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GRADIENTSENSINGINEUKARYOTICCELLS:MECHANISMAND SPATIOTEMPORALDYNAMICS By KULANDAYANKASISUBRAMANIAN ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT OFTHEREQUIREMENTSFORTHEDEGREEOF DOCTOROFPHILOSOPHY UNIVERSITYOFFLORIDA 2004 Copyright2004 by KulandayanKasiSubramanian Dedicatedtomylovelyfamily,GuruandDeivam. ACKNOWLEDGMENTS Iwishtoconveymysincerethanksandappreciationtomymentor,Dr.Atul Narang,forhisguidance,constantencouragementandconstructivecriticism.Iwouldlike tothankmyco-advisor.Dr.GerryShawforhispatientguidanceandencouragement.I wouldalsoliketothankthemembersofmycommittee.Dr.RangaNarayanan,Dr.Lewis Johns,Dr.RichDickinsonandDr.DanielPurich,fortheiradviceandavailability.Itake thisopportunitytothankDr.OrionWeiner,Harvard,forhissupportwiththeHL60cell line.IalsowishtothankDr.SergeiPilyuginforhisadviceregardingthemodelanalysis. Iwouldliketothankmygroupmembers,Shakti,Eric,Ved,Jason,Greg,Ashely, Brentandothergraduatestudentsinthedepartmentfortheirsupportandfriendship.Iam verygratefultothemembersoftheShawlab,CuiandSilas,fortheirhelpandlaboratory assistance.IowemanythankstoSaurabh,LyleandFieldfortheirhelpduringthecourse ofthework.IamgratefultoTimVaught,MicroscopyCoreFacility,forhelpwith microscopy.SpecialthanksgotoSriks,KK,Thamba,Hari,Omar,Jeff,Maddy,Subby, Vijay,Subbu,Bulletandallmyotherfriendsfortheirconstantsupportandhelp. Finally,Iwanttoexpressmydeepgratitudeandloveformyparents,brothersand otherfamilymembersfortheirunwaveringsupportandencouragement. IV 58 TABLEOFCONTENTS Page ACKNOWLEDGMENTS iv LISTOFTABLES viii LISTOFFIGURES ix ABSTRACT xi CHAPTER 1 GRADIENTSENSINGINEUKARYOTICCELLS 1 Introduction 1 IdentificationoftheFirstPolarizedComponent 4 SpatiotemporalDynamics 7 PolarizedSensitivity 8 AdaptationandSpontaneousPolarization 9 ProposedmodelforGradientSensing 10 2 MODELFORGRADIENTSENSING 1 SignalTransductionPathway:ThePhosphoinositideCycle 15 Model 1 Receptor-ligandBinding 18 AdaptationSubsytem 19 PolarizationSubsytem 19 BoundaryandInitialConditions 21 DimensionlessEquations 22 ModelEquations 23 Simulations 24 FastResponses 25 Phosphoinositidelocalization 27 Uniquelocalization 29 Thequasisteadystateasafunctionofthechemoattractantstimulus 30 Polarizedsensitivity 33 SlowResponses 38 Adaptation 38 Spontaneouspolarization 39 v 3 MATHEMATICALANALYSIS 43 SimplifiedPolarizationSubsystem 44 EvolutionofthePhosphoinositideLocalization 46 FirstPhase 47 SecondPhase 48 ConstructionoftheNon-homogenousSteadyState 48 OuterSolution 49 InnerSolution 56 ZerothOrderApproximationtoCompleteSolution 59 InstabilityoftheHomogenousSteadyStatewith RespecttoChangeink{ 61 4 EXPERIMENTALANALYSIS 66 ThePositiveFeedbackLoop 67 Protocols 71 CulturingandDifferentiationofHL60cells 71 TransientTransfectionbyElectroporation 71 MotilityAssays 72 Preparationofcells 72 Livecellimaging 72 FixingandImmunostaining 73 Results 75 BiophysicalExperiments 75 Spatiotemporaldynamicsofgradientsensing 75 Spontaneouspolarizationasafunctionofthe chemoattractantconcentration 76 BiochemicalExperiments 77 RoleofPI5KinthePositiveFeedbackLoop 77 ConstructionoffluorescentPI5KIamutantsandCoronin 78 AssayingtheefficacyofthefluorescentconstructsinCos7cells 79 ExpressionanddistributionofPI5KIain<7HL60cells 81 PI5KIaregulatesPIP3polarityinafHL60cells 82 5 CONCLUSIONS 85 FastResponses 85 SlowResponses 86 vi APPENDIX A NOMENCLATURE 89 B ORDERSOFMAGNITUDEOFPARAMETERS 91 LISTOFREFERENCES 93 BIOGRAPHICALSKETCH 99 vii 1- 2- LISTOFTABLES Table page 1.Modelsofgradientsensing 12 1.Dimensionlessparametervaluesusedinsimulations 24 A-l.Modelparameters 89 A-2.Greeksymbols 90 viii LISTOFFIGURES Figure page 1-1.Thefivephasesofthechemotacticcycle 1 1- 21--2.Signalinginvolvedingradientsensing 3 1-3.Theprincipleofthefluorescentimagingexperiments 5 1-4.Simplemodelforlocalizationofphosphoinositides 6 1-5.Schematicrepresentationofthespatiotemporaldynamics 7 6.Dependenceonpre-existingpolarity 8 1.Thephosphoinositidecycle 16 22--2.Thekineticschemeofthemodel 17 23--3.Phosphoinositidelocalization 26 2-4.Uniquelocalization 29 2-5.Variationofthequasi-steadystateasafunctionofchemoattractantstimulus 31 2-6.Responseofapre-existingphosphoinositidepeaktoanewgradient 34 2-7.SimulationofdatainshowninFigure1-6 36 2-8.Adaptationinresponsetoauniformincreaseinthe chemoattractantconcentration 38 9.Spontaneouspolarizationinresponsetoauniformbut noisychemoattractantprofile 40 1.Steadystatesofthereducedmodel 46 3-2.Nullclinedn/dr= 0 47 3-4.Phaseportraitsforeachbranchof/{n,i)=0 50 3-5.Patchedphaseplanefortheoutersolution«(£) 52 IX . 3-6.Outersolutionn{^) 56 3-7.Heteroclinicorbit(AB)representinginnersolutionn'(y) 59 33--8.Zerothordersolutions(dashedlines)incomparison 4- withthenumericallycalculatedcompletesolution(solidlines) 60 3-9.PlotsofDet(Mo)andTrace(Mo)asparameterKfisvaried 63 10.Turingbifurcationofthehomogenousstudystate 64 1.Kineticschemeofthehypothesizedpositivefeedbackloop 67 4-2.Componentsofthepositivefeedbackloopinaresting cellandinapolarizedcell 68 4-3.SpatiotemporaldynamicsofPIP3localizationinc/HL60cells 74 4-4.Spontaneouspolarizationasafunctionofchemoattractantconcentration 76 4-5.Quantificationofpolarity 77 4-6.DistributionofendogenousPI5KIainPDGFstimulatedCos7cells 79 4-7.RuffleinhibitioninPI5KIaA?itransfectedCos7cells 79 4-8.ExpressionanddistributionofPI5KIainrfHL60cells 80 4-9.OverexpressionofPI5K 81 4-10.Controlexperiment 82 4-11.InhibitionofPI5K 83 4-12.PI5KregulationofPIP3polarization-Quantificationofstudy 83 x