshort, z1 5 mminlength (average ¼ 2.7 mm),andstructurestypicalofthelengthofaxonalmicrotubules (manytensofmicronslong),werenotobservedtomove.Itwasthussuggested thatshortmicrotubulesareconveyedrapidlybutintermittentlybyslowaxonaltransportwhilelongmicrotubulesarestationary(Baas,Nadar,&Myers,2006).
Severalobservationssupporttheviewthatlongmicrotubulesarenottransported inaxons.Forexample, Chang,Svitkina,Borisy,andPopov(1999) usedspecklemicroscopytorevealindividualaxonalmicrotubulesinlivingaxons,andnoneofthese polymerswasobservedtomove.Inanotherapproach,microtubuleplusendswere taggedwithfluorescenttip-bindingproteinsandthenimagedtoseewhetherthe polymersmoved.Itisexpectedthattheplusendswilladvanceasthemicrotubules elongate.Iftheyalsoundergotransport,thentherateofadvancewillexceedthatdue tomicrotubuleelongationalone.However,innocasewasthisobserved(Kim& Chang,2006;Ma,Shakiryanova,Vardya,&Popov,2004).Asthesestudiesimaged largenumbersofmicrotubules,ifmicrotubuletransportoccurred,eveninfrequently, itshouldhavebeendetected.Thus,theconclusionthatsuchtransportdoesnotoccur isreasonable.However,thisneedstobequalifiedasthestudiesdidnotrestrict analysestomicrotubulesofparticularlength,butexaminedanypolymerthatcould bedetected.Asmostaxonalmicrotubulesaremanytensofmicronsinlength (Bray&Bunge,1981),thefindingsreasonablyapplytosuchlongpolymers. Whethertheyapplytoshortmicrotubulesisunknown,andgiventheresultsby theBrownandBaaslabsdiscussedabove,theyverywellmaynot.
AkeyquestioninthestudiesbytheBrownandBaaslabsiswhetherthemoving tubulin-containingstructuresareinfactshortmicrotubules.Giventhattubulin
Black,Trojanowski,&Lee,2007;Roy,Winton,Black,Trojanowski,&Lee,2008).
ThesestudiesfocusedonthinaxonsinwhichtheGFP-taggedSCbproteinsappeared asoccasionaldiscretepunctaaboveamorediffusedistribution.Whilemanypuncta remainedstationaryduringimaging,somemovedatratescomparabletoFC(z1 2 mm/s).Suchmovementswererelativelyinfrequentandtheywereinterruptedby pausesofvariableduration.Furthermore,whilemostpunctamovedanterogradely, somemovedretrogradely.TheseresultsshowedthatSCbproteinscanmovebidirectionallyinastop-and-gomanner.Itwasarguedthattheoverallslowrateoftransport reflectedtheaverageforthepopulationofthetimespentmovingrapidlyandthe timespentpausing.
Thekeyfindingsofthesestudiesnotfullyappreciatedatthetimederivedfrom directcomparisonsofthetransportbehaviorofSCbandFCcargoesinindividual axons.Neuronscoexpressingred-tagged a-synuclein(andlatersynapsin-1(Tang etal.,2013))andgreen-taggedsynaptophysin,anintegralmembraneproteinof FC,wereimagedsimultaneouslytorevealtheirtransport.Synaptophysinappeared assmallpunctaandexhibitedtypicalFCbehaviorasreportedbyothers,withsynaptophysinpunctamovingfrequentlyandhighlypersistent.Thiswasinmarked contrasttotheinfrequentandlesspersistentmovementsofSCbpuncta.However, SCbandsynaptophysinpunctaexhibitednearlyidenticaltransportvelocitiesduring boutsofmovement,andfurthermore,indualimaginganalyses,allmovingSCb punctamovedtogetherwithsynaptophysin.ThislatterfindingwasstrikingandsuggestiveofalinkagebetweenthemovementofSCbproteinsandFCcargoes.SubsequentworkbyRoyandcolleaguesdemonstratedtheimportanceofthislinkageto thetransportofatleastsomeSCbproteins.
TofurtherdissectthemechanismsofSCbtransport,RoyandcolleaguesdevelopedanassayforSCbtransportinculturedneuronsusingphotoactivatablevectors inwhichbulkcargomovementandparticledynamicscouldbevisualizedwithhigh resolution(Scottetal.,2011;Tangetal.,2012,2013).Thesestudiesfocusedonthree SCbproteins,synapsin-1a,calmodulin-dependentkinaseIIa,and a-synuclein. Whiletheseproteinshavedistinctivetransportkinetics,Iwillfocusontheircommonalities.Theresultsobtainedshowthatthebulkoftheseproteinsmoveswith ananterogradebiasatarateof z0.01 mm/s,whichissimilartotheratesreported forSCbproteinsbasedonpulse-labelingstudies.Thetransportrequiresmicrotubules,microtubulemotors,andATP.
IthasnotbeenpossibletovisualizediscretemovementswithinthebulkpopulationofSCbproteinspresumablybecauseindividualmovementsaretoobrieftocaptureand/orthevectoriallymovingproteinsdonotstandoutfromtheirneighborsthat arejustdiffusing.However,aminorsubpopulationoftheseSCbproteinsappearsas discreteparticlesthatexhibitintricatetransportkinetics.Duringtheirmovement, transportratesarerelativelyfast, z1 2 mm/s,butthedurationofmovementisvariable,rangingfromafewsecondstoafewtensofseconds(theoriginallive-cellimagingstudiesby Royetal.(2007) focusedonthisminorsubpopulationofSCb cargoes).Itisassumedthatmovementwithinthewaveexhibitstransportkinetics similartotheseparticles,butformuchshorterdurations.Simulationswere
