Peters EC, Gossett AJ, Goldstein B, Der CJ, Reiner DJ (2013).

15 Pages·2013·1.67 MB·English

INVESTIGATION
Redundant Canonical and Noncanonical
Caenorhabditis elegans p21-Activated Kinase
Signaling Governs Distal Tip Cell Migrations
Eldon C.Peters,* AndreaJ.Gossett,†BobGoldstein,†ChanningJ.Der,*,‡andDavidJ.Reiner*,‡,1
*LinebergerComprehensiveCancerCenter,†DepartmentofBiology,and‡DepartmentofPharmacology,Universityof
NorthCarolina,ChapelHill,NorthCarolina27599
ABSTRACT p21-activated kinases (Paks) are prominent mediators of Rac/Cdc42-dependent and KEYWORDS
-independentsignalingandregulatesignaltransductionandcytoskeletal-basedcellmovements.Weused RhoG
thereproduciblemigrationsoftheCaenorhabditiselegansgonadaldistaltipcellstoshowthattwoofthe Dock180
three nematode Pak proteins, MAX-2 and PAK-1, function redundantly in regulation of cell migration but ELMO
are regulated by very different mechanisms. First, we suggest that MAX-2 requires CED-10/Rac function MIG-2
and thus functions canonically. Second, PIX-1 and GIT-1 function in the same role as PAK-1, and PAK-1 RAC-2
interactionwithPIX-1isrequiredforPAK-1activity;thus,PAK-1functionsnoncanonically.ThehumanPak- PAK-2
Pix-Git complex is central to noncanonical Pak signaling and requires only modest Rac/CDC-42 input.
Unlike the human complex, our results suggest that the C. elegans Pak-Pix-Git complex requires PAK-1
kinase domain activity. This study delineates signaling network relationships in this cell migration model,
thus providing potential further mechanistic insights and an assessment of total Pak contribution to cell
migration events.
The p21-activated kinases, or Paks, are a group of Ste20-related of binding Cdc42 and Rac, Group B Paks are not activated upon
kinases that function in signal transduction and regulation of cyto- GTPasebindingandprobablyhavefunctionsdistinctfromtheGroup
skeletalorganization.Theyaredefinedbysharedkinasedomainho- APaks(Arias-RomeroandChernoff2008).Instead,theRacorCdc42
mologyandtheabilitytobeboundandregulatedbyCdc42andRac GTPasemaybeinvolvedinsubcellulartargetingoftheGroupBPaks,
smallGTPases(Bokoch2003;Hofmannetal.2004).Rhofamilysmall butPakactivityispromotedbysomeindependentmechanism.Both
GTPases, including Cdc42 and Rac, are regulated by their guanine groupsarethoughttoregulatethecytoskeletonandcellmovements,
nucleotide binding state; guanine nucleotide exchange factors (GEFs) buttheGroupAPaksarefarbetterstudied.Inadditiontoactivities
stimulateGTPloadingandhenceactivation,whereasGTPase-activating mediated by the kinase domain, Group A Paks also can function as
proteinsacceleratetheintrinsicGTPaseactivitytohydrolyzeguanosine akinase-andRac-Cdc42-independentscaffoldforacomplexcontain-
triphosphate (i.e., GTP) to guanosine diphosphate (i.e., GDP) and ing the exchange factor Pix and the G-protein2coupled receptor
inactivate the protein(Wennerberg and Der2004). kinase interactor Git (Loo et al. 2004; Parnas et al. 2001). Their
MammalianPaksaredividedintotwogroups:GroupA(Pak1,2, kinase-independentactivitiesarethoughttobeGTPaseindependent,
3)andGroupB(Pak4,5,6).Cdc42orRacfrequentlyactivatesGroup althoughperhapstheGTPaseaidssubcellularlocalization.Inaddition,
APaks.Althoughtheypossessap21-bindingdomain(PBD)capable kinase-independentbutRac-dependentPakhasbeenshowntofunc-
tionasascaffoldforPDKandAktsignaling(Higuchietal.2008).
Group A Paks have been associated with a variety of human
Copyright©2013Petersetal. diseases,includingoncogenesisandmetastasisincancer(reviewedin
doi:10.1534/g3.112.004416 Kumar et al. 2006), Alzheimer’s disease (McPhie et al. 2003; Zhao
ManuscriptreceivedSeptember13,2012;acceptedforpublicationNovember27,2012
This is an open-access article distributed under the terms of the Creative et al. 2006), and X-linked nonsyndromic mental retardation (Allen
CommonsAttributionUnportedLicense(http://creativecommons.org/licenses/ et al. 1998; Bienvenu et al. 2000; Gedeon et al. 2003). This latter
by/3.0/),whichpermitsunrestricteduse,distribution,andreproductioninany functionis thought toinvolve Pakinthe context ofthe Pak-Pix-Git
medium,providedtheoriginalworkisproperlycited.
complex rather than canonical Rac-Pak signaling (Kutsche et al.
1Correspondingauthor:LinebergerComprehensiveCancerCenterand
2000). Together, these Pak-based disease etiologies, along with the
Departmentof,Pharmacology,UniversityofNorthCarolina,ChapelHill,NC
27599-7295.E-mail:[email protected] regulation of cytoskeleton and cell projections by Paks, argue that
Volume3 | February 2013 | 181
Paks arecritical regulatorsof cell movementsand are importantfor functionupstreamofMAX-2,whereasthePIX-1/PixandGIT-1/Git
the understanding of many diseases. However, the extensive redun- orthologs are required for PAK-1 branch activity. Similar genetic
dancyinherentinsixmammalianPakgenessuggeststhatPakstudies relationships have been previously described (Lucanic and Cheng
insimpler metazoans, which have fewerPaks,couldshedimportant 2008),althoughwearriveatsomewhat-differentmechanisticconclu-
lightontotalPakcontributiontocellbiology. sions.WealsoshowthattheCED-2,-5,/-12Dock/ELMOnoncanon-
Caenorhabditis elegans contains three Pak proteins (Chen et al. icalRacGEFcomplexislikelytoactivateCED-10inDTCmigration.
1996;Hofmannetal.2004);PAK-1sharesallknownsequencemotifs DespitetheapparentroleofPAK-1inaPak-Pix-Gitcomplex,which
with Group A Paks, PAK-2 is more similar to Group B Paks, and waspreviouslydescribedas beingkinase-independent,ourdatasug-
MAX-2, although closest in sequence identity to Group A Paks in gestthatPAK-1kinasedomainactivityisrequiredforPAK-1branch
thePBDandkinaseregions,doesnotshareotherN-terminalregulatory function.Furthermore,geneticanalysissuggestsamodestcontribution
sequence motifs typical of Group A Paks. PAK-1 binds CDC-42 and ofCED-10/RactoPAK-1branchactivity,suggestingthatunlikepre-
CED-10/RacandduringmorphogenesiscolocalizeswiththeseGTPases viouslydescribed,thePak-Pix-GitcomplexispartiallyRac-dependent.
attheplasmamembraneofepithelialcells(Chenetal.1996).Lossof WealsoidentifyothereventsduringC.elegansdevelopmentthatre-
MAX-2 causes modest disruptions of axonal pathfinding and loss of dundantlyrequireMAX-2andPAK-1.
PAK-1greatlyenhancesthesedefects,butlossofPAK-1alonecauses
noobviousdefects(Lucanicetal.2006).Consistentwiththefrequently MATERIALSAND METHODS
observedantagonismbetweenRac/Cdc42andRho,inDrosophilaPak
andRhoareantagonistic(VlachosandHarden2010),andPakpartic- C.elegansculturing
ipates with Rac and Cdc42 inmigrationsof epithelial sheets (Harden Handling,maintenance,andnomenclatureofC.elegansstrainswereas
et al. 1999). Use of multiple signals and multiple GTPase effectors is described(Brenner1974;Horvitzetal.1979).Allstrainswerederived
complex in growth cone migration (Demarco and Lundquist 2010; fromtheN2(Bristol)wildtypeandwereculturedonEscherichiacoli
Lucanic et al. 2006; Lundquist et al. 2001; Norris et al. 2009; Quinn strainOP50.Animalsweregrownina23(cid:1)incubator,whichwefound
et al. 2008; Shakir et al. 2006, 2008; Soto et al. 2002; Struckhoff and tobetheoptimaltemperatureforbacteriallymediatedRNAi.Asum-
Lundquist2003)andepithelialmorphogenesis(Gallyetal.2009;Patel maryofkeymutationsusedinthisstudyisshowninTable1.
etal.2008;Zhangetal.2011).Distaltipcellmigration,analyzedhere,
mayprovideasimplermodelforPakpathwayrelationships. ScoringDTCmigrationphenotypes
In this study we analyze the roles of C. elegans Pak proteins in Hermaphroditesweregrownat23(cid:1)andscoredatthelateL4stageon
migration of the distal tip cells (DTCs) of the gonad. DTCs are so- 3% agar pads using a Nikon microscope equipped with Differential
maticgonadalcellswhosemigratorypathduringlarvaldevelopment Interference Contrast (DIC) Nomarski optics. The DTC migration
dictatestheshapeofthematuregonad(Hedgecocketal.1987;Kimble path was inferred from the final position of the two fully developed
andHirsh 1979).Startingventrallynearthe anterior/posterior(A/P) gonadarms,andfordatastorageforeachanimal,theshapeofboth
mid-pointinthebody,thetwoDTCsmigrateanteriorlyorposteriorly gonad arms wassketched. For eachgonad arm,the entiretrajectory
on the surface of the basement membrane, turn dorsally, then turn wastracked,includingZaxischanges.Usingthistechniquewefound
again to migrate back to the A/P mid-point, thus forming a highly thatwecouldreproduciblydistinguishbetweenfolded-overandtrun-
reproducible inverted-U shape. During migration, the DTC remains catedgonadarms.Inpreviousstudiesauthorssuggestthateacharm
connected to its gonad arm, and the gonad proceeds with germline migratesindependentlyoftheother,sothateacharmconstitutesan
development.BecausethepreciserouteofDTCmigrationcanbein- individualdatapoint(Nishiwaki1999).
ferredafterthefactbythefinalshapeofthematuregonad(Nishiwaki Scoring DTC migration revealed two general phenotypic classes:
1999),DTCmigrationisanexcellentsystemforstudyingthegenetic (1)pathfindingdefects,wheretheDTCsmigratedthenormaldistance
regulationofcellpathfindingandmigration. but made inappropriate turns; and (2) truncation defects, where the
Here we describe striking synergy between max-2 and pak-1 in DTCmigrationstoppedimmediatelyafterthedorsalturn.Inaddition,
DTC migration. Loss of both results in pathfinding errors and in- some DTCs exhibited both pathfinding and truncation defects. A
completely extended gonad arms, suggesting that these redundant DTC was scored as wild type if it extended from the vulva, turned
Paksarecriticalforbothappropriatedirectionalityofmigrationand dorsally,thenreturnedtothemidline,forminga“U”shapeinone
migration itself. Genetically, we show that CED-10/Rac is likely to plane. Also scored as wild type were gonads with minor shape
n Table1 Mutantallelesusedinthisstudyandtheireffectsonproteinfunction
Gene Allele Lesion ProposedFunctionalEffect Reference
pak-1 ok448 Exon7-10D(outofframe) Kinasedeleted,regulatoryintact Lucanicetal.2006
tm403 Exon1-7D(in-frame) Regulatorysequencesdeleted, Zhangetal.2011
kinasedomainintact(lforgf?)
max-2 nv162 Exon1-4D(incl.ATG/startingMet) Nullforfull-lengthisoform Lucanicetal.2006
cy2 G340E Lossofkinasefunction Lucanicetal.2006
pak-2 ok332 Exon1-7D Putativenull Fujikietal.2010
ced-10 n1993 V190G Reducedfunction ReddienandHorvitz2000
n3246 G60R Alteredfunction(gfordn) ReddienandHorvitz2000;Shakiretal.2006
mig-2 mu28 W60stop Putativenull Zipkinetal.1997
pix-1 ok982 Exon12-14D Probablestrongloss LucanicandCheng2008
gk416 Exon1-4D Probablestrongloss LucanicandCheng2008
git-1 tm1962 Exon8-9D Probablestrongloss LucanicandCheng2008
182 | E.C.Petersetal.
perturbationsthatappearatlowfrequencyinallgenotypesobserved, secondcrosswasperformedonmax-2RNAiplates,andnon-Uncnon-
including the wild type. Included in this category are gonads in DpyhermaphroditeL4progenywerescoredforDTCmigrationdefects.
whomthedistalarmdipsventrallyattheendofitsmigration,distal
armswhosemigrationisslightlyshort,andminordorsalextension
Plasmidsused
defectsthatcauseslightripplesintheresultinggonad.Wealsonotethat
Bacterially mediated RNAi used genomic or cDNA sequence
allstrainsderivedfromtheN2Bristolwildtypehadabaselineof10–
insertionsinthepPD129.36(a.k.a.L4440)backbone.Thoseannotated
15%pathfindingdefects,althoughnotruncationdefectswereobserved.
withchromosomallocationswerefromtheAhringerlibrary(Kamath
Wedefine“pathfinding”defectsasgonadsthathavemadeincorrect
et al. 2001). Pre-existing RNAi plasmids used were: gfp (Zand et al.
turnsduringdevelopment.Weobservednoreproduciblepatternsasso-
2011), pop-1 (I-1K04), pak-1 (X-3E04), max-2 (II-8F19), git-1 (X-
ciated with specific genotypes, and there were many different ways in
5I19),unc-22(IV-6K06),andrac-2(IV-7L24).Ourconstructedfeed-
whichthegonadscouldmis-migrate,includingfailuresofallturnsand
ing RNAi plasmids, pEP2.7 (L4440+ced-10): We digested pEL319
mostpossibleinappropriateturns.AtlowfrequencyweobservedaDTC
(Lundquist et al. 2001) with BglI/NcoI to remove rrf-3, leaving only
migratingpasttheA/Pmidline,althoughnotasignificantdistance.
ced-10cDNA,andthenitwasKlenowtreatedandbluntligated.pEP1
A truncation defect describes gonads with an earlytermination of
(L4440+mig-2): we used DJR611/612 (ATGTCTTCACCGTCGAGG
migration.Thisphenomenoncanbebrokendownintothreecategories:
CAG/CGTCGCGCAAATCGAGTTTGG) from C. elegans genomic
(1)ashortarm,wherethegonadextends,butmakesnodorsalturn;(2)
DNA to amplify a 970-bp region between exons 1 and 3 of
adorsal“hockeystick,”wherethegonadstopsjustafterthedorsalturn; mig-2 andcloned itintoL4440digested withNotI/SalI.
and (3) no arm, where the gonad does not extend at all. The large Markers and experimental plasmids for transgenics, i.e., pMH86
majorityoftruncationsobservedwerethe“hockeystick”variety,where (dpy-20 (+)), pPD118.33 (P ::gfp): The pJK590 plasmid (P )
myo-2 lag-2
theDTCmakesthedorsalturnbutstops.Manytruncatedgonadswere (Blelloch et al. 1999) uses the 3-kb sequence upstream of the lag-2
swollen at the distal end, as if there were not sufficient room in the startcodon(59tttttaaattctcat–caaatttgccttt39)andcontainsamutated
truncatedgonadtocontainthegermlineproliferationtherein. BsmI site within the promoter to assist in cloning. pCM15.2 (P ::
lag-2
TheN2wild-typestrainshowedminorpathfindingdefectsatlow cdc-42):Weamplifiedcdc-42cDNAfromplasmidyk1443h8byusing
penetrance. We examined N2 Bristol stocks from several different DJR549/550 (ATGCAGACGATCAAGTGCGTCGTCG/TTAGAGA
laboratories and the CB4856 wild isolate from Hawaii and observed ATATTGCACTTCTTCTTC) and cloning it into pCM14.1 (P ::
lag-2
defectsataveragepenetranceof12%.Mostdefectswereminor,butall chw-1) digested with AgeI/NotI (V. Muñiz-Medina, C. J. Der, and
strainshadrareanimalswithsignificantpathfindingdefects.Wean- D. J. Reiner, unpublished data). We mutagenized pCM15.2 with
alyzedplatesmadeindifferentlaboratoriesandincubatorsindifferent DJR517/518tocreatepCM15.3(P ::cdc-42Q61L)andmutagenized
lag-2
buildingsandneversawanappreciabledifferenceinbasaldefectrates. pCM15.2withDJR623/624togeneratepCM15.4(P ::cdc-42S17N).
lag-2
We hypothesize that this phenomenon is not restricted to the N2 pCM13.1 (P ::ced-10): ced-10 cDNA was amplified from plasmid
lag-2
strainandisageneralfeatureofC.elegansDTCmigration. pEL319 using DJR545/546 (ATGCAAGCGATCAAATGTGTCG
TCG/TTAGAGCACCGTACACTTGCTCTTTTTGG)wasclonedin-
Bacteriallymediated RNAiprotocol topCM14.1digestedwithAgeI/NotI.WemutagenizedpCM13.1with
RNAimethodswerederivedfromdescribedprotocols(Kamathetal. DJR513/514 to create pEP3.2 (Plag-2::ced-10 Q61L). pEP12.1 (Plag-2::
2003;Timmonsetal.2001).HT115bacteria,transformedwithL4440 pak-1(+)): we amplified pak-1 cDNA from yk1619a12 by using
plasmidcontainingcodingregionforthegeneofinterest,wereplated DJR631/629 (AGGCTTGCCAAAATGAAAGCTTTCTCATCG/TTA
for no more than 16 hr on LB media containing tetracycline and TGAGTTGCTAGCTTCGGCGATGC) and cloning it into pCM14.1
ampicillin. Overnight cultures in LB plus ampicillin were grown for digestedwithKpnI/NotI.WemutagenizedpEP12.1withDJR678/679
nomorethan12hr(longergrowthcoulddecreaseRNAiefficacy).A to generate the disrupted Pix binding mutant pEP12.2 (Plag-2::pak-1
totalof80mLofeachfeedingRNAiculturewasspottedontotwoNG (234A,235A)) and mutagenized pEP12.1 with DJR674/675 to generate
platescontaining1mMIPTGand50mg/mLcarbenecillin.Depend- thekinasedeadmutantpEP12.3(Plag-2::pak-1(324R)).pEP15.1(Plet-858::
ingonthefecundityofthestrain,fourtoeightlateL4hermaphrodites pak-1(+)):WedigestedpPD118.25withNheItoremovegfpandthen
religated to create pEP10.1. We amplified pak-1 cDNA from
were picked to a plate for each feeding RNAi clone. Animals were
yk1619a12 by using DJR631/629 (as described previously) and
pickedtoRNAiplatesusingmineraloiltominimizetransferofcon-
cloned into pEP10.1 digested with KpnI/SpeI. Sequencing primers
taminatingOP50bacteria.Animalsweretransferredafter24hrtothe
forpPD118.25wereDJR655/656andforpJK590mutwereDJR654/
secondplatefromthesamefeedingRNAiculture.L4hermaphrodite
660. The sequences of plasmids and oligonucleotides are available
progenywerescored48hrafterthetransfer.
uponrequest.
Everyexperimentincludedpositiveandnegativecontrolsofpop-1-
and gfp-directed RNAi, respectively. In our hands under optimal condi-
tions,pop-1-directedRNAiinduced100%embryoniclethality.Experiments Transgene generation
whereanimalsescapedpop-1-inducedlethalitywerediscarded.AllRNAi Markers (concentrations) used included: pA2132 (unc-119 (+)) at
experiments were compared with gfp-directed RNAi baselines to control 50 ng/mL; pMH86(dpy-20(+)) at20ng/mL; pPD118.33 (Pmyo-2::gfp)
forpossiblemigrationeffectsofengagingtheRNAiresponse. at5ng/mL;Plag-2::ced-10(orcdc-42)at5ng/mL;andPlag-2::pak-1at
5or10ng/mL.
pak-1dominance test
We tested potential dominance of pak-1 alleles by comparing max-2 Mutations used
(RNAi); pak-1(tm403)/+ with max-2(RNAi); pak-1(ok448)/+ animals. Weusedthefollowingmutations:pak-1(ok448),pak-1(tm403),max-2
pak-1 is on the X chromosome, so we performed this experiment by (nv162), max-2(cy2), mig-2(mu28), pix-1(ok982), pix-1(gk416), git-1
crossingN2malestotm403homozygotestogeneratetm403/0hemizy- (tm1962), ced-10(n1993), ced-10(n3246), ced-1(e1756), ced-5(n1812),
gousmales,whichwerethencrossedintodpy-8(e130);unc-6(e78).This ced-12(n3261), unc-33(e204), hIn1[unc-54(h1040)], unc-29(e193),
Volume3 February2013 | RedundantPaksinCellMigration | 183
dpy-24(s71), unc-43(n498n1179), dpy-8(130), unc-6(e78), unc-119 whereanotherPakgenewasmutated.Atamorphologicallevel,double
(ed3), dpy-20(e1282). Before performing double loss-of-function loss-of-functionanimalsappearedwildtypeexceptforonephenotype;
analysis, we outcrossed pak-1 alleles ok448 and tm403 for 10· and greaterthan90%ofanimalsdisplayedlightpatchesinvariouspartsof
5·,respectively).max-2allelesnv162andcy2werebothoutcrossed4·. the body between the pharynx and anus (Figure 1, A and B). This
patchyphenotypeisoftenseenwithdefectsinmigrationofthegonadal
Primersforgenotyping DTCs(Nishiwaki1999).Normally,thetwoDTCsofthesomaticgonad
To detect deletions for outcrossing the alleles we used the following migrate in mirror image U-shapes. Each arm of the mature gonad
primers: pak-1(ok448): DJR684.f1 CAGTACACAAAACCGAAA occupiesalateralhalfofthebodycavity,whereastheintestine,which
GAGGAGG, DJR685.f2 GTTGGTAAAAGTTAGGGATGACCCC, appears dark in the dissecting microscope, occupies the other half.
DJR686.r1 TTCTCTTACCTTGAACAACAAAGTCATGG; pak-1 Thus,becauseanimalsontheplategenerallyareviewedfromtheside,
(tm403):DJR636.f1ATGAGGGCATGTAATACACAAGTACCG, in the wildtype one observes the uninterrupted darkbandof the in-
DJR637.f2 TTGCATGCTTATTCTCACGCATCACC, DJR638.r1 testinerunningthelengthoftheanimal,andthecleargonadarmsare
GAATCTCTTCCAGGGAAGTCGGG; max-2(nv162) (Lucanic et al. notvisible.However,intheeventof defectiveDTCpathfinding,por-
2006): nv162.f1 CCGGCAGGAAGACTATATGACTC, nv162.r1 tionsofthegonadcanoverlap,occupyingboththeleftandrightplanes,
CACAAAGAGGGAAGAAGATCCTC, nv162.r2 CCTTCTTCTGA from the lateral perspective, and thus displace the intestine. This in-
TCGGCAAGACTG; max-2(cy2) (Lucanic et al. 2006): cy2.f1 testinaldisplacementresultsinclearpatchesthatareeasilydetectedby
CGGCAGTGTTGTCTCCACAACAT, cy2.r1, GCCGAGCAGCAC dissectingmicroscope,andthisphenotypehasbeenshowntobestrong
GTTGTCACTCT,cy2eliminatesaBspEIsiteintheamplicon;git-1 enoughtoisolatemutantsingeneticscreens(Nishiwaki1999).
(tm1962): DJR667.f1 CGATCTCAGTTCTCCCGAAAGG, DJR668.f2 By DIC/Nomarksi microscopy, we recorded highly penetrant DTC
GCAAAATGAGGAGCTGACAAAGTG, DJR669.r1 GGTGGCATAA migrationdefectsinmax-2;pak-1doubleloss-of-functionanimalscom-
CTCTTTCTGGAGT; pix-1(gk416): DJR661.f1 CCCCGCAAAAGA pared with few defects in the single mutant (Figure 1C). We observed
GACCCAGAG, DJR662.f2 TCGGAAAAGGAGTACATGCAAAGC, a wide range of pathfinding defects, but with one exception (see Pak
DJR663.r1 GAAACAGTGAGCTACTCGCCCC; and pix-1(ok982): controlsbothDTCpathfindingandmigrationexecution,below)noclass
DJR664.f1 GGACTCAGTGAGCCACTCCGG, DJR665.f2 CATCCCC of defect was more representative than another, and both the anterior
CAGGAAACTCGGAT,DJR666.r1CAATGCGGGGCTCGAACC. andposteriorgonadarmswereaffectedequally.Weanalyzedtwoalleles
of each gene in double loss-of-function combinations by targeting the
other Pak gene with RNAi; all observed mutation-RNAi combinations
RESULTS
conferredstrongsyntheticDTCmigrationdefects,withnoquantitative
PAK-1andMAX-2redundantly controlDTCmigration orqualitativedifferencesbetweenthegeneticcombinations(Figure1C).
To analyze the functions of Pak kinases during development, we As negative controls, single-mutant animals subjected to gfp-directed
disruptedPakactivitybyRNAi-targetingonePakgeneinabackground RNAiwerenotsignificantlydifferentfromanimalsgrownwithoutRNAi.
Figure 1 max-2 and pak-1 are redundant for DTC
migration.Bright-fieldphotomicrographsof(A)max-
2(nv162) and (B) max-2(nv162); pak-1(RNAi) late L4
animals.Inbothanimalsanteriorisleftandventralis
down.Ablackarrowindicatesthesemicircularclear
patchformedbythedevelopingvulvaanduterusat
thelateL4stage,andwhitearrowsindicateinappro-
priateclearpatchescausedbygonadoverlap.(C)All
pair-wise combinations of max-2 and pak-1 disrup-
tion show strong synthetic DTC migration and go-
nad shape defects. Number in columns is the
numberofgonadarmsscored.pak-1singlemutants
onnegativecontrolgfp-directedRNAiwerenotsig-
nificantlydifferentfromthewildtype(5–15%defec-
tiveinallexperiments,seeFigure3Bforexample),
whereasmax-2lossoffunctionmutationsmaycause
DTCmigrationdefectsthatareslightlyelevated;cy2
butnotnv162ongfp(RNAi)wassignificantlydiffer-
ent than both ok448andtm403 (P , 0.05). For all
gfp vs. pak-1 or max-2 RNAi comparisons (paired
columns),P,0.0001byFisher’sexacttest.Noerror
barsareshownbecausethesearepooledassays.
184 | E.C.Petersetal.
AllelesusedinthisstudyarelistedinTable1.pak-1(ok448)deletes n Table2 pak-1(tm403)butnotpak-1(ok448)isweaklydominant
mostofthekinasedomainanddisruptssubsequentreadingframeand
Genotype %DTCDefect(N)
should therefore be null for PAK-1 kinase function. pak-1(tm403)
gfp(RNAi);pak-1(ok448)/+ 10.2(168)
deletes most of the N-terminal regulatory sequences, including the
gfp(RNAi);pak-1(tm403)/+ 12.5(160)
PBD, the autoinhibitory domain, the Pix-binding site, the acidic re-
max-2(RNAi);pak-1(ok448)/+ 17.3(228)
gionandmanyofthepotentiallyregulatoryPXXPSH2-bindingsites, max-2(RNAi);pak-1(tm403)/+ 26.5(148)
but frame is retained and a functional kinase should be made. Such
ok448/+ortm403/+animalsweretestedfordominanteffectsbyplatingongfp-
atruncatedproteincouldresultinagain-of-functionortoxicproduct
directed (control) or max-2-directed (experimental) RNAi and assessing fre-
(see pak-1(tm403) may be weakly semidominant, below). max-2 quencyofDTCmigrationdefects.tm403/+;max-2(RNAi)issignificantlydifferent
(nv162) deletes the first four exons of the gene, including the start thantm403/+;gfp(RNAi)(P,0.05,Fisher’sexacttest).DTC,distaltipcells;N,
codon,andthusrepresentsanullforfull-lengthpakfunction.max-2 numberofgonadarmsscored.
(cy2) is a point mutation altering a conserved residue in the kinase
domain, suggesting that it causes strong kinase loss of function turnswereobservedateverystageofDTCmigration.Wehypothesize
(Lucanic et al. 2006). We note that both pak-1 and max-2 express that these phenotypes reflect a general requirement for redundant
multiple isoforms due to alternative splicing or use of alternative pro- max-2 and pak-1 function in communicating positional information
moters, including shorter isoforms altogether lacking the N-terminal fromtheextracellularenvironmentintothemigratingcell.
regulatory regions (wormbase.org WS228). Furthermore, mammalian Second, a large subset of mutant animals had prematurely
GroupAPakshavebeenshowntohavekinase-independentfunctions terminatedDTCmigrations,wheremigrationwasarrested/truncated
(see Putative PAK-1 partners GIT-1 and PIX-1 are redundant with justaftertheDTCmadethedorsalturnhalfwaythroughthewild-type
MAX-2,below).Thus,mutationsthateliminatecertaindomainsofthe migration path (Figure 2, C and D). Generally, the resulting gonads
proteinmaystillretaindevelopmentalfunctions,andnoneofthemuta- wereofotherwisenormalshape,althoughweobservedbulgingofthe
tionscharacterizedthusfarislikelytobenullforallgenefunctions. gonad arm toward the distal end of the gonad (Figure 2D). Table 3
displaysthefrequenciesofthetwoclassesofphenotypes.Wenotethat
pak-1(tm403)may beweaklysemidominant the high frequency of DTC truncations likely obscures underlying
The pak-1(tm403) allele deletes the N-term regulatory sequences in- pathfinding defects, thereby depressing our estimate of pathfinding
frameand,duetode-regulation,mightproduceagain-of-functionor defects in genotypes with frequent truncation defects. We speculate
otherwise toxic protein. Thus tm403 might be predicted to be semi- thatwithoutthetruncationdefect,mostDTCswouldstillhavepath-
dominant. We tested this prediction genetically. gfp(RNAi); pak-1 finding defects. The fertility of truncated gonad arms could not be
(tm403)/+andgfp(RNAi);pak-1(ok448)/+hermaphroditeshadsimilar, assessed,because(1)theywerefrequentlypairedinthesameanimal
wild-type levels of DTC migration defects. However, max-2(RNAi); withnon-truncatedgonadarms,and(2)weobservedseveresterility
pak-1(tm403)/+ and max-2(RNAi); pak-1(ok448)/+ were significantly inallanimalsinwhichbothPakpathwaysweredisrupted(seePAK-1
different, with almost 27% of max-2(RNAi); pak-1(tm403)/+ gonad andMAX-2arerequiredforfertility,below),eveninanimalsinwhich
armshavingDTCmigrationdefectscomparedwith17%inthecontrol neithergonadarmwastruncated.
(Table2;P,0.05,Fisher’sexacttest).Theseobservationssuggestthat Althoughasmallminorityoftruncatedgonadarmsfailedtomake
pak-1(tm403) might encode a toxic, semidominant protein. As addi- the dorsal turn, generally the point of migration arrest was re-
tional support for this hypothesis, we were unable to transgenically producible; most truncated DTCs truncated immediately after the
rescuemax-2(RNAi);pak-1(tm403)DTCmigrationdefectswithwild- dorsalturn,resultingin ashapelike ahockeystick.Totestwhether
type PAK-1 protein, whereas we could rescue max-2(RNAi); pak-1 DTC migration rate was diminished, we scored DTCs at the young
(ok448) (see PAK-1 is cell autonomous and requires kinase activity adultstageratherthantheusualL4stagetogiveDTCsadditionaltime
andPIX-1-interactionsequences,below).Despitethispredicteddiffer- to migrate, but we observed no differences in the frequency of
enceinconsequencesofthetwopak-1alleles,pak-1(tm403)andpak-1 truncated gonads, nor were truncated gonads migrated any farther.
(ok448)homozygotesonmax-2-directedRNAiconferredDTCmigra- Therefore, we speculate that loss of max-2 and pak-1 results not in
tion defects of similar strength (Figure 1C). Interestingly, the ced-10 generallycompromisedcellmigration,whichwepredictwouldresult
(n1993) hypomorphic allele and n3246 weak gain-of-function allele in gonads truncated at many points but rather disrupts response to
(Shakir et al.2006)onpak-1(RNAi) also haveroughly similar affects aspecificmigratorycue,i.e.,thatwhichguidestheDTCbacktothe
on DTC migration [Figure 3; ced-10 null is maternal effect lethal A/Pmidpointoftheanimal nearthedevelopingvulva.
(Kinchenetal.2005)andwasnotusedforthisanalysisfortechnical Below we describe genetic analyses of other components of the
reasons].Becauseitisoutsideofthescopeofthisstudy,wehavenot MAX-2 and PAK-1 signaling pathways. In all cases in which we
pursuedfurtherthepotentiallytoxicnatureofpak-1(tm403). disrupted the two redundant pathways, we observed accompanying
pathfinding defects and truncated migrations (Table 3). This obser-
PakscontrolbothDTCpathfinding vationsuggeststhatthesamegeneticpathwayscontrolbothprocesses.
andmigrationexecution PreviousworkdescribedalteredDTCmorphologyinpak-1single
Weobservedtwogeneralclassesofgonadmorphologydefectsinmax- mutants(LucanicandCheng2008).Wescoredpak-1(ok448)andpak-
2; pak-1 double loss-of-function animals: pathfinding and migration 1(tm403) mutants blindly and were unable to detect reproducible
execution (defects that are referred to here as “truncation.”) We hy- DTCmorphologydefectsrelativetothewildtype.Wedonotknow
pothesize that these defects reflect two distinct functional roles for thesourceofthisdiscrepancy.
max-2andpak-1inDTCmigration.First,abroadvarietyofpathfind-
ingdefectswereobserved(atypicalexampleisshowninFigure2B,but CED-10/RacandtheDock/ELMORacGEFcomplex
all possible permutations were observedatlow frequency).Nosingle function upstreamofMAX-2
defectwasrepresentativeofthemax-2;pak-1double,butinsteadmany Basedonextensivemammalianwork,wehypothesizedthatCED-10/
possible misguidance defects were represented, and aberrant DTC Rac would be required to activate PAK-1 and/or MAX-2, and we
Volume3 February2013 | RedundantPaksinCellMigration | 185
plex,weproposethatthisRacGEFactivatesCED-10tocontrolDTC
migration,andourgeneticdataarguethatCED-10thensignalsprin-
cipallythroughMAX-2/Pak.ThatCED-10/RacsignalsthroughMAX-2
butnotPAK-1 waspreviouslydeduced, although the analysiswasnot
extendedtotheDock/ELMOcomplexanddidnotincludemultiple
allelesof pak-1,max-2,and ced-10(Lucanicand Cheng2008).
The synergy between loss of CED-5/12 or CED-10 and loss of
PAK-1,butnotMAX-2,suggeststhattheCED-10/Racproteinsignals
primarily through MAX-2, whereas PAK-1 functions in a parallel
signaling branch. However, one observation complicates this simple
interpretation. CED-10/Rac was previously described as being re-
quired for DTC migration (Wu and Horvitz 1998), and we found
that ced-10(n1993) or ced-10(n3246) grown on gfp-directed RNAi
had DTC migration defects well above background levels (Figure
3A). Moderate defects in DTC migration were previously described
formutationsdisruptingtheDock/ELMORacGEF(Gumiennyetal.
2001;ReddienandHorvitz2000;WuandHorvitz1998).Wesimilarly
Figure2 Lossof max-2andpak-1causeserrors inbothpathfinding observed mismigrated DTCs in ced-2(n1994), ced-2(e1752), ced-5
andmigrationcompletion.ShownareDICphotomicrographsofsingle (n1812), ced-12(tp2), and ced-12(n3261) backgrounds. Loss of these
gonadarms,lateralview.Inallpanels,lateL4animalswereusedand proteinshadsimilareffectsonDTCtruncation(Table3)(Gumienny
whitearrowspointtothedevelopingvulva.Blacklinestrackthepath et al. 2001; Reddien and Horvitz 2000; Wu and Horvitz 1998). The
of DTCmigrationinferredbyterminalgonadshape,andthedotted contrast in frequency of gonad arm truncation in pak-1 or max-2
lineindicatestrackofthegonadthatisoutofthefocalplane.Black singlemutantscomparedwithced-2,-5,-10,and-12mutantanimals
lines end with an arrow for a completed migrations and a circle for leadsustohypothesizethatCED-10anditsGEFarerequiredinboth
a truncated migrations. (A) Wild-type, U-shaped gonad arm. (B2D)
Pakpathwaybranches,thoughprobablytodifferingdegrees.
max-2(nv162); pak-1(ok448) animals from double-heterozygote
parents. (B) Gonad arm with a typical pathfinding error, where
CED-10signalsthoughMAX-2, butthecontribution
the distal arm reversed. (C) Truncated gonad arm with a typical,
“hockey stick” morphology, where the distal turn is made but sub- ofother GTPasesisunclear
sequent migration fails. (D) An atypical truncated gonad arm where Therobustsyntheticphenotypeofmax-2;pak-1doubleloss-of-function
somemigrationbacktowardthevulvalisaccomplished.Forboth(C) animalsprovidesanopportunitytodissecttheregulationof these
and(D),typicalwideningofthegonadatthedistalendisvisible. Pak proteins in vivo and thereby understand their function in cell
n Table3 Disruptionofbothpak-1andmax-2branchesis
tested this model genetically by pair-wise knockdown of putative
requiredforsignificantDTCtruncationdefects
pathwaycomponents.Bothced-10mutationsexaminedhadsynthetic
interactions with pak-1 but not max-2-directed RNAi (90% vs. 45– % %
Row Genotypes Pathfinding Truncation N
60%;Figure3A). pak-1(ok448) and pak-1(tm403) animals had mod-
eratesyntheticdefectswhengrownonced-10-directedRNAi,whereas 1 N2onGFP 11.8 0.0 160
max-2(nv162) was not enhanced (~40% and 60% vs. ,20%; Figure 2 pak-1(ok448);gfp(RNAi) 13.3 0.0 158
3B).ced-10-directedRNAiwasonlymodestlyeffectiveinallcontexts, 3 pak-1(ok448);max-2(RNAi) 29.4 73.1 160
4 pak-1(tm403);gfp(RNAi) 11.0 0.0 154
even though we included rrf-3 sequences in the RNAi vector to hy-
5 pak-1(tm403);max-2(RNAi) 1.0 91.8 146
persensitizeanimalstoced-10-directedRNAi(Simmeretal.2002).
6 max-2(nv162);gfp(RNAi) 14.6 1.2 158
An atypical Rac-specific Dock/ELMO GEF complex has been
7 max-2(nv162);pak-1(RNAi) 25.0 80.7 140
described in both mammals and nematodes, and the corresponding
8 max-2(cy2);gfp(RNAi) 26.1 1.5 130
CED-2/5/12complexactivatesCED-10/Racincellcorpseengulfment. 9 max-2(cy2);pak-1(RNAi) 5.0 97.1 140
Single mutants for each of these genes, as well as ced-10, have been 10 ced-10(n1993);gfp(RNAi) 38.1 18.1 160
described as having moderately penetrant DTC migration defects 11 ced-10(n1993);pak-1(RNAi) 14.3 92.2 154
(Gumiennyet al. 2001; Reddien andHorvitz2000; Wu andHorvitz 12 ced-10(n3246);gfp(RNAi) 38.7 42.5 106
1998).WeanalyzedmutationsintheDock/ELMOcomplexincom- 13 ced-10(n3246);pak-1(RNAi) 3.0 98.0 100
bination with loss of pak-1 or max-2. Double-mutant combinations 14 pix-1(ok982);gfp(RNAi) 16.3 0.0 80
15 pix-1(ok982);max-2(RNAi) 1.3 96.3 80
between ced-5 or ced-12 and pak-1 yielded animals with the patchy
bright-field phenotype indicative of DTC migration defects, whereas 16 git-1(tm1962);gfp(RNAi) 7.5 0.0 80
17 git-1(tm1962);max-2(RNAi) 12.5 81.3 80
double-mutantcombinationsbetweenced-5orced-12andmax-2were
nodifferentthansinglemutants(Table4).Wevalidatedtheseobser- Eachgonadarmevaluatedwasindependentlyscoredfordefectsinpathfinding
ortruncation,soinprinciplethepathfindingandtruncationcolumnscantotal
vationswithpak-1andmax-2-directedRNAi:lossofced-2,ced-5,or
greater than 100% (e.g., see row 9). Greater frequency of truncation defects
ced-12conferredstrongsyntheticDTCmigrationdefectsincombina- correlates with lower frequency of pathfinding defects, presumably because
tion with pak-1(RNAi) but not max-2(RNAi). These results suggest defects in distal arm migration were more frequent than defective proximal
that the RacGEF complex required for CED-10 activation in cell armmigration,andthustruncatedDTCmigrationsobscureunderlyingpathfind-
ingdefects.Thereissomeoverlapbetweencategories(i.e.,truncatedgonad
corpse engulfment also functions in the same pathway as CED-10
armscanbemis-migrated)andthustotalpercentdefectcanbegreaterthan
in DTC migration because it has the same genetic interactions with
100%(e.g.,rows7,9,11,13).DTC,distaltipcells;N,numberofgonadarms
max-2andpak-1.GivenpublisheddataabouttheDock/ELMOcom- scored.
186 | E.C.Petersetal.
Figure 3 CED-10 acts primarily
throughMAX-2andnotPAK-1.Num-
berofgonadarmsassayedisshownin
each column. (A) Both ced-10 alleles
examined had significant baseline
DTC defects and showed strong syn-
theticdefectswithpak-1-butnotmax-
2-directed RNAi. (B) The wild-type,
pak-1 andmax-2animalswere grown
onced-10-directedRNAi.RNAiofced-
10inducesanadditionaldefectinthe
N2 wild-type background that is sig-
nificant (P , 0.001), but in all of
our experiments the effects of ced-
10-directed RNAi were significantly
weaker than defects seen for ced-10
hypomorphic n1993 and weak gain-
of-function n3246 alleles. The two
pak-1 alleles but not max-2(nv162)
are synthetic with ced-10(RNAi). (C)
RNAi of pak-1, max-2, or ced-10 en-
hance the previously described DTC
migrationdefectofmig-2(mu28)(P,
0.003), arguing that mig-2 functions
in parallel to the two Pak pathways.
(D) Mutations in git-1 and pix-1 are
stronglysyntheticwithRNAitargeting
max-2 but not pak-1. (E) Mutations in
max-2 but not pak-1 are synthetic
with RNAi targeting git-1. Statistics
weregeneratedviaFisher’sexacttest.
Noerrorbarsareshownbecausethese
arepooledassays.
migration. Paks are generally thought of as effectors of Rho family 2encodesaputativehomologofmammalianRhoG,aCdc-42-and
smallGTPases,specificallyRacandCdc42.C.eleganscontainsasin- Rac-relatedbutfunctionallydistinctmemberoftheRhofamilythat
gle Cdc42homolog, CDC-42,andtwo Rachomologs,CED-10and generally interacts with effectors different than those of Rac or
RAC-2. CDC-42 and CED-10 are very highly conserved with their Cdc42(deBakkeretal.2004;Wennerberg andDer2004).
mammaliancounterpartsandareabsolutelyconservedintheSwitch rac-2(RNAi) caused weak synergy in the max-2(nv162) but not
1effector-bindingregion,butRAC-2containsdivergentresiduesin max-2(cy2) backgrounds (24.6% and 13.8%, respectively, P , 0.02).
theSwitch1region.Basedongeneticanalysis,CED-10appearstobe Therac-2(ok326)mutationdidnotconfersinglemutantDTCdefects
the major Rac homolog in C. elegans, whereas RAC-2 may play or synthetic interactionsincombination withpak-1 or max-2 RNAi
a minor, redundant role (Lundquist et al. 2001). In addition, mig- (combinationwithpak-1ormax-2RNAi,9.4and12.2%,respectively).
n Table4 TheDock/ElmoRacGEFcomplexisredundantwithpak-1butnotmax-2
Genotypes + ced-10(n1993) ced-5(n1812) ced-12(n3261) ced-1(e1735)
+ + Fertile,wkpatchy Fertile,wkpatchy Fertile,wkpatchy Fertile
pak-1(ok448) Fertile Sterile,strpatchy Sterile,strpatchy Sterile,strpatchy Fertile
pak-1(tm403) Fertile Sterile,strpatchy Sterile,strpatchy Sterile,strpatchy Fertile
max-2(nv162) Fertile Fertile,Unc Fertile,wkpatchy Fertile,wkpatchy Fertile
max-2(cy2) Fertile Fertile,Unc ND ND ND
Likeced-10(n1993),ced-5(n1812)andced-12(n3261),whichencodecomponentsoftheDock/ElmoRacGEFcomplex,causedsyntheticDTCmigrationandfertility
defectsincombinationwithpak-1,butnotmax-2.Animalswerescoredbyinspectionunderdissectingmicroscope:“Str”is“strong,”“wk”is“weak,”and“Unc”is
“uncoordinated.” ced-1(e1735),which regulates cell corpse engulfment through adifferent pathway, wasincludedas a negative control. Asdescribed,max-2
mutationsalsocauseasevereuncoordinatedphenotypeintheced-10(n1993)background(LucanicandCheng2008),butnotinced-5andced-12backgrounds
(ourobservations),arguingthataGEFotherthanCED-2/5/12functionstoactivateCED-10inneuriteoutgrowth.ND,notdetermined.
Volume3 February2013 | RedundantPaksinCellMigration | 187
Previous studies (Lucanic and Cheng 2008; Lundquist et al. 2001) involvedinadifferentaspect of DTC migration.Thisobservation is
suggest that RAC-2 plays a modest role in DTC migration, but our noteworthybecause MIG-2/RhoGhas been describedasfunctioning
results are weak or negative. This discrepancy in a relatively mild upstream of the Dock/ELMO RacGEFthat activates CED-10/Rac in
phenotypecouldbeduetoexperimentaldifferences. nemotodesandmammalianapoptoticcellcorpseengulfment(deBakker
cdc-42-directedRNAiconfersstrongembryoniclethality,andsur- etal.2004)andthusmighthavebeenpredictedtofunctioningsimilarly
vivinganimalsaregrosslydefective ingonad organization as well as to this group of proteins. To summarize, MIG-2/RhoG functions in
showingdisruptionofearlysomaticgonadpolarity(Welchmanetal. DTC migration, but does not have clearly interpretable genetic inter-
2007). Previously, cdc-42-directed DTC tissue-specific RNAi did not actionswithothersignalingcomponents.
perturb DTC migration on its own. However, cdc-42+pix-1 but not Ifmoleculesfunctioninalinearpathway,ectopicactivationofthe
cdc-42+max-2 double DTC-specific RNAi caused a weak synthetic upstream component might allow genetic dissection of pathway
phenotype (Lucanic and Cheng 2008). The strength of this effect colinearity.Weexpressedwild-typeorQ61L-mutatedCED-10spe-
was weaker than the strong synthetic phenotype observed between cificallyinDTCsusingthelag-2promoter.Q61Lmutationsortheir
pak-1 and max-2, perhaps due to variability in RNAi efficacy from equivalent activate all Rho and Ras small GTPases by inactivating
genetogene.Alternatively,perhapscontributionofCDC-42to their intrinsic GTPase catalytic activity, thus locking the protein in
MAX-2ismodest.ButtheconclusionwasthatCDC-42playsarole the GTP-bound active state. Ectopic CED-10(Q61L) caused defec-
inparalleltothePak-Pix-Gitcomplex,perhapsupstreamofMAX-2 tive DTC migrations (Figure 4A), arguing that mutationally acti-
(Lucanic andCheng2008). vated CED-10 protein has biological activity in migrating DTCs.
Totestthishypothesiswithadifferentmethodology,weexpressed However, loss of pak-1 or max-2 failed to abrogate the activated
mutant CDC-42(T17N) protein specifically in DTCs using the lag-2 CED-10phenotype (Figure 4A),arguing thatCED-10does notex-
promoter,whichdrivesexpressioninDTCsandnotsurroundingcells clusivelyuseasinglePakeffectorpathway,butrathercontributesto
(Blellochetal.1999).This17NmutationiswellcharacterizedinRas both, consistent with our arguments based on the aforementioned
and Rho small GTPases as conferring potent dominant-negative ac- ced-2/-5/-10/-12singlemutants.
tivity,andthemaincaveatwiththisreagentisthatitcouldbeoverly Similarly,ectopicexpressionofCDC-42(61L)causedDTCmigra-
strong by sequestering GEFs required for activation of other Rho tiondefects.Alternatively,CED-10orCDC-42couldengageeffectors
family members in addition to CDC-42. DTC-specific CDC-42 otherthanPakswhenectopicallyexpressedinDTCs,butendogenous
(T17N)causednoDTCmigration defectsandwas not pursuedfur- protein does not do so. This result may seem to contradict our ob-
ther(datanotshown). servationthatCDC-42(dn)causesnoDTCdefect,butwearguethat
We also tested the MIG-2/RhoG contribution to Pak-mediated ectopic CDC-42iscapable ofengagingPakeffectors andmay doso
DTC migration. RhoG does not bind and activate Pak proteins in elsewhere during development. PAK-1 loss weakly suppressed the
mammalian cells (Wennerberg et al. 2002), but by yeast two-hybrid totalDTCdefectbutnotspecificallypathfindingortruncation,while
assayMIG-2/RhoGmayinteractwithMAX-2/Pak(Fujikietal.2010). MAX-2lossweaklysuppressedthetruncationdefect(Figure4B).We
Alongwiththe previouslyobservedmoderateDTC migrationdefect hypothesizethatectopic,activatedCDC-42canengagebothPakpro-
inmig-2nullmutantanimals(Lundquistetal.2001),wefoundthat teinssimultaneously,andthuscanonlybesuppressedweaklybyloss
lossofmax-2,pak-1,orced-10weaklyenhancedmig-2mutantdefects ofsinglePaks.
(Figure 3C), but not with the very strong synergy observed between
the max-2 and pak-1 pathway branches. Although in general ced-10 PutativePAK-1partnersGIT-1andPIX-1areredundant
(RNAi) efficacy was modest, pak-1- and max-2-directed RNAi con- withMAX-2
ferredstrongloss-of-functionphenotypes.Thus,theweakinteraction Our pair-wise genetic analysis suggests that PAK-1 has a modest re-
between mig-2and pak-1 ormax-2 argues thatmig-2 probably does quirement for CED-10/Rac activation. This conclusion is consistent
not play a significant role in either of these pathways, but rather is withapreviouslydescribed,partiallyGTPase-independentfunctionof
Figure 4 Activated CED-10/Rac and
CDC-42 disrupt DTC migration. (A)
DTC-directed expression of gain-of-
function (gf), mutationally activated
CED-10(Q61L) causes pathfinding and
truncationdefects.(B)DTC-directed
expression of gain-of-function, muta-
tionally activated CED-10(Q61L) also
causes pathfinding and truncation
defects.Lossofeitherpak-1ormax-2
fails to rescue the activated CED-10
defects,whereasthelossofpak-1sup-
presses the truncation but not path-
finding defect of activated CDC-42,
andlossofmax-2modestlysuppresses
bothdefectsofactivatedCDC-42.Sta-
tisticsweregeneratedviaFisher’sexact
test,andonlysignificantdifferencesare
shown. No error bars are shown be-
causethesearepooledassays.
188 | E.C.Petersetal.
Group A Paks in cell culture. In addition to its role as a Rac/Cdc42 ure5A).Totestwhethertransgenedosewasrelevant,wealsoinjected
effector, Pak can function as a scaffold for a protein complex that at10ng/mL,buttherescueresultsweresimilartothoseatthelower
includes Git1 and Pix (Loo et al. 2004). This Pak-Pix-Git complex concentration. Therefore, we conclude that a significant portion of
may not require GTPase input for activation, though Cdc42 and Rac PAK-1 function inDTC migrationis cell-autonomous, as predicted.
arethoughttohelptargetthecomplextodiscretesubcellularcompart- We speculate that rescue is incomplete because PAK-1 functions in
ments,andthuscontributesomewhattooptimalcomplexactivity.Itis a multiprotein complex, where stoichiometry may be important for
alsonotablethatthemammalianPak-Pix-Gitcomplexdoesnotrequire function. Thus, perhaps transgenic overexpression of PAK-1 titrates
activityofthePakkinasedomain.Thesignalingoutputofthiscomplex crucial PAK-1 binding proteins away from the complex, thus com-
isunclear,thoughclearlybiologicallyimportant(Zhangetal.2011). promisingfunctionandpreventingfullrescue.Alternatively,perhaps
TheC.elegansgenesgit-1andpix-1arewell-conservedorthologs not all PAK-1 function is cell autonomous, or a different isoform
of Git1 and Pix, respectively (Dyer et al. 2010; Lucanic and Cheng providessomecriticalpak-1functionintheDTCs.
2008).TotestwhethertheputativeGTPase-independentroleofPAK-1 WesimilarlytestedP ::pak-1(+)rescueofpak-1(tm403),thein-
lag-2
in DTC migration is GIT-1- and PIX-1-dependent, we used pair- frametruncationofthePAK-1N-terminalregulatorysequencesthat
wisegeneticknockdownslikethoseusedtocharacterizePAK-1and isweaklydominant(seeabove).WefoundthatDTC-directedexpres-
MAX-2parallelism.Wefoundthatoutcrossedstrainscontaininggit- sionofpak-1(+)failedtorescuepak-1(tm403)(Figure5A).Thetrans-
1(tm1962), pix-1(gk416), or pix-1(ok982) conferred no DTC defects gene used was the same used for rescuing pak-1(ok448), so we are
alone(orongfp-directedRNAi;Table3)andwerestronglysynthetic confident that wild-type protein is expressed. This result reinforces
with max-2(RNAi) but not pak-1(RNAi). Conversely, mutants for ourhypothesisthatpak-1(tm403)encodesatoxic,perhapsdominant-
max-2 showed strong synthetic defects in combination with git-1- negativeorgain-of-functionproteinproduct.
directed RNAi, but pak-1 mutants did not (Figure 3, D and E). We further used DTC-directed rescue of PAK-1 to test the mo-
Furthermore, mutants for ced-10 were synthetic with git-1(RNAi) lecularrequirementsofPAK-1intheputativePak-Git-Pixcomplex.If
(data not shown). These results indicate that git-1 and pix-1 are PAK-1functionsaspartofsuchacomplex,thePix-bindingdomainof
redundant with max-2 and ced-10 but not pak-1, and therefore we PAK-1,whichishighlyconservedwithitsmammaliancounterparts,
hypothesizethatPAK-1functionsaspartofaPak-Git-Pixcomplex shouldberequiredforrescue.Therefore,weconstructedaP ::pak-1
lag-2
describedinothersystems. (R234A,P235A)“Pixdead”plasmid,guidedbypreviousbindingstud-
iesofhumanPak1andPixinteractions,inwhichresiduesofPAK-1
PAK-1is cellautonomousandrequireskinaseactivity essentialforPix-bindingweremutated(Danielsetal.1999).Usingthe
andPIX-1-interactionsequences same assay as for pak-1(+) rescue, we found that the “Pix dead”
Because a major function of mammalian Pak proteins is to regulate construct failed to rescue the synthetic max-2(RNAi); pak-1(ok448)
thecytoskeleton,wehypothesizedthatPAK-1functionscellautono- DTC migration phenotype (Figure5B), arguing that PAK-1 binding
mously in the DTCs to control their pathfinding and extension. To toPIX-1isnecessaryforPAK-1functioninDTCs.Togetherwiththe
test this model, we expressed the wild-type pak-1A isoform cDNA requirement for pix-1 and git-1 for pak-1 branch activity, this result
behindthelag-2promoter,whichdrivesexpressioninDTCsbutnot indicatesthatPAK-1functionsinacomplexwithPIX-1andGIT-1to
surrounding cells (Blelloch et al. 1999). We crossed P ::pak-1 regulateDTCmigration.
lag-2
(+)-containing transgenes marked with P ::gfp into the pak-1 Previous mammalian studies suggest that the kinase domain of
myo-2
(ok448) background, which we predict to be null for PAK-1 kinase PakisdispensableforPakfunctionwiththePak-Git-Pixcomplexand
function. We then subjected the strain to max-2-directed RNAi and thePakscaffoldingactivityforphosphatidylinositol3kinaseandPDK
scoredarray-bearingvs.nonarray-bearinganimals(thelatterprovided (Higuchi et al. 2008). Therefore, we predicted that DTC-directed
an internal control for RNAi efficacy and a baseline for statistical expressionofpak-1cDNAwithamutatedkinasedomainwouldstill
comparisontosiblingarray-bearinganimals).TheDTC-directedex- beabletorescuethemax-2(RNAi);pak-1(ok448)syntheticDTCmi-
pression of the pak-1(+) cDNA, injected at 5 ng/mL, rescued the gration defect. We constructed a P ::pak-1(K324R) “kinase dead”
lag-2
syntheticDTCmigrationdefectwithmax-2,althoughnotfully(Fig- (KD)plasmid,guidedbypreviousbiochemicalandfunctionalassays
Figure5 PAK-1(+)butnotPAK-1(KD)
orPAK-1(Pixdead)rescuePAK-1null.
(A) DTC-directed expression of pak-1
(+)(isoformA)cDNApartiallyrescues
the DTC migration defect of pak-1
(ok448) but not pak-1(tm403) animals
grown on max-2-directed RNAi. (B)
Neither the KD nor pix-interaction
dead pak-1 cDNAs were able to res-
cuetheDTCmigrationdefectofpak-1
(ok448) animals grown on max-
2-directed RNAi. For rescue experi-
ments, array-bearing animals were
scoredinparalleltononarray-bearing
siblings as an internal control. Statis-
ticsweregeneratedviaFisher’sexact
test.Noerrorbarsareshownbecause
thesearepooledassays.
Volume3 February2013 | RedundantPaksinCellMigration | 189
ofhumanGroupAPakproteins(Manseretal.1997;Sellsetal.1997). and9,respectively).Perhapsthisobservationindicatesthatbothtm403
Surprisingly,wefoundthatDTC-directedexpressionofpak-1(K324R) andcy2encodeproteinsthataretoxicforfull-lengthDTCmigration
failedtorescue(Figure5B).Therefore,wehypothesizethatthePak- but are less severe than putative null alleles for embryonic morpho-
Pix-GitcomplexrequiresPakkinaseactivitytoperformitsfunctionin genesis.Alternatively, unlikemax-2(nv162),which disruptsthetrans-
DTCmigration. lational start of the predictedlongbut notshort isoform, max-2(cy2)
disruptskinasdomainfunction,presumablyinbothisoforms.Soper-
Pakproteinsarerequiredforembryonicmorphogenesis hapstheMAX-2kinasedomainisrequiredinmultipleisoformsduring
To further characterize Pak-dependent DTC migration defects, we DTC migration,butnot as strongly requiredforembryonic develop-
builtpair-wisedoublemutantstrainswithmutationsinpak-1(ok448 mentandfertility.
andtm403)andmax-2(nv162andcy2).Allalleliccombinationswere PAK-1isaclearorthologofmammalianGroupAPaks(Pak1,2,
lethal or subviable and therefore impossible to maintain in culture, 3), with all known mammalian sequence motifs conserved in the C.
with severely compromised fertility (see PAK-1 and MAX-2 are re- elegansprotein(Chenetal.1996;Hofmannetal.2004).Bysequence
quired for fertility, below) and frequent embryonic morphogenesis analysis, MAX-2 is Group A-like, but stereotypical Group A Pak
defects.Thislatterdefectwasnotexaminedingreatdetail,butcursory sequence motifs are missing, notably the PXXP SH2 domain recog-
inspection revealed embryos in which the epithelium had failed to nition motifs and the PIX binding domain (Lucanic et al. 2006).
enclose, thus resulting in gross disorganization of tissue types and Additionally, the MAX-2 PBD, required for binding by activated
gutcellsontheexterioroftheembryo(Sotoetal.2002).Wecannot RacorCdc42,islessconservedthanthatofPAK-1andthemamma-
ruleout defectsin cell fate, but differentiated pharynx and gut were lianGroupAPakproteins.C.elegansalsocontainsaprotein,pak-2,
visible. Rare surviving embryos that hatched had variable surface that is an ortholog of mammalian Group B Pak proteins (Hofmann
bulgesconsistentwithmorphogeneticdefectsinventralenclosureor etal.2004;Lucanicetal.2006).Toexaminethetotalcontributionof
elongation(ChisholmandHardin2005).Inaddition,theserare,sur- Pakproteinstoembryogenesis,weanalyzedsingle,double,andtriple
vivingdouble-mutantanimalshadseverelocomotiondefects,whereas Pak loss of function. We observed that pak-2(ok332) single mutants
pak-1 single-mutant locomotion was wild type, and max-2 single- and pak-1(ok448) single mutants had significant but relatively low-
mutant locomotion was only mildly defective (Lucanic et al. 2006). level embryonic lethality (P , 0.0001; Figure 6). We constructed
Thelethalityandlocomotiondefectswerematernallyrescued;double- apak-2(ok332);pak-1(ok448)double-mutantstrain,andobservedsig-
homozygous mutant F progeny from a double-heterozygote parent nificantlymorelethalitythaneitherthepak-1orpak-2single-mutant
2
could be readily selected based on the patchy phenotype from DTC strains(P,0.0001).Growthofpak-1(ok448)single-mutantanimals
migration defects. These patchy animals moved well and had no on max-2-directed RNAi caused no change in baseline lethality, but
morphogeneticdefects,althoughtheyhadfewornoprogeny.Wealso growthofpak-2(ok332)singlemutantorpak-2(ok332);pak-1(ok448)
observed maternal rescue of morphogenetic and locomotion defects double-mutant animals on max-2-directed RNAi significantly sup-
when the parent was homozygous mutant for one Pak, and hetero- pressedlethalitycomparedwiththegfp-directedRNAicontrolgroup
zygousmutantfortheotherPak,arguingthatbothmax-2andpak-1 (P , 0.0001 and P = 0.04, respectively; Figure 6). Together, these
arematernallyrescued.Weconcludethatearlydevelopmentalevents observationssuggestthatpak-1andpak-2areredundantforembry-
likeembryonicmorphogenesisandaxonpathfindingrelyonmaternal onicmorphogenesisandfertility,andperhapsthatmax-2antagonizes
PAK-1andMAX-2contribution,whereaslaterdevelopmentalevents pak-2inthisprocess.Sincepak-2didnotcontributetoDTCmigra-
likeDTCmigrationrelyonzygoticgeneproduct. tion,wedidnotfurtherpursuepak-2analysis.
During these experiments, we found that only one of the four Additionally, pak-2(ok332); pak-1(ok448) double-mutant animals
possible pair-wise double mutants yielded viable progeny, albeit at weresubstantiallysmallerthanwildtypeasconfirmedbyblindphe-
averylowrate.max-2(cy2);pak-1(tm403)animalshad223progeny notypicscoring.Neitherpak-1(ok448)norpak-2(ok332)singlemutant
per generation, enough that the strain could be maintained but not animals were of abnormallysmallstature, suggesting that pak-1 and
frozen. Double mutant animals for other allelic combinations were pak-2 are redundant for regulation of body stature. Body stature is
isolated, butthe strainswere inviable.However, upon picking many controlledbyTGF-b signaling(Savage-Dunn2005),soperhapsPAK-1
double-mutantanimalsforeachgenotypiccombination,rarehealthier andPAK-2functioninthatprocess.
clonesofanimalsaroseafteravariablenumberofgenerations,andthe
resultingstrainswerequalitativelymorerobust.Thesehealthierclones PAK-1andMAX-2arerequired forfertility
presumably resulted from spontaneous second site mutations that We observedthatmax-2; pak-1double-mutantanimalswere mostly
compensatedfortheseverityofthemax-2;pak-1double-mutantem- sterile.Wesimilarlyobservedthatmax-2orpak-1single-mutantani-
bryonic lethalityand sterility. Alternatively, perhaps background en- malsgrownonRNAitargetingthereciprocalPak-encodinggenewere
hancermutationswere lost,thoughoutcrossedstrainswere usedfor mostlysterile.Asidefromaberrantpathfindingandshortenedgonad
theconstructions.UnlikethepreviousPakmutantstudy(Lucanicand arms,thesegonadslookedmostlynormalbyDIC.Theonlydefectwe
Cheng2008)wedidnotanalyzedouble-mutantDTCphenotypesof observedwasthatmanyofthematureoocytesweresmallerthantheir
thesehealthierisolatesbecausewewereconcernedaboutthepotential wild-type counterparts. Late L4 single mutant animals picked to
caveats of additional mutations in unknown genes. Because the RNAi, whoseprogeny would be sterile, werethemselvesquitefertile
healthier clones arose at relatively high frequency, we predict that andremainedsothroughouttheiradultlife,suggestingthatthepoint
a large group of genes can be mutated to weakly ameliorate the ingermlinedevelopmentinwhichbothpak-1andmax-2arerequired
max-2;pak-1embryoniclethalityandsterility. isbeforethelateL4stage,orthePakproteinsperdure.
Thefactthatcy2;tm403canbeculturedwithoutsecondsitemuta- To determine the cause of the extensive sterility, we fixed whole
tions whereas other allelic combinations could not argues that these animalsatvariousstagesfromL4tomid-adultandstainedthemwith
two alleles are not null (see above). We note that pak-1(tm403) and DAPItovisualizeDNA.Wethusanalyzedallpair-wisemax-2;pak-1
max-2(cy2)havethemostseveredegreeofgonadarmtruncationwhen mutant-RNAi combinations and observed no genotypic differences.
incombinationwithRNAioftheredundantPakgene(Table3,lines5 As controls, we compared double loss of function to single mutants
190 | E.C.Petersetal.