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Wayne State University
Wayne State University Theses
1-1-2017
Synaptotagmin C2b Ca2+-Binding Loops ImposeDistinct Exocytosis PhenotypesMichael W. SchmidtkeWayne State University,
Follow this and additional works at: https://digitalcommons.wayne.edu/oa_theses
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This Open Access Thesis is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in WayneState University Theses by an authorized administrator of DigitalCommons@WayneState.
Recommended CitationSchmidtke, Michael W., "Synaptotagmin C2b Ca2+-Binding Loops Impose Distinct Exocytosis Phenotypes" (2017). Wayne StateUniversity Theses. 585.https://digitalcommons.wayne.edu/oa_theses/585
SYNAPTOTAGMINC2BCA2+-BINDINGLOOPSIMPOSEDISTINCTEXOCYTOSISPHENOTYPES
by
MICHAELW.SCHMIDTKE
THESIS
SubmittedtotheGraduateSchool
ofWayneStateUniversity,
Detroit,Michigan
inpartialfulfillmentoftherequirements
forthedegreeof
MASTEROFSCIENCE
2017
MAJOR:BIOLOGICALSCIENCES
(Cellular,Developmental,&Neurobiology)
ApprovedBy:
_______________________________________Advisor Date
ii
DEDICATION
Tomylovingwife,Christinaandourlittlebunintheoven.
iii
ACKNOWLEDGMENTS
Iwould first like to thankmy committeemembers. In particular, I am grateful tomy
research advisor, Dr. Arun Anantharam, for his intellectual support, research funding, and
willingnesstotakemeonasagraduatestudentinhislab.Iamalsoindebtedtomythesisadvisor,
Dr.KarenBeningo,forherguidanceandmotivationduringthepreparationofmythesisandoral
presentation. Additional thanks go to Dr. DavidNjus andDr. Joy Alcedo for their thoughtful
suggestionspertainingtomywritinganddataanalysis.Furthermore,IamgratefultoDr.Edward
GolenbergandMs.RoseMaryPriestoftheWayneStateUniversitybiologydepartmentfortheir
commitmenttostudentsuccessandadministrativeassistance.
Specialthanksgoouttomyformerlabcolleagues,Dr.TejeshwarRaoandMr.PeterDahl,
forthemanyconstructiveconversationsandlatenightsspentworkingtogether.Iwouldnotbe
whereIamtodaywithoutyourencouragementandcamaraderiethroughtheyears.
Finally,Iamgratefultomyfamilyfortheirpersistentsupportandunwaveringconfidence
inmy abilities. Thank you tomy parents,Mark and Julie Schmidtke, for instilling inme the
integritytohandleanysituation,andtomywife,Christina,forhavingthepatiencetodealwith
thetrialsandtribulationsofgraduatestudentlife.
iv
TABLEOFCONTENTS
Dedication......................................................................................................................................ii
Acknowledgements.......................................................................................................................iii
ListofFigures................................................................................................................................vi
ListofTables.................................................................................................................................vii
Chapter1–BackgroundInformation
OverviewofExocytosis.......................................................................................................1
TypesofExocytosis.............................................................................................................1
StepsofRegulatedExocytosisandMajorMolecularPlayers.............................................4
TheAdrenalChromaffinCell............................................................................................11
ACloserLookattheCalciumSensorSynaptotagmin.......................................................14
SeminalSynaptotagminFindingsandResultingQuestions..............................................16
PrefacetoThesisResearch...............................................................................................21
Chapter2–Methods
Site-DirectedMutagenesisofSynaptotagmin-1C2BLoops.............................................25
IsolationandTransfectionofBovineChromaffinCells....................................................29
TIRFandpTIRFMicroscopy..............................................................................................30
ImageAcquisition.............................................................................................................35
LiveImagingofChromaffinCells......................................................................................36
ImageAnalysis..................................................................................................................37
Chapter3–ResultsandDiscussion
Results..............................................................................................................................39
v
Discussion.........................................................................................................................47
Conclusions......................................................................................................................55
References....................................................................................................................................57
Abstract........................................................................................................................................72
AutobiographicalStatement........................................................................................................74
vi
LISTOFFIGURES
Figure1.1:ModesofExocytosis.....................................................................................................4
Figure1.2:SNAREComplexStructure............................................................................................7
Figure1.3:ChromaffinCellLargeDense-CoreVesicles(LDCVs)..................................................13
Figure1.4:SynaptotagminStructure...........................................................................................15
Figure1.5:ComparisonofSynaptotagminC2BDomainStructures.............................................23
Figure2.1:Site-DirectedMutagenesisPrimerDesign..................................................................26
Figure2.2:SynaptotagminC2BDomainAlignmentsandChimeraConstructs............................28
Figure2.3:TotalInternalReflectionFluorescence(TIRF)Microscopy.........................................31
Figure2.4:VisualizingMembraneTopologywithMembrane-IntercalatingDiD.........................32
Figure2.5:VisualizingExocytosiswithpH-SensitivepHluorin.....................................................35
Figure3.1:SampleImageSequencesofSynaptotagminConstructs...........................................40
Figure3.2:PersistenceofSynaptotagminChimerasatFusionSites............................................42
Figure3.3:Post-FusionElevationsinP/SRatio............................................................................44
vii
LISTOFTABLES
Table2.1:Site-DirectedMutagenesisPrimerSequences.............................................................26
Table2.2:NumberofCellsandFusionEventsAnalyzed..............................................................38
Table3.1:Chi-SquareAnalysisofProteinPersistence.................................................................39
Table3.2:AverageProteinPersistenceFollowingFusion............................................................41
Table3.3:Chi-SquareAnalysisofElevatedP/SDurations............................................................45
Table3.4:AverageDurationofElevatedP/SRatiosFollowingFusion.........................................46
1
CHAPTER1–BACKGROUNDINFORMATION
OverviewofExocytosis
Exocytosisand themolecularmachines thatare involved in carryingout thisessential
cellularprocess,arecomplexandpoorlyunderstood.Initsmostgeneralsense,exocytosisisthe
processbywhichacellreleasesaportionofitscontentstotheexternalsurfaceofitsmembrane.
This is achievedby the traffickingand subsequentmergingof cytoplasmic,membrane-bound
vesicleswiththeplasmamembrane.Exocytosisiscriticalforthesurvivalofnearlyallcelltypes,
where its precise role is defined by the vesicular contents. For example, synaptic vesicles in
neuronscontainsmall,water-solubleneurotransmittersthatareusedtotransmitsignalsfrom
oneneurontoanotherthroughoutthebody.Similarly,aspecialclassofneuroendocrinecells
found inthepancreascontainvesiclespre-loadedwiththepeptidehormone insulin,which is
released into the blood stream to help sequester glucose. However, exocytosis is not used
exclusivelyforthereleaseofcargo,itisalsousedformaintainingcomponentsofthecell’splasma
membrane. Various lipidmolecules andmembrane associated proteins that form the vesicle
membranewillbecomecontinuouswiththeplasmamembraneduringexocytosis.Forexample,
shuttlingofcellsurfacereceptorsatasynapseisoneoftheprimarywaysthatneuronscantune
theirresponsetoincomingsignals(long-termpotentiation).Theprocessofexocytosisshouldnot
be confused with the conceptually similar, yet functionally opposite process of endocytosis,
whichinvolvestheuptakeofextracellularmaterialsintothecytoplasmofacell.
TypesofExocytosis
Exocytosis can be broadly classified as either “constitutive exocytosis” or “regulated
exocytosis”.Constitutiveexocytosis,isaprocessthatoccursrelativelycontinuouslywithincells
2
anddoesnotrequireanyexternallyinduced“trigger”(Burgoyne&Morgan,1993).Thisformof
exocytosismaintainstheappropriatelipidandproteincontentofacell’splasmamembrane,and
releasesextracellularmatrixproteins(Albertsetal.,2008).Duetotheimportanceofmaintaining
cell membrane composition, constitutive exocytosis is found in essentially all cell types
throughoutanorganism.Incontrast,regulatedexocytosis isdefinedbytherequirementfora
stimulatingsignalto“trigger”thefusionofvesicleandplasmamembranes(Burgoyne&Morgan,
1993). This triggering signal induces a sharp rise in the concentrationof intracellular calcium
(Ca2+)anddirectsthemolecularmechanismsleadingtofusionofvesicleandplasmamembranes.
Forthisreason,regulatedexocytosisissometimesreferredtoasCa2+-dependentexocytosis.In
intact tissue, the coordination of a triggering signal with a rise in intracellular Ca2+ requires
specializedreceptorsandionchannels.Regulatedexocytosisistypicallyemployedfortherelease
of biologically active signalingmolecules from highly specialized cell types (e.g. neurons and
neuroendocrinecells),becausethetriggeringrequirementallowsfortighttemporalregulation
ofagivensignal.
During regulated exocytosis, once intracellular Ca2+ levels have spiked, the temporal
dynamicsofvesiclefusionwiththeplasmamembranecanvary.Basedontheirtiming,eventsare
describedaseither“synchronous”or“asynchronous”.Synchronousreleasereferstotheportion
ofCa2+-inducedexocytoticeventsthatoccurwithintensofmillisecondsofatransientspikein
Ca2+(i.e.theseeventsare“synchronized”withtheCa2+spike).Ontheotherhand,asynchronous
releaseeventsoccurafterameasurabledelayandmaycontinuetotakeplaceforuptoaminute
ormore(Chowetal.1992;HaglerandGoda,2001;Yoshiharaetal.,2010;Raoetal.2014).In
general,mostexocytoticevents(asmuchas90%inneurons)areobservedtobesynchronous
3
(KaeserandRegehr,2014).However,manystudieshavesuggestedthatinneuroendocrinecells,
suchasadrenalchromaffincells,thetemporalcouplingisnotastight,resultinginmorebalance
between synchronousandasynchronous fusionevents (Chowetal. 1992;Voetsetal., 1999,
Schonnetal.,2008;Lefkowitzetal.2014).Thebasisforthisdifferenceintimecourseisnotwell
understood,butsomehavespeculatedthatitislinkedtovesiclepopulationssensingincoming
Ca2+differently.Forexample,thiscouldbeexplainedbyvesiclesdifferingintheirproximityto
Ca2+ entry points (CaV channels) and/or utilizing Ca2+ sensing proteinswith different binding
affinities(Lietal.,1995;Chowetal.,1996;Kaeser&Regehr,2015).
Finally,aftervesicleshaveinitiallyfusedwiththeplasmamembrane,thereisyetanother
distinctionthatcanbemadebetweenthemodesofexocytosis.Theclassic,or“Fullcollapse”,
fusionmodeoccurswhenthevesiclemembranerapidlyflattensintotheplasmamembrane,with
the inner leafletof the vesiclemembranebecoming continuouswith theouter leafletof the
plasmamembraneandconsequentlyreleasingallvesiclecontentstotheextracellularspace.In
contrast, “kiss-and-run” (also called “cavicapture”) exocytosis occurs when a fusing vesicle
remainslargelyintact,withonlyanarrowfusionporeformingbetweenthevesiclelumenand
extracellularspace.Followingthepersistenceofthisfusionpore,thesevesiclesmayeventually
undergofullcollapseintotheplasmamembraneasdescribedabove,oralternativelytheirpore
mayresealallowingthevesicleandremainingcargotoberecycledbackintothecytoplasm(the
defining property of “kiss-and-run”; Figure 1.1). It has been suggested that “kiss-and-run”
exocytosismaybeusedbysecretorycellsasameansforconservingenergyandlimitingcontent
releaseduringbasalconditions(i.e.homeostasis),while“full-collapse”mayoccurwhenthereis
an immediate need for abundant secretion (e.g. during a stress response). However, the
4
molecular mechanisms underlying these contrasting modes of exocytosis are not fully
understoodandwillbeafocusofthisthesis.
Figure1.1:ModesofExocytosis.Followingfusionofvesicle(red)andplasmamembranes(black),exocytosiscanproceedviatwocontrastingmodes.The“classic”viewofexocytosis(scenario1)involvesthevesiclemembranerapidlyandcompletelycollapsingintotheplasmamembrane,releasingallcargofromthevesicle.However,itisnowknownthatsomeexocytoticeventsarecharacterizedbythepersistenceofanarrow fusion pore (scenario 2) before the vesicle either fully collapses into the plasmamembrane,orisendocytosedbackintothecellretainingaportionofitscargo(kiss-and-run).
StepsofRegulatedExocytosisandMajorMolecularPlayers
Regulatedexocytosisinadrenalchromaffincellsoccursinfourstages,andwhilemanyof
the molecular players that orchestrate this process are known, a number remain to be
discovered.Thesestagesandselectknownplayersaredescribedbelow.
Step1:Docking
Thefirststageofexocytosisinvolveslocalizingsecretoryvesiclestothecytoplasmicregion
immediatelyadjacenttotheplasmamembrane(i.e.thesubplasmalemmalspace).Moststudies
classifyvesiclesasdockedbasedonamorphologicaldefinition.Onthisbasis,vesiclesthatare
locatedwithinapproximately100nmor lessfromtheplasmamembraneandexhibitminimal
5
lateral motion are considered “docked” (Steyer et al., 1997; Verhage & Sorensen, 2008). In
adrenalchromaffincells,thetypicaldensitydistributionofvesiclesisappreciablyhigherinthis
subplasmalemmalspaceascomparedtotherestofthecytoplasm,withonestudyestimatingas
muchasa20-foldincreaseinvesicledensityinthisregion(Steyeretal.,1997).
OneofthebestunderstoodmoleculesinvolvedinvesicledockingistheproteinMunc18-
1.Munc18-1 isacytosolicproteinfound inchromaffincellsandneuronsthatbindswithhigh
affinitytotheSNAREproteinSyntaxin-1(discussedbelow)(Hataetal.,1993).Chromaffincells
lackingMunc18-1havea10-foldreductioninthenumberofmorphologicallydockedvesiclesand
reducedlevelsofSyntaxin-1(Voetsetal.2001b).Syntaxin-1isatransmembraneproteinlocated
on the inner surface of the plasmamembrane and is one of the three essential SolubleN-
ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE) proteins involved in
exocytosis.CorroboratingitsroleinvesicledockingisthefindingthatSyntaxin-1cleavageresults
inanapproximately7-fold reductionofdockedvesicles (deWitetal.,2006).Currentmodels
suggestthatMunc18-1bindingtoSyntaxin-1actstoclampthefusion-promotingSNAREcomplex
insuchawaythatvesiclesremain“tethered”neartheireventualfusionsites(Gulyas-Kovacset
al.,2007;Verhage&Sorensen,2008;Sudhof&Rothman,2009).
Step2:Priming
In comparison to the visually perceivable process of docking, the priming step of
exocytosisismoredifficulttodiscern.Whiletheexactproportionvariesbetweenstudies,it is
wellestablished thatonlya subsetof the total vesiclesdockedat theplasmamembranewill
undergoexocytosis.Primingconverts vesicles fromsimplybeingdockedat themembrane to
becomingreleasecompetent(i.e.capableofundergoingfusionwiththeplasmamembraneupon
6
experiencingatriggeringspikeinintracellularCa2+).Thismeansarelease-competentvesicleis
notreadilydiscernableuntilithasundergoneexocytosis.
Oneoftheearliestobservationsregardingprimingofvesicleswastheneedforadenosine
triphosphate(ATP)toconvertmorphologicallydockedvesiclesintoreleasecompetentvesicles
(Holzetal.,1989).OneoftheproposedrolesforATPintheprimingprocessinvolvesresettingof
the post-fusion SNARE complex via the ATPase, NSF. The core components of the fusion
machineryare formedbythree“SNARE” (orSNAPReceptor)proteins.Twoof theseproteins,
Syntaxin-1andSynaptosome-AssociatedProteinof25kDa(SNAP-25),arelocatedontheplasma
membrane (t-SNAREs), whereas the third protein, Synaptobrevin-2 (also called VAMP-2 for
Vesicle-AssociatedMembraneProtein-2)isfoundinsertedintothevesiclemembrane(v-SNARE;
Figure1.2A).Thezipperingofα-helicaldomainswithintheseproteinsprovidesthespecificity
and driving force for completing the process of exocytosis (Rothman 1994). Following a full-
collapsefusionevent,thethreeSNAREproteinsarefullyzipperedtogetherontheinsideofthe
plasma membrane in what is termed the ternary cis-SNARE complex. Because this is an
energetically favorable state, the SNARE componentswill remain twisted together, and thus
unusableforfutureexocytosis,unlessdisassembledbyanoutsidesource.Disassemblyofthecis-
SNARE complex is achieved by the combined actions of cytoplasmic α-SNAP and N-
ethylmaleimide-SensitiveFactor(NSF).NSFisanATPasethatutilizesATPhydrolysistounzipthe
α-helicesoftheSNAREproteins,thusreturningthemtotheirpre-fusiontransstate(Sollneret
al.,1993;Hay&Scheller,1997).Thetimingofthisrearrangementprocesshasbeendebatedand
may differ between cell types, but evidence from both chromaffin cells and PC12 cells (an
immortalized chromaffin cell line) suggests that ATP-dependent NSF-mediated SNARE
7
disassemblyoccursaspartoftheprimingstepleadinguptoexocytosis(Banerjeetetal.,1996;
Xuetal.,1999).
Figure1.2:SNAREComplexStructure.(A) Proposed arrangement of the SNARE complex between vesicle and plasmamembranes.Synaptobrevin(darkred)isthesolevesicularSNARE(v-SNARE),withitsN-terminusinsertedintothevesiclemembrane.Syntaxin(yellow)andSNAP-25(green)arethetargetSNARES(t-SNARES),providingspecificity forsitesofexocytosisontheplasmamembrane.SyntaxincontainsanN-terminaltransmembranedomain,whereasSNAP-25isanchoredtotheinnerleafletoftheplasmamembraneviapalmitoylation.(B)AsingleunitoftheSNAREcomplexcontainsonecopyeachofSynaptobrevinandSyntaxin,alongwith twocopiesofSNAP-25.Theprimarydriving force forexocytosis results from zippering of the four-helix bundle formed by a single α-helicalcontributionfromeachofthefourSNAREmolecules.
A
B
8
A second proposed role for ATP during vesicle priming is through the biosynthesis of
Phosphatidylinositol 4,5-bisphosphate (PIP2) from precursor molecules. PIP2 is a negatively
charged lipidmolecule that is foundon the inner leafletof theplasmamembraneat sitesof
exocytosis,andservesasacontext-specificsignal forbindingofeffectorproteins (Holzetal.,
2000; Alberts et al., 2008). Synthesis of PIP2 involves the sequential conversion of
Phosphatidylinositol(PI)toPhosphatidylinositol4-phosphate(PIP)andthentoPIP2bytheATP-
dependent activity of Phosphatidylinositol 4-kinase (PI4KI) and Phosphatidylinositol 4-
phasphate-5-kinase(PIP5KI),respectively.PropertraffickingofPIP2totheplasmamembranealso
involves the actions of Phosphatidylinositol Transfer Protein (PITP), amolecule shown to be
requiredforATP-dependentvesiclepriming(Hay&Martin,1993).AnearlystudybyWiedemann
etal.showedthatinhibitionofPIP2,eitherdirectlyorbyblockingsynthesis,resultedinfewer
releasecompetentvesiclesinchromaffincells(1996;Martin,2012).Morerecentstudieshave
confirmed the requirement for PIP2 in vesicle priming and identified candidate effector
molecules,includingCa2+-DependentActivatorProteinforSecretion1(CAPS-1)andMunc13-1/2.
ThesemoleculesbindtobothPIP2andSNAREproteins,possiblyservingtostabilizetheSNARE
complex prior tomembrane fusion (Xu et al., 1999;Martin, 2012; Kabachinski et al., 2014).
Furthermore,biochemicalinhibitionofeitherCAPS-1orMunc13willreducethepoolofrelease
competentvesicles(Klenchin&Martin,2000).
Step3:Triggering
Oncevesicleshavebecomereleasecompetent,theysitpoisedattheplasmamembrane
until an appropriate triggering signal arrives. This triggering signal is ultimatelymanifest as a
sharp rise in intracellular Ca2+ levels. In adrenal chromaffin cells, this Ca2+ spike results from
9
upstream signaling by acetylcholine (ACh) and Pituitary Adenylate Cyclase-Activating Peptide
(PACAP), culminating in the opening of voltage-gated Ca2+ (CaV) channels in the plasma
membrane(Smith&Eiden,2012).Ca2+entrytriggersexocytosisbyactingonmembersofthe
Ca2+-bindingSynaptotagmin(Syt)proteinfamily(Perinetal.,1990;Broseetal.,1992;Tucker&
Chapman,2002).AlthoughtherearemanyisoformsofSyt,chromaffincellsonlyexpressSyt-1
and Syt-7 (Schonn et al., 2008). These Syt isoforms are transmembrane proteins found on
secretoryvesiclesandcontaintwocytoplasmicCa2+-bindingdomains(seebelow).LossofSyt-1
and/or Syt-7 will reduce synchronous and asynchronous Ca2+-triggered vesicle fusion
respectively,implicatingtheseproteinsastheCa2+sensorsforregulatedexocytosis(Geppertet
al.,1994;Voetsetal.,2001a;Schonnetal.,2008;Bacajetal.,2013).
OnceboundbyCa2+,Sytmayact topromote thedownstreamfusionprocess through
multiplemechanisms.First, there isevidencethatCa2+-boundSytcanbindwith,andpartially
insert itself into,membranes containing PIP2 (Martens et al., 2007; Lynch et al., 2008). This
interactionmay inducepositiveplasmamembranecurvaturethatpromotesmergingwiththe
closelyapposedvesiclemembrane(Huietal.,2009).Secondly,Ca2+-boundSythasbeenshown
to bind the t-SNARE proteins Syntaxin-1 and SNAP-25 as well as the cytoplasmic protein
Complexin.MultiplestudieshaveshownthatmutationsdisruptingtheCa2+-mediatedbindingof
Syt-1tot-SNAREsinhibitsSNARE-inducedvesiclefusionbothinvitro(Bhallaetal.,2005;2006)
andinvivo(Martensetal.,2007;Lynchetal.,2008).Thus,ithasbeenproposedthatSyt-SNARE
bindingmay facilitateassemblyor stabilizationof theSNAREcomplexas itdrivesmembrane
fusion.EvidencealsosuggeststhatthesolubleproteinComplexinplaysanimportantroleduring
Syt-mediated triggering of exocytosis. It is believed that Complexin acts as a “clamp”,
10
simultaneously binding the t-SNARE Syntaxin-1 and v-SNARE Synaptobrevin-2 to prevent
premature zipperingofα-helices and concomitant vesicle fusion. Consistentwith thismodel,
Ca2+-boundSytmayactbybindingComplexinandremovingitfromtheSNAREcomplex,thereby
releasingtheinhibitory“clamp”andallowingfusiontoproceed(Tokumaruetal.,2008).These
mechanismsarenotmutuallyexclusive,andthereforemightacttogethertotriggerexocytosis.
Recently,athirdmechanismfortriggeringoffusionwasproposedbasedonthetendencyforSyt
toformhomo-oligomers invitro.ThisexplanationsuggeststhatpriortoCa2+entry,aring-like
chain of Syt molecules could act as a spacer between the vesicle and plasma membranes,
preventingfurtherzipperingoftheSNAREcomplexuntiltheringdisassemblesinthepresenceof
Ca2+(Wangetal.,2014).However,thismechanismhasyettobeproveninvivo,andtherefore
requiresfurtherinvestigationtoassessitsvalidity.
Step4:Fusion
The final andmost conspicuous step of exocytosis is the actual fusion of vesicle and
plasmamembranes.Asdiscussedabove,myriadevidencesupportsthetheorythatmembrane
fusionisspecifiedanddrivenbytheenergeticallyfavorablezipperingofα-helicaldomainswithin
thetransmembranev-andt-SNAREproteins–aconceptreferredtoasthe“SNAREhypothesis”
(Rothman,1994).CrystalstructureanalysisoftheSNAREcomplexrevealedthatthecytoplasmic
portioniscomposedoffourα-helices;Synaptobrevin-2andSyntaxin-1eachcontributeasingle
helix,whereastwocopiesofSNAP-25contributeonehelixeach(Suttonetal.,1998;Figure1.2
B).Whilethisfour-helixbundleservesasabasicunitoftheSNAREcomplex,itisstillunclearhow
manycopiesofthisstructureareassociatedwithatypicalexocytoticevent.Priortofusion,v-
SNAREsandt-SNAREsarefoundonseparatemembranes,andthereforereferredtoasatrans-
11
SNAREcomplex.Followingα-helixzipperingandfull-collapseofthevesiclemembraneintothe
plasmamembrane,allSNAREsarelocatedtogetheronthesamemembrane,referredtoasthe
cis-SNAREcomplex(alsocalledtheternarycomplexsinceitiscomposedofthreetightlycoiled
proteinspecies).Disassemblyofthecis-complexisrequiredbeforeSNAREscanberecycledto
theirrespectivemembranesforuseinanotherroundofexocytosis,andthisisachievedbythe
combined actions of the cytoplasmic proteins NSF and α-SNAP (Sollner et al., 1993; Hay &
Scheller,1997).
Itwasoriginallythoughtthatoncethevesicleandplasmamembranesinitiallyfusedwith
oneanothertoformafusionpore,exocytosiswouldalwaysproceedrapidlywithfull-collapseof
thevesicleintotheplasmamembrane(Heuseretal.,1979;Chowetal.,1992).However,more
recentworkhasestablishedacontrastingmodeknownas“kiss-and-run”fusion,wherethefusion
poremaypersistfortens-of-secondsbeforeresealingasaformofendocytosis(Fesceetal.,1994;
Taraskaetal.,2003;Harataetal.,2006;Raoetal.,2014).Manyproteinshavebeensuggestedto
influence the rate of fusion pore expansion (or the lack thereof), including the Ca2+ sensor
Synaptotagmin.However,thebasisforhowSytinfluencesporeexpansion,andthusfusionmode,
iscurrentlyunknown.
TheAdrenalChromaffinCell
Chromaffincells,namedbyAlfredKohnfortheirchromiumsaltaffinity(usedforstaining),
are theprimary cell type found in the adrenalmedulla. These cells are best known for their
physiologicalroleinproducingandsecretingthecatecholaminesepinephrine(adrenaline)and
norepinephrineinthecontextofthemammalian“fight-or-flight”responseoriginallydescribed
by Walter B. Cannon. Chromaffin cells serve as the primary effector in the Hypothalamo-
12
Splanchnico-Adrenomedullary (HSA) axis, receiving input from the greater splanchnic nerves
(part of the sympathetic nervous system) (Carmichael & Winkler, 1985). In response to
acetylcholine (ACh)andPituitaryAdenylateCyclase-ActivatingPeptide (PACAP)signaling from
thesplanchnicnerves,chromaffincellsreleaseavarietyofphysiologicallyrelevantsubstances
viaboth synchronousandasynchronousexocytosis (Voetsetal., 1999; Smith&Eiden2012).
Thesesubstancesinclude:epinephrine(Epi),norepinephrine(NE),neuropeptideY(NPY),tissue
plasminogenactivator(tPA),enkephalin,andchromograninsA&B(CgA,CgB)(Lundbergetal.,
1979; Carmichael&Winkler, 1985; Coupland, 1989). The functional roles of thesemolecules
rangefromincreasingheartrateandbloodpressure(Epi&NE),tobreakingdownbloodclots
(tPA),andeveninducinganalgesia(enkephalin).Substancestobereleasedfromchromaffincells
areloadedintolargedense-corevesicles(LDCVs),namedfortheirlargesize(~300nm)relative
tosynapticvesiclesandvisuallydarkenedcenterresultingfrompeptideaggregations(e.g.CgA).
LDCVsoccupymostofthecytoplasm,andsinglechromaffincellshavebeenestimatedtocontain
upwardsof30,000individualvesicles(Carmichael&Winkler,1985;Figure1.3).
13
Figure1.3:ChromaffinCellLargeDense-CoreVesicles(LDCVs).Electronmicrographfromfreezefractureofachromaffincellrevealingtherelativesizeandhighdensity of large dense-core vesicles (LDCVs) in the cytosol. Nucleus (white dashed circle)surroundedbythecytosolpackedfullofLDCVs(orangearrows)withapproximatediametersof~300nm.ImagecredittoWolfgangSchmidtoftheUniversityofInnsbruck,Austria.
ThesizeandabundanceofLDCVsinchromaffincells,alongwiththerelativeeasewith
whichthesecellscanbeisolatedandmaintainedinprimaryculture,makethechromaffincellan
idealmodelforstudyingthepropertiesofexocytosis.Theirlargesizeandlackofmorphological
processesmake chromaffin cells easier to viablydissociate from living tissueas compared to
neurons(Kolski-Andreacoetal.,2007).Onceisolatedin2-dimensionalculture,thesecellstake
on a roughly spherical shape that makes them amenable to a variety of imaging and
electrophysiologicaltechniques,suchaspatchclamping(Fenwicketal.,1982;Anantharametal.,
14
2010).Furthermore,chromaffincellscansurviveincultureforaweekormore,allowingthemto
beisolatedinlargebatchesandsubsequentlyusedinmultipleexperiments.
ACloserLookattheCalciumSensorSynaptotagmin
As discussed above, Synaptotagmin (Syt) is widely accepted to be the primary Ca2+-
sensingproteinthattriggersregulatedexocytosisinneuronsandneuroendocrinecells(Broseet
al.,1992;Sudhof&Rizo,1996;Tucker&Chapman,2002).Sytwas first identifiedasp65 ina
proteinscreenofsynapticvesiclesfromratneurons(Perinetal.,1990).Themostsalientfeature
ofp65wasitsabilitytobindCa2+ionsandacidicphospholipids.Bindingsitesforthesemolecules
arelocatedintwocytoplasmicdomainsthatshareahighdegreeofsequencehomologywiththe
Ca2+-bindingC2domainofproteinkinaseC(PKC).Todate,asmanyas17isoformsofSythave
beenidentifiedinthemammaliangenome,withallfamilymemberssharingacommonstructure
containinganN-terminaltransmembranedomain,followedbythetandemC2domains(referred
to as C2A and C2B) connected by a short linker region (Sudhof& Rizo, 1996; Sudhof, 2002;
Gustavsson&Han,2009;Bacajetal,.2013;Figure1.4,A-C).
15
Figure1.4:SynaptotagminStructure.(A) Simplified domain structure of Synaptotagmin, showing the relative position of thetransmembranedomain(red)andCa2+-bindingC2AandC2Bdomains(yellow),eachcontaining
A
B
CB
16
three Ca2+-binding loops (green). A short linker connects the C2A and C2B domains (green).AminoacidrangesarelistedforSyt-1basedonChapmanetal.,1996andSudholf&Rizo,1996.(B)RibbondiagramofSyt-1.Thetransmembranedomain(TMD;red)isasingleα-helix,whereastheC2domainsareeachcomposedofaβ-sandwich(yellow)withCa2+-bindingloopsextendingoutfromtheedge(green).BoundCa2+ionsaredepictedasredspheres.ImagemodifiedfromChapman,2008.(C)PositionofSytinthevesiclemembraneatanexocytoticsite.TheC2AandC2Bdomainshangintothecytosol,poisedtointeractwiththeplasmamembraneandSNAREcomplexuponCa2+binding.ImagefromChapman,2002.
EachC2domainofSytiscomposedoftwostackedβ-sheets(i.e.aβ-sandwich)withthree
flexibleloopsextendingoutfromthetopandbottom(Suttonetal.,1995;Sudhof&Rizo,1996).
TheCa2+-bindingabilityofSytisgenerallyattributedtotheinteractionofpositivelychargedCa2+
ionswithfivenegativelychargedasparticacid(Asp,D)residuesfoundinthetoploopsofeachC2
domain(Fernandez-Chaconetal.,2002).BindingofSyttoanionicphospholipidsalsoappearsto
be dependent on these Asp residues and likely occurs due to the electrostatically favorable
“bridge”thatCa2+ionsformbetweenthenegativelychargedphospholipidheadgroupsandC2
domain Asp residues (Zhang et al., 1998). Furthermore,membrane binding is thought to be
enhancedbyotheraminoacidresiduespresent intheC2 loopsthatpossesseitherapositive
charge(Lys,Arg)orhydrophobicsidechain(Met,Phe,Val,Ile)(Frenandez-Chaconetal.,2002).
ItisworthmentioningthatnotallSytisoformsbindCa2+orphospholipidswiththeirC2domains,
andthis is likelyduetotheobservedabsenceofthecriticalAspresidues(Gustavsson&Han,
2009).
SeminalSynaptotagminFindingsandResultingQuestions
Following thediscovery and structural characterizationof Syt, a numberof important
studieswere conducted that paved theway for the current thesis research. Althoughmany
isoformsofSytarefoundinthemammaliangenomeandexpressedinvariouspartsofthebody,
17
thefindingthatonlySyt-1andSyt-7areexpressedinthephysiologicallyimportantchromaffin
cell(Schonnetal.,2008)raisedanimportantquestion–aretheseisoformsinterchangeablein
theirphysiologicalroles,ordotheypossessuniquepropertiesallowingthemtobeemployedin
divergentcontexts?Sugitaetal.(2002)werethefirsttocomparetherelativeCa2+affinitiesof
variousSytisoformsinvitro,concludingthatSyt-7showed~10-foldhigheraffinityforCa2+than
Syt-1.ThisfindingwastakenonestepfurtherbyBhallaetal.(2005)whocomparedtheabilityof
theSyt-1andSyt-7cytoplasmicdomains(whichincludebothC2AandC2B)topromoteSNARE-
mediatedvesicle fusion invitro.Usingtheir fusionassay, theyobservedthatSyt-7stimulated
membranefusioninthepresenceofroughly400-foldlessCa2+thanSyt-1,indicatingthatatleast
in vitro these isoforms can couple exocytosis to drastically different concentrations of Ca2+.
Furthermore,concurrentresearchbythesamelabprovidedevidencethatSyt-1dissociatesfrom
lipidsurfacesmorethan10xfasterthanSyt-7invitro,suggestingthatSyt-1andSyt-7mayact
separatelytomediatesynchronousandasynchronousreleaserespectively(Huietal.,2005).In
thewakeoftheseinvitrobiochemicalstudies,Schonnetal.(2008)providedearlyevidencefor
thistheorybyinvestigatingtheeffectsofSyt-1orSyt-7knockoutonvesiclereleasekineticsusing
culturedmousechromaffincells.TheyfoundthatSyt-1KOresultedinasubstantialdecreasein
fast,synchronousexocytosis(inagreementwithVoetsetal.,2001a),whileSyt-7KOledtoan
increaseinthefastcomponent(mostlikelyduetothelossofslower,asynchronousexocytotic
events).
Theseearlystudiesprovidedthefirsthintsatapotentialmechanismforhowchromaffin
cells might utilize their two versions of Syt to tune the release kinetics of vesicular cargo.
However, taken together these findings also raised a series of new questions regarding the
18
molecularregulationofexocytoticfusionmodesinchromaffincells.Forexample,doindividual
LDCVscontaincopiesofbothSyt-1andSyt-7,oraretheseisoformssortedtodifferentvesicle
populations?Ifthelatteristrue,dothedistinctbiochemicalCa2+andlipid-bindingpropertiesof
each isoform translate into differences in how (i.e. fusion mode) and when each vesicle
population is activated to undergo exocytosis? Furthermore, if there are differences in the
preferredfusionmodeofSyt-1versusSyt-7,doesthisalterthereleaserateofbiologicallyactive
cargomoleculesfromvesicles?
Tobegintoaddressthesequestions,Zhangetal.(2011)performedastudyexaminingthe
localizationofSytisoformsandtheirassociatedfusionmodeinpheochromocytoma(PC12)cells
– a cell linederived from tumorigenic chromaffin cells isolated from the rat adrenalmedulla
(Greene&Tischler,1976).Althoughderivedfromnormalchromaffincells,PC12cellsdiffer in
thattheyexpressfourisoformsofSyt,includingSyt-1,-4,-7,and-9(Fukudaetal.,2001;Tucker
etal.,2003;Ahrasetal.,2006).ThestudybyZhangetal.foundthatthesefourSytisoformsare
largely co-sorted inPC12 cells,withmore than60%co-localizationbetweenall isoformpairs
except Syt-1/Syt-7 which showed significantly less overlapping distribution (~45%). More
strikingly,thestudyalsofoundthatSyt-1andSyt-7appeartodifferintheirfusionkinetics.Using
totalinternalreflectionfluorescence(TIRF)microscopy(describedbelow),theauthorsobserved
threetypesoffusionevents:thosethatdisplayedrapid(<2sec)lossoffluorescently-taggedSyt
fromfusionsitesfollowingexocytosis,thosewherelossofSytoccurredbutwasslower(>2sec),
and those where Syt fluorescence remained elevated indefinitely. Syt-1 fusion events were
characterizedbyfasterlossofSytproteinfromsitesofexocytosis,whereasSyt-7eventsprimarily
displayed either slow loss or persistence of protein. The authors attributed these kinetic
19
differencestovesiclesundergoingcontrastingmodesoffusionbasedonwhichSytisoformthey
possessed.OneimportantcaveatofthisstudyisthefactthatitwasconductedinPC12cellsas
opposedtoprimarychromaffincells.Aswithanyimmortalizedcellline,PC12cellsarenolonger
undertheselectionpressures imposedbyresidence inan intactorganism,andtherefore it is
possible thatdecadesof subculturinghave resulted inadditionalgenomicalterationsbeyond
thosethatledtotheirtumorigenicproperties.Forexample,thiscouldexplainwhytwoadditional
Syt isoforms are expressed in these cells and/orwhy these isoforms show a high degree of
isoformco-sorting.
Rao et al. addressed the above concern by studying the localization and exocytotic
phenotypeofSyt-1andSyt-7 inprimarychromaffincells isolatedfrombovineadrenalglands.
Usinganantibody-basedapproach,theyfoundthatSytisoformswereindependentlysortedin
thesecellstoafargreaterdegreethaninPC12cells,withlessthan10%ofvesiclesexpressing
bothisoforms(Raoetal.,2014).Furthermore,overexpressionoffluorescentSyt-pHluorinfusion
proteinsdidnotelevatethedegreeofco-localization.Afterestablishingthedifferentialsorting
ofSytisoforms,thegrouplookedattheCa2+requirementsforstimulatingexocytosisfromeach
vesiclepopulation.
As mentioned earlier, Ca2+ entry into chromaffin cells occurs via voltage-gated Ca2+
channels intheplasmamembrane.Whilethevoltagethresholdforopeningthesechannels is
achieved through acetylcholine and PACAP receptor activation in vivo, this threshold can be
surmounted incellculturestudiesbybathingcellswithsolutionscontainingelevatedcations,
suchasK+.SolutionscontaininghigherK+concentrationsresult inagreater influxofCa2+ into
cells(Fulop&Smith,2007).UsingarangeofsolutionscontainingvariousconcentrationsofKCl,
20
Raoetal.showedthatmoderatestimulationwith25mMKCl(resulting inamildrise inCa2+)
specificallyfavoredexocytosisbySyt-7containingvesicles,whilestrongerdepolarizationwith56
mMKClledtoaslightpreferenceforSyt-1fusionoverSyt-7.Thisfinding,confirmedbyadditional
methodsinafollow-upstudy(Raoetal.,2017),supportstheideathatSyt-1andSyt-7endow
theirresidentvesiclepopulationswithdistinctCa2+sensitivities,inagreementwiththeirdistinct
biochemistries(Sugitaetal.,2002;Bhallaetal.,2005).
Raoetal.alsocomparedthefusionphenotypeofSyt-1versusSyt-7vesiclesinthreeways
using polarized TIRF (pTIRF) microscopy. This technique allows for simultaneous, real-time
imagingofplasmamembranetopographyandfluorescentproteinbehavior(Anantharametal.,
2010;seemethodssection foracompletedescriptionof this technique).First,examiningthe
persistenceofSytatfusionsites,itwasfoundthatSyt-1rapidlydiffusedwhileSyt-7remained
clusteredforaminuteormore(consistentwiththePC12findingsofZhangetal.,2011).Second,
usingacombinationofmembranetopologyandrapidbathsolutionpHswitching,theyshowed
thatfusionporesassociatedwithSyt-7eventstendedtoremainconstrictedforaminuteormore,
whereasthoseassociatedwithSyt-1quicklyexpandeduntilthevesiclemembraneflattenedinto
theplasmamembrane(i.e.full-collapse).NearlyathirdofallSyt-7eventsresultedinendocytosis
(i.e.kiss-and-run)duringtheimagingperiod,whileonly8%offusingSyt-1vesicleswereobserved
tofollowthesamefate.Finally,trackingthereleaserateoftheabundantLDCVcargoprotein
ChromograninB(CgB)revealedthatitisreleasedsignificantlyfasterfromSyt-1asopposedto
Syt-7vesicles.Recently thiseffectofSyt isoformsoncargoreleaserateshasbeenreaffirmed
usingtheappreciablysmallercargomoleculeNeuropeptideY(NPY)(Raoetal.,2017).
21
Insummation,thestudybyRaoetal.(2014),alongwiththoseprecedingit,havepainted
a potential picture of how the calcium sensing protein Synaptotagmin influences fusion
phenotype, and how this influencemay be used by chromaffin cells to tune their secretory
responsebasedonthedegreeofsympatheticnervoussystem(i.e.HSAaxis)activation.Whenan
organismisinastateofrest,HSAaxisactivityisminimal,meaningthesplanchnicnerveswillbe
releasingrelativelysmallquantitiesofAChandPACAPontotheirpostsynapticchromaffincells.
Asaresult,thechromaffincellswillexperienceonlymildrisesinintracellularCa2+,whichwould
favor exocytotic release from Syt-7-bearing LDCVs. These vesicular release events are
predominantlycharacterizedbyaconstrictedfusionporewhichlikelylimitsthediffusionrateof
cargo (i.e. biologically active signalingmolecules suchasepinephrine). Furthermore,manyof
thesereleaseeventswillendupundergoingendocytosis(i.e.kiss-and-run),whichmayserveas
awayofconservingresourcesassociatedwithvesiclebiogenesis,packagingofcargomolecules,
andclathrin-mediatedretrievalofvesiclematerialsfromtheplasmamembrane.
Conversely,whenanorganism ischallengedbyastressor,HSAactivityandsplanchnic
nerveoutputwillincreasedramatically,resultinginlargeCa2+spikeswithinchromaffincells.In
thiscontext,secretionwilloccurfrombothSyt-7andSyt-1vesicles.Themassivedemandsofthis
fight-or-flight scenario take priority over being stingy with vesicle materials and cargo, and
thereforefull-collapsefusionmediatedbySyt-1vesicleswillbeadvantageousfortheimmediate
needto,forexample,upregulateheartrateandbloodpressure(viaEpiandNE)andincreasepain
tolerance(viaenkephalins).
PrefacetoThesisResearch
22
Establishing the independentsortinganddivergent secretoryphenotypesofSyt-1and
Syt-7 in chromaffin cells was a major step in understanding the molecular regulation of
exocytosis.However,thesefindingsunveilednewquestionsregardinghowSyt isoformsexert
influenceonthefusionmodeofLDCVs.Fromamolecularstandpoint,oneofthemostimportant
questions iswhatdomain(s)ofSytareresponsible fortheirdifferentialassociationwith long-
livedversustransientfusionpores.Consideringtheirwell-establishedrole inbindingCa2+and
phospholipids,andthestarkdifferencethatSyt-1andSyt-7display inthisrespect,theSytC2
domainsariseasaprimarycandidateforregulatingfusionporedynamics.
BoththeC2AandC2BdomainsofSytarenecessaryfortriggeringexocytosis,however
theirrespectiverolesappeartodiffersomewhat.Forexample,studieshavesuggestedthatCa2+
bindingtoC2Bplaysamoreimportantroleinplasmamembraneengagementandstabilization
of fusion pores, whereas Ca2+ binding to C2A may act more like a permissive “switch” for
exocytosistooccur(Tucker&Chapman,2002;Bai&Chapman,2004;Segoviaetal.,2010;Striegel
etal.,2012).Furthermore,aknockoutstudyconducted inhippocampalneuronsshowedthat
replacingwild-typeSyt-1withachimericversioncontainingthefullSyt-7C2Bdomain(inplace
ofSyt-1C2B)couldnotrescuetherobustfastcomponentofneurotransmitterrelease,suggesting
thatdisparitiesbetweenisoformsinthisdomainaccountforthephenotypicdifferencesinfusion
mode(Xueetal.,2010).
Asdiscussedabove, theprimary structures responsible forCa2+binding toSytare the
three flexible loops extending out from the top of each C2 domain β-sandwich (Fernandez-
Chaconetal.,2002).Figure1.5showsacomparisonoftheC2BdomainsofSyt-1(AandD)and
Syt-7 (BandE), illustrating that theC2B loopsofSyt-7cancoordinateanadditionalCa2+ ion.
23
Therefore,myresearchaimedtostudytheeffectsofmutatingtheCa2+-bindingloopsoftheSyt-
1C2BdomaintomakethemsynonymouswiththecorrespondingloopsfoundinSyt-7.Tothis
end,thephenotypiceffectsofindividualloopconversions,aswellascollectiveconversionofall
threeloops,wereinvestigatedbyimagingindividualexocytoticeventsassociatedwithchimeric
Syt-1:Syt-7mutants.Basedon the fusionmodepreferenceofeach isoformand the fact that
mutations were performed in a Syt-1 background, phenotypically relevant loop conversions
shouldbemanifestasanincreaseinthepersistenceoffusionporesandpossiblythechimericSyt
proteinsthemselves.Theresultsofthisstudyaredescribedbelow.
AC
BA
EB
DB
CB
24
Figure1.5:ComparisonofSynaptotagminC2BDomainStructures.(A)RibbondiagramoftheSyt-1C2BstructureasdeterminedbyNMRspectroscopywithCa2+-binding loops labeled. (B)Syt-7C2BdomainstructureasdeterminedbyX-raycrystallographywithCa2+-bindingloopslabeled.(C)OverlayofSyt-1(orange)andSyt-7(blue)C2BstructuresfromX-raycrystallographydata.Ca2+ionsaredepictedasyellowspheres.(DandE)EnlargedviewoftheCa2+-bindingpocketofSyt-1 (D) andSyt-7 (E)basedonX-raycrystallography.Aminoacidresidues implicated inCa2+bindingare labeledandhighlighted in red.Syt-7 residuenumbersdepictedin(E)areoffsetby-6relativetothecorrespondingpositionsinSyt-1duetodifferencesin the upstream sequence. For example,M296 in (E) corresponds toM302 in (D). Note theadditionalCa2+ionboundbySyt-7.FigurereconstructedfromXueetal.2010.
25
CHAPTER2–METHODS
Site-DirectedMutagenesisofSynaptotagmin-1C2BLoops
Overall,theC2BdomainsofSyt-1andSyt-7aremoderatelysimilar intheiraminoacid
sequence.However,thissimilarityisevenmorepronouncedinthethreeCa2+-bindingloopsof
theC2Bdomain(Figure2.2A).Therefore,site-directedmutagenesiswasanappealingmethod
forconvertingtheSyt-1C2BloopstomatchthoseofSyt-7.
To perform this mutagenesis, three sets of primers were designed, with each set
containingaforwardandreverseoligonucleotidethattargetedasingleC2Bloop(IntegratedDNA
Technologies,Coralville, IA).Theseprimerscontainedacentral sequencematching theentire
correspondingSyt-7 loop, flankedby12-15nucleotideshomologous to theSyt-1bases in the
adjacent5’and3’regionsoutsideoftheloop(Figure2.1;Table2.1).Forwardandreverseprimers
weredesignedtooverlapintheirbindingtothecomplementaryDNAstrandscontainingtheloop
ofinterest.Thisallowedfortheuseofinversepolymerasechainreaction(PCR)toamplifythe
entireSyt-containingplasmid.InversePCRwascarriedoutinaBio-RadT100thermalcycler(Bio-
Rad Laboratories, Hercules, CA) using CloneAmp HiFi PCR Premix (Clontech Laboratories,
MountainView,CA).Thethermalcyclerprotocolincludedaninitialheatactivationstep(98°C–
1min)followedby35cyclesofdenaturation(98°C–10sec),annealing(55-58°C–10sec),and
extension(72°C–5mins).Afterthelastcycle,afinalextensionstepwasadded(72°C–5mins)
beforetheproductswereheldat4°Cuntilfurtheruse.Wild-typeSyt-pHluorinplasmidDNAused
as a PCR template (and for control experiments) was a gift from Dr. Edwin R. Chapman
(DepartmentofNeuroscience,UniversityofWisconsin–Madison).
26
Figure2.1:Site-DirectedMutagenesisPrimerDesign.Binding sites for mutagenesis primers (purple) within the Syt-1 C2B domain (entire domainshown).ForwardandbackwardprimersweredesignedtocompletelyoverlapforinversePCR.TheDNAsequencesoftheC2BCa2+-bindingloopsaredepictedinred.
Table2.1:Site-DirectedMutagenesisPrimerSequences.DNAsequencesandspecificSyt-1basepairtargetsofallmutagenesisprimers.Theredportionofeachprimer sequence contains themutagenicbasesof the correspondingSyt-7C2B loop,while theblackportionscorrespond to the flankingC2B regions inSyt-1used to“clamp” theprimertotheSyt-1template.ThesubscriptnumberintheprimernameindicatestheC2Blooptargetedbyeachprimerset.
Inverse PCR productswere digestedwith themethylation-specific endonucleaseDpnI
(NewEnglandBiolabs,Ipswich,MA).DuetoitsrequirementformethylatedDNA,DpnIwillonly
27
cleavetemplateDNApresentinthereactionmixture(inthiscasewild-typeSyt-1),becausethis
DNAhasbeenpreviouslyreplicatedinE.colicontainingtheDammethylaseenzyme.Following
DpnI digestion and heat inactivation, the remaining PCR products were transformed into
competent DH5α cells according to the manufacturers recommendations, although with an
additional two hours of incubation (Invitrogen Corporation, Carlsbad, CA). Six transformants
wereisolatedfromcoloniesgrownovernightonAmpicillin-selective(Amp+)agarmedia.Selected
transformantsweretransferredtoliquidAmp+mediaandagainincubatedovernightina37˚C
shaker. The following day, DNA was isolated using the QIAprep Spin Miniprep Kit (Qiagen
Company,Hilden,Germany),andafterquantificationthepurifiedDNAwaspreparedforSanger
sequencingwithprimerstargetingtheSyt-1C2Bdomain(AppliedGenomicsTechnologyCenter,
WayneStateUniversity–SchoolofMedicine,Detroit,MI).
DNA stocks containing the proper nucleotide substitutions were then subjected to a
secondroundofSangersequencingwithanarrayofprimersdesignedtogiveareadoutofthe
entire open reading frame containing the Syt-pHluorin fusion construct. This readout was
comparedtothewild-typeSyt-1constructsequencetoensurethatmutagenesiswasrestricted
to thedesiredC2B loop.Threesingle-loopSytchimerasweregenerated:Syt1:7-C2B1,Syt1:7-
C2B2, and Syt1:7-C2B3 (subscript numbers corresponding to the C2B loop mutated in each
construct).Theresultingaminoacidsubstitutionsforeachconstructareasfollows:Syt1:7-C2B1:
K301A,V304I,L307T;Syt1:7-C2B2:N333R,T334N;Syt1:7-C2B3:Y364K,I367L,G368S,K369R.The
triple-loop chimera Syt1:7-C2B1,2,3 contains all the above mutations and was generated by
performingtwoadditionalroundsofmutagenesis;first,Syt1:7-C2B1wasusedasatemplateto
mutateC2Bloop2,andthenthisproduct(Syt1:7-C2B1,2)wasusedasthetemplateinasecond
28
reactiontomutateC2Bloop3.ThefullC2Baminoacidsequencesforthesechimerasaredepicted
inFigure2.2,AandB.
Figure2.2:SynaptotagminC2BDomainAlignmentsandChimeraConstructs.(A)AlignmentoftheC2BdomainaminoacidsequencesofSyt-1,Syt-7,andthefourchimericSytconstructsusedinthecurrentstudy.AminoacidshighlightedinredindicateadeviationfromtheWTSyt-1sequence.ThegrayshadedregionsdenotetheCa2+-bindingloops.SubscriptnumbersintheconstructnameindicatewhichC2Bloop(s)hasbeenmutatedtomatchWTSyt-7.Amino
AC
BC
29
acidrangesbasedontheSyt-1sequencearelistedbeloweachloop(seeFigure1.4foralldomainranges).(B)SimplifieddepictionoftheSyt1:7chimerasillustratingtheregionscorrespondingtoeitherWTSyt-1(green)orWTSyt-7(yellow).
IsolationandTransfectionofBovineChromaffinCells
Adrenal glands of adult cows (Bos taurus) were obtained from JBS Packing Company
(Plainwell,MI)onthedayofslaughterandkeptoniceduringtransit.Theadrenalglandswere
cleaned inanon-divalent salinesolution (145mMNaCl,5.6mMKCl,15mMHEPES,5.6mM
Glucose,100Units/mlPenicillin-Streptomycin),theninjectedwithapre-warmedmixtureof2:1
Liberase TH and TL (Sigma-Aldrich, St. Louis,MO) andplaced in a beakerwithin a stationary
incubator at 37°C. After 18minutes, the glandswere transferred to a clean beaker and the
digestionwasrepeatedwithafreshenzymesolution.Followingthisstep,theglandswerecut
openandthemedullarytissuewasscrapedoffintoacollectiontubeusingascalpel.Thepooled
tissuefromallglandswasmincedbyhandinasterilepanwithscalpelsfor10min,thenincubated
inaglassspinnerflaskwithpre-warmed2:1LiberaseTHandTLmixtureat37°Cfor30minutes.
After incubation,thecell/tissuemixturewasfirstrinsedthrougha400µmmeshscreenusing
non-divalent saline solution and then centrifuged at 800 rpm to pellet chromaffin cells.
Supernatantwasdiscarded,andtheprocedurewasrepeatedtwomoretimeswitha250µmand
150µmscreen.
Atthispoint,cellswereresuspendedin1mlofBufferRfromtheNeontransfectionkit
(ThermoFisherScientific,Waltham,MA)andcountedmanuallyusingahemocytometer.Aliquots
of suspended cells were made according to the manufacturer’s recommendations and
electroporatedwith15µgoftheappropriateDNAconstructviaasingle1100mVpulseapplied
for40millisecondsusingtheNeontransfectionsystem(ThermoFisherScientific,Waltham,MA).
30
Electroporatedcellswere immediatelyplatedon35mmglass-bottomdishes(refractive index
1.51;World Precision Instruments, Sarasota, FL) coatedwith poly-D-lysine and collagen at a
densityof106cellsperdish.Disheswerepre-warmedwithDulbecco’sModifiedEagleMedium:
NutrientMixtureF-12(DMEM/F12)supplementedwith10%fetalbovineserum(ThermoFisher
Scientific,Waltham,MA)andamixtureof100Units/mlPenicillin-Streptomycin(ThermoFisher
Scientific,Waltham,MA)and23.8µg/mlGentamicin(Sigma-Aldrich,St.Louis,MO).Platedcells
wereincubatedin5%CO2at37°Cforaminimumof48hourspriortoimagingtoensurerobust
fusion protein expression. Experiments were completed within two days following this
incubationtime.
TIRFandpTIRFMicroscopy
Background
Total Internal Reflection Fluorescence (TIRF) microscopy has been used in biological
applicationssincetheearly1980’s(reviewedbyAxelrodetal.,1984).Intheory,thismicroscopy
methodutilizesthephysicalpropertyof lighttoreflect,ratherthanundergorefraction,when
encounteringaninterfacebetweentwosubstanceswithdifferentrefractiveindices(e.g.aglass
coverslipandanaqueousbiologicalsample).Importantly,thistotalinternalreflectionwillonly
occuriftheincominglightstrikestheinterfaceatoraboveacriticalangle(θc)describedbySnell’s
law:
θc=sin-1(n2/n1)
Inthisequation,n1representstherefractiveindexofthefirstmaterialthatlightwillpass
through (e.g. the glass coverslip), and n2 is the index of the second material (e.g. a cell
membrane).AlthoughmostlightwillbereflectedbackintothefirstmaterialduringTIRF(and
31
thusawayfromthesample),someoftheincominglightwillstillpropagatethroughtothesecond
mediumintheformofan“evanescentwave”.TheadvantageofTIRFmicroscopyisbasedonthe
factthatthisevanescentwavedecaysexponentiallyasitentersthesecondmaterial,meaning
onlyobjectsrelativelyclosetothesampleinterface(within~100nm)willbeilluminated.This
effectivelyincreasesimagequalitybyeliminatingbackgroundilluminationofstructuresfurther
into the sample. In the case of TIRF microscopy, the illuminating light source(s) are usually
wavelength-specificlasersusedtoexcitefluorophore-linkedmoleculeswithinacell(Figure2.3).
Figure2.3:TotalInternalReflectionFluorescence(TIRF)Microscopy.TheuseofTIRFmicroscopyforimaginglivecellsinculture.OneormorelaserlinesareguidedintotheobjectiveofaninvertedmicroscopeataTIRFangle(θT)greaterthanthecriticalangle(θc) described by Snell’s law relating the refractive indices of the glass coverslip and cellmembrane(seetext).Thisresultsinthepropagationofanexponentiallydecaying“evanescentwave” into the cell thatwill selectively excite fluorophoresnear the coverslip (i.e. in the cellmembraneorsubplasmalemma).ImagefromOckenga,2012.
ImagefromOckenga,2012
32
Overtheyears,TIRFmicroscopyhasbeenadaptedtomeetvariousexperimentalneeds,
including the incorporation of polarized light sources (pTIRF) (Taraska & Almers, 2004;
Anantharametal.,2010).Propersplittingandpolarizationofa laserresults inonebeamthat
specificallyexcitesfluorophoresorientedinthesameplaneasthecoverslip-sampleinterface(s-
polarlized; s-pol), and another beam that excites fluorophores orientedperpendicular to the
interface(p-polarized;p-pol).Thissetupisadvantageousforimagingtopologicalchangesinthe
plasmamembraneusingmembrane-intercalatingdyes (e.g.DiD,DiI),because their transition
dipolemoment(i.e.theplanetheycanbeexcitedin)isroughlyparalleltothemembranesurface
they sit within (Axelrod, 1979). Therefore, during an exocytotic event, the s-pol beam will
primarilyexcitedyeintheplasmamembranesurroundingafusionpore,whereasthep-polbeam
willexcitedyeinthe“walls”ofasecretoryvesicleandfusionpore(Figure2.4,AandB).
AC
BA
33
Figure2.4:VisualizingMembraneTopologywithMembrane-IntercalatingDiD.(A) Orientation of a DiD molecule intercalated into a lipid bilayer. Membrane insertion ismediated by two long, lipophilic hydrocarbon chains. The resulting absorption and emissiontransitiondipolemoments(dmn)arethereforeroughlyparalleltothesurfaceofthemembranethemoleculeisresidingwithin.(B)TheorientationofDiDcanbeexploitedtotrackmembranetopologybyusingapolarizedexcitationlightsource.561-nms-polarizedlight(s-pol)willprimarilyexciteDiDintheplasmamembraneparalleltotheglasscoverslip,withasmallcontributionfromdye marking the back of fused vesicles (because these molecules are much deeper in theevanescent field; left image). Conversely, p-polarized light (p-pol) will selectively excite DiDorientedperpendiculartothecoverslipinmembraneformingthewallsofafusedvesicle(orangemolecules,rightimage).
Usingtheemissionintensityvalueselicitedfroms-andp-polexcitation,twomeaningful
parameterscanbecomputed.TheP/Sintensityratioatafusionsitereportsonthedegreeof
membranecurvature.Ahigherratio indicatesthatmoreofthestainedmembraneisoriented
perpendiculartotheplasmamembranesurface,andisthereforecharacteristicofvesiclesthat
areatthepointofhavingmergedwiththeplasmamembraneandformedafusionpore.When
thisvesicleeventuallyundergoesfull-collapse,orconverselyresealsandundergoesendocytosis
(kiss-and-run), the ratio will drop back down closer to its pre-fusion value as the plasma
membraneflattensandthevesicleleavestheevanescentfield.Asecondparameter,theP+2S
value,hasbeenshowninpreviousstudiestocorrespondwiththetotalamountofdyeinaregion
but is also influencedbyhowdeep thedyemoleculesare in relation to theevanescent field
(Anantharametal.,2010;2012).Atleastintheory,anincreaseinP+2Svaluesduringexocytosis
couldcorrespondtoavesiclethatisconnectedtotheplasmamembranebyanarrowfusionpore,
asthiswouldserveasanaccumulationpointfordyenearthecoverslip(wheretheevanescent
fieldisstrongest).AdecreaseinP+2Scouldthereforeresultfromdyediffusingintoadepression
formedbythemembraneofacollapsingvesicleandthusmovingfurtherawayfromthecoverslip.
34
ExperimentalpTIRFWavelengthsandFluorophores
The thesis research presented herewas conducted using the same pTIRFmicroscopy
systemasRaoetal.(2014).Thissystememploystwosolid-statelasers(CoherentInc.,SantaClara,
CA); a 488-nm laser used to excite the Synaptotagmin-linked fluorescent protein pHluorin
(described below), and a 561-nm laserwhose output is split into twoorthogonally polarized
beamsusedtoselectivelyexcitethemembrane-intercalatingdyeDiD(InvitrogenCorporation,
Carlsbad,CA)basedonitsorientation(seeabove).Thecurrentstudyfocusedprimarilyonthe
persistenceof topological changes (i.e.elevatedP/S ratios)andpHluorinsignatures following
membranefusionmediatedbySytchimeras.
The fluorescentproteinpHluorin (pHl)wasused for thecurrent study,because itspH
sensitivityservesasaconspicuousindicatorofexocytoticevents.pHlwasdevelopedfromGreen
FluorescentProtein(GFP),andshowsmaximalfluorescenceemissionbetweenpHvaluesof7.0-
8.0,whilebeingalmostentirelyquenchedinmoreacidicenvironments(pH~5.5)(Miesenbocket
al.,1998).ThesepHvaluescoincidecloselywiththeconditionsinlivingchromaffincells,where
theinternalpHoflargedense-corevesicles(LDCVs)ismaintainedat~5.5byvesicularATPases,
whilethesurroundingextracellularenvironmentismoreneutralat~7.4.Basedonthesefeatures
and the vesicular localization of Synaptotagmin, this study used a fusion protein with pHl
attachedtotheN-terminusofSyt,thusconfiningthefluorophorewithintheacidicvesiclelumen
priortoexocytosis(Figure2.5).
35
Figure2.5:VisualizingExocytosiswithpH-SensitivepHluorin.Left:Priortoexocytosis,pHluorin(pHl)fluorescenceisquenchedduetotheacidityofthevesiclelumen(pH~5.5)maintainedbyvesicular-ATPases.Center:Uponfusion,theformationofafusionpore results in rapid neutralization of the lumen and a concomitant increase in pHluorinfluorescence.Right:pHlinvesiclesthatareendocytosedviakiss-and-runwillagainbequenchedasthevesiclelumenisre-acidified.ImagereproducedfromKavalali&Jorgensen,2014.
Usingthissetup,dockedvesiclesawaitingexocytosisemitlittle-to-nofluorescencewhen
exposedtothe488-nmlaser.However,onceexocytosisbeginsandafusionporeopensbetween
the vesicle lumen and extracellular fluid, the luminal pH rapidly neutralizes, causing pHl
molecules exposed to 488-nm light to fluorescebrightly. This fluorescencewill persist at the
fusion site until one of two outcomes occur; either (1) the vesicle collapses into the plasma
membrane(i.e.classicexocytosis)andtheSyt-pHlmoleculesdiffuseawayfromthefusionsite,
or(2)thefusionporereseals,andthevesicleundergoesendocytosis(i.e.kiss-and-run)following
whichitwillre-acidify(therebyquenchingthefluorescenceonceagain).
ImageAcquisition
36
Thethreeincominglaserlines(488-nm,s-pol561-nm,p-pol561-nm)werealignedina
single60x(1.49numericalaperture)objectiveonanOlympusIX81invertedmicroscope(Olympus
Corporation,CenterValley,PA).Illuminationofthesamplewasachievedusingrapidcyclingof
LambdaSCshutters(SutterInstruments,Novato,CA)tosequentiallyallowthetransmissionofa
singlebeamontothesample.OutputemissionwascapturedusinganAndoriXonEMCCDUltra
897 camera (Andor Technology, Concord, MA) controlled by MetaMorph imaging software
(MolecularDevices,Sunnyvale,CA).Collectively,thissetupallowedforimageacquisitionat~2.5
framespersecondforeachchannel,andproducedatotalopticalmagnificationof~200x,which
translatedintoapixelsizeof~80nmintheanalyzedimages.
LiveImagingofChromaffinCells
Priortoimaging,mediawasremovedandcultureplatescontainingchromaffincellswere
washedthreetimeswithabasalsalinesolution(basalPSS)containing:145mMNaCl,5.6mM
KCl,2.2mMCaCl2,0.5mMMgCl2,5.6mMglucose,15mMHEPES,pH7.4.Cellmembraneswere
thenlabelledbyadding10μlofDiDto2mlofbasalPSSandswirlingthissolutionontheplate
for~15-20secatroomtemperature.Followingthisstep,thedyesolutionwaspouredoffand
plateswerewashedthreemoretimestoremoveexcessdye.Individualcellswereselectedfor
imagingiftheyshowedbothDiDstainingandpHluorinexpression(thoughthiswasusuallyvery
weakpriortocellstimulation).Duringimaging,plateswerebathedinbasalPSSandthetargeted
cellwasselectivelystimulatedusingsolutionsadministeredthroughaperfusionneedle(100µm
innerdiameter;ALAScientific Instruments,Westbury,NY)positioned justoutside the fieldof
viewusingamicromanipulator.Cellswereperfusedsequentiallywith5secofbasalPSS,75sec
ofelevatedK+(56mMKCl)PSStotriggerexocytosis,and10secofbasalPSSagaintoclearthe
37
stimulatingsolutionfromtheperfusionneedle.TheelevatedK+PSSsolutioncontained:95mM
NaCl,56mMKCl,5mMCaCl2,0.5mMMgCl2,5.6mMGlucose,15mMHEPES,pH7.4.Perfusion
timeswereregulatedviasoftwarecoupledtoelectromagneticvalves(ALAScientificInstruments,
Westbury,NY)andsynchronizedwiththestartofimageacquisition.Experimentswerecarried
outunderambientairconditions,withthetemperaturetypicallyrangingbetween24-26°Cin
theimagingroom.
ImageAnalysis
Acquiredimagestacksforeachchannel(488-nm,s-pol561-nm,p-pol561-nm)werefirst
processedusingacustomIDLsoftwarescriptwrittenbyDanielAxelrod(DepartmentofPhysics,
UniversityofMichigan–AnnArbor).This softwareperformsthree important functions: (1) It
spatiallyalignstheimagescapturedfromallchannelsateachtimepointforagivencell,thereby
allowingtheusertoselecta~270nmregionofinterest(ROI)aroundsinglefusioneventsacross
allchannelsatonce.(2) Itnormalizesthep-polands-pol imagestacksagainstRhodamine6G
imagestakenbytheuseronthesamemicroscopesetup(seebelow).(3)Thesoftwaregenerates
compositeimagesofthepixel-by-pixelp/sintensityratiosandp+2svaluesacrossthecellforeach
timepoint.Normalizationofp-ands-polimagesusesaRhodamine6Gsolution(Sigma-Aldrich,
St.Louis,MO),whichisassumedtohaverandomlyorientedtransitiondipolemoments.Thisis
an important step for factoring out orientation biases imposed by the optical setup, and
essentiallycorrectsforsmalldifferencesintheintensityoftheincominglaserbeamsthatcould
otherwiseskewtheP/Sratio.
Foreachcellanalyzed,thefinaloutputfromtheIDLsoftwareisaseriesoftextdocuments
with the averaged (across the ROI) fluorescence intensity at every time point during image
38
acquisition.AseparatedocumentiscreatedforthepHlemissionintensities,P/Sratios,andP+2S
valuesineachROI,andthisisdoneforallROIsselectedwithinagivencell.Thesevalueswere
then transferred to an excel document and graphed. Exocytotic events (i.e. ROIs) were only
includedintheanalysisiftheyshowedasignificantchange(>10%)inallthreevalueswithinfive
frames (~2 sec) of the fusion frame, as indicated by the spike in pHl emission intensity. As
expected,pHlandP/Sratiovaluesalwaysincreasedfollowingfusion,whereasP+2Svalueseither
increasedordecreased.The10%significancethresholdusedinthisstudywasbasedonasimilar
(albeit slightly less stringent) thresholdof7%usedbyRaoetal. (2014), and this changewas
measuredinrelationtotheaverageparametervalueinthethreeframespriortofusion(i.e.the
pre-fusion baseline). All reported durations reflect the amount of time a given parameter
remained above this 10% threshold following the initial rise. The number of cells and total
numberofeventsacrossallcellsforeachconstructarereportedinTable2.2.
Table2.2:NumberofCellsandFusionEventsAnalyzed.Eventswereselectedbasedonthecriteriadescribedabove.Foragivencell,eventswereincludedintheanalysisifthefluorescencesignalofpHl,P/S,andP+2Schangedbyatleast10%(relativetothepre-fusionbaseline)withinfiveframesofthespike inpHl indicatingvesiclefusion.Thenumberofeventsreportedinthetablereflectshowmanytotaleventsforeachconstructwerepooledtogetherinthequantitativeanalysisbasedonmeetingthiscriterion.
39
CHAPTER3–RESULTSANDDISCUSSION
Results
ThePost-FusionPersistenceofChimericSynaptotagminsDiffersfromWild-Type
Thespatialandtemporaldynamicsoffusionmachineryproteinscandiffersubstantially,
providing clues to their respective roles in the fusionprocess. For instance,Raoetal. (2014)
comparedthepost-fusionpersistenceofpHluorin-taggedSynaptotagmin(Syt)isoformsatsites
ofexocytosis.Inthecurrentstudy,thiswasinitiallyinvestigatedforcontroldata(i.e.wild-type
[WT] Syt-1 and Syt-7) to validate the previously published findings on the behavior of these
isoforms. Itwas foundthatWTSyt-7pHlremainedpunctateatsitesofexocytosis for tensof
seconds,whereasWT Syt-1 pHl signalwas rapidly lost (Figure 3.1). Plotting the durations of
numerousindividualfusioneventsacrossmultiplecellsrevealedahighlydivergenttrend,where
87%ofSyt-7eventswerecharacterizedbypersistentaggregationoftheproteinatthefusionsite
formorethan20seccomparedtoonly27%ofSyt-1eventssuggestingdifferentfunctionsfor
theseisoforms(Figure3.2,A-C).
Table3.1:Chi-SquareAnalysisofProteinPersistence.Pairwise Chi-Square tests for homogeneitywere used to assesswhether protein persistencedistributionsdifferedsignificantlybetweenSytconstructs.Allindividualeventsforeachconstructwere binned into three groups based on how long the pHluorin signal remained elevated
40
following fusion (0-20 sec, 20-40 sec,or>40 sec). ThedistributionsofWTSyt-7and chimeraSyt1:7C2B1,2,3differedsignificantlyfromWTSyt-1,whereasSyt1:7C2B2andSyt1:7C2B3didnot.ThisanalysiscouldnotbeperformedonSyt1:7C2B1,becausethedataforthiscomparisondidnotmeetthesecondofthetworequiredassumptionsforaChi-Squaretest(allexpectedvalues≥1andnomorethan20%ofexpectedvalues<5).
Figure3.1:SampleImageSequencesofSynaptotagminConstructs.Sampletime-lapseimagesequencesofindividualfusioneventsfromtheindicatedSytconstructs.The top row for each construct shows the pHluorin emission used to determine the time ofvesicle fusion (t=0 sec)and trackproteinpersistence.Themiddle row (P/S)andbottomrow(P+2S) report on thepixel-by-pixelmembrane curvature andDiD concentration, respectively.NotetheelevatedpersistenceofSyt1:7C2B1,2,3proteinandmembranecurvatureincomparisontoWTSyt-1.Scalebar,800nm.
To identify what portion of the Syt-isoforms contributed to these differences, the
chimerasweretestedinthisassayandcomparedtothewild-typeisoforms.Itwasfoundthatthe
single loop Syt chimeras, Syt1:7-C2B1, Syt1:7-C2B2, and Syt1:7-C2B3 all showed a less
41
dichotomous trend, with protein persisting for >20 sec in 33%, 38%, and 39% of events,
respectively(Figure3.2C).Binningofpersistencedurationsforalleventsintothreegroups(<20
sec,20-40 sec,>40 sec)enabled the comparisonofeach chimericdistributionwithWTSyt-1
(Table3.1).Althoughthiscomparisondidnotshowsignificanceattheα=0.05significancelevel,
itwasevidentfromplottingalldurationsthatthesechimerasgenerallyexhibitedanintermediate
phenotype,withmorebalancebetweeneventsshowingpersistenceversusrapidlossofprotein
(Figure3.2A).Thisismoreclearlyobservedbycomparingtheaveragetimeofpersistenceforthe
middle50%ofdurations (i.e.durationswithin the innerquartile range, IQR).Performing this
calculation,Syt-1hadanaveragepersistenceof10.8sec(SEM±1.14sec),whileSyt-7averaged
56.2sec(SEM±2.04sec).Allthreesingleloopchimerasshowedamodestincreaseinthisvalue
fromtheirSyt-1sourceDNA,withaveragesof13.4sec(SEM±1.16sec),14.2sec(SEM±1.65
sec),and16.4sec(SEM±0.93sec),respectively(Figure3.2B;Table3.2).Thissuggeststhatsingle
C2BloopconversionsbetweenSyt-1andSyt-7haveasmallbutnoticeableeffectonpersistence
oftheproteinatfusionsites.
Table3.2:AverageProteinPersistenceFollowingFusion.Values indicatetheaverageduration(andSEM)ofelevatedpHluorinsignal (>10%ofthepre-fusionbaseline)foreventsthatfellwithintheinnerquartilerange(seeFigure3.2B).
42
Figure3.2:PersistenceofSynaptotagminChimerasatFusionSites.(A) Scatterdotplotsofproteinpersistence forall individual fusioneventsbasedonpHluorinemissionprofiles.DurationsindicatetheamountoftimethepHlsignalremainedabovethe10%thresholdfollowingitsinitialriseatthetimeoffusion.Asdescribedinthetext,someofthehighervaluesreflecttruncateddurationsduetotheendofimageacquisitionoccurringbeforethesignaldroppedbelowthe10%threshold.Notetheclusteroflong-durationeventsforchimeraSyt1:7C2B1,2,3 in comparison toWT Syt-1. (B) Box plots depicting the inner quartile range (IQR) ofdurationsandtheaveragedurationcomputedfromalleventswithintheIQR.(C)Graphreportingthepercentageofalleventsforeachconstructthatshowedproteinpersistenceinexcessof20sec based on the pHl fluorescence signal. Again, note the intermediate phenotype of Syt1:7C2B1,2,3.
SynaptotagminCa2+-bindingLoopsAlsoInfluenceFusionPoreExpansion
InadditiontotrackingthepersistenceoftheSytchimerasatfusionsites,polarizedtotal
internalreflectionfluorescence(pTIRF)microscopywasusedtoinvestigatethebehaviorofthe
AC
CA
BA
43
vesicularandplasmamembranesfollowinginitialfusion.Usingthissystem,topologicalchanges
indicativeofavesiclefusingwiththeplasmamembranearereportedasasharpriseintheP/S
fluorescenceratio (seemethods).Thisvalue remainselevateduntileither (1) the fusionpore
dilatesandthevesiclemembraneflattensintotheplasmamembrane(i.e.full-collapsefusion),
or(2)thefusionporereseals(i.e.kiss-and-runendocytosis)andthevesicleisbroughtoutofthe
evanescentfield.
ConsistentwithRaoetal.(2014),thisstudyfoundthatincontrolcells,elevatedP/Sratios
persistedfar longerforWTSyt-7-mediatedexocytoticeventsthanforWTSyt-1events.Given
thattheSytchimerasweregeneratedinaSyt-1background(Figure2.2),functionallyrelevant
loopswapswereexpectedtoresultinfusioneventswhereP/Sremainedelevatedlongerthan
forWTSyt-1.Interestingly,allsingleloopchimerasshowedanincreaseintheaverageduration
ofelevatedP/Sratios(Figure3.3,AandB).However,thefinite imagingtimeandvariation in
wheneventsoccurredfollowingcellstimulationledtosomeofthelongerdurationvaluesbeing
truncated(i.e.becausetheelevatedP/Sratiopersistedbeyondtheendoftheimageacquisition
period).Therefore,itismorereliabletocalculateaveragetimesforeachconstructbasedonlyon
valueswithintheinnerquartilerange(IQR).ChimerasSyt1:7-C2B1andSyt1:7-C2B2showedonly
amodestincreaseinelevatedP/Sdurationfrom7.3sec(SEM±0.94sec)forWTSyt-1to13.9sec
(SEM±2.91sec)and15.3sec(SEM±1.63sec),respectively.Ontheotherhand,swappingofC2B
loop3(chimeraSyt1:7-C2B3)appearedtohaveamoresubstantialinfluenceonprolongingthe
decayofP/Sratios,withanaveragedurationof27.6sec(SEM±2sec)comparedto38.6sec(SEM
±2.74sec)forWTSyt-7(Figure3.3B;Table3.4).UsingChi-SquareanalysistocompareallP/S
44
durations for each chimera against WT Syt-1 revealed that the difference between the
distributionofSyt1:7-C2B3andWTSyt-1washighlysignificant(p<0.001;Table3.3).
Figure3.3:Post-FusionElevationsinP/SRatio.(A)ScatterdotplotsoftheelevatedP/Sratiodurationforallindividualfusionevents.DurationsindicatetheamountoftimetheP/SratioinanROIremainedabovethe10%thresholdfollowingits initialriseatthetimeoffusion.Asdescribedinthetext,someofthehighervaluesreflecttruncateddurationsduetheendofimageacquisition(seepanelC).Notetheclustersoflong-durationeventsforchimerasSyt1:7C2B2,Syt1:7C2B3,andSyt1:7C2B1,2,3incomparisontoWTSyt-1.(B)Boxplotsdepictingthe innerquartilerange(IQR)ofelevatedP/SdurationsandtheaveragedurationcomputedfromalleventswithintheIQR.(C)Graphindicatingthepercentageofalleventsforeachconstruct inwhichP/Sremainedelevatedbeyondtheendofthe imageacquisitionperiod.Syt1:7C2B1,2,3wasindistinguishablefromWTSyt-7inthisrespect.(D)GraphreportingthepercentageofalleventsthatshowedelevatedP/Sratiodurationsinexcessof20secbasedonDiDemission.
AC
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45
Table3.3:Chi-SquareAnalysisofElevatedP/SDurations.PairwiseChi-SquaretestsforhomogeneitywereusedtocomparethedistributionsofelevatedP/SratiosbetweenSytconstructs.AllindividualeventsforeachconstructwerebinnedintothreegroupsbasedonhowlongtheP/Sratioremainedelevatedfollowingfusion(0-20sec,20-40sec,or>40sec).ThedistributionsofWTSyt-7,Syt1:7C2B3,andSyt1:7C2B1,2,3differedsignificantlyfromWTSyt-1.ThisanalysiscouldnotbeperformedonSyt1:7C2B1,becausethedataforthiscomparisondidnotmeetthesecondofthetworequiredassumptionsforaChi-Squaretest(allexpectedvalues≥1andnomorethan20%ofexpectedvalues<5).
ElevationsinP/SoutlastedtheimagingperiodmorefrequentlyforSyt-7events(~34%)
thanforSyt-1(<10%)(Figure3.3C).ChimeraSyt1:7-C2B3eventsbehavedmorelikeSyt-7inthis
regard,with~25%ofallanalyzedeventspersistingbeyondtheendofacquisition.Binningevents
basedona20-secthresholdrevealedasimilartrend.ForchimeraSyt1:7-C2B3,50outof87events
(~57%)exhibitedaP/Ssignallastinglongerthan20sec,whichresembledthecaseofWTSyt-7
(~71%)morethanWTSyt-1(22%)(Figure3.3D).Unexpectedly,P+2Svalues(reportingonoverall
dyeconcentrationatthefusionsite)showedapost-fusionincrease(versusadecrease)fornearly
alleventsacrossconstructs.Thisrangedfrom88%ofWTSyt-1eventsto97%ofWTSyt-7,with
all chimericproteins falling in themiddleat~95%(datanot shown).Thiswillbediscussed in
greaterdetailbelow.Overall, these findings suggest that theSyt-7C2B loopscan individually
impede theexpansionof theexocytotic fusionpore,with themost significant impacton this
behaviorresultingfromloop3.
46
Table3.4:AverageDurationofElevatedP/SRatiosFollowingFusion.Valuesindicatetheaverageduration(andSEM)ofelevatedP/Sratios(>10%ofthepre-fusionbaseline)foreventsthatfellwithintheinnerquartilerange(seeFigure3.3B).
ChimeraSyt1:7-C2B1,2,3BehavesthemostlikeWild-TypeSyt-7
The fact that individual C2B loop swaps appeared to influence exocytosis raised the
questionofwhetherswappingallthreeloopstogetherwouldfurtherpushthesecretorybehavior
ofSyt-1towardsthatofSyt-7.Thiswasaddressedbyanalyzingthephenotypeofthetripleloop
mutantSyt1:7-C2B1,2,3,inwhichallthreeCa2+-bindingloopsinSyt-1C2Bwerereplacedwiththose
ofSyt-7.ThetypicalbehaviorofthisproteininrelationtoWTSytsandtheloop1mutantcanbe
seen in the sample time-lapse images shown in Figure 3.1. Regarding protein persistence
following fusion, the single loop chimeras did not strongly favor a single phenotype (i.e.
persistenceorrapiddiffusion),whereasSyt1:7-C2B1,2,3wasmorereminiscentofWTSyt-7,with
~64%ofeventscharacterizedbypersistenceexceeding20sec(Figure3.2,AandC).Theaverage
residencetimeforthemiddle50%ofdurations,31.2sec(SEM±2.76sec),wasalsoshiftedfurther
fromWTSyt-1thanthesingleloopchimeras(Figure3.2B;Table3.2).
47
Furthermore, the triple loop chimera had the greatest influence on post-fusion
membranetopology.EventsthatfellwithintheIQRhadanaverageP/Srisethatlastedfor36sec
(SEM±3.56sec),nearlyaslongasWTSyt-7(38.3±2.99sec)(Figure3.3,AandB;Table3.4).
Likewise,more than 60% of Syt1:7-C2B1,2,3 events showed a P/S elevation exceeding 20 sec
(comparetoabove),with~34%ofallelevationsoutlastingtheimagingperiod(roughlythesame
asforWTSyt-7;Figure3.3,CandD).Thedurationdistributionsforpersistenceofbothprotein
and membrane topology were highly significant between Syt1:7-C2B1,2,3 and WT Syt-1 (p <
0.0005;Tables3.1and3.3).Takentogether,theseresultssuggestthattheaminoacidswithinthe
SytC2BCa2+-binding loops influenceboth thepersistenceofSytat fusionsitesand thepost-
fusionexpansionoftheexocytoticfusionpore.Furthermore,theeffectsofswappinginallthree
Syt-7C2Bloopstogetheraremorepronouncedthanthatofanyindividualloopswap.
Discussion
Thisstudyfocusedonthemolecularbasisforthepreviouslyobserveddifferencesinthe
behaviorofSyt-1andSyt-7 inadrenalchromaffincells.To thiseffect,chimericproteinswere
generatedinwhichtheCa2+-bindingloopsintheSyt-1C2Bdomainwerereplacedbyloopsfrom
thesameregioninSyt-7.Thedatapresentedabovesuggestthatthegeneticmakeupofthese
loopsinfluencesatleasttwoofthedifferentialpropertiesbetweenisoformsduringexocytosis;
namely, thepersistenceof the Syt protein and thebehavior of the fusionpore.All chimeras
showedatleastaslightshiftfromtheSyt-1phenotypetowardsthatofSyt-7,withthetripleloop
mutant(Syt1:7-C2B1,2,3)behavingmostlikeSyt-7.Theeffectsofsingleloopswapsdidnotappear
tobestrictlyadditivewhencomparedtothetripleloopmutant,andthismayreflectthefactthat
C2domainCa2+bindingresultsfromthecoordinatedarrangementofmultipleloopstoforma
48
Ca2+-bindingpocket,ratherthaneachloopinteractingwithindependentCa2+ ions(Figure1.5;
Shaoetal.,1996;Sudhof&Rizo,1996).
InadditiontothefindingsofRaoetal.(2014),increasedpersistenceandaggregationof
Syt-7 following fusion was confirmed more recently using the super-resolution method of
StochasticOpticalReconstructionMicroscopy(STORM;Rao,2016).Thefindingspresentedhere
suggest that Ca2+-mediated binding specifically by the C2B domain influences how long Syt
remains clustered at sites of exocytosis. This clusteringmay result from Syt interactingwith
phospholipids in the membrane, other fusion machinery proteins, and/or forming homo-
oligomerswithitself.ItwasrecentlydemonstratedinvitrothatthecytosolicfragmentofSyt-7
(containing C2A and C2B) dissociates from liposomes containing negatively-charged
phosphatidylserine(PS)farmoreslowlythanSyt-1whenCa2+ischelatedoutofthesystem(Rao,
2016).Therefore,itisreasonabletopredictthatconversionoftheSyt-1C2Bloopswouldslow
therateofmembranerelease,whichisconsistentwithwhatwasobservedregardingdiffusion
of the Syt1:7 chimeras. Syt1:7-C2B1,2,3 showed the most pronounced reduction in diffusion,
remainingpunctateforroughly20seclongerthanWTSyt-1onaverage(Figure3.2;Table3.2).
TrackingmembranetopologyusingpTIRFrevealedasimilarlystrikingtrend.P/Sratios
remainedelevatedfarlongerfollowingfusioneventsmediatedbySyt1:7-C2B3andSyt1:7-C2B1,2,3
thanWTSyt-1(Figure3.3;Tables3.3and3.4).Asmentionedabove,P/Sratiosincreaseasaresult
ofDiDreorientationduringvesicleandplasmamembranefusion.AsubsequentdecreaseinP/S
wouldintheoryoccurduetooneoftwocontrastingoutcomes;eitherfusionporedilationand
concomitantvesiclecollapse,orfusionporeclosure(endocytosis)followedbytraffickingofthe
vesicleoutoftheevanescentfield.ThissuggeststhatinthecaseoftheSyt1:7chimeras,fusion
49
poreswereeithermoreoftendelayed in their expansion, or they tended to reseal,with the
vesicleremainingincloseappositiontotheplasmamembrane.Inotherwords,theC2Bloopsof
Sytappeartoexertcontroloverthelikelihoodoffusionporesundergoingrapidexpansion,with
theSyt-7loopsmoreofteninhibitingporeexpansion.FurtherinsightcanbedrawnfromthepH-
switchingexperimentbyRaoetal.(2014),inwhichkiss-and-runendocytosiswasobservedfar
morefrequentlyforSyt-7thanSyt-1.Basedonthis,itwouldbereasonabletopredictthatthe
Syt1:7 chimeras (particularly Syt1:7-C2B1,2,3) would also show higher rates of kiss-and-run
endocytosiscomparedtoWTSyt-1,althoughthisremainstobetested.
ComputersimulationsandempiricalworkhavesuggestedthatchangesinP+2Scanalso
beusedtoassesswhetherfusionporesareexpandingorremainingconstricted.Intheory,P+2S
values should correspond to the relative dye concentration in a region confoundedwith the
depthofthatregionwithintheevanescentfield.Basedonthis,exocytoticeventscharacterized
by a persistently constricted fusion pore and/or endocytosiswould be predicted to show an
increase in P+2S, while events that undergo fusion pore expansion should show a transient
decrease inP+2S(seemethods). Inthecurrentstudy,P+2Svalueswereobservedto increase
followingnearlyallfusionevents(~95%),includinginWTcontrols.Furthermore,thedurationsof
thesechangeswereinsomecasessubstantiallydifferentfromthedurationofP/Sperturbations
(datanotshown).Whileseveralfactorscouldinfluencethis(includingthestateofthefusionpore
ordepthofendocytosedvesicles),themostlikelyexplanationmightbebasedonthefactthat
DiD,insomecases,exhibitedphotoactivationduringimageacquisition.Thiscouldbeseeninthe
correspondingrawimagestacks,andoccasionallyresultedinintensityversustimeplotswhere
P+2S steadily increased from the beginning until the end of the imaging period. However,
50
photoactivation of DiD should not have influenced P/S ratios, as both p- and s-pol elicited
emissionswouldchangeproportionatelywhenthisoccurred.
TherelationshipbetweenpHlpersistenceandP/Scurvaturecouldleadonetoquestion
whether this correlation was simply a byproduct of bleed through fluorescence signatures
betweenimagingchannels(488nm,561nmp,561nms).However,thiswasnotthecase,as
emissionspectraforpHlandDiDarehighlydivergent,withnearlynopHlemissionexceeding600
nm,andthelowestwavelengthDiDemissionbeginningat~625nm(Shenetal.,2014;Invitrogen
Corporation,Carlsbad,CA).Additionally,thesignalsforpHlandDiDoftendifferedconsiderably
indurationforindividualeventsandcouldbereadilydistinguishedinrawimages(e.g.pHlwould
beelevatedfor3.6sec,whileP/Sremainedhighfor45.6sec).Thisuncouplingwasobservedwith
all constructs, and suggests that while Syt persistence might contribute to maintaining
membranecurvature,thetwoparametersarenotalwaysintighttemporalsynchronization.
The C2B domain of Syt has been previously implicated in influencing fusion pore
dynamics.Wangetal.(2006)foundthatapointmutationinthethirdSyt-1C2Bloop,D363N,
resultedin~50%fewerfusioneventsinwhichthefusionporedilatedafterforming.Themutation
utilized intheWangetal. studyeffectivelyneutralizesoneof theconservedasparticacid (D)
residuesfoundintheCa2+-bindingpocketofbothWTSyt-1andSyt-7.Datafromthecurrentstudy
suggests that other non-conserved amino acid residues in the Ca2+-binding pocket can also
impact the ability of Syt C2B to influence fusion pore dilation. One potential source for this
influenceistheadjacentresidueatposition364.Syt-1possessesatyrosine(Y)residueinthis
position containing a hydrophobic, aromatic side chain, while Syt-7 (and therefore chimeras
Syt1:7-C2B3andSyt1:7-C2B1,2,3)hasalysine(K)residuewithahydrophilic,positively-chargedside
51
chain(Figure2.2).Thedifferentialpropertiesoftheseaminoacidsidechainscouldtheoretically
result in proteins with the K364 genotype forming tighter electrostatic interactions with
negatively-charged lipids (e.g. PIP2, Phosphatidylcholine, Phosphatidylserine) near the fusion
pore.
FurtherinsightcanbedrawnfromthepresumptivelocationoftheadditionalCa2+binding
siteinSyt-7C2B(Figure1.5).Thisthirdbindingsiteistheresultofcontributionsfromaserine(S)
andarginine (R) residuewithinC2B loop3 (positions368and369 respectively inFigure2.2).
Thesepositions inSyt-1areoccupiedbyaglycine (G)and lysine (K) residuerespectively.The
substitutionofthesetwoaminoacids(particularlythehydroxyl-containingserine)inSyt1:7-C2B3
andSyt1:7-C2B1,2,3,mayunderliethesignificantlylowerlikelihoodoftheirfusionporestorapidly
collapse(Figure3.3;Table3.3).
Xueetal.(2010)performedasimilarSytchimerastudyinSyt-1knockoutneuronswitha
slightlydifferentapproachthanthepresentstudy.Theyfirstgeneratedachimerainwhichthe
Syt-1 C2B domain was replaced entirely with the Syt-7 C2B domain, and then introduced
mutationsthatrevertedportionsofthedomainbacktotheirSyt-1genotype.Replacingtheentire
C2B domain resulted in a significant reduction in the amplitude of post-synaptic electrical
signaling (consistentwith adecrease in rapid full-collapse fusionevents typical ofWTSyt-1).
NoneofthereversionstheyintroducedintoC2Bcouldrescuethelossoftherobustrapidfusion
component.However,itisinterestingtonotethatthisstudyspecificallyavoidedrevertingthe
C2B Ca2+-binding loops, lending further support to the idea that these loops play a role in
governingfusionmode.
52
Studies have also established the ability of Syt C2B to undergo partial membrane
penetrationviainsertionofcriticalhydrophobicresiduesintotheplasmamembrane(Baietal.,
2002;Fernandez-Chaconetal.,2002;Baietal.,2004a).InSyt-1,theseresiduesarevaline(V)and
isoleucine (I) at positions 304 (loop1) and367 (loop3), respectively. Interestingly, Syt-7 has
substitutionsatbothpositions(V304IandI367L),andalthoughthesubstitutedaminoacidsare
alsohydrophobic,theymaystilldifferintheirdegreeofpenetrationandrelativeaffinitybased
on their structural differences. Bai et al. (2002) showed in vitro that Syt-1 C2B membrane
penetrationisonlyenabledbythepresenceoftheC2Adomain,andothermorerecentstudies
havesuggestedthatitisisoform-specificdifferencesinC2A,ratherthanC2B,thatunderliethe
disparatemembranebindingcharacteristicsofSyt-1andSyt-7(Brandtetal.,2012;Vermaas&
Tajkhorshid,2017).Therefore,additionalexperimentsusingC2Achimerasshouldbeconducted
toaddresstherelativecontributionsofthesedomainstomembranehandling.
Homo-oligomerizationisanothercommonlyreportedpropertyofSytthatmightexplain
theobserveddifferencesinpersistenceandfusionporestability.AstheroleofSytintriggering
exocytosisbegantobestudied,oneofthefirstobservationsresearchersmadewasthatSytcould
formhomo-oligomersinthepresenceofCa2+andanionicphospholipids,andthatthisproperty
wasspecificallydependentontheC2Bdomain(Sugitaetal.,1996;Chapmanetal.,1996;Wuet
al.,2003;butalsoseeUbachetal.,2001).Therefore,differencesintheC2Bsequencebetween
Syt-1 and -7 might translate into differences in how avidly these isoforms aggregate with
themselves. For example, multiple studies have shown that point mutations altering select
tyrosine(Y)or lysine(K)residuesintheSyt-1C2Bdomainimpedeoligomerizationandoverall
exocytosis invitro (Chapmanetal.,1998;Littletonetal.,2001).Asnotedabove,apotentially
53
significantsubstitution intheSyt1:7-C2B3chimera istheY364Kmutation,whichmighthavea
similarinfluenceonoligomerizationofSytsurroundingfusionpores.Evidencealsoshowsthat
Syt can form oligomers in a Ca2+-independent manner, potentially through N-terminal
interactions(Baietal.,2000;Wangetal.,2014).Althoughintheorythisinteractioncouldaffect
fusiondynamics,thedatapresentedinthecurrentstudyonlysupportaroleforC2B-mediated
interactions,astheN-terminiofallchimeraswereleftunchangedfromtheirSyt-1parentDNA.
Oligomerization of Syt has also been implicated as ameans for clustering other vital
componentsofthefusionmachinery.InthepresenceofCa2+,Syt-1canbindthet-SNAREproteins
SyntaxinandSNAP-25,andtheseinteractionshavebeenshowntobeC2B-dependentandcritical
forexocytosistoproceedcorrectly(Sollneretal.,1993;Chapmanetal.,1995;Chapmanetal.,
1996;Zhangetal.,2002;Baietal.,2004b;Lynchetal.,2008).Therefore,ithasbeensuggested
thatSytC2B-mediatedoligomerizationmightcreateascaffoldfortherecruitmentandproper
assemblyofSNAREcomplexesbetweenthevesicleandplasmamembrane invivo(Littletonet
al., 2001). Furthermore, Syt-SNAREbinding also appears to influence fusionpore stability, as
impairment of t-SNARE binding inhibits fusion pore expansion and promotes kiss-and-run
exocytosis(Lynchetal.,2008).ThisraisesthequestionofwhetherSyt-1andSyt-7differintheir
affinity for binding SNAREs. For example, the tendency of chimeras Syt1:7-C2B3 and Syt1:7-
C2B1,2,3(aswellasWTSyt-7)tomediateeventsinwhichthefusionporefailstorapidlyexpand
couldbepartiallyduetoweakerinteractionswithSNAREcomplexes.
Evenintheabsenceofoligomerization,interactionsbetweenmoleculesofSytandother
effector proteins can alter fusion pore stability. Research shows that Syt-1 can directly bind
Complexin (Cpx) -1 and -2 associated with SNARE complexes, and that this interaction is
54
enhancedbyCa2+(Tokumaruetal.,2008;Xuetal.,2013;Dharaetal.,2014).Currentmodels
suggest that Cpx acts as a fusion clamp, binding the SNARE complex to prevent premature
exocytosis,andthatSytmightdisplaceorinduceconformationalchangesinCpxtoreleasethe
clamp and promote fusion. Cpx overexpression has also been shown to impede fusion pore
expansion–aneffectthatismimickedbySyt-1knockoutinchromaffincells(Dharaetal.,2014).
ThesefindingspaintapictureinwhichCpxandSyt-1workinoppositiontooneanothertoeither
stabilizeordestabilizethefusionpore,respectively.However,interactionsbetweenCpxandSyt-
7havenotbeenwellcharacterized,raisingthepossibilitythatSyt-7ismorepermissiveofCpx-
mediatedfusionporerestrictionthanSyt-1.
How and why would organisms use multiple Synaptotagmin isoforms that impart
divergentoutcomesonsecretoryvesicles?PerhapsthebestpossibilityisthatSyt-1andSyt-7are
usedtofinetunetheenergeticcostsofsecretionfromadrenalchromaffincellsbasedonthe
current environmental context.Mild sympathetic nervous system (SNS) activity should favor
secretionfromSyt-7vesicles,whereasrobustSNSactivationduringperiodsofstressshouldalso
promoteSyt-1secretion(Fulop&Smith,2007;Raoetal.,2014).Theadvantageofthisschemeis
thatmild Syt-7 release (presumably constituting themajorityof anorganism’s life)wouldbe
characterizedmoreoftenbyaconstrictedfusionporeandkiss-and-runexocytosis,whereonlya
limitedamountofcargo(primarilysmallcatecholaminesversuslargerneuropeptides)wouldbe
releasedintothecirculatorysystem.Additionally,thestructuralcomponentsofthevesicle(i.e.
phospholipidsandintegralproteins)wouldremainlargelyintactandcapableofrapidrecycling
foradditionalroundsofexocytosis(Fulopetal.,2005).However,whenastressfulcircumstance
is perceived, the adrenal glands would be able to quickly effect physiological changes by
55
emptyingthefullcontentsofvesiclesintothebloodstreamviaarapidlyexpandingfusionpore.
Furthermore, the current study provides evidence that relatively small genetic alterations in
critical domains of Syt can lead to gradated changes in the secretory phenotype. This may
thereforehighlightapathforpastandfutureevolutionarydiversificationofSytisoformsinother
tissues.
Asidefromchromaffincells,Sytdiversitycouldplayaregulatoryroleinothercelltypes.
Forexample,hippocampalneuronsareknowntoexpressbothSyt-1andSyt-7,andithasbeen
shownthatstress induceschanges inmRNA levelsofSyt inhippocampal tissue (Sugitaetal.,
2001;Thomeetal.,2001).Deficitsinlearningandmemoryfunctionarewell-characterizedeffects
of chronic stress, and are likely due to impairment of long-term potentiation (LTP) in the
hippocampus(Kimetal.,1996).OnemechanismimplicatedintheprocessofLTPismodulation
ofpresynapticfusionpores.Specifically,thistheorysuggeststhattheconversionofglutamate-
associatedfusioneventsfromanon-expandingtorapidlyexpandingporephenotypeincreases
the signaling activity at a given synapse (the definition of LTP) by activating an additional
postsynaptic receptor type (Choi et al., 2000; Choi et al., 2003). Therefore, an attractive
hypothesiswouldbethatLTPinthehippocampusresultsfromacombinationofdown-regulating
Syt-7and/orup-regulatingSyt-1tomodifythepredominantfusionporephenotype.
Conclusions
Thecurrentstudyprovidesevidencethat theSynaptotagminC2BdomainCa2+-binding
loopsinfluencethepersistenceofSytatfusionsitesandthelikelihoodofvesiclefusionpores
undergoing rapidexpansionduringexocytosis.Thiswas investigated in livingchromaffin cells
throughpolarizedTIRFmicroscopyusingchimericSytproteinsinwhichtheC2BloopsofSyt-1
56
werereplacedbythoseofSyt-7.SubstitutionofallthreeC2Bloopsresultedinapronouncedshift
in theSyt-1phenotype towards thatofWTSyt-7.However, thisdidnot fullyaccount for the
difference observed between wild-type isoforms, suggesting that other domains should be
investigated for their relative contribution to these behaviors. Additionally, future research
should address whether swapping the C2B loops also leads to higher rates of kiss-and-run
exocytosisandslowingofcargorelease,ashasbeendocumentedforWTSyt-7.Nonetheless,the
resultspresentedhereprovidenovelandimportantinsightsintohowSynaptotagmin,aprotein
previously thought to act only in the steps leading up to fusion,may also play a role in the
molecularregulationoffusionporeexpansionduringthelaterstagesofexocytosis.
57
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ABSTRACT
SYNAPTOTAGMINC2BCA2+-BINDINGLOOPSIMPOSEDISTINCTEXOCYTOSISPHENOTYPES
by
MICHAELW.SCHMIDTKE
August2017
Advisor:Dr.KarenBeningo
Major:BiologicalSciences(Cellular,Developmental,&Neurobiology)
Degree:MasterofScience
Regulatedexocytosisfromchromaffincellsintheadrenalmedullaplaysacriticalrolein
maintainingorganismalhomeostasis.Intheabsenceofstress,thesecellsreleasephysiologically
relevantsubstancesintothebloodstreamonlyinlimitedquantities,whereasstressfulconditions
resultinarapiddelugeofsignalingmoleculesused,forexample,toincreaseheartrateandpain
tolerance. Although the cellular mechanisms governing the switch from low-level to stress-
inducedsecretionarenotwellunderstood,recentevidencehasimplicatedtheexocytoticCa2+-
sensingproteinSynaptotagmin(Syt)inthisrole.
TwoisoformsofSytareexpressedinchromaffincells(Syt-1andSyt-7),andeachissorted
toadifferentsecretoryvesiclepopulation.FusioneventsmediatedbySyt-7occurundermilder
stimulationconditionsandresultinslowerreleaseofvesiclecargothroughanarrowfusionpore
thatoftenreseals(“kiss-and-run”exocytosis).Conversely,Syt-1eventsrequirestrongercellular
stimulation(characteristicofastressresponse)andtypicallyresultinvesiclesfullycollapsinginto
theplasmamembranetorapidlyreleaseallcargo.Furthermore,Syt-7remainsclusteredatfusion
siteswhileSyt-1rapidlydiffusesaway.
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Although thedistinctphenotypesofSyt-1andSyt-7mayexplainhowchromaffincells
tunetheirsecretoryresponsetodealwithintermittentperiodsofstress,asignificantgapinthis
field is understanding the intermolecular basis for these phenotypic differences. The current
studyaimedtoaddressthisgapbyusingpolarizedTotalInternalReflectionFluorescence(pTIRF)
microscopytocomparethephenotypesofvariousSytchimeras,containingportionsofbothSyt-
1andSyt-7,withthebehaviorofthewild-type(WT)proteins.TheCa2+-bindingloopsoftheSyt
C2B domain were a prime candidate to target based on this domain’s established role in
exocytosisandCa2+-mediatedinteractionswithothermolecules.ChimerascomposedofaSyt-1
backgroundwithindividualC2BloopsconvertedtomatchSyt-7showedamildincreaseinSyt
persistenceatfusionsitesandslightinhibitionoffusionporeexpansionwhencomparedtoWT
Syt-1.TheseeffectswereenhancedsubstantiallywhenallthreeC2Bloopswereconvertedtothe
Syt-7 genotype, suggesting that the coordinated action of these loops serves as a major
determinantoftheexocytoticfusionmode.
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AUTOBIOGRAPHICALSTATEMENT
MICHAELW.SCHMIDTKE
MichaelreceivedhisBachelorofSciencein2011fromtheUniversityofMichigan–Ann
Arborwithaconcentrationinecologyandevolutionarybiology.Followinggraduation,heworked
as an ecological field research technician and volunteered in the mammals division at the
UniversityofMichiganMuseumofNaturalHistory.Healsospenttimeworkingasabiological
science technician for the U.S. Forest Service in 2013. Michael’s interest in behavioral
endocrinology and stress led him toWayne StateUniversity in the fall of 2014 to study the
molecularmechanicsofadrenalglandsecretion.