University of Wyoming
Wyoming Scholars Repository
Honors Theses AY 16/17
Undergraduate Honors Theses
Spring 5-13-2017
The Limiting Effect of Cytoplasmic Volume on
Microtubule Dynamics
Jacob Zumo
[email protected]
Follow this and additional works at: http://repository.uwyo.edu/honors_theses_16-17
Recommended Citation
Zumo, Jacob, "The Limiting Effect of Cytoplasmic Volume on Microtubule Dynamics" (2017). Honors Theses AY 16/17. 53.
http://repository.uwyo.edu/honors_theses_16-17/53
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TheLimitingEffectofCytoplasmicVolumeon
MicrotubuleDynamics
JacobZumo
Spring2017
B.S.inMolecularBiologyandMicrobiology
HonorsProgram
PrincipleInvestigator:Dr.JesseGatlin,MolecularBiology
UniversityofWyoming
Abstract:
Mitotic spindles play a key role in cellular division. These structures, which are composed of
dynamic filaments called microtubules, are responsible for separation and segregation of
chromosomesduringmitosis.Spindlesmustbethecorrectshapeandsizetoinsurefidelityof
this process, however, their formation and assembly are still not entirely understood. For
example,themechanismsthatgovernspindleshapeanddetermineindividualspindlesizefora
givencelltypearestillunknown.Basedonevidencefromrecentstudiesofspindlescaling,in
whichspindlesizeeffectivelyscaledwithcellsize,wehypothesizethatwithinsmallcytoplasmic
volumes,spindlebuildingblocksbecomelimitingandtherebylimitspindlesize.Thishypothesis
predictsthatmicrotubuledynamicswithinthespindlewillbeadverselyaffectedbychangesin
cytoplasmic volume. By combining cell-free cytoplasmic extracts, microfluidics, and confocal
microscopy,wehopetomeasurechangesinmicrotubuledynamicstoelucidatetherelationship
betweencellvolumeandspindlesizewithinthecell.
Background:
Cellulardivisionisatopicmostindividualscanconceptualize.Foracelltofaithfullyreplicateand
divide,itmustensurethatthenewdaughtercellsaregivenequalamountsofthesamegenetic
materialthatwasproducedduringDNAreplication.Mitoticspindlesplayakeyroleincellular
division. They are responsible for the segregation of chromosomes into daughter cells during
mitosis. These spindles are composed of microtubules, filamentous polymers made up of
heterodimeric protein subunits called tubulin (Feit et al, 1971). Microtubules are inherently
dynamicfilamentsinthesensethattheymustgrowandshortentofunction,e.g.tobindtoand
separate the chromosomes during mitosis. The dynamics of microtubules are determined by
several different parameters including growth rate, shrinkage rate, and the frequencies of
transitionsbetweenthetwostates(AkhmanovaandSteinmetz,2015).Itmakessensethatthese
dynamicsmightaffectspindlesize.Indeed,therateatwhichmicrotubulesgrow,thetubulinmass
thathasbeenincorporatedintothemicrotubules,andtheaveragelengthofthemicrotubulesall
affectspindlesize.Forexample,withinXenopuslaevis,abalanceofmassisrequiredbetween
therate of tubulinentering spindleandthatoftubulinleavingspindleassembly(Reberetal,
2013).
Inordertomaintainhomeostasis,everylivingcellmustbeabletoregulatethesizeofitsinternal
partsregardlessofitsownsize.Thegeometricrelationshipistermedscalingandcomesintwo
forms:isometricscalingandallometricscaling.Whentwoobjectsthataregeometricallysimilar,
forexampleasmallequilateraltriangleandalargerequilateraltriangle,theyaresaidtoexhibit
isometry.Isometricscalingwouldmeanthattherelationshipsbetweenvolumeandsurfacearea
would remain constant between different sizes of cells because the cells are geometrically
similar.Allometryreferstoachangeinshapeinresponsetoachangeinsize.Thismeansthat
allometric scaling would result in not only a change in size, but shape. Scaling occurs at the
extremeofcellsizes-bacteriaassmallas1µmindiameterandX.laevisblastomeres,whichcan
beupto1.2mmindiameter(some1000timeslarger)bothmustscaletheirreplicationmachinery
asameansforsurvival.Ineukaryotes,failureofchromosomeseparationcanleadtoaneuploidy,
which can lead to problems like Down’s Syndrome and cancer (Fang and Zhang, 2011). This
scalingchallengeisessentialfornormaldevelopment.Spindlesmustbeinthecorrectsizeand
shapeinordereffectivelyensurefidelityofthisprocess.Dynamicsofmicrotubulesaffectspindle
sizeandshape(DumontandMitchison,2009).Itisgenerallyobservedthat,upuntilapoint,as
celldiameterincreases,sodoesspindlelength(Wühretal,2008).Thisistrueupuntilacertain
point where relationship plateaus (Wühr et al, 2008). This suggests that to the relationship
between cell size and spindle size could be regulated by limiting components, changes in
cytoplasmic composition that accompany developmental progression, or negative feedback
inducedbystericallyconstrainingspindlesinsmallvolumes.
Itisknownthatenzymesareinvolvedintheregulationofspindlesizehoweverthisprocessisnot
entirelyunderstood(Reberetal,2013;Petry,2016).Currently,a“limitingcomponent”modelis
generallyaccepted(Goodetal2013,Hazeletal2013).Thismodepositsthatforasystemof
microtubules(e.g.aspindle)isbuiltinsmallcellularenvironment,thecytoplasmicconcentrations
ofspindlecomponentsandenzymesrequiredtoassemblethemwillbemoregreatlyaffected
thaninalargercellbuildingthesamestructure.Forexample,theenzymeXMAP215,whichis
partofafamilyofproteinsthatpromotesmicrotubulegrowth,candirectlycatalyzetheaddition
upto25tubulindimerstothegrowingendofthemolecule(Brouhardetal,2008).Thisleadsto
thegeneralquestionofnotonlyhowdoescellsizedeterminespindlesize,butalsotherateat
which spindles are formed. We hypothesized that if spindle assembly occurs in a larger cell
volume,thenthesespindleswillbeformedatafasterratethancellsofasmallervolumebecause
they would be able to maintain a saturating number of enzyme molecules per growing
microtubuleend.Inasmallcellularvolume,therelativestartingconcentrationofthatenzyme
mightstillbethesame,howeverthenumberofenzymemoleculespermicrotubulegrowingend
wouldbesub-saturating,resultinginaslowerrateofmicrotubuleassembly.Cellsofdifferent
volumeswouldstillhavethesameconcentrationsofthisenzyme.However,oncemicrotubules
begintoassemble,andtheenzymesthatbindthemareeffectivelypulledoutofsolutiontoadd
tubulin subunits to growing ends, lowering the degree of saturation of this enzyme on those
growingends(Reberetal,2013;Brouhardetal,2008).
X.laeviseggextractshavealongandextensivehistoryforuseasmodelcell-freesystems.In
1985, Lohka and Maller demonstrated that nuclear envelope breakdown, chromosome
condensation,andspindleassemblycouldallbestudiedivvitrousingextractsofamphibianeggs
(LohkaandMaller,1985).Thissystemhasbeenusedtoresearchseveralfundamentalbiological
processes ranging from analyzing the effect cigarette smoke has on the inhibition of CFTR
expression,tostudyingstabilityoftheDNAreplicationfork(Moranetal,2014;Hashimotoand
Costanzo,2011).
X.laevisfrogswereinjectedwithhormonessotheycouldlayeggs(asdescribedinDesaietal,
1999).Oncetheeggswereharvested,theywerewashedwithbufferandde-jellied,andpacked
into a test tube to remove any excess buffer. A crushing spin produced a stratified and
fractionatedextractfromwhichthecytoplasmiclayerwascollected.Thiscytoplasmicextractis
whatweusedtovisualizeandanalyzemicrotubulegrowth.Thereareseveraladvantagestousing
thiscell-freesystem.Itallowsforeasycontrollingofinvitroprocesses,withproteinbiosynthesis
beingespeciallyimportantforthisexperiment.Additionally,theabsenceofacellularmembrane
usuallyallowsforafasterreactionthenmicrobialsystemswithcellularmembranes(Zhuetal,
2013;MartíndelCampoetal,2013).
Dropletmicrofluidicsallowustoperformlaboratoryoperationsonverysmallscales(Mashaghi,
etal,2016).Wecouldtakeourcytoplasmicextractandemulsifyitwithasurfactantandoilphase.
Asthenameimplies,thisisdoneatamicroscopicscale,atasizewhichwecancontrol.Fillinga
singledevicecanprovidethousandsofindividualextractdroplets.Thisprovidesuswithflexibility
tosearchunderthemicroscopeforthedropletswithspecificconditionsweareinterestedin
studying.
Weutilizedthissystemtotestourpredictionthatlargercellularvolumeswillresultinafaster
rateofmicrotubulegrowthandultimatelylargerspindles.Byusingcell-freecytoplasmicextracts,
wedidnothavetorelyongeneticinvivocontrolofcellsize.Byinsertingfluorescentlylabeled
proteins we could visualize and analyze microtubule growth rates using confocal microscopy.
Usingdropletmicrofluidics,wecouldcontrolthesizeoftheextractdropletsinwhichmicrotubule
structureswereassembled,whichisanalogoustocontrollingthevolumeofthecell.
MaterialsandMethods:
Extractandproteinsolutionpreparation-500µLofcytoplasmicextractfromtheeggsofX.laevis
was prepared according to Desai and kept on ice (Desai et al, 1999). A genetically encoded
microtubule,consistingofthemicrotubulebindingdomainoftheTauprotein(Elieetal,2015)
was fused to mCherry (mCherry-Tau), expressed and purified in E. coli, and stored at a stock
concentrationof180µM(workingconcentrationwas~1µM;Mooneyetal,inpress),wasused
to visualize microtubules emanating from artificial microtubule organizing centers (aMTOCs;
Fieldetal,2017;TsaiandZheng,2005)usedataconcentrationtoachieve0to1aMTOCsper
extractdroplet.End-bindingprotein1fusedtoGFP(EB1:56µM)wasaddedtothemixtureata
1:100dilution.Both100xcalciumat40mMand100xcycloheximideat35mMweredilutedtoa
a1:100workingconcentration.Thisextractmixturewaskeptoniceuntildropletscouldbemade.
Assembly of droplets- A microfluidic-based platform for confining spindle assembly to desired
dropletsizeaccordingtoHazeletal,2013.Picosurfsurfactantwasusedtosuspendtheextract
mixture,andresponsibleforformingtheextractintodroplets.Differentsizeddropletdevices
wereusedtoallowforsmallandlargedropletsizesandvolumes.1mLNormJectsyringeswere
usedtoinjecttheextractmixtureandthesurfactantatprecisevolumesutilizingsyringepumps
(CetoniCorp).Thesyringesanddropletdeviceswerekeptoniceuntilreadyforuse.Thedevices
werefilledinaroomat0°Ctolimitthetemperature-sensitiveassemblyuntilreadytobeviewed
ontheconfocalmicroscope.ThemixingoftheextractandsurfactantataT-junctionwithinthe
deviceallowedforthesuspensionofextracttobecomestabilizeddropletsofrelativelysimilar
size.Afterfillingthedevicewithdroplets,thetubeswereremovedandtheholeswerecovered
withnailpolishtoactasaseal.Thedevicewasplacedonicepriortoviewingundertheconfocal
microscope.600µLofextractallowedtwotothreedevicestobefilledatonce.Thisexperiment
utilizeda63µLdevice,anda113µLdevicetoensuredropletsofdifferentsizesandvolumescould
beanalyzed.
Imagining spindle formation- Imaging of the spindle assembly was completed on a confocal
microscopeat60xmagnificationunderoilimmersion.Time-lapseswerecompletedat2.5second
intervalsforthreeminutes.EB1proteintrackingwasdonewiththemTrackJpluginontheimage
analysissoftwareFiji.
Figure1:A,theprocessedeggextractinwhichaMTOCs,mCherry-Tau,andEB1-GFPwereadded.B,adiagramofthe
T-junctiondevicesusedtocreatethedroplets(Gatlin,2017).Anoilphasecontainingsurfactantwaspumpedintothe
devicethroughoneinlet,whiletheextractandproteinsolutionenteredfromanother.AftermeetingattheTjunction,thesurfactantemulsifiedtheextractintodroplets.Cshowsthedropletsbeingformedasviewedfroma
microscopeunderthe20xobjectivelens.DdemonstratestherelationshipofEB1andtubulinusingimagescaptured
bytheconfocalmicroscope.EB1protein(pseudocoloredred)attachedtothegrowingendsofthemicrotubules
(pseudocoloredgreen).Thesefluorescentlytaggedproteinsweretrackedtoanalyzemicrotubuleassemblyvelocity.
Data:
MicrotubulegrowthratesfromindividualaMTOCsweremeasuredintwodropletsofdifferent
sizes(Fig2).Thefirstdroplethadadiameterof62µm,andtheseconddroplethadadiameterof
86µm.Manualtrackingwasusedtomeasurethevelocitiesoftwentygrowingmicrotubuleends
usingtheMtrackJplugininFiji(NIH).Themeanvelocityofallofthecomets’averagevelcocities
wasdeterminedforeachdroplettoassesaveragemicrotubuleassemblyvelocityforeachdroplet
Figure2:A-BsingleimagesobtainedviaconfocalmicroscopyofEB1andtubulinofthe62µmdroplet,respectively.CDsingleimagesobtainedviaconfocalmicroscopyofEB1andtubulinofthe86µmdroplet,respectively.Etheaverage
velocitiesofthemicrotubuleassemblybetweenthetwodropletsizes.
size.The62µmdroplethadanaveragemicrotubuleassemblyvelocityof20.706µm/minute,with
astandarddeviationof0.0537.The86µmdroplethadanaveragemicrotubuleassemblyvelocitiy
of 20.571µm/minute, with a standard deviation of 0.0396 (Tables A-B). A two-tailed t-test
reportedthevalueof0.9suggeststhatthereisnostatisticaldifferencebetweentheaverage
microtubleassemblyvelocityofthesetwodroplets.
TablesA-B:Minimum,maximum,andaveragevelocitiesof20differenttrackedEB1cometsforthe
62µmand86µmdroplets.
Discussion:
We sought to determine the effect cell size has on the rate of microtubule assembly. As
discussed, the limiting component model suggests that smaller cells would assemble
microtubules at a slower rate than larger cells. The absolute concentration of enzymes and
tubulinrequiredformicrotubulegrowthwoulddepletedfasterinadropletwithasmallervolume
thanadropletwithalargevolume.Alargecellwouldhavealargecytoplasmwithmorevolume
thanasmallcell.Whenmicrotubulesareconstructedinthecell,thismeansthattherewouldbe
moreenzymesandresourceswithinthecytoplasmthatarereadilyavailablecomparedtocells
ofasmallervolume.Whenexperimenting,weworkedtotestthismodelwhileoperatingunder
acoupleassumptionsthatwerenotassessed.First,webelievedthateachsizeddropletwould
havethesamerelativeinitialconcentrationofbuildingblocks,especiallyXMAP215,requiredfor
microtubule assembly. Secondly, we assumed that the number of growing microtubule ends
wouldbethesameforeachsystem,i.e.thatthisparameterwouldnotscalewithcytoplasmic
volume.ThismeansthatthesamenumberofXMAP215moleculesthatwerebeingdrawnoutof
solutiontofacilitatemicrotubuleassemblywouldbethesameforeachgrowingmicrotubuleend
despitedropletsize.Aspreviouslystated,eachmoleculeXMAP215canadd25tubulindimersto
thegrowingendofthemicrotubulesperbindingevent(Brouhardetal,2008),whichweassumed
wastrueofeachdropletsize.
Weexpectedtheaveragemicrotubuleassemblyvelocityofthesmallerdroplettobeslowerthan
thelargerdroplet.Theresultsofthisexperimentarenotwhatwepredicted.Thisprovidesus
informationonwheretotaketheexperimentsinthefuture.Itispossiblethatthedifferencein
sizebetweenthetwodropletsinthisexperimentwasnotsubstantialenoughtomakeastatistical
difference in microtubule assembly velocity. Previous research has suggested the maximum
velocityofgrowthofamicrotubule,suchthatoccurswhenallpossibleXMAP215bindingsitesat
thegrowingendouroccupied,isbetween20-25µm/minuteThisisclosetowhatwasobserved
inbothdropletsizesexploredinthesestudies.Thismightsuggestthatallmicrotubuleendsare
saturatedeveninthesmallerdroplet,however,thisisinconsistentwithpreviousresultsfrom
the Gatlin lab (unpublished data). Additionally, we would expect more growing ends to be
producedbyaspindlerelativetoasingleaMTOC.ThiswouldimpactthenumberofXMAP215
pulledfromsolutionandwhethereachgrowingendisfullysaturatedwithXMAP215molecules.
Thiscouldexplaindiscrepancieswithpublishedspindlescalingdatawhichshowthatspindles
begintoscalewithcellsizewhendiametersarelessthan~150um(Goodetal,2013;Hazeletal,
2013).Futureexperimentscouldnotonlytestsmallerandlargerdropletsizesthantheones
usedforthisexperiment,butalsoincreasethenumberofdropletsanalyzedforthisrelationship.
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