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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