Segmentedandpolarity-markedmicrotubulesareveryusefulformanydifferenttypesofinvitroassays.Segmentedmicrotubulesaremicrotubuleswithabrightseedanddimelongatedsegmentsonbothends.Polaritymarkedmicrotubulesaremicrotubuleswithabrightseedandadimelongatedsegmentonlyononeend--theplusend.SelectiveelongationofoneendisachievedbyinclusionofNEM-treatedtubulin,acompetitiveinhibitorofminusendpolymerization.MorecomplexmicrotubulesubstratessuchasGDPmicrotubulelatticescappedwithsegmentsofGMPCPPtubulincanalsobepreparedbyplayingaroundwithinvitropolymerizationconditions.Also,segmentedandpolaritymarkedmicrotubulescanbemadefromdifferentcolortubulinsinsteadofdifferentintensityofasinglecolortubulinasdescribedhere.

Notethatthepreciseratiosoflabeledtounlabeledtubulindescribedheremayneedtobeadjusteddependingonthelabeledtubulinprep.DescribedhereiswhathasworkedwellforususingtetramethylrhodamineNHSester-labeledtubulin(stoichiometryoflabeling~1.4).

• I.Solutions&Supplies
• II.SegmentedTaxolMicrotubules
• III.PolarityMarkedTaxolMicrotubules
• IV.SegmentedGMPCPPMicrotubules
• V.PolarityMarkedGMPCPPMicrotubules

Backtoprotocols

I.Solutions&Supplies

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

100mMGTP

100mMGMPCPP

Taxol:10mMstock;200µM,20µMand2µMdilutionallinanhydrousDMSO;soldundertradename"Paclitaxel"bySigma

BrightGMPCPPSeedMix(2mg/ml;1partrhodaminetubulinto2partsunlabeledtubulin;preparedandstoredat-80¡Casdescribedabove)

NEMGTP-Tubulin(preparedbytreatingrecycledtubulin(~5-15mg/ml)inBRB80+0.5mMGTPwith1mMNEM(N-ethylmaleimide;fromafresh50mMstockinwaterpreparedjustbeforeuse)for10"at0¡C,quenchingtheNEMwith8mMb-mercaptoethanolfor10"at0¡C,freezingaliquotsinliquidnitrogenandstoringat-80¡C.

NEMCPP-Tubulin(preparedbytreatingrecycledtubulin(~5-15mg/ml)inBRB80+0.5mMGMPCPPwith1mMNEM(freshlypreparedasa50mMstockinwater)for10"at0¡C,quenchingNEMwith8mMb-mercaptoethanolfor10"at0¡C,freezingaliquotsinliquidnitrogenandstoringat-80¡C.

II.SegmentedTaxolMicrotubules

1.PolymerizebrightGMPCPPseedmixat37¡Cfor15"-30".

2.Onicepreparethedimelongationmixconsistingof15µMtubulin(1partrhodaminetubulinto10partsunlabeledtubulin)in1XBRB80,1mMDTT,1mMGTP.Incubateat0¡Cfor5"and(optionally)clarifyat90Kfor5"at2¡CinaTLA100rotor.

3.Incubatedimelongationmixat37¡Cfor1".Add1/10-1/20volumeofGMPCPPseedsandmixgently.Incubateat37¡Cfor20".GMPCPPseedsarecold-lABIleandwilldepolymerizeonice.Therefore,onlyaddtheseedsaftertheelongationmixhaswarmedup.

4.Addtaxolstepwiseto20µM.

5.Thesegmentedtaxol-stabilizedmicrotubulescanbepelletedoveraglycerolcushionandresUSPended,oruseddirectlyafterdilution.Alldilutionsoftaxol-stabilizedMTsshouldbedoneintobufferscontaining10-20µMtaxol.

III.PolarityMarkedTaxolMicrotubules

1.PolymerizebrightGMPCPPseedmixat37¡Cfor15"-30".

2.Onicepreparethedimpolarelongationmixconsistingof15µMtubulin(1partrhodaminetubulinto10partsunlabeledtubulin)and12µMNEMGTP-tubulinin1XBRB80,1mMDTT,1mMGTP.Incubateat0¡Cfor5"and(optionally)clarifyat90Kfor5"at2¡CinaTLA100rotor.

3.Incubatedimpolarelongationmixat37¡Cfor1".Add1/10-1/20volumeofGMPCPPseedsandmixgently.Incubateat37¡Cfor20".

4.Addtaxolstepwiseto20µM.

5.Thepolarity-markedtaxol-stabilizedmicrotubulescanbepelletedoveraglycerolcushionandresuspended,oruseddirectlyafterdilution.Alldilutionsoftaxol-stabilizedMTsshouldbedoneintobufferscontaining10-20µMtaxol.

IV.SegmentedGMPCPPMicrotubules

1.PreparedimGMPCPPElongationMix:10µM(1mg/ml)1:9rhodaminelabeled:unlabeledtubulinin1XBRB80,1mMDTT,0.5mMGMPCPP.Incubateonicefor5"-10",spin90K5"inTLA100at2¡C,freezeinliquidnitrogenin10µlaliquots(orusefresh).

2.ThawGMPCPPbrightseedmixbyadding9volofwarm(37¡C)BRB80+1mMDTT(resultingin2µMtubulinfinal)andincubateat37¡Cfor30"-45".

3.ThawCPPelongationmixandstoreonice.Diluteasfollowsonice:17µlBRB80+1mMDTT3µlCPPelongationmix(Thisresultsinelongationof~1.5µMCPP-tubulin)

4.IncubatedilutedCPPelongationmixat37¡Cfor20secbeforeadding2µlofpolymerizedbrightCPPseeds.

5.Incubateat37¡Cfor1-2hours.Pelletandresuspendorusedirectly.

V.PolarityMarkedGMPCPPMicrotubules

1.PreparedimGMPCPPpolarelongationmix:10µM(1mg/ml)1:9rhodaminelabeled:unlabeledtubulinand8µMNEMCPP-Tubulinin1XBRB80,1mMDTT,0.5mMGMPCPP.Incubateonicefor5"-10",spin90K5"inTLA100at2¡C,freezeinliquidnitrogenin10µlaliquots(orusefresh).

2.ThawGMPCPPbrightseedmixbyadding9volofwarm(37¡C)BRB80+1mMDTT(2µMtubulinfinal)andincubateat37¡Cfor30"-45".

3.ThawCPPpolarelongationmixandstoreonice.Diluteasfollowsonice:17µlBRB80+1mMDTT3µlCPPPolarElongationMix(Thisresultsinelongationof~1.5µMCPP-tubulin)

4.IncubatedilutedCPPpolarelongationmixat37¡Cfor20secbeforeadding2µlofpolymerizedbrightCPPseeds.

5.Incubateat37¡Cfor1-2hours.Pelletandresuspendorusedirectly.