st. jude presentation 2016
TRANSCRIPT
G.TravisTaborDepartmentofBiology
AppalachianStateUniversity,BooneNC28608St.JudeNURS2016
Actin ECMInvadopodia
• Actin-dense,lipid-richventralprotrusionsofthecellmembrane• ExertmotiveforceonandmediateremodelingofECM• Putativelyinvolvedinthemetastasisofcertaincancers
InvadopodiaStructureandFunctioninSrc-3T3CellsF-actin Nuclei Gelatin
CancerCell2005,7(2),155-165.2
• SrcTyrosinekinasesubstratecontaining5SH3domains• ScaffoldsmachineryrequiredforECMadhesionanddegradation• PXdomainassociateswithlipids(PtdIns(3,4)P2)• SH3likelyinteractswithproteins(Nck,WASp,Grb2)• InducesinvadopodiadevelopmentandECMremodelingactivityinLNCaPcells
Tks5LocalizationandFunctionwithinInvadopodiaJ.BiolChem.2003,278(19),16844-16851.J.CellSci.2009,122(15),2727-2740.Prostate2014,74(2),134-148.
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PX SH3A SH3B SH3C SH3D SH3EY
SpliceSiteB+/-28aa
PX SH3A SpliceSiteBSpliceSiteA
LocationoftheTwoSpliceSitesinTks5
SpliceSiteA+/-15aa
• Twoalternativesplicesites(AandB)giverisetofourspliceforms:(short),A,B,andAB
WehypothesizethattheTks5spliceformsdifferentiallyinduceinvadopodiaactivity,thusmakingTks5alternativesplicingusefulfortrackingtheprogressionofprostatecancersinpatients.
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Harvestcells
Incubatecellsfor48hoursat37°Cina12-welldishcontaininggelatin-coatedcoverslipsanda6-welldishforlysates
HarvestslipsStain
NormalizegelatindegradationtonumberofnucleiusingImageJ
+mTks5plasmid
Transfection
+
WesternBlot
Tks5
GAPDH
PreparelysatesProteinassay
J.Vis.Exp.2012,66,e4119.MethodsMol.Biol.2011,769,111-136.
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ComparisonofGelatinDegradationInducedbyOverexpressionofEachmTks5SpliceForminLNCaPCells
Mock Short A
B AB Src-3T3Tks5
GAPDH
Mock Short A B AB
SpliceForm
Nuclei Gelatin
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• 9-11imagesanalyzedforeachtreatment• Tks5-A,B,andABinducedsignidicantlymoregelatindegradationthan(short)whenoverexpressedinLNCaPcells
0200400600800100012001400160018002000
Mock Short A B AB
MeanGelatinDegradation
(areaunits/cell)
mTks5SpliceForm(n=3)(n=3) (n=3) (n=3) (n=2)
(p=0.022)
(p=0.034)
(p=0.010)
*
**
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PX SH3A
PX SH3A SpliceSiteBSpliceSiteA
+++
+
=Short=A=B=AB
• SYBRGreenSystem• Ampliconlengths:281-282bp• Meltingtemperatures:59.6-60.6°C(IDTOligoAnalyzer)• Noprimerdimerinteractionspredicted(ThermoFisher)
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CloningStrategiesforGeneratinghTks5Constructs
A. hTks5(short)wasgeneratedbydeletingSpliceSiteBfromhTks5-BB. hTks5-AandABweregeneratedbyinsertingSpliceSiteAintohTks5(short)
andBrespectively
A
B
SH3A SpliceSiteB
Tks5-B
pcDNA3
SH3A
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MW Sh A B AB + MW
pcDNA3-hTks5-B+
hTks5PrimerSet
• Predictedamplicon:281bp• Annealingtemperature=51°C,30cycles• 2%agarosegel(0.5XTBE,5E(-5)%ethidiumbromide)
AnalysisofhTks5RT-PCRPrimerSpecidicity
300bp
+=PrimerSetTargetingConstitutiveExon
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-0.200
0.000
0.200
0.400
0.600
0.800
1.400
1.200
1.000
60 65 70 75 80 85 90 95
Temperature(°C)
Derivative
• SuperimposedanalysesofstandardcurveandHeLacDNAqRT-PCRproducts• Nosignidicantoff-targetartifacts/dimers• LowexpressionatRNAlevel(~8.98±0.35copies/ngtotalRNA)
MeltingCurveAnalysisofhTks5(short)PrimerSpecidicity
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G=GAPDHPrimerSet(35cycles)
MW Sh A B AB G MW
LNCaPcDNA+
hTks5PrimerSet
PreliminaryRT-PCRAnalysisofTks5SplicinginLNCaPCells
300bp
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• SeeminglylowmRNAexpression,consistentwithwesternblot• Moreinvasiveprostatecancerlines(PC3,DU145)mayexhibitdifferent
splicingprodiles
Tks5
GAPDH
Mock Short A B AB
SpliceForm
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1. ThethreelongerTks5spliceformsinducedsignidicantlymoregelatindegradationthantheshortformwhenexogenouslyexpressedinLNCaPcells.
2. AqRT-PCRstrategyfordeterminingtheTks5spliceformexpression
prodileofhumancarcinomacelllineshasbeendevelopedandiscurrentlybeingassessedforitsvalidity.
3. IfTks5alternativesplicingisfoundtobeatissue-specidic
phenomenonindicativeoftumormalignancy,theaforementionedqRT-PCRstrategymayprovetobeanoveldiagnostictool.
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• JoeyH.Breeyear,cloningofmTks5constructs• MatthewC.Sanderson,futureqRT-PCRanalysis
• DarrenF.Seals,researchmentor• AppalachianStateUniversity,primaryfunding
• ASUOfdiceofStudentResearch,UndergraduateResearchAssistantshipaward
• ASUHonorsCollege,PartnershipBoardResearchFundaward
• Drs.SheaTubertyandEceKaratanofAppState,reagents• Dr.ClareAbramofUCSF,someoriginalmTks5constructs
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