Purification of recombinant human and Drosophila septin

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Purification of recombinant human and Drosophilaseptin hexamers for TIRF assays of actin-septin filament

assemblyManos Mavrakis, Tsai Feng-Ching, Gijsje H. Koenderink

To cite this version:Manos Mavrakis, Tsai Feng-Ching, Gijsje H. Koenderink. Purification of recombinant human andDrosophila septin hexamers for TIRF assays of actin-septin filament assembly. Methods in Cell Biol-ogy, Elsevier, 2016, Septins, 136, pp.199. �10.1016/bs.mcb.2016.03.020�. �hal-01363593�

https://hal.archives-ouvertes.fr/hal-01363593

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Runningtitle:

Invitroreconstitutionofactin-septinfilamentassembly

Title:

Purification of recombinant human and Drosophila septin

hexamersforTIRFassaysofactin-septinfilamentassembly

ManosMavrakis1*,Feng-ChingTsai2,3andGijsjeH.Koenderink2*

1 Aix Marseille Université, CNRS, Centrale Marseille, Institut Fresnel, UMR 7249,

FacultédesSciencesSaint-Jérôme,13013Marseille,France

2FOMInstituteAMOLF,SciencePark104,1098XGAmsterdam,TheNetherlands

3 Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, CNRS

UMR168, 75005, Paris, France; SorbonneUniversités, UPMCUniv Paris 06, 75005,

Paris,France

*Correspondingauthors:

e-mailaddress:[email protected];[email protected]

Keywords:cytoskeleton,reconstitution,fluorescencemicroscopy,septins,actin

Abstract

Septins are guanine nucleotide binding proteins that are conserved from fungi to

humans. Septins assemble into hetero-oligomeric complexes and higher-order

structures with key roles in various cellular functions including cell migration and

division. The mechanisms by which septins assemble and interact with other

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cytoskeletalelementslikeactinremainelusive.Apowerfulapproachtoaddressthis

question is by cell-free reconstitution of purified cytoskeletal proteins combined

withfluorescencemicroscopy.Here,wedescribeproceduresforthepurificationof

recombinant Drosophila and human septin hexamers from Escherichia coli and

reconstitution of actin-septin co-assembly. These procedures can be used to

compareassemblyofDrosophilaandhumanseptinsandtheirco-assemblywiththe

actincytoskeletonbytotalinternalreflectionfluorescence(TIRF)microscopy.

INTRODUCTION

Since their discovery in budding yeastmore than forty years ago (Hartwell, 1971;

Hartwell, Culotti, Pringle, & Reid, 1974), septin GTP-binding proteins have been

shown tobepresent inall eukaryotesexceptplants (Nishihama,Onishi,&Pringle,

2011; Pan, Malmberg, & Momany, 2007). Although several groups encountered

mammalian septin genes during their studies in the early 1990s (for example,

(Nakatsuru,Sudo,&Nakamura,1994)),the identificationandfunctionalanalysisof

Drosophila septins (Fares, Peifer, & Pringle, 1995; Neufeld & Rubin, 1994)

established that septin family proteins existed in animals and not only in budding

yeast, and also provided strong evidence for roles of septins not related to

cytokinesis. Soon after came the first isolation of native septin complexes from

Drosophilaembryonicextractsusinganimmunoaffinityapproach(Fieldetal.,1996),

which provided the first evidence that septin heteromeric complexes are able to

polymerize into filaments in vitro. This study was followed by the isolation of

endogenous heteromeric septin complexes from yeast extracts with a similar

immunoaffinityprotocol(Frazieretal.,1998),aswellasfrommammals,bothbyrat

brainfractionation(Hsuetal.,1998)andbyimmunoisolationfrommousebrainand

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HeLacells(Kinoshita,Field,Coughlin,Straight,&Mitchison,2002).Morerecently,a

new protocol was reported for the purification of endogenous septin complexes

from Drosophila embryonic extracts with a fractionation approach (Huijbregts,

Svitin,Stinnett,Renfrow,&Chesnokov,2009).

Understandingtherequirementandthepreciseroleofdifferentseptinproteinsfor

theformationandstabilityofseptincomplexes,aswellasfortheircapacitytoform

filamentsnecessitated the co-expressionof septins inheterologous systems. Thus,

work during the 2000s focused on the co-expression of recombinant septins in

bacteriaandininsectcells,usinggeneticallyencodedtagsonone(inmoststudies)

ortwoseptinstofacilitateproteinpurification.Buddingyeastseptincomplexeswere

purifiedfrombacteria(Bertinetal.,2008;Farkasovsky,Herter,Voss,&Wittinghofer,

2005;Garciaetal.,2011;Verseleetal.,2004),mammalianseptincomplexeswere

isolatedfrombacteria(Sheffieldetal.,2003;Sirajuddinetal.,2007)andinsectcells

(Kinoshita et al., 2002), C. elegans septin complexes frombacteria and insect cells

(John et al., 2007), and Drosophila septin complexes were also expressed in and

purifiedfrombacteria(Huijbregtsetal.,2009;Mavrakisetal.,2014)andinsectcells

(Huijbregts et al., 2009). Biochemical analysis and electron microscopy of the

purified recombinant complexes in these studies, aswell as ofmammalian septin

complexes immuno-purified (Sellin, Sandblad, Stenmark, & Gullberg, 2011) or

affinity-purified(Kim,Froese,Estey,&Trimble,2011)frommammaliancellcultures,

together with the crystal structure of a human septin complex (Sirajuddin et al.,

2007),havealtogetherestablishedthatseptinsfromallorganismsformrod-shaped

complexes containing two, three or four septinswith each present in two copies,

forming a tetramer (C.elegans), hexamer (Drosophila and human) or octamer

(buddingyeastandhuman),respectively.

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Thecombinationofelectronmicroscopy (EM)with invitroreconstitutionofseptin

filamentassembly in low-saltconditions(<100mMKCl)usingrecombinantpurified

complexeshasbeen instrumentaland isstilloneofthemostpowerfulapproaches

for deciphering how septin protomers assemble into filaments and how filaments

organizeintohigher-orderstructures.ThemaindrawbackofEMapproachesisthat

they provide snapshots of septin organization and do not allow studies on how

freely-diffusing septin protomers dynamically assemble, grow and organize into

filament assemblies. Fluorescence-based assays using purified recombinant septin

complexespresentgreatpotential in thisaspectsincetheycancombinemolecular

specificity(whentaggingspecificseptinsubunits)withawiderangeoffluorescence-

basedtechniquesthatenablestudiesacrossdifferentspatialandtemporalscales.

Atpresentthereisonlyahandfulofreportsusingfluorescentlytaggedrecombinant

septincomplexesforstudyingseptinassembly,almostexclusivelyforbuddingyeast

septins.Threeapproachesarebeingusedforfluorescenttagging:(1)geneticfusion

of a specific septin subunitwith a fluorescentprotein, suchasmEGFPormCherry

(Bridges et al., 2014; Sadian et al., 2013), (2) genetic fusion of a specific septin

subunitwithaSNAP-tagand its furtherderivatizationwith fluorescentdyes (Renz,

Johnsson,&Gronemeyer,2013),or(3)chemicalconjugationat lysinesorcysteines

ofpurifiedcomplexeswithAlexaFluordyes(Booth,Vane,Dovala,&Thorner,2015;

Mavrakisetal.,2014).TherecentdevelopmentofaTIRFimagingassayusingpurified

GFP-taggedyeastseptinoctamersonsupported lipidbilayers (Bridgesetal.,2014)

providedthefirstfluorescencemicroscopy-basedquantitativeassayforstudyingthe

kineticsofyeastseptinfilamentassembly,highlightingthepromiseofthisapproach.

The mechanisms that control septin assembly into complexes and higher-order

filamentousstructuresandtheregulationofseptinstructuresandtheirdynamicsare

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still largely unclear. Especially little in vitro work has been done on human and

Drosophilaseptins,soitremainsunknownhowanimalseptinassemblydiffersfrom

budding yeast septin assembly. In addition, septin interactions with other

cytoskeletal components remain elusive. Here, we describe procedures to purify

recombinant Drosophila and human septin hexamers from Escherichia coli. We

describe procedures for fluorescent tagging (using either genetic GFP fusions or

chemical conjugation with organic dyes), and provide protocols for in vitro

reconstitution of actin and septin assembly in surface assays amenable to high-

resolutionimagingbyTIRFmicroscopy.

1. Cloning strategy for recombinant septin complex production in

bacteria

To isolate stoichiometric hexameric septin complexes we combine the extended

pET-MCN(pETMulti-CloningandexpressioN)seriesasaseptinco-expressionsystem

(Diebold, Fribourg, Koch, Metzger, & Romier, 2011) with a two-tag purification

scheme.Tothisend,weusetwovectors,pnEA-vHandpnCS.pnEA-vHharborsthe

central subunitof thehexamer (DSep1 forDrosophila septinsorhSep2 forhuman

septins)underthecontroloftheT7promoter,withaTEV-cleavable6xHis-tagfused

to itsN-terminus.pnCSharborstheothertwoseptingenes(DSep2andPeanut for

Drosophila septins,orhSep6andhSep7 forhumanseptins)under thecontrolofa

singleT7promoter(Figure1).UsingappropriatePCRprimerswefusetheC-terminus

of the terminal subunit of the hexamer (Pnut for Drosophila septins or hSep7 for

human septins) to a noncleavable eight-amino-acid Strep-tag II (WSHPQFEK, 1058

Da).

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To generate the pnCS vector harboring two septin genes under the control of a

single promoter, we use bidirectional cloning to concatenate two pnCS vectors

encodingone septin geneeach. Each septin gene in thepnCS vector is flankedby

SpeIon the5' sideof theMCSandbyXbaIon the3' side.Using standard cloning

procedures (Green & Sambrook, 2012) we double-digest the donor plasmid with

SpeI/XbaI,ligatetheinserttoSpeI-digestedacceptorplasmidandselecttheclones

withthecorrectinsertorientationusingrestrictionanalysis(Figure1).

We have used this strategy for successful coexpression of both Drosophila and

humanseptinhexamers.TheflexibilityofthepET-MCNseries(detailedin(Dieboldet

al.,2011))enablesrapidscreeningtotesthowthepositionofthetags,theorderof

septin genes, or their expression under a single ormultiple promoters affects the

quantityandstoichiometryoftheresultingcomplex.

[InsertFigure1here]

Figure1.Overviewofcloningstrategyforco-expressingrecombinantseptinsusingthepnEA-vHand

pnCS vectors of the pET-MCN series (Diebold et al., 2011). Here this strategy is used for the

generationof recombinantDrosophila (DSep1-DSep2-Pnut)andhuman (hSep2-hSep6-hSep7) septin

hexamers.

2.Expressionofrecombinantseptincomplexesinbacteria

We co-express His6-hSep2, hSep6 and hSep7-Strep for producing human septin

hexamers (hSep2-hSep6-hSep7), and His6-DSep1, DSep2 and Pnut-Strep for

producingDrosophilaseptinhexamers(DSep1-DSep2-Pnut).

Materialsandreagents

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Solutionsarepreparedinwaterunlessstatedotherwise.

• plasmidsforco-expressionofseptins:

Drosophilaseptins:His6-DSep1inpnEA-vH,DSep2/Pnut-StrepinpnCS

DrosophilaGFP-labeledseptins:His6-DSep1inpnEA-vH,mEGFP-DSep2/Pnut-Strepin

pnCS

humanseptins:His6-hSep2inpnEA-vH,hSep6/hSep7-StrepinpnCS

• E.coliBL21(DE3)competentcells(200131,AgilentTechnologies),-80°C

• Ampicillin(A0166,Sigma),100mg/mL,-20°C

• Spectinomycin(S4014,Sigma),100mg/mL,-20°C

• LBmedium(L3022,Sigma),RT

• LBagar(L2897,Sigma),RT

• LBagarplatescontainingbothampicillinandspectinomycinat100µg/mLeach,

4°C

• SOCmedium(S1797,Sigma),-20°C

• TerrificBroth(091012017,MPBiomedicals),RT

• IPTG(EU0008-C,Euromedex),1M,-20°C

• 2LErlenmeyerflasks

Day0.Heat-shocktransformationofbacteria

We use standard procedures (Green & Sambrook, 2012) to co-transform E.coli

BL21(DE3) competent cellswith the twoplasmidsencoding all three septin genes,

andgrowcoloniesonLBagarplatesat37°Covernight.

Day1.Bacterialpre-culture

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1.Usingasterilepipettetip,selectasinglecolonyfromyourLBagarplate.Dropthe

tip into liquid LB containingbothantibiotics (1000xdilutionof theantibiotic stock

solutions)andswirl.Calculatethevolumeofthepre-culturegiventhatyouwilluse

1/50ofthetotalvolumeofthecultureforinoculation.

2.Incubatethebacterialcultureat37°Cfor12-16hinashakingincubatortoprepare

your pre-culture. For long-term storage of the co-transformed bacteria, mix your

pre-culturewithglyceroltomakea50%v/vglycerolstockandstoreitat-80°C.

Day2.Bacterialcultureforproducingdark(unlabeled)septincomplexes

The C-termini of both Peanut (when co-expressedwith DSep1 and DSep2) and of

hSep7 (when co-expressed with hSep2 and hSep6) are prone to degradation by

bacterialproteasesduringproteinexpression(Mavrakisetal.,2014).Thepresence

of the Strep-tag II helps isolate complexes containing full-length Pnut/hSep7. To

minimize degradation, we developed a protocol whereby each bacterial cell

produces less protein (short induction time) but with less degradation. We

compensate for the smaller protein yield per cell by using Terrific Broth to grow

bacteria at 37°C to a high density before induction.We typically prepare 6-8 L of

culture.

1. Add 700 mL Terrific Broth containing both antibiotics (1000x dilution of the

antibioticstocksolutions)intoeach2LErlenmeyerflask.

2.Inoculateeachflaskwithyourpre-culture(1/50oftheculturevolume).

3.Incubateat37°Cinashakingincubator(220rpm)untiltheOD600reaches2-3(this

shouldtake3h-4h30).

4.InduceproteinexpressionbyaddingIPTGto0.5mMfinal.Allowcellstogrowfor3

hat37°C.

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5.Collectcellsbycentrifugingat2,800gand4°Cfor10min.Poolbacterialpelletsin

a50mLFalcontubeandstoreat-80°C.Ifyouwanttoproceeddirectlywithprotein

purification,storethetubeat-80°Cforatleast30min,thencontinuewithcelllysis.

Days2-3.BacterialcultureforproducingGFP-labeledseptincomplexes

TominimizeproteindegradationandalsoallowGFPtofold,wegrowcellsat37°Cto

alowdensityandtheninduceexpressionat17°Covernight.Wetypicallyprepare8-

10 L of culture. We minimize exposure to light by covering the incubator with

aluminumfoilandkeepingthelightsoff.

1.Inoculateflaskswithyourpre-cultureasdescribedfordarkseptinproduction.

3.Incubateat37°Cinashakingincubator(220rpm)untiltheOD600reaches0.6-0.8

(thistakes2h30-3h).

4. Induce protein expression by adding IPTG to 0.5mM final. Allow cells to grow

overnight(16h)at17°C.

5. Collect and store bacterial pellets as described for dark septin production. The

pelletsshouldbeyellow-greenishconfirmingthepresenceofGFP.

3. Purification and characterization of recombinant septin complexes

frombacteria

We use a two-tag purification scheme (His6-tag on the central subunit i.e.

DSep1/hSep2andaStrep-tagIIontheterminalsubuniti.e.Pnut/hSep7)inorderto

select for complexes with full-length Pnut/hSep7 and to minimize isolation of

substoichiometric septin complexes (Figure 2). The nickel affinity column isolates

His6-DSep1/hSep2-containing complexes, whereas the Strep-Tactin (engineered

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streptavidin) affinity column further isolates those complexes that also contain

Pnut/hSep7-Strep thus heterohexamers. A final gel-filtration step helps remove

aggregates and isolate hexamers.We use the same protocol for purifying human

septin hexamers (hSep2-hSep6-hSep7) and Drosophila septin hexamers (DSep1-

DSep2-Pnut).WhenpurifyingmEGFP-taggedseptins,weminimizeexposuretolight

bycoveringcolumnswithaluminumfoilandkeepinglightsoff.

[InsertFigure2here]

Figure2.Schematicoverviewofthetwo-tagpurificationschemefor isolatingstoichiometrichuman

septin hexamers (the same applies to Drosophila septins by analogy). See text for details.

Understandingtheprinciplesofseptincomplexassemblyisanintensetopicofinvestigation,andthe

presenceandstabilityofintermediatecomplexeshasnotbeenfullydocumented.Weshowselected

populations of human septin complexes in the cell lysate (monomers, homo- and heterodimers,

heterotrimers andhexamers) basedon the isolation and characterizationof such recombinant and

nativecomplexes(Kim,Froese,Xie,&Trimble,2012;Mavrakisetal.,2014;Sellinetal.,2011;Serrao

et al., 2011; Sheffield et al., 2003; Sirajuddin et al., 2007; Sirajuddin, Farkasovsky, Zent, &

Wittinghofer,2009;Zent,Vetter,&Wittinghofer,2011;Zent&Wittinghofer,2014).



Forthelysisbuffer:

• Tris-HClpH8,1M,RT

• KCl,4M,RT

• OmniPur®Imidazole(5710-OP,MERCKMillipore),1M,RT

• MgCl21M,RT

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• PMSF (78830, Sigma), 100 mM in ethanol, -80°C, dilute into lysis buffer

immediatelybeforeuse

• Lysozyme(5934-D,Euromedex),50mg/mL,-20°C

• cOmplete™ProteaseInhibitorCocktailTablets(11697498001,Roche),4°C:use1

tabletfor50mLoflysisbuffer

• MgSO4,2M,RT

• DNaseI(10104159001,Roche),2g/L,-20°C

Forproteinpurification:

• d-Desthiobiotin (D1411, Sigma), powder, 4°C: add to 2.5mM final in StrepTrap

elutionbufferimmediatelybeforeuse

• DTT,1M,-20°C:addto5mMfinalingelfiltrationbufferimmediatelybeforeuse

• HisTrapFFcrude5-mLcolumn(17-5286-01,GEHealthcare)

• StrepTrapHP1-mLcolumn(28-9075-46,GEHealthcare)

• HiLoad16/600Superdex200pgcolumn(28-9893-35,GEHealthcare)

• ÄKTAFPLCsystem

Forproteinconcentration:

• Amicon Ultra 4 mL Centrifugal Filter Units with membrane NMWL of 30 kDa

(UFC803024,Millipore)

• TritonX-1005%v/v,4°C

Day1.Septincomplexpurification

Filterallbuffersthrough0.22-µmfilters.Performallpurificationstepsat4°C.Keep

aliquotsfromtheelutionpeaksaftereachpurificationstepforSDS-PAGE/Coomassie

stainingandWesternblotsformonitoringproteinintegrityandenrichmentofseptin

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complexes. Measure the protein concentration after each purification step using

absorbance measurements at 280 nm for monitoring protein loss during the

purificationprocess.

1.Resuspendthebacterialpellet in ice-cold lysisbuffer (50mMTris-HClpH8,500

mMKCl,10mMimidazolepH8,5mMMgCl2,0.25mg/mLlysozyme,1mMPMSF,

cOmplete™proteaseinhibitorcocktail,0.01g/LDNaseI,20mMMgSO4).Weuse100

mLoflysisbufferforabacterialpelletfroma6Lculture.Lysecellsonicewithatip

sonicatorusing5cyclesof30s"ON",30s"OFF"(30%amplitude).

2.Clarifythelysatebycentrifugationat20,000gfor30minat4°C.

3. Load thesupernatantonaHisTrapFFcrudecolumnequilibratedwith5column

volumes(CV)of50mMTris-HClpH8,500mMKCl,10mM imidazolepH8,5mM

MgCl2.Washwith7CVofthesamebuffer,thenwashwith7CVof50mMTris-HClpH

8, 500 mM KCl, 20 mM imidazole pH 8, 5 mMMgCl2 to remove nonspecifically

bounduntaggedproteins.

4.EluteHis6-DSep1/hSep2-containingcomplexeswith7CVof50mMTris-HClpH8,

500mMKCl,250mMimidazolepH8,5mMMgCl2.Collect1-mLfractions.Poolall

fractionscontainedintheelutionpeak(typically10-15mL).

5.LoadthepooledfractionstoaStrepTrapHPcolumnequilibratedwith5CVof50

mMTris-HClpH8,300mMKCl,5mMMgCl2.WeusetwoStrepTrapHPcolumnsin

tandem.Washwith7CVofthesamebuffer.

6.ElutePnut/hSep7-Strep-containingcomplexeswith7CVof50mMTris-HClpH8,

300mMKCl, 5mMMgCl2,2.5 mM desthiobiotin. Collect 1-mL fractions. Pool all

fractionscontainedintheelutionpeak(typically5mL).

7. Load thepooled fractions to a Superdex 200HiLoad16/60 columnequilibrated

with1.2CVof50mMTris-HClpH8,300mMKCl,5mMMgCl2,5mMDTT.Elute

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with 1.2 CV of the same buffer. Elute for 0.25CV before collecting 1-mL fractions.

Pool the fractions corresponding to the elution peak (typically 10 mL) and

concentrate using passivated Amicon concentrators (see below). Measure the

concentration using the elution buffer as a blank, prepare 10- or 20-µL aliquots,

flash-freeze purified septin complexes in liquid nitrogen and store at −80°C. This

purificationprotocoltypicallyyields1-2mgofstoichiometricseptinhexamers,which

weconcentrateto10-15µM(about3-5mg/mL).

Wecalculateseptincomplexconcentrationfromabsorbancemeasurementsat280

nm. We compute extinction coefficients from the amino acid sequences using

ExPASy at http://web.expasy.org/protparam/, assuming two copies of each full-

lengthseptin(tagsincluded)perhexamer.

darkDrosophilaseptins (His6-DSep1/DSep2/Pnut-Strep):

306.9kDa,1g/L=3.3µM,ε=0.545L.g-1.cm-1at280nm(assumingallCysreduced)

mEGFP-taggedDrosophilaseptins (His6-DSep1/mEGFP-DSep2/Pnut-Strep):


darkhumanseptins(His6-hSEP2/hSEP6/hSEP7-Strep):


Day2a.Concentrationofpurifiedseptincomplexes

Septinstendtobestickyandadsorptiontothemembraneoftheconcentratorleads

to significant yield loss. To improve recovery of septins during the concentration

step,wepassivatetheconcentratorsbytreatingthemwith5%v/vTritonX-100.

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1.Wash the concentrator by fillingwithwater and spinning the liquid through at

4,500gfor10min.Removeresidualwaterthoroughlybypipetting.

2.Filltheconcentratorwith5%v/vTritonX-100.Incubatefor2hatRT.

3.RemovetheTritonX-100solution.Rinsethedevice3or4timesthoroughlywith

water and finally spin through for 5min at 4,500 g. The passivated device is now

readytouse.

4. Add gel filtration elution buffer and spin through for 5 min at 4,500 g to

equilibratetheconcentratormembrane.

5. Add the gel filtration eluate and spin through until reaching the desired

volume/concentration. Do cycles of spinning of 20min at 4,500 g. Between two

cycles,checkthevolumeoftheproteinsolution,refillwiththeremainingsolution,

mixtheproteinsolutionverygentlybypipettingupanddownandremovetheflow

through.KeepanaliquotforSDS-PAGE.

Day2b.Characterizationofpurifiedseptincomplexes

Weanalyzeseptinpreppurityandproteinintegrityby12%SDS-PAGEandWestern

blot.ForDrosophilaseptins,weusemouse4C9H4anti-Pnut(1:100,Developmental

Studies Hybridoma Bank), rat anti-DSep1-95 (1:500) (Mavrakis et al., 2014) and

guineapiganti-DSep2-92(1:500)(Mavrakisetal.,2014).Forhumanseptins,weuse

goat anti-hSep2 (1:500, Santa Cruz Biotechnology, sc-20408), rabbit anti-hSep6

(1:500,SantaCruzBiotechnology,sc-20180)andrabbitanti-hSep7(1:200,SantaCruz

Biotechnology, sc-20620). For both Drosophila and human septins, we use HRP-

conjugated anti-Penta-His (1:10,000, Qiagen) and HRP-conjugated anti-Strep-tag

(1:10,000,AbDSerotec).

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hSep2 antibodies recognize the N-terminus of hSep2, whereas hSep6 and hSep7

antibodies recognize the C-terminus of hSep6 and hSep7, respectively. Pnut

antibodies recognize the N-terminus of Peanut (Mavrakis et al., 2014), whereas

DSep1-95andDSep2-92antibodiesrecognizetheN-terminusofDSep1andDSep2,

respectively(Mavrakisetal.,2014).WecombinetheseantibodieswiththeHis-and

Strep-tag antibodies that recognizeN- and C-termini, respectively, to examine the

integrityofeachseptinintheprepbyWesternblot.

Thepurityof theseptinprepscanbe further testedbymassspectrometryboth in

solution and from protein bands excised from the gels.We found the N-terminal

methioninesofallthreeDrosophilaseptinscleavedinthebacterialpreps.Theonly

proteinweidentifiedinourprepsbesidesseptinswasthebacterialchaperoneDnaK

whichrunsat70kDainSDS-PAGE(Mavrakisetal.,2014).

Finally, we evaluate the quality of each septin preparation in terms of oligomer

composition and hexamer content by transmission electronmicroscopy combined

with 2D single particle image analysis (Figure 3), as described in the chapter by

AurelieBertin.

4.LabelingseptinsforTIRFimaging

4a.Generatingseptin-GFPfusions

ForTIRFimagingoffluorescentDrosophilaseptins,fusemEGFPtotheN-terminusof

DSep2andgenerateapnCSvectorharboringbothmEGFP-DSep2andPnut-Strep,as

detailed above. Co-express His6-DSep1 with mEGFP-DSep2/Pnut-Strep to produce

and purify fluorescent septin hexamers, His6-DSep1/mEGFP-DSep2/Pnut-Strep, as

detailedabove.

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[InsertFigure3here]

Figure 3. Characterization of purified septin complexes by SDS-PAGE (unlabeled and GFP-labeled

Drosophilaseptinhexamers,leftandrightpanelsinA,respectively)andby2Dsingleparticleanalysis

ofelectronmicroscopyimages(B,seechapterbyAurelieBertin).

4b.ChemicallabelingofpurifiedseptincomplexeswithAlexaFluordyes

Septinscanbelabeledatprimaryamines(ε-aminogroupoflysinesandN-terminus)

usingAlexaFluor488succinimidylesters.Weisolatelabeledseptinsinfilamentous

form, to ensure that hexamers are still polymerization-competent after labeling.

Different septin complexes and septins from different species can polymerize to

different extents to higher-order structures of different sizes,whichmight require

optimizationoftheincubationandcentrifugationtimesdetailedbelow.


• purified septins in50mMTris-HClpH8, 300mMKCl, 5mMMgCl2, 5mMDTT

(septinbuffer)

• Alexa Fluor 488 carboxylic acid, succinimidyl ester (NHS ester) (A20000,

Invitrogen),-20°C,protectedfromlight

• Tris-HClpH8,1M,RT

• Hepes-KOHpH8,1M,4°C,storedinthedark

• PD-10columns(GEHealthcare)

Day1.ReactionwithNHSesterandpelletingofpolymerization-competentseptins

17

1.ToexcludereactivityofNHSesterswithamineandthiolgroupsofcompoundsin

the septinbuffer (TrisandDTT),dialyze septins into50mMHepes-KOHpH8,300

mMKCl,5mMMgCl2.

2.Preparea20mMstockoftheNHSesterinDMSOimmediatelybeforestartingthe

reaction. Add to dialyzed septins at 4:1 molar ratio (dye:septins), mix gently and

incubatefor1hatRTinthedark.QuenchthereactionbyaddingTristo20mMTris-

HClpH8final.

3.Add50mMTris-HClpH8,5mMMgCl2,5mMDTTtodiluteKClto50mMfinaland

polymerizeseptinsfor1hatroomtemperature.Topelletpolymerization-competent

Alexa-taggedseptins,centrifugeat100,000gfor3hat4°C(weuseaTLA100.3rotor

with 1.5-mL tubes). The resulting pellet will be yellow-greenish confirming the

reactionwithAlexaFluor488(AF488)NHSesters.

4. Carefully remove the supernatant and add 0.5mL ice-cold septin buffer to the

AF488-septinpellet.Allowthepellettoresuspendoniceovernightinthedark.Mix

verygentlythenextmorningbypipettingupanddown.

Day2.SeparationofAF488-septinsfromunreactedAF488withaPD-10column

1.Pre-coolandequilibrateaPD-10columnwith25mLseptinbufferanddiscardthe

eluate.

2.Add0.5mLoftheresuspendedAF488-septins.Oncethesolutionhasenteredthe

column,add2.0mLseptinbuffer.Discardtheeluate.

3. Elute with 3.5 mL septin buffer in 0.5-mL fractions. AF488-septins elute in

fractions2-4(1.5mLintotal).Measuretheproteinconcentrationandcalculatethe

degreeoflabeling.Usethevaluesprovidedbythemanufacturerforthedye(λmax,ε,

correction factor at 280 nm) to correct for the contribution of the dye to the

18

absorbanceat280nm.Prepare10-or20-µLaliquots,flash-freezeAF488-septins in

liquidnitrogenandstoreat−80°Cinthedark.

5.BuildingflowcellsforTIRFmicroscopy

Tovisualizeactinfilamentsandseptinsbytotalinternalreflectionfluorescence(TIRF)

microscopy, we design flow cells having 6 rectangular shaped channels with

approximatedimensionsof22mminlength,2.5mminwidthand0.1mminheight

(Figure 4). Approximately 10 µL of a protein solution can be loaded into each

channel.

[InsertFigure4here]

Figure 4. A flow cell assembled by melting strips of Parafilm between a cleaned and passivated

coverslipandaglassslide.


lH2O2(35%inwater,95299,Sigma),4°C

lNH4OH(30%inwater,320145,Sigma),RT

lDichlorodimethylsilane(DDS),(40140,Sigma),RT

lTrichloroethylene(TCE),(251402,Sigma),RT

lPluronic®F-127(P2443,Sigma),10%inDMSO,RT

TocompletelydissolvePluronic®F-127powderinDMSO,wewarmupthesolution

to45°C.Forflowcelltreatment,wediluteto1%(w/v)inF-bufferrightbeforeuse.

lParafilm(PM996,Bemis)

lMicroscopeglassslide(24×60mm,thickness#1,0.15mm,MenzelGläser)

lMicroscopecoverslips(22×40mm,thickness#1,0.15mm,MenzelGläser)

19

5a.Cleaningandsilanizingmicroscopeglassslides/coverslips

Toremoveorganicresiduesfromtheglasssubstratesoftheflowcells,wecleanthe

microscopeslidesandcoverslipsinabase-piranhasolution.Additionally,thepiranha

treatment generates free hydroxyl groups on the glass substrates, making them

hydrophilic.Topreventnonspecificproteinadhesiontothesurfaces,wecoatthem

withahydrophobiclayerofdimethylsilane.

1. LoadmicroscopeglassslidesandcoverslipsintoaTeflonrack.

2. PlacetheTeflonrackintoaglassbeakerfilledwithMilli-Qwater.Rinsetheglass

slidesandcoverslipstwice,5mineach,withMilli-Qwaterinabathsonicator.

3. TransfertheTeflonracktoanewglassbeaker.Theglassbeakershouldbe large

enoughtocovertheglassslidesandcoverslipswiththebase-piranhasolution(Milli-

Qwater,30%NH4OHand35%H2O2ata5:1:1volumeratio).Inafumehood,fillthe

glassbeakerwithfivepartsMilli-Qwater,heatonahotplateto80°C.Addonepart

NH4OHand thenslowlyaddonepartH2O2.Gentlymix the solutionbymoving the

Teflon rack up-and-down in the beaker. Once the piranha solution reaches a

temperature of 60°C, bubbles form in the solution, indicating an active reaction.

Allowthesolutiontoreactfor30min.

Tip: The base-piranha solution is a dangerous reaction, thus wear gloves and

safetygoggles,andavoidspills.

Tip: Make fresh base-piranha solution for each use because the solution

decomposes.Theusedsolutioncanbeleftinthehoodovernight.Oncethereaction

hascompleted,onlywaterisleft,whichcanbesafelydiscardeddownthesink.

4. Rinsetheslidesandcoverslipsasinstep2.

20

5. Toactivate thehydroxylgroupsonglass substrates for the silanization reaction,

transfer theslidesandcoverslips toaglassbeaker filledwitha0.1MKOHsolution

andsonicatefor15mininabathsonicator.

Note:Ifneedbe,thesubstratescanbestoredinthe0.1MKOHsolutionforuptoone

month,otherwiseweproceedtothefollowingsteps.

6. Transfer the slides and coverslips to a glass beaker filled with Milli-Q water,

sonicatefor5min,blow-drycompletelywithnitrogengas,andsilanizeimmediately.

7. Inafumehood,prepareasolutionof0.05%v/vDDSinTCEinaglassbeakerthat

islargeenoughtocovertheslidesandcoverslipswiththesolution.

Note:BothDDSandTCEarevolatileandtoxic,thusweargloves,donotinhaleand

workalwaysinthefumehood.

8. Transfer the slides and coverslips into the glass beaker filledwith the DDS/TCE

solutionandincubatefor1h,withoutstirring.

Note:CarefullydisposeusedDDS/TCE solutionaccording to local laboratory safety

regulations.

9. Transfer the slides and coverslips in a glass beaker filled with methanol and

sonicatefor15mininabathsonicator.

10. Blow-dry completely the slides and coverslipswithnitrogen gas and store in a

cleansealedcontainerforuptooneweek.

5b.Constructingflowcells

HerewedescribehowtoconstructaflowcellbymeltingstripsofParafilmbetween

aglassslideandacoverslip.

1. Blow-dryasilanizedglassslidewithnitrogengas.

21

2. Cut a piece of Parafilm that is large enough to cover the glass slide. Place the

Parafilmlayerontheglassslideandpressfirmlytoensurethelayeradherestothe

glassslide.ThethicknessoftheParafilmlayersetstheheightoftheflowchannels.

3. UseascalpeltocuttheParafilmlayeralongtheedgesoftheglassslide,trimming

awayexcessParafilmalongtheedges.

4. Useascalpeltocut2.5mmwidestripsoftheParafilmlayeralongtheshortedge

oftheglassslide.Cut11strips,centeredwithrespecttotheglassslide.Cutfirmlyto

ensuretheedgesoftheadjacentstripsarewellseparated.

5. Removeevery second stripbyusinga forceps. Thewidthof the removed strips

setsthewidthoftheflowchannels.

6. Placeasilanizedglasscoverslipontopofthestrips,centeredwithrespecttothe

glassslide.

7. Place the assembly on a hot plate set at 120°C. Once the Parafilm strips are

melted,usea forcepsandgentlypress thecoverslip tomaximize thecontactarea

betweenthestripsandthecoverslip.Donotapplytoomuchpressurethatcanbreak

thecoverslip.Removetheassemblyfromthehotplateandleaveittocoolatroom

temperature.

Tip: While melting the assembly, air bubbles can appear at the contact surface

between the Parafilm strips and the glass coverslip. Apply gentle pressure on the

coverslip with a forceps to remove the air bubbles, which could cause leakage

betweenadjacentchannels.

8. Flow1%Pluronic®F-127intochannels,incubatefor5minandrinsewiththeactin

polymerizationbuffer(F-buffer)(seesection6bforthecompositionofF-buffer).The

flowcellisreadytouse.

22

Note: Silanized glass substrates are hydrophobic,which hampers inflowof sample

solutions. We thus treat channels with Pluronic F-127 to render the surface

hydrophilicenoughtobeabletoflowintheaqueoussolution.

Note: Pluronic® F-127 is a copolymer composed of a hydrophobic block of

polypropyleneglycol flankedbytwohydrophilicblocksofpolyethyleneglycol, thus

havingsurfactantproperties.

6.Invitroreconstitutionofactin-septinfilamentassembly



lDTT(D0632,Sigma),1M,-20°C

lMgATP,100mM,-80°C

Note: ToobtainMgATP,preparea200mMsolutionofNa2ATP (ATPdisodiumsalt

hydrate,A2383,Sigma)bydissolvingNa2ATPpowderinMilli-Qwaterandadjusting

thepHofthesolutiontopH7.4usingNaOHsolution.MixtheNa2ATPsolution1:1

witha200mMsolutionofMgCl2toobtain100mMMgATP.

lMethylcellulose(M0512,Sigma),1%(w/v),RT

lProtocatechuate acid (PCA) (03930590-50MG, Sigma), 100 mM in water and

adjustedtopH9usingNaOH,-80°C

lProtocatechuate 3,4-dioxygenase (PCD) (P8279-25UN, Sigma), 5µM in 50% v/v

glycerol,50mMKCl,100mMTris-HClpH8,-80°C

lTrolox(238813-1G,Sigma),100mM,-20°C

Topreparea100mMTroloxsolution:

1. Dissolve0.1gofTroloxpowderin430µLmethanol

23

2. Add3.2mLofMilli-Qwater

3. Add360µLof1MNaOH(Thesolutionturnsyellowish)

lVALAP(amixtureofvaseline,lanolinandparaffinwithequalweight),RT

6a.Preparingproteinsolutions

G-actin ispurified fromrabbit skeletalmusclebya standardprocedure includinga

finalgelfiltrationsteponaHiPrep26/60SephacrylS-200HRcolumn(GEHealthcare)

(Pardee&Spudich,1982).G-actin is storedat -80°C inG-buffer (2mMTris-HCl,pH

7.8,0.2mMNa2ATP,0.2mMCaCl2,and2mMDTT).G-actinisfluorescentlylabeled

withAlexa Fluor®594 carboxylic acid, succinimidyl ester (AF594-G-actin) (Soares e

Silvaetal.,2011)andisalsostoredat-80°CinG-buffer.Darkandfluorescentseptins

are stored in septin buffer as detailed above. Before use, aliquots of actin and

septinsarethawedoniceandclearedfor5minat120,000gatroomtemperaturein

aBeckmanairfuge.Finally,proteinconcentrationsaredeterminedbymeasuringthe

absorbanceoftheproteinsolutionsat280nm,usinganextinctioncoefficientof1.1

L.g-1.cm-1forG-actin(1g/L=23.8µM).Proteinsarekeptoniceandusedwithinone

week.

6b.Buffersandcomponents

The final assay buffer (referred to as F-buffer, or actin polymerization buffer)

containsthefollowingcomponents:

l20mMimidazole-HCl,pH7.4

l50mMKCl

l2mMMgCl2

l0.1mMMgATP

24

l1mMDTT

l1mMTrolox

l2mMPCA

l0.1 µMPCD

l0.1%(w/v)methylcellulose

Note:Whenmixingproteinswiththeabovecomponentstoreachthecompositionof

theF-buffer,takeintoconsiderationthattheseptinbuffercontains300mMKCland

5mMMgCl2.

Note:Troloxisincludedinthesolutiontoquenchtripletstatesandthustoprevent

photobleachingdue to the reactionsof the triplet stateswithoxygen free radicals

(Cordes, Vogelsang, & Tinnefeld, 2009). PCA-PCD is a substrate-enzyme pair that

scavengesoxygenfreeradicalsandthusminimizesphotobleaching (Shi,Lim,&Ha,

2010).

Note:ForTIRF imaging,weemploya0.1%(w/v)solutionofmethylcellulose,which

exerts an entropic force pushing actin-septin filaments towards the surface to

facilitateobservationbyTIRFimaging.However,werecommendtouseatmost0.2%

(w/v) methylcellulose, since higher methylcellulose concentrations induce the

formationofactinbundles(Popp,Yamamoto,Iwasa,&Maeda,2006).

6c.Reconstitutingactin-septinfilamentassembly

Here we describe a procedure to prepare samples in which actin and septins co-

polymerize. To visualize actin and septins,wedopeactinwithAF594-G-actin (10%

molar label ratio) and septinswithAF488-septins orGFP-septins (10%molar label

ratio) (Figure 5). We prepare protein samples with a final volume of 10 µl. We

25

provideasanexamplethesamplepreparationforco-polymerizing1µMactinwith1

µMseptins.

1. Prepare a “master buffer” containing 5-fold higher concentrations of all the

componentsofF-bufferapartfromPCD,takingintoaccountthecontributionofKCl

andMgCl2fromtheseptinsolutionthatwillbeaddedintothefinalmixturetoreach

thedesiredseptinconcentration.

2. Mix labeled and unlabeled G-actin to a final concentration of 5 µM in G-buffer

witha10%molarlabelratio.

Note:GiventhatdenseG-actinsolutions(>=5mg/ml)arequiteviscousanddifficult

tomix,wediluteG-actintoanintermediateconcentration.

3. Mixlabeledandunlabeledseptinstoafinalconcentrationof6 µMinseptinbuffer

witha10%molarlabelratio.

4. InoneEppendorftube,add4.1µLofMilli-Qwater,2µLofmasterbuffer(5x),0.2

µLofPCDand1.7µLoflabeledseptinsandmixwell.

Tip:We typicallydilute theseptin solution6-fold into the finalmixture inorder to

obtain50mMKCl fromtheseptinbuffer.Whenperforminga seriesof samples in

which actin concentration stays constant but septin concentration varies, one can

prepare septin solutionshaving6-foldhigher concentrations than the finaldesired

concentrationsbydilutingseptins inseptinbuffer,andthuspipette1.7µLofeach

septindilutionineverysample.

5. InanotherEppendorftube,place2µLoflabeledactin.

26

6. Loadtheseptinmixturefromstep4intotheG-actincontainingtube,mixthetwo

solutions thoroughlybyaspiratingupanddown3 timesand immediately load the

mixedsolutionintooneflowchannel.

Note: Given that G-actin polymerizes into F-actin immediately when mixed with

salts,itisimportanttoperformtheabovestepquickly.

7. Seal the two open ends of the channels with VALAP using a cotton-tipped

applicator.

Note:Beforeuse,meltVALAPattemperaturesexceeding80°C.Wetypicallykeepa

smallbeakerwithliquidVALAPonahotplate(120°C).

8. Incubatethesamplesforatleast1houratroomtemperaturetoensurecomplete

actinpolymerizationbeforeobservation.

Toprepareseptin filaments in theabsenceofactin (Figure6),we followthesame

procedureasabove,butreplacetheG-actinsolutionwithG-buffer.

For experiments with preformed actin filaments at 1 µM, we first pre-polymerize

actin at 24 µM (10% molar label ratio) in F-buffer for at least 1 hour at room

temperatureinthedark.Wethenfollowthesameprocedureasabove,butprepare

themaster buffer by taking into account that pre-polymerized F-actin contains 50

mMKCland2mMMgCl2.

[InsertFigure5here]

Figure 5. TIRF images of in vitro reconstituted actin-septin co-assembly, showing AF594-actinwith

10%molar label ratio (left), AF488-Drosophila septinswith 10%molar label ratio (middle) and the

composite image (right). TheconcentrationsofactinandDrosophila septinsare1µMand0.1µM,

27

respectively.Undertheseconditions,theseptinsarepredominantlypresentashexamers(Mavrakiset

al.,2014).Scalebar,10µm.

[InsertFigure6here]

Figure6.TIRFimagesofAF488-Drosophilaseptinbundlesat1µM(A)andofGFP-taggedDrosophila

septin bundles at 1 µM (B). The spotty appearance of AF488-labeled bundles suggests that the

effectivelabellingstoichiometryisbelowthenominalratioof10%.Scalebars,10µm.

7.TIRFmicroscopy

We image actin-septin filament assembly near the surface of the passivated

coverslips by TIRF microscopy, which is ideally suited to provide a high signal-to-

noiseratiofor invitrosurfaceassays.Samplesare imagedwithaNikonApoTIRF×

100/1.49NAoilobjectivemountedonanEclipseTimicroscope(Nikon)using491nm

and 561 nm laser lines and imaged with a QuantEM 512SC EMCCD camera

(Photometrics).We generally use exposure times of 100-200ms andoptimize the

laserpowerof the488nmand561nm laser lines tomaximize the signal-to-noise

ratiowhileminimizingphotodamage(evidentfromtheoccurenceofsevering)ofthe

actinandseptinfilaments.

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Figurelegends

Figure1.Overviewofcloningstrategyforco-expressingrecombinantseptinsusingthepnEA-vHand

pnCS vectors of the pET-MCN series (Diebold et al., 2011). Here this strategy is used for the

generationof recombinantDrosophila (DSep1-DSep2-Pnut)andhuman (hSep2-hSep6-hSep7) septin

hexamers.Seetextfordetails.

Figure2.Schematicoverviewofthetwo-tagpurificationschemefor isolatingstoichiometrichuman

septin hexamers (the same applies to Drosophila septins by analogy). See text for details.

Understandingtheprinciplesofseptincomplexassemblyisanintensetopicofinvestigation,andthe

presenceandstabilityofintermediatecomplexeshasnotbeenfullydocumented.Weshowselected

populations of human septin complexes in the cell lysate (monomers, homo- and heterodimers,

31

heterotrimers andhexamers) basedon the isolation and characterizationof such recombinant and

native complexes (Kim et al., 2012; Mavrakis et al., 2014; Sellin et al., 2011; Serrao et al., 2011;

Sheffield et al., 2003; Sirajuddin et al., 2007; Sirajuddin et al., 2009; Zent et al., 2011; Zent &

Wittinghofer,2014).

Figure 3. Characterization of purified septin complexes by SDS-PAGE (unlabeled and GFP-labeled

Drosophilaseptinhexamers,leftandrightpanelsinA,respectively)andby2Dsingleparticleanalysis

ofelectronmicroscopyimages(B,seechapterbyAurelieBertin).

Figure4.AflowcellassembledbymeltingstripsofParafilmbetweenacoverslipandaglassslide.

Figure5.TIRF imagesof invitro reconstitutedactin-septinco-assembly.Theconcentrationsofactin

andDrosophilaseptinsare1µMand0.1µM,respectively.Scalebar,10µm.

Figure6.TIRFimagesofAF488-Drosophilaseptinbundlesat1µM(A)andofGFP-taggedDrosophila

septinbundlesat1µM(B).Scalebars,10µm.

Acknowledgments

We thankM. Kuit-Vinkenoog for G-actin purification and F. Iv for septin purification. The research

leading to these results has received funding from CNRS, from two PHC Van Gogh grants (no.

25005UAandno.28879SJ,ministèresdesAffairesétrangèresetdel’Enseignementsupérieuretdela

Recherche), and from the European Research Council under the European Union's Seventh

FrameworkProgramme(FP/2007-2013)/ERCGrantAgreementn.[335672].

Documents

Purification of recombinant human and Drosophila septin