• I.Solutions&Supplies
• II.PrepolymerizationClarification
• III.GTPPolymerization
• IV.TaxolPolymerization
• V.GMPCPPPolymerization
• VI.DeterminingConcentrationofGMPCPP/TaxolMTs

Backtoprotocols

I.Solutions&Supplies

BRB80(1X):80mMPIPES,1mMMgCl₂,1mMEGTA,pH6.8withKOH(generallymadeasa5Xstockandstoredat4¡C)

100mMGTP

100mMGMPCPP

Taxol:10mMstock;100µM,10µMand1µMdilutions(and/or200µM,20µMand2µMdilutions)allinanhydrousDMSO;Taxolissoldunderthetradename"Paclitaxel"bySigma

RecycledTubulin

LabeledTubulin(optional)

II.PrepolymerizationClarification

Althoughnotabsolutelynecessary,werecommendmixingtubulinandnucleotidesin1XBRB80for5"oniceandthenclarifyingthismixusingaTLA100rotorfor5"at90Kat2¡C.WeespeciallyrecommendthisclarificationwhenpolymerizationincludesGMPCPPand/orhighlylabeledfluorescenttubulins.Wealsorecommendaclarificationspinonthawedlabeledtubulinspriortomicroinjection.

III.GTPPolymerization

1.Onicemixunlabeledtubulinandlabeledtubulinatanappropriateratioin1XBRB80with1mMDTTand1mMGTP.Incubateat0¡Cfor5".

2.ClarifymixinTLA100rotorat90Kfor5"at2¡C.

3.Collectsupernatantandincubateat37¡C.Ifthetubulinconcentrationis2mg/mlorhigher,assemblywillproceedrapidlytosteadystate(~30").Iftheconcentrationislower,nucleationcanbelimitingandtheprecisekineticsofapproachtosteadystateisdifficulttopredictandwilldependontheamountofactivetubulininyourmix.Formanyexperiments,weaddseedstosurmountthenucleationbarrier,therebyspecificallyassayingelongation--forthisweroutinelymakeGMPCPPseeds,washoutanyfreeGMPCPPandaddasmallvolumeoftheseeds(~1/20-1/50vol)afterthepolymerizationmixhasbeenat37¡Cfor1".Axonemesorcentrosomescanalsobeusedasnucleatingstructures.Ifthepurposeislabelingorrecyclingthetubulin,polymerizationispromotedbyadditionofDMSOorglycerolasdescribedabove.

IV.TaxolPolymerization

1.Onicemixunlabeledtubulinandlabeledtubulinatanappropriateratioin1XBRB80with1mMDTTand1mMGTP.Incubateat0¡Cfor5".

2.ClarifymixinTLA100rotorat90Kfor5"at2¡C.Incubatesupernatantat37¡Cfor1"-2".

Nowtherearetwooptions:

I.Addtaxolstepwisetoequimolarasfollows(for1mg/mltubulin):

(Pipetinthetaxolandimmediatelyflickthetubetomixitin)

Add1/10vol1µMtaxol;Incubateat37¡Cfor5"-10"

Add1/10vol10µMtaxol;Incubateat37¡Cfor5"-10"

Add1/10vol100µMtaxol;Incubateat37¡Cfor15"

Pelletmicrotubulesoverawarm40%glycerolinBRB80cushioninaTLA100,100.2or100.3rotor,aspirateandwashsample/cushioninterface,rinsepelletandresUSPendinwarmBRB80+1mMDTT+10-20µMtaxol(taxolshouldbeatleastequimolarandpreferablyinexcesstothetubulin)

Note:Iftaxolisaddedallatonceitwillcausetubulinprecipitation!Ifpolymerizing2mg/mltubulin,use2µM,20µMand200µMsteps

ORII.Add10%(v/v)DMSOandincubateat37¡Cfor20"-30"

Pelletandresuspendmicrotubulesasdescribedabove

ForDMSOpolymerizationitisbesttohavehightubulinconcentrations(5-10mg/ml)intheoriginalmixbeforeaddingDMSO.However,MTsmustberesuspendedafterpelletingwithequimolartaxol.

A"quick-and-dirty"taxolpolymerizationmethod(popularinmotorlabswheretaxolmicrotubulesareusedassubstratesformotility/ATPaseassays):

1.ThawrecycledtubulinstoredinIB(generally5-20mg/ml)

2.Addequalvolume2XBRB80+2mMDTT+2mMGTP+20%DMSO

3.Incubateat37¡Cfor20"-30"

4.Pelletmicrotubulesandresuspendasdescribedabove

Ifthereislabeledtubulininthemix,dilutethemicrotubulesto1-10µg/mlandcheckunderafluorescentmicroscope.Taxol-stABIlizedmicrotubulescanbeshearedbydilutingthemto~100µg/mlandthenpassingthemthrougha27gneedle~5-6times.Alldilutionsoftaxol-stabilizedmicrotubuleshouldbedoneintobufferscontaining10-20µMtaxol.

V.GMPCPPPolymerization

GMPCPPisthebestcurrentGTPanalogfortubulinpolymerization.Itsmajorlimitationislackofcommercialavailability.Inthepresenceofpotassiumascounterion,GMPCPPisveryslowlyhydrolyzedwithinthemicrotubulelattice,andisessentiallynon-hydrolyzablewithinthetimecourseofmostexperiments.Inthepresenceofsodiumascounterion,GMPCPPishydrolyzedslightlyfasterinthelattice--thishydrolysisisacceleratedtremendouslybytreatmentwithglycerol.GiventhisinformationontheeffectofbuffercounterionsonGMPCPPstabilitywithinmicrotubulelattices,wealwaysusepotassiumcounterionbuffersforallourmicrotubulework.GMPCPPisapotentnucleatorofmicrotubules.Therefore,attubulinconcentrationsof1mg/mlorhigher,verynumerousandshortmicrotubulesareformedinthepresenceofGMPCPP.IflongerGMPCPPmicrotubulesaredesired,nucleationcanbelimitedbydilutingthetubulinto~2-3µM(0.2-0.3mg/ml).Wegenerallymakea1-3mg/mlCPPtubulinmixandstoreitat-80¡Cinsmallaliquots.DirectlypolymerizingthismixresultsinshortGMPCPPseeds.DilutingthemixwhilethawingitresultsinformationoflongerCPPmicrotubules.

1.Onicemixunlabeledtubulinandlabeledtubulin(1-3mg/mlfinal)atanappropriateratioin1XBRB80with1mMDTTand0.5-1mMGMPCPP.Incubateat0¡Cfor5"-10".

2.ClarifymixinTLA100rotorat90Kfor5"at2¡C.Freezesupernatantin5-10µlaliquotsinliquidnitrogenandstoreat-80¡C.

3A.ToformshortGMPCPPseeds,transferatubefromthefreezertoa37¡Cbath.Incubate15"-20"at37¡C.Diluteto150-200µlwithwarmBRB80+1mMDTT,pellettheseedsinaTLA100rotor(90K5"at25-30¡C),discardsupernatantandresuspendpelletin1-2XthestartingvolumeofBRB80+1mMDTT.ThisprocessremovesfreeCPPandanyunpolymerizedtubulin.Seedsaregenerallyaddedat1/20-1/50voltoapolymerizationmixcontainingtubulinand1mMGTP.Giventhe~10XhigheraffinityoftubulinforGTPversusGMPCPP,theamountofGMPCPPaddedfromaseedmixatthesedilutionsisinsignificant.Therefore,weoftenaddseedsdirectlyintoapolymerizationmixwithoutdilution/sedimentation/resuspension.

3B.ToformlongGMPCPPmicrotubules,thawaCPPmixtubebyaddinginenoughwarmBRB80+1mMDTTsuchthatthefinaltubulinconcentrationis2-3µM(pipetin37¡CBRB80+1mMDTT,mixbygentlypipetingupanddownuntilthefrozenseedmixpelletisthawed,thenplacein37¡Cbath).Incubateat37¡Cfor30"orlonger.FreeCPPcanberemovedasdescribedin3AortheCPPmicrotubulescanbeuseddirectlyforassays.

VI.DeterminingConcentrationofGMPCPP/TaxolMTs

ToaccuratelyestimatetheconcentrationoftubulinpolymerinGMPCPP/Taxolpolymerizations,MTsarepelletedandresuspendedinbufferwithoutfreenucleotide.AsmallamountoftheresuspendedMTsarethendilutedintoabuffercontainingCaCl₂onicetoinducedepolymerizationandtubulinconcentrationdeterminedbytheA₂₈₀.HereisaprotocolforGMPCPPMTs(identicalfortaxolMTs,exceptthattheresuspensionbufferwillhave10-20µMtaxol):1.Pelletpolymerizationmixture(2mg/ml)90K5"inTLA100at35¡C.2.RemovesupeasthoroughlyaspossIBLe.3.Resuspendpelletin80%ofstartingvolumeusing37¡CBRB80+1mMDTTuntilhomogenous(requirequitealotofpipetingupanddown)4.Dilute10µlresuspendedMTsinto90µlofBRB80+50mMKCl+5mMCaCl₂andincubateonicefor10"(dilute10µlBRB80+1mMDTTsimilarlyasablank).5.After10"at0¡C,readA₂₈₀againstblankandcalculateconcentrationusinganextinctioncoefficientof115,000M^-1cm^-1.