Gradient sensing in eukaryotic cells: mechanism and spatiotemporal dynamics

2004·4.2 MB·English

by  Subramanian, Kulandayan Kasi

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