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1-1-2017

Synaptotagmin C2b Ca2+-Binding Loops ImposeDistinct Exocytosis PhenotypesMichael W. SchmidtkeWayne State University,

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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

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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

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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

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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.

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