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HAL Id: hal-01363593https://hal.archives-ouvertes.fr/hal-01363593
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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�
1
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:manos.mavrakis@univ-amu.fr;g.koenderink@amolf.nl
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
2
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
3
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.
4
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
5
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).
6
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
7
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
8
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.
9
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
10
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).
Materialsandreagents
Solutionsarepreparedinwaterunlessstatedotherwise.
Forthelysisbuffer:
• Tris-HClpH8,1M,RT
• KCl,4M,RT
• OmniPur®Imidazole(5710-OP,MERCKMillipore),1M,RT
• MgCl21M,RT
11
• 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
12
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
13
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):
361.6kDa,1g/L=2.8µM,ε=0.584L.g-1.cm-1at280nm(assumingallCysreduced)
darkhumanseptins(His6-hSEP2/hSEP6/hSEP7-Strep):
285.7kDa,1g/L=3.5µM,ε=0.565L.g-1.cm-1at280nm(assumingallCysreduced)
Day2a.Concentrationofpurifiedseptincomplexes
Septinstendtobestickyandadsorptiontothemembraneoftheconcentratorleads
to significant yield loss. To improve recovery of septins during the concentration
step,wepassivatetheconcentratorsbytreatingthemwith5%v/vTritonX-100.
14
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).
15
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.
16
[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.
Materialsandreagents
• 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.
Materialsandreagents
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
Materialsandreagents
Solutionsarepreparedinwaterunlessstatedotherwise.
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.
References
Bertin,A.,McMurray,M.A.,Grob,P.,Park,S.S.,Garcia,G.,3rd,Patanwala,I.,...Nogales,E.(2008).Saccharomycescerevisiaeseptins:supramolecularorganizationofheterooligomersandthemechanismoffilamentassembly.ProcNatlAcadSciUSA,105(24),8274-8279.doi:10.1073/pnas.0803330105
Booth,E.A.,Vane,E.W.,Dovala,D.,&Thorner,J.(2015).AForsterResonanceEnergyTransfer(FRET)-basedSystemProvidesInsightintotheOrderedAssemblyofYeastSeptinHetero-octamers.JournalofBiologicalChemistry,290(47),28388-28401.doi:10.1074/jbc.M115.683128
Bridges,A.A.,Zhang,H.,Mehta,S.B.,Occhipinti,P.,Tani,T.,&Gladfelter,A.S.(2014).Septinassembliesformbydiffusion-drivenannealingon
28
membranes.ProcNatlAcadSciUSA,111(6),2146-2151.doi:10.1073/pnas.1314138111
Cordes,T.,Vogelsang,J.,&Tinnefeld,P.(2009).OnthemechanismofTroloxasantiblinkingandantibleachingreagent.JAmChemSoc,131(14),5018-5019.doi:10.1021/ja809117z
Diebold,M.L.,Fribourg,S.,Koch,M.,Metzger,T.,&Romier,C.(2011).Decipheringcorrectstrategiesformultiproteincomplexassemblybyco-expression:applicationtocomplexesaslargeasthehistoneoctamer.JStructBiol,175(2),178-188.Retrievedfromhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=21320604
Fares,H.,Peifer,M.,&Pringle,J.R.(1995).LocalizationandpossiblefunctionsofDrosophilaseptins.MolBiolCell,6(12),1843-1859.Retrievedfromhttp://www.ncbi.nlm.nih.gov/pubmed/8590810
Farkasovsky,M.,Herter,P.,Voss,B.,&Wittinghofer,A.(2005).Nucleotidebindingandfilamentassemblyofrecombinantyeastseptincomplexes.BiolChem,386(7),643-656.doi:10.1515/BC.2005.075
Field,C.M.,al-Awar,O.,Rosenblatt,J.,Wong,M.L.,Alberts,B.,&Mitchison,T.J.(1996).ApurifiedDrosophilaseptincomplexformsfilamentsandexhibitsGTPaseactivity.JCellBiol,133(3),605-616.Retrievedfromhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8636235
Frazier,J.A.,Wong,M.L.,Longtine,M.S.,Pringle,J.R.,Mann,M.,Mitchison,T.J.,&Field,C.(1998).Polymerizationofpurifiedyeastseptins:evidencethatorganizedfilamentarraysmaynotberequiredforseptinfunction.JCellBiol,143(3),737-749.Retrievedfromhttp://www.ncbi.nlm.nih.gov/pubmed/9813094
Garcia,G.,3rd,Bertin,A.,Li,Z.,Song,Y.,McMurray,M.A.,Thorner,J.,&Nogales,E.(2011).Subunit-dependentmodulationofseptinassembly:buddingyeastseptinShs1promotesringandgauzeformation.JCellBiol,195(6),993-1004.doi:10.1083/jcb.201107123
Green,M.R.,&Sambrook,J.(2012).MolecularCloning:ALaboratoryManual(FourthEdition):ColdSpringHarborLaboratoryPress.Hartwell,L.H.(1971).Geneticcontrolofthecelldivisioncycleinyeast.IV.Genes
controllingbudemergenceandcytokinesis.ExpCellRes,69(2),265-276.Retrievedfromhttp://www.ncbi.nlm.nih.gov/pubmed/4950437
Hartwell,L.H.,Culotti,J.,Pringle,J.R.,&Reid,B.J.(1974).Geneticcontrolofthecelldivisioncycleinyeast.Science,183(4120),46-51.Retrievedfromhttp://www.ncbi.nlm.nih.gov/pubmed/4587263
Hsu,S.C.,Hazuka,C.D.,Roth,R.,Foletti,D.L.,Heuser,J.,&Scheller,R.H.(1998).Subunitcomposition,proteininteractions,andstructuresofthemammalianbrainsec6/8complexandseptinfilaments.Neuron,20(6),1111-1122.Retrievedfromhttp://www.ncbi.nlm.nih.gov/pubmed/9655500
Huijbregts,R.P.,Svitin,A.,Stinnett,M.W.,Renfrow,M.B.,&Chesnokov,I.(2009).DrosophilaOrc6facilitatesGTPaseactivityandfilamentformationoftheseptincomplex.MolBiolCell,20(1),270-281.doi:10.1091/mbc.E08-07-0754
John,C.M.,Hite,R.K.,Weirich,C.S.,Fitzgerald,D.J.,Jawhari,H.,Faty,M.,...Steinmetz,M.O.(2007).TheCaenorhabditiselegansseptincomplexisnonpolar.EMBOJ,26(14),3296-3307.doi:10.1038/sj.emboj.7601775
29
Kim,M.S.,Froese,C.D.,Estey,M.P.,&Trimble,W.S.(2011).SEPT9occupiestheterminalpositionsinseptinoctamersandmediatespolymerization-dependentfunctionsinabscission.JCellBiol,195(5),815-826.doi:10.1083/jcb.201106131
Kim,M.S.,Froese,C.D.,Xie,H.,&Trimble,W.S.(2012).Uncoveringprinciplesthatcontrolseptin-septininteractions.JournalofBiologicalChemistry,287(36),30406-30413.doi:10.1074/jbc.M112.387464
Kinoshita,M.,Field,C.M.,Coughlin,M.L.,Straight,A.F.,&Mitchison,T.J.(2002).Self-andactin-templatedassemblyofMammalianseptins.DevCell,3(6),791-802.Retrievedfromhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12479805
Mavrakis,M.,Azou-Gros,Y.,Tsai,F.C.,Alvarado,J.,Bertin,A.,Iv,F.,...Lecuit,T.(2014).SeptinspromoteF-actinringformationbycrosslinkingactinfilamentsintocurvedbundles.NatCellBiol,16(4),322-334.doi:10.1038/ncb2921
Nakatsuru,S.,Sudo,K.,&Nakamura,Y.(1994).MolecularcloningofanovelhumancDNAhomologoustoCDC10inSaccharomycescerevisiae.BiochemBiophysResCommun,202(1),82-87.doi:10.1006/bbrc.1994.1896
Neufeld,T.P.,&Rubin,G.M.(1994).TheDrosophilapeanutgeneisrequiredforcytokinesisandencodesaproteinsimilartoyeastputativebudneckfilamentproteins.Cell,77(3),371-379.Retrievedfromhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8181057
Nishihama,R.,Onishi,M.,&Pringle,J.R.(2011).Newinsightsintothephylogeneticdistributionandevolutionaryoriginsoftheseptins.BiolChem,392(8-9),681-687.doi:10.1515/BC.2011.086
Pan,F.,Malmberg,R.L.,&Momany,M.(2007).Analysisofseptinsacrosskingdomsrevealsorthologyandnewmotifs.BMCEvolBiol,7,103.doi:10.1186/1471-2148-7-103
Pardee,J.D.,&Spudich,J.A.(1982).Purificationofmuscleactin.MethodsCellBiol,24,271-289.Retrievedfromhttp://www.ncbi.nlm.nih.gov/pubmed/7098993
Popp,D.,Yamamoto,A.,Iwasa,M.,&Maeda,Y.(2006).Directvisualizationofactinnematicnetworkformationanddynamics.BiochemBiophysResCommun,351(2),348-353.doi:10.1016/j.bbrc.2006.10.041
Renz,C.,Johnsson,N.,&Gronemeyer,T.(2013).AnefficientprotocolforthepurificationandlabelingofentireyeastseptinrodsfromE.coliforquantitativeinvitroexperimentation.BMCBiotechnol,13,60.doi:10.1186/1472-6750-13-60
Sadian,Y.,Gatsogiannis,C.,Patasi,C.,Hofnagel,O.,Goody,R.S.,Farkasovsky,M.,&Raunser,S.(2013).TheroleofCdc42andGic1intheregulationofseptinfilamentformationanddissociation.Elife,2,e01085.doi:10.7554/eLife.01085
Sellin,M.E.,Sandblad,L.,Stenmark,S.,&Gullberg,M.(2011).Decipheringtherulesgoverningassemblyorderofmammalianseptincomplexes.MolBiolCell,22(17),3152-3164.doi:10.1091/mbc.E11-03-0253
Serrao,V.H.,Alessandro,F.,Caldas,V.E.,Marcal,R.L.,Pereira,H.D.,Thiemann,O.H.,&Garratt,R.C.(2011).Promiscuousinteractionsofhumanseptins:
30
theGTPbindingdomainofSEPT7formsfilamentswithinthecrystal.FEBSLett,585(24),3868-3873.doi:10.1016/j.febslet.2011.10.043
Sheffield,P.J.,Oliver,C.J.,Kremer,B.E.,Sheng,S.,Shao,Z.,&Macara,I.G.(2003).Borg/septininteractionsandtheassemblyofmammalianseptinheterodimers,trimers,andfilaments.JournalofBiologicalChemistry,278(5),3483-3488.doi:10.1074/jbc.M209701200
Shi,X.,Lim,J.,&Ha,T.(2010).Acidificationoftheoxygenscavengingsysteminsingle-moleculefluorescencestudies:insitusensingwitharatiometricdual-emissionprobe.AnalChem,82(14),6132-6138.doi:10.1021/ac1008749
Sirajuddin,M.,Farkasovsky,M.,Hauer,F.,Kuhlmann,D.,Macara,I.G.,Weyand,M.,...Wittinghofer,A.(2007).Structuralinsightintofilamentformationbymammalianseptins.Nature,449(7160),311-315.doi:10.1038/nature06052
Sirajuddin,M.,Farkasovsky,M.,Zent,E.,&Wittinghofer,A.(2009).GTP-inducedconformationalchangesinseptinsandimplicationsforfunction.ProcNatlAcadSciUSA,106(39),16592-16597.doi:10.1073/pnas.0902858106
SoareseSilva,M.,Depken,M.,Stuhrmann,B.,Korsten,M.,MacKintosh,F.C.,&Koenderink,G.H.(2011).Activemultistagecoarseningofactinnetworksdrivenbymyosinmotors.ProcNatlAcadSciUSA,108(23),9408-9413.doi:10.1073/pnas.1016616108
Versele,M.,Gullbrand,B.,Shulewitz,M.J.,Cid,V.J.,Bahmanyar,S.,Chen,R.E.,...Thorner,J.(2004).Protein-proteininteractionsgoverningseptinheteropentamerassemblyandseptinfilamentorganizationinSaccharomycescerevisiae.MolBiolCell,15(10),4568-4583.doi:10.1091/mbc.E04-04-0330
Zent,E.,Vetter,I.,&Wittinghofer,A.(2011).StructuralandbiochemicalpropertiesofSept7,auniqueseptinrequiredforfilamentformation.BiolChem,392(8-9),791-797.doi:10.1515/BC.2011.082
Zent,E.,&Wittinghofer,A.(2014).HumanseptinisoformsandtheGDP-GTPcycle.BiolChem,395(2),169-180.doi:10.1515/hsz-2013-0268
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].
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