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Small GTPases
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RhoG and Cdc42 can con ibu e o Rac-dependen
lamellipodia o ma ion h ough WAVE egula o y
complex-binding
Ma hias Schaks, He mann Dö ing, F ieda Kage, Anika S e en, Thomas
Klünemann, Wul Blanken eld , The esia S adal & Klemens Ro ne
To ci e his a icle: Ma hias Schaks, He mann Dö ing, F ieda Kage, Anika S e en, Thomas
Klünemann, Wul Blanken eld , The esia S adal & Klemens Ro ne (2019): RhoG and Cdc42 can
con ibu e o Rac-dependen lamellipodia o ma ion h ough WAVE egula o y complex-binding,
Small GTPases, DOI: 10.1080/21541248.2019.1657755
To link o his a icle: h ps://doi.o g/10.1080/21541248.2019.1657755
© 2019 The Au ho (s). Published by In o ma
UK Limi ed, ading as Taylo & F ancis
G oup
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BRIEF REPORT
RhoG and Cdc42 can con ibu e o Rac-dependen lamellipodia o ma ion
h ough WAVE egula o y complex-binding
Ma hias Schaks
a,b
, He mann Dö ing
a,b
, F ieda Kage
a,b
, Anika S e en
b
, Thomas Klünemann
c
,
Wul Blanken eld
c
, The esia S adal
b
, and Klemens Ro ne
a,b
a
Di ision o Molecula Cell Biology, Zoological Ins i u e, Technische Uni e si ä B aunschweig, B aunschweig, Ge many;
b
Cell Biology,
Helmhol z Cen e o In ec ion Resea ch, B aunschweig, Ge many;
c
S uc u e and Func ion o P o eins, Helmhol z Cen e o In ec ion
Resea ch, B aunschweig, Ge many
ABSTRACT
Cell mig a ion equen ly in ol es he o ma ion o lamellipodial p o usions, he ini ia ion o which
equi es Rac GTPases signalling o he e open ame ic WAVE egula o y complex (WRC). While Rac-
ela ed RhoG and Cdc42 can po en ly s imula e lamellipodium o ma ion, so a p esumed o occu by
ups eam signalling o Rac ac i a ion, we show he e ha he la e can be bypassed by RhoG and
Cdc42 gi en ha WRC has been a i icially ac i a ed. This e idence a ises om gene a ion o B16-F1
cells simul aneously lacking bo h Rac GTPases and WRC, ollowed by econs i u ion o lamellipodia
o ma ion wi h speci ic Rho-GTPase and di e en ially ac i e WRC a ian combina ions. We conclude
ha o ma ion o canonical lamellipodia equi es WRC ac i a ion h ough Rac, bu can possibly be
uned, in addi ion, by WRC in e ac ions wi h RhoG and Cdc42.
ARTICLE HISTORY
Recei ed 2 Ap il 2019
Re ised 13 Augus 2019
Accep ed 15 Augus 2019
KEYWORDS
A p2/3 complex; CRISPR/
Cas9; Rho-GTPase;
ilopodium; lamellipodium;
blebbing; s ess ib e;
mig a ion; p o usion
In oduc ion
Cell mig a ion is essen ial o many physiological and
pa hological p ocesses, such as emb yonic de elopmen ,
immuni y and me as asis [1–3]. P o usion o he plasma
memb ane o enable cell mig a ion is commonly achie ed
by he o ma ion o hin b anched a ays o ac in ilamen s,
called lamellipodia [4,5]. Small GTPases o he Rac amily
(i.e. Rac1, Rac2, Rac3 in mammals) a e essen ial o lamel-
lipodia ini ia ion and main enance, a leas in pa , by
ac i a ion and con inuous in e ac ion h ough wo binding
si es on S a-1 (o i s o hologue PIR121) embedded in o
he e open ame ic WAVE egula o y complex (WRC)
[6,7]. The closes ela i es o Rac GTPases a e RhoG and
Cdc42. Bo h ail o ini ia e lamellipodia in ib oblas s lack-
ing Rac exp ession [6], bu can clea ly p omo e Rac-
dependen lamellipodia o ma ion. This ac i i y is hough
o de i e om c oss alk in ol ing dis inc Rac-GEF com-
plexes [8–11]. The inabili y o RhoG and Cdc42 o ini ia e
lamellipodia has hi he o been hough o be due o a lack
o su icien in e ac ion wi h WRC. This is because bo h
RhoG and Cdc42 show a bes a weak in e ac ion wi h S a-
1/PIR121 [6,12], and Cdc42, as opposed o Rac1, ails o
ac i a e na i e WRC in i o [13].
Aside om S a-1 (o PIR121), WRC is composed o
ou addi ional subuni s –WAVE2 (o i s pa alogues
WAVE1/WAVE3), he S a-1/PIR121 in e ac o Nap1 (o
Hem1 in he hema opoie ic sys em), Abi1 (o Abi2/Abi3)
and HSPC300 [14–17]. The S a-1 subuni is ‘ ansinhibi -
ing’ he A p2/3 complex-ac i a ing, so called WCA domain
loca ed on he C- e minal end o WAVE p o eins, and Rac
binding o S a-1 ou compe es his inhibi o y in e ac ion o
elease he WCA domain, making i accessible o ac in and
A p2/3 complex binding [18]. We ha e ecen ly shown
ha he wo a o emen ioned Rac binding si es on S a-1/
PIR121 [18,19] a e essen ial o allos e ic ac i a ion o
WRC in cells [7]. Howe e , in spi e o he p e iously
p oposed sa e box model equi ing wo keys o allow o
WRC ac i a ion o occu [19], we ha e ound su p isingly
speci ic physiological unc ions o he wo si es in li e cells.
Whe eas he low a ini y A si e is c ucial o ac i a ion
in i o, he high a ini y D si e is con ibu ing o he
e iciency o lamellipodial p o usion, bu by no means as
impo an o WRC ac i a ion as he A si e. Aside om he
appa en c i ical unc ion o Rac in WRC ac i a ion, i is
less well es ablished i o i so o wha ex en Rac-WRC
in e ac ions also d i e WRC ec ui men o and accumula-
ion in he lamellipodium. Fo ins ance, we ha e also ound
ha lamellipodia o ma ion can be ini ia ed, in p inciple,
wi hou di ec WRC-Rac in e ac ions once WRC is en-
de ed ac i e, assuming a leas ha in oduced, espec i e
CONTACT Klemens Ro ne [email p o ec ed] Di ision o Molecula Cell Biology, Zoological Ins i u e, Technische Uni e si ä
B aunschweig, B aunschweig, Ge many
The supplemen al da a o his a icle can be accessed he e.
SMALL GTPASES
h ps://doi.o g/10.1080/21541248.2019.1657755
© 2019 The Au ho (s). Published by In o ma UK Limi ed, ading as Taylo & F ancis G oup
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion-NonComme cial-NoDe i a i es License (h p://c ea i ecommons.o g/licenses/by-nc-
nd/4.0/), which pe mi s non-comme cial e-use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed, and is no al e ed, ans o med, o buil
upon in any way.
mu a ions o bo h A and D si es in o ac i e WRC abolished
i s in e ac ion wi h Rac en i ely [7,19]. This is consis en
wi h he ac ha dele ing he CAAX-box in Rac1, which is
c ucial o plasma memb ane associa ion, does impai , bu
no abolish lamellipodia o ma ion in Rac1 knockou (KO)
ib oblas s [6]. In spi e o he absence o an unequi ocal,
al e na i e mechanism o WRC ec ui men , hese da a
sugges ha ac i a ionandlamellipodial a ge ing o
WRC migh po en ially be sepa able. An example o such
a sepa a ion clea ly cons i u es he ela ed GTPase Cdc42,
which media es ac i a ion o i sdowns eame ec o s
FMNL2 and −3 as p e equisi e o hei lamellipodial a ge -
ing [20,21], which howe e can ully occu wi h ac i e
a ian s comple ely lacking GTPase in e ac ion su aces
([22] and unpublished da a). The e o e, e ec o ec ui -
men o lamellipodia is possible, in p inciple, wi hou
engagemen o a gi en GTPase in spi e o i s es ablished
ele ance in e ec o ac i a ion. He e, we ha e de eloped
no el cell lines o compa e he capabili y o lamellipodia
o ma ion by ac i a ed WRC in he absence e sus p esence
o endogenous Rac GTPases.
Resul s
Rac –WRC in e ac ions a he plasma memb ane
a e dispensable o lamellipodia o ma ion
We sough o es i plasma memb ane inse ion o he
p enyl g oup o Rac1 is equi ed o lamellipodia induc-
ion in B16-F1 cells. Fo his, we exp essed cons i u i ely
ac i e (Q61L), myc- agged Rac1 o an iden ical cons uc
lacking he CAAX-box essen ial o C- e minal p enyla-
ion in B16-F1 cells lacking Rac1/2/3 (clone#1 [7]). In
analogy o ou p e iously published expe imen s
employing ib oblas s gene ically dele ed o Rac1 [6],
dele ion o he CAAX-box educed, bu did no abolish
lamellipodia o ma ion in hese condi ions. Mo e speci-
ically, hese s uc u es we e induced wi h o e all sligh ly
educed equency, and he majo i y o hem ( oughly
60%) appea ed o be imma u e, acco ding o he p e-
iously es ablished ca ego iza ion o unde de eloped
lamellipodia [7]. In con as , he majo i y o cells exp es-
sing ull leng h, cons i u i ely ac i e (Q61L) Rac1 ha -
bou ed ully de eloped lamellipodia (Figu e S1(a,b)).
These da a sugges ha he same e ec s seen in Rac1
−/−
ib oblas s [6] we e no cell- ype speci ic, and could no
po en ially be explained by emnan s o Rac2 o −3
p o ein exp essed pe haps a unde ec able le els om
espec i e genes no a ge ed in hese ib oblas s.
In analogy, we assembled WRCs in S a-1/PIR121 KO
cells (clone #3) ha bou ing a S a-1 a ian media ing con-
s i u i e WRC ac i a ion, bu lacking unc ional Rac bind-
ing si es (A + D si e WCA* [7], and Figu e S1(e)). In his
cons uc , speci ic poin mu a ions abolished he binding o
Rac o he A si e (C179R/R190D) and D si e (Y967A)
[7,18,19], whe eas he WCA* mu a ion (L697D/Y704D/
L841A/F844A/W845A) in he WH2- and C- egion (W
and C) con ac si es o S a-1 p e en ed he ‘ ansinhibi o y’
binding o S a-1 o he WCA domain o WAVE, which is
hen eleased o ac i a ing A p2/3 complex [7,18]. We
ound ha S a-1/PIR121 KO cells (clone #3) ha bou ing
A + D si e WCA*-mu a ed WRC can s ill escue lamelli-
podia o ma ion, albei a s ongly comp omised equency
[7]. Once o med hough, and al hough comp omised,
hese lamellipodia can s ill accumula e WRC a hei ips
(Figu e S1(c), see also [7]), and media e con inuous p o-
usion ha is less smoo h hough han wi h lamellipodia
d i en by WRCs ha bou ing WT S a-1 (Figu e S1(d)). No
su p isingly, a e age p o usion eloci ies o hese a e
examples o comp omised lamellipodia we e educed as
compa ed o hose media ed by wild ype S a-1 (Figu e S1
( )). Toge he , all hese da ase s hus sugges ha al hough
help ul, con inuous Rac-WRC in e ac ions a he plasma
a e no absolu ely obliga o y o lamellipodium p o usion.
Gene a ion o a cell line allowing u he dissec ion
o he Rac-WRC signalling module
Nex we asked whe he Rac is essen ial o WRC-
media ed lamellipodia o ma ion solely because o i s
essen ial unc ion in WRC ac i a ion o because o se -
ing addi ional unc ions. To es his, we had o de elop
cell sys ems in which endogenous WRC o Rac p o eins
could be eplaced by ac i e a ian s o each o unc ional
de iciency mu an s in a combina o ial ashion. In p e-
ious wo k, we had es ablished cell lines lacking ei he
Rac1/2/3 o unc ional WRC (S a-1/PIR121-null), in
which o - a ge e ec s po en ially caused by CRISPR/
Cas9-media ed genome edi ing we e excluded by
a combina ion o analysing mul iple, independen ly gen-
e a ed clones as well as by escue o pheno ypes wi h
exogenous Rac and S a-1, espec i ely [7]. In he cu en
wo k, we ex ended his app oach o gene a e a no el cell
line dis up ed o all i e genes (S a-1/PIR121+ Rac1/2/3
KO#3/11; Figu e 1(a)). The la e now allows deciphe -
ing Rac/WRC signalling in mo e de ail. In hese cells,
lamellipodia o ma ion is s ikingly dependen on exo-
genous exp ession o bo h S a-1 and Rac1. While nei he
exp ession o EGFP as con ol, EGFP-S a-1 no myc-
Rac1L61 alone acili a ed lamellipodia o ma ion in
hese cells, co- ans ec ion o EGFP-S a-1 and myc-
Rac1L61 po en ly es o ed lamellipodia, indica ing he
p esence o lamellipodia in hese cells o s ic ly equi e
bo h Rac and S a-1 (Figu e 1(b,c)). No ably, Rac1L61
exp ession in hese WRC-de icien cells also caused
plasma memb ane blebbing (Figu e 1(b) o
2M. SCHAKS ET AL.
ep esen a i e image), eminiscen o ou p e ious
obse a ions upon Rac mic oinjec ion upon WRC sub-
uni knockdown [15]. This pheno ype was obus and
occu ed a high equency (45 ± 13% in Rac1L61
exp essing cells s. 13 ± 12% in EGFP-exp essing con-
ols), al hough a p ecise, mechanis ic unde s anding o
Figu e 1. Gene a ion o S a-1/PIR121+ Rac1/2/3 KO cells.
(a) Wes e n blo ing o dis inc cell lines o p obe o exp ession le els o endogenous S a-1/PIR121 and/o Rac GTPases. (b) Cell
mo phologies o S a-1/PIR121+ Rac1/2/3 KO cells (clone #3/11) exp essing espec i e cons uc s, as indica ed. Panels in op ow show
s ainings o he ac in cy oskele on wi h phalloidin, and middle and/o bo om ow images show luo escence o he same cells de i ed om
ei he EGFP o an i-myc an ibody s ainings, as indica ed. (c) Quan i ica ion o lamellipodial pheno ypes. EGFP-S a-1 WCA* deno es
a cons uc ende ing WRC cons i u i ely ac i e due o mu a ions elie ing he au oinhibi o y in e ac ion o S a-1 wi h he C- e minal WCA-
domain (hence WCA* o ac i e WCA) o WAVE [7,18]. Lamellipodial ac in ne wo ks ha we e gene ally small, na ow, i egula o displayed
mul iple u les we e de ined as ‘“imma u e lamellipodia”’, as opposed o egula , ully de eloped lamellipodia. n gi es numbe o cells
analysed, da a co espond o a i hme ic means ± SEM om a leas h ee independen expe imen s. S a is ical signi icance was assessed o
di e ences be ween pe cen ages o cells wi h ‘no lamellipodia’pheno ype. ***p < 0.001 ( wo-sample, wo-sided - es ).
SMALL GTPASES 3
he phenomenon is cu en ly lacking. To es i cons i-
u i e ac i a ion o WRC would be su icien o igge
lamellipodia o ma ion in he absence o Rac, we
exp essed EGFP- agged S a-1 WCA*. Su p isingly, how-
e e , EGFP- agged S a-1 WCA*, lacking he need o
Rac-media ed ac i a ion o WRC was s ill incapable o
d i ing lamellipodia o ma ion in his cell line and con-
di ions. I compa ing his esul wi h cons i u i ely
ac i e WRCs comp omised in Rac binding, bu gi ing
ise o ine icien lamellipodia o ma ion in S a-1/
PIR121-KO cells ([7] and Figu e S1(c,d)), wo heo e ical
explana ions o his disc epancy a e hinkable: Fi s ly,
he A + D si e-mu a ed, ac i e WRC used in Figu e S1
can s ill ine icien ly bind o endogenous Rac p o eins
p esen in hese cells. Al e na i ely, Rac possesses addi-
ional, essen ial unc ions absen in cells ha bou ing
ac i e WRC bu lacking endogenous Rac p o eins (as
in Figu e 1(b,c)). Fu u e expe imen s will ha e o dis in-
guish be ween hese wo possibili ies.
Rac- ela ed Rho GTPases ail o ac i a e WRC, bu
can subs i u e o Rac in he p esence o ac i a ed
WRC
Up o his da e, he li e a u e con ains con lic ing
esul s conce ning he ele ance o he closes ela-
i es o Rac GTPases in mammals, in pa icula
RhoG, bu o a ce ain ex en also Cdc42. In spi e
o p ominen s udies es ablishing unc ions o RhoG
in signalling complexes ope a ing ups eam o Rac
[10–12], RhoG has also al eady been concluded o
con ibu e o ib oblas mig a ion independen o Rac
ac i a ion [23], al hough i has emained unclea how
ha migh occu mechanis ically [24]. O no e, and
again in ull acco dance wi h ou p e iously pub-
lished ib oblas da a [6], ou Rac1/2/3-de icien B16-
F1 melanoma line ailed en i ely o o m lamellipodia
e en upon exp ession o cons i u i ely ac i e RhoG
(Figu e 2(a,b)). Iden ical esul s we e ob ained wi h
o e exp essed, cons i u i ely ac i e Cdc42 bu no
Rac1, which obus ly es o ed lamellipodia o ma ion
(Figu e 2(a,b)), as expec ed [7]. Aside om he
incapabili y o Cdc42 o induce lamellipodia, we
ound a p ominen induc ion o s ess ib es in
hese condi ions (Figu e 2(a); 89 ± 2% o ans ec ed
cells), which was no seen wi h RhoG (2 ± 3%). Such
an ac i i y is adi ionally s ill mos ly a ibu ed o
RhoA/B/C ac i i y in he li e a u e, and hus no
ollowed up u he in he con ex o he cu en
s udy. Howe e , since ou p e ious e o s allowed
us o expe imen ally sepa a e Rac-media ed WRC
ac i a ion om o he po en ial unc ions ([7] and
see abo e), we wonde ed whe he we migh –in
analogy o Rac –be able o es ablish connec ions
be ween RhoG o Cdc42 ac i i ies and WRC unc ion
independen o Rac-media ed WRC ac i a ion. Fo
his, we explo ed RhoG- o Cdc42-d i en ac in
Figu e 2. Rac- ela ed GTPases RhoG and Cdc42 ail o induce lamellipodia in he absence o Rac exp ession.
(a) Cell mo phologies o Rac1/2/3 KO cells (clone #1) exp essing EGFP- agged GTPases, as indica ed. (b) Quan i ica ion o lamellipodial
pheno ypes was pe o med as desc ibed o Figu e 1(c). n gi es numbe o cells analysed, da a co espond o a i hme ic means ± SEM om
a leas h ee independen expe imen s. S a is ical signi icance was assessed o di e ences be ween pe cen ages o cells wi h ‘no
lamellipodia’pheno ype. ***p < 0.001 ( wo-sample, wo-sided - es ).
4M. SCHAKS ET AL.

cy oskele on emodelling in cells exclusi ely ha bou -
ing ac i e WRC bu lacking endogenous Rac GTPases
(see Figu e 1). And indeed, co- ans ec ion o ac i e
(Q61L), myc- agged RhoG o Cdc42 oge he wi h
S a-1 WCA* led o pa ial escue o lamellipodia
o ma ion in S a-1/PIR121+ Rac1/2/3 KO cells
(clone #3/11) (Figu e 3(a,c) o ep esen a i e images
and quan i a ions, espec i ely). While RhoG was
able o induce imma u e lamellipodiainmo e han
40% o ans ec ed cells and e en occasional, ully
de eloped lamellipodia, Cdc42 caused lamellipodia
o ma iononlyinasmallsub ac iono cells(see
Figu e 3(a), as e isks, and Figu e 3(c) o quan i ica-
ion). Al hough he low equency o Cdc42-media ed
induc ion o (a leas imma u e) lamellipodia in cells
co-exp essing S a WCA* could no be igno ed, i was
no signi ican ly di e en s a is ically om cells co-
exp essing cons i u i ely ac i e Cdc42 wi h wild ype
S a-1 (no shown). This esul was ob ained wi h
wo-sided, wo-sample - es , bu when using
Fishe ’s exac es also app op ia e o compa ing
wo expe imen al g oups (Cdc42 co-exp ession wi h
S a-1 WT e sus WCA*) alling in o wo ca ego ies
(wi h o wi hou lamellipodia), he di e ence
be ween hem u ned ou o be s a is ically signi ican
(p = 0.023). Mo eo e , bo h RhoG and Cdc42 we e
capable, in p inciple, o d i ing accumula ion o S a-
1 WCA* a p o usion si es gi en ha lamellipodia
we e o med (Figu e 3(b)). Speci ic S a-1 WCA*
en ichmen upon co-exp ession o bo h RhoG and
Cdc42 was also con i med by linescan analyses o
espec i e images (Figu e 3(b)). In con as , co-
an ec ion o S a-1 WCA* wi h myc- agged, cons i-
u i ely ac i e RhoA (G14V) ailed o s imula e
lamellipodia o lamellipodia-like s uc u es in all
cells analysed (96 cells om 3 independen expe i-
men s; Figu e 3(a,c) o quan i ica ion). This was
consis en wi h he lack o any accumula ion o
EGFP- agged S a-1 WCA* a he pe iphe y o hese
cells (Figu e 3(b), igh panel), in spi e o cons i u-
i ely ac i e RhoA clea ly being unc ional in his
case, as e idenced by he expec ed, p ominen s imu-
la ion o s ess ib es (Figu e 3(a), igh panels).
As al eady men ioned in he con ex o s a is ical
analysis, he abili y o RhoG and Cdc42 o induce
lamellipodia in hese expe imen s was s ic ly depen-
den on cons i u i e WRC ac i i y in his cell line
( h ough using S a-1 WCA*), as bo h RhoG and
Cdc42 ailed o induce lamellipodia when co-
exp essed wi h WT-S a-1 (Figu e 3(c)). Finally, addi-
ional mu a ion o he wo Rac binding si es on con-
s i u i ely ac i e WRC comple ely p e en ed (in case
o Cdc42) o a leas s ongly inhibi ed (in case o
RhoG) he induc ion o lamellipodia in hese condi-
ions (Figu e 3(a,c)), indica ing ha he ac ion o
RhoG/Cdc42 may be explained by di ec , (Rho-
GTPase binding su ace-dependen ) in e ac ion wi h
WRC. In line wi h his, immunop ecipi a ion expe i-
men s showed clea ly de ec able in e ac ions o RhoG
and Cdc42 wi h S a-1 WCA*, albei somewha weake
pe haps han obse ed o Rac1 (Figu e 3(d)).
Assuming ha he desc ibed, po en ial RhoG/Cdc42 -
WRC in e ac ions may be di ec , we also wonde ed
whe he hese GTPases migh p e e en ially a ge he
A o he D si e p e iously es ablished as in e ac ion
su aces on WRC wi h Rac1 [19]. Due o he low
equency o Cdc42-media ed lamellipodia o ma ion
in his assay (Figu e 3), we ocused on RhoG in hese
expe imen s compa ed o Rac1 used as con ol. We
again co- ans ec ed ou cell line lacking endogenous
WRC plus all Rac-GTPases wi h he indi idual A and
D si e mu an s o WCA* S a-1 and cons i u i ely
ac i e Rac1 (as con ol) e sus RhoG. While mu a ing
ei he he A o D si e caused signi ican impai men o
lamellipodia o ma ion induced by ac i e Rac1, albei
o dis inc easons, as expec ed ( o compa ison o
analogous, bu no iden ical expe imen s, see e [7].),
mu a ing he A si e did no cause a s a is ically sig-
ni ican educ ion o RhoG-media ed lamellipodia o -
ma ion in his assay. In con as , ab oga ion o D si e
unc ion caused a se e e educ ion o lamellipodia
(Figu e S2(a)), i ually iden ical o he le el obse ed
upon simul aneous mu a ion o bo h si es (Figu e 3
(c)). F om his, we concluded ha he po en ial, pe -
haps di ec WRC-RhoG in e ac ion is la gely media ed
by he D si e o S a-1.
All hese esul s p omp ed us o compa e by addi-
ional in silico analyses he pu a i e binding su aces
o RhoG and Cdc42 wi h he D si e o S a-1, which
also cons i u es he high a ini y binding si e o Rac
GTPases [19]. Sequence alignmen s e ealed ha wo
(incaseo RhoG)o h ee(incaseo Cdc42)amino
acids di e ed om Rac1 in he pu a i e binding
in e ace (Figu e S2(b)). Howe e , nei he o hese
esidues caused signi ican changes in elec os a ic
su ace po en ials (Figu e S2(c)). RhoA, on he con-
a y, showed ob ious di e ences in su ace elec o-
s a ic po en ial in he pu a i e binding in e ace,
pa icula ly caused by he glu amine o aline sub-
s i u ion a posi ion 33 in RhoA, he analogous sub-
s i u ion o which in Rac1 (E31V) appa en ly
in e e ed wi h p ope lamellipodia o ma ion [7,25].
Toge he , hese da a sugges ha bo h RhoG and
Cdc42 can speci ically in e ac wi h WRC, in p inci-
ple, and in a physiologically ele an manne once
WRC has been ac i a ed by Rac1.
SMALL GTPASES 5
Figu e 3. Induc ion o lamellipodia by RhoG o Cdc42 in conjunc ion wi h ac i a ed WRC, bu in he absence o Rac exp ession.
(a) Rep esen a i e images o S a-1/PIR121+ Rac1/2/3 KO cells (clone #3/11) exp essing EGFP-S a-1 WCA* oge he wi h myc- agged, small
GTPases, as indica ed. T ans ec ed cells iden i ied by myc-s aining (as e isks) we e analysed conce ning hei cell mo phologies ( op ow, ac in
ilamen s aining wi h phalloidin) and he capabili y o accumula e ac i e WRC (S a-1 WCA*) a pe iphe al lamellipodia (b). Measu emen s shown
in b we e pe o med along line scans as shown in he images p o ided in a (g een lines). (c) Quan i ica ion o lamellipodial pheno ypes, done as
desc ibed o Figu e 1(c). n gi es numbe o cells analysed, and di e en ially colou ed columns a e a i hme ic means ± SEM om a leas h ee
independen expe imen s. S a is ical signi icance was assessed o di e ences be ween he pe cen ages o cells wi h ‘no lamellipodia’pheno ype.
***p < 0.001 ( wo-sample, wo-sided - es ). (d) S a-1/PIR121 KO cells (clone # 3) we e co- ans ec ed wi h mChe y-S a-1 WCA* and ei he EGFP o
cons i u i ely ac i e (Q61L), EGFP- agged small GTPases, lysed and subjec ed o immunop ecipi a ion agains EGFP. No e he comple e absence o
immunop ecipi a ion o S a-1 upon co-exp ession o EGFP alone (nega i e con ol), and signi ican in e ac ions o S a-1 WCA* wi h Rac-1, RhoG
and Cdc42. (e) Model o how RhoG and Cdc42 migh egula e WRC and lamellipodia o ma ion.
6M. SCHAKS ET AL.
Discussion
Al hough puzzling ini ially, we ha e p e iously ound and
con i med he e using dis inc expe imen al sys ems ha
Rac may no ha e o associa e wi h he plasma memb ane
in o de o ac i a e o ec ui WRC du ing lamellipodia
ini ia ion and main enance. Ye , and no inconsis en
wi h his iew, Rac GTPases emain o be obliga o y o
bo h WRC ac i a ion and lamellipodia o ma ion, as cells
lacking bo h endogenous Rac GTPases and WRC can
only o m lamellipodia upon addi ional, speci ic manip-
ula ion. To ou su p ise, we es ablish o he i s ime
he e ha lamellipodia o ma ion is possible, in p inciple,
wi hou Rac GTPases, gi en ha cells lack he need o
WRC ac i a ion (because i is al eady ac i a ed o does
no need o be ac i a ed), and ha hey o e -exp ess
cons i u i ely ac i e a ian s o ei he RhoG o Cdc42.
In o he wo ds, o e exp ession o RhoG, and o a lesse
ex en Cdc42 can cause he accumula ion o cons i u i ely
ac i e WRC e en in he comple e absence o endogenous
Rac GTPases, p esumably causing WRC-dependen
A p2/3 complex ac i a ion. This sugges s ha aside
om p e iously es ablished signalling c oss alk be ween
Rac GTPases and RhoG/Cdc42 [8–10], he la e may
di ec ly con ibu e o he main enance and/o ac i i y o
WRC a p o uding lamellipodia edges once Rac has
managed o ac i a e indi idual WRCs. I is hus hinkable
ha RhoG and Cdc42 may di ec ly suppo Rac signalling
by pa ially aking o e Rac unc ions conce ning WRC
posi ioning i espec i e o WRC ac i a ion.
Consis en wi h his iew, we ha e p e iously ound
a e age u no e imes o WRC subuni s a he p o-
uding plasma memb ane ha ce ainly i he hypo h-
esis o con inuous ac i a ion e en s o A p2/3
complexes, media ed by indi idual WRC uni s in
a Rho GTPase binding-dependen ashion [7]. Slow
u no e and molecula c owding o indi idual WRCs
a he memb ane likely con ibu es o e icien A p2/3
ac i a ion a hese si es (see also [26,27]), las , no leas
because e icien A p2/3 complex ac i a ion was p e-
iously demons a ed o in ol e simul aneous engage-
men o wo WCA domains [28–30]. The e o e, i
seems plausible ha e icien A p2/3 complex ac i a ion
a he lamellipodium ip coincides wi h WRC clus e -
ing, wi h he la e being a ec ed, a leas in pa , by
GTPase signalling.
The desc ibed, di ec con ibu ion o RhoG and
Cdc42 o WRC-media ed ac in emodelling is also
ound o occu in spi e o a bes weak o en i ely non-
speci ic in e ac ions o RhoG and Cdc42 wi h wild ype
WRC [6,12], as we show he e ha he si ua ion changes
d ama ically i cons i u i ely ac i e WRC is used, which
can be p ecipi a ed qui e e icien ly by bo h Rac- ela ed
GTPases. Mo eo e , we also ound ha a leas he
in e ac ion o RhoG wi h WRC is la gely media ed
h ough he D si e o S a-1 in WRC, and no he
A si e (Figu e S2(a)), es ablished p e iously o be c u-
cial o WRC ac i a ion by Rac1 [7]. This hen could
explain he comple e ailu e o RhoG (and Cdc42) o
ac i a e WRC in he absence o endogenous Rac
GTPases (Figu es 2 and 3(c)). All hese conside a ions
a e also con i med by sequence alignmen s and s uc-
u al conside a ions conce ning e ec o in e ac ion
su aces p esen on Rac1, RhoG and Cdc42 e sus
RhoA (Figu e S2). Fu u e s uc u al s udies will be
needed o solidi y he hypo hesis o di ec in e ac ions
and explain why –i con i med o be di ec – he
compa ably obus in e ac ion o RhoG and Cdc42
wi h S a-1 obse ed he e canno occu wi h inac i e
WRC and/o ansla e in o WRC ac i a ion.
Al hough he binding e iciency o RhoG and Cdc42 o
ac i e WRC appea ed compa able in immunop ecipi a-
ion expe imen s, he e was clea ly measu able di e ences
be ween he e iciency o he ou pu esponse (lamellipo-
dia in his case), he p ecise easons o which emain o
be de e mined. Ye , he as majo i y o bo h RhoG and
Cdc42-dependen lamellipodia o med in hese condi-
ions (absence o endogenous Rac GTPases) we e s ill
imma u e, clea ly illus a ing he ele ance o Rac in o -
ma ion and u no e o hese s uc u es beyond i s es ab-
lished, essen ial unc ion in WRC ac i a ion.
The no el cell sys em lacking endogenous Rac and
WRC p o eins (B16-F1 S a-1/PIR121+ Rac1/2/3 KO
clone #3/11) also ha bou s he po en ial o emphasizing
p e iously men ioned, bu less well s udied pheno ypes
caused by ac i e, small GTPases, indica i e o he
commonly es ablished, bu ch onically unde es ima ed
complexi y a ising om Rho-GTPase c oss alk [31,32].
Fo ins ance, wi hou WRC, Rac ailed o induce lamel-
lipodia, bu no in equen ly caused plasma memb ane
blebbing much less common o B16-F1 cells exp essing
endogenous WRC and hus capable o lamellipodia
o ma ion. Simila obse a ions we e p e iously
epo ed o o e exp ession o he Rac e ec o loop
mu an F37A [33], which we now conside o be
impai ed in d i ing lamellipodia o ma ion as a esul
o comp omised WRC in e ac ion [7]. Whe he his
blebbing ac i i y a ises om in insic, Rac-speci ic ea-
u es (i.e. equi ing di ec Rac-e ec o binding) o is
amo e indi ec esul o c oss alk o RhoA/B/C signal-
ling emains unclea . Mo eo e , ou p elimina y obse -
a ions also indica ed ha Cdc42 ac i i ies can
p ominen ly unnel in o con ac ile s ess ib e o ma-
ion i Rac signalling (o lamellipodia o ma ion) is
missing. This could likely be media ed h ough signal-
ling o MRCK kinases, p e iously shown o coope a e
SMALL GTPASES 7
wi h Rho-ROCK signalling [34–36], which hi he o
appea ed less p e alen in ou cell sys ems wi hou
comp omised lamellipodia o ma ion (see [37,38]). As
he lamellipodia esponse seen wi h he Cdc42 –
(ac i e) WRC combina ion was much less p ominen
han seen o he RhoG –(ac i e) WRC couple, i is
emp ing o specula e ha he s ess ib e induc ion
pheno ype men ioned abo e migh in e e e wi h
mo e obus lamellipodia o ma ion. This migh be
explained pe haps by he widely accep ed and long-
s anding an agonism be ween p o usion- (lamellipo-
dia) e sus con ac ili y-dependen (s ess ib es) p o-
cesses [39,40]. Fu u e expe imen s will ha e o e eal
whe he his assump ion is co ec .
Wha e e he case, ou da a add o he iew ha
sys ema ic gene a ion and side-by-side compa ison o
Rho GTPase and e ec o knockou s in he same pa -
en al cell line will con inue o un old mechanis ic
insigh s in o he in icacies o Rho signalling and c oss-
alk ele an o ac in emodelling p ocesses.
Ma e ials and me hods
Cell cul u e
B16-F1 cell line was pu chased om ATCC (CRL-6323,
sex:male). B16-F1 de i ed S a-1/PIR121 KO cells (clone
#3), as well as Rac1/2/3 KO cells (clone #1) we e as
desc ibed [7]. B16-F1 cells and de i a i es we e cul u ed
in DMEM (4.5 g/l glucose; In i ogen), supplemen ed wi h
10% FCS (Gibco), 2 mM glu amine (The mo Fishe
Scien i ic) and penicillin (50 Uni s/ml)/s ep omycin
(50 µg/ml) (The mo Fishe Scien i ic). B16-F1 cells we e
ou inely ans ec ed in 35 mm dishes (Sa s ed ), using
0.5 µg DNA in o al and 1 µl Je P ime o con ols, and
1 µg DNA in o al and 2 µl Je P ime o B16-F1-de i ed
knockou cells. A e o e nigh ans ec ion, cells we e pla-
ed on o acid-washed, laminin-coa ed (25 µg/ml) co e slips
and allowed o adhe e o a leas 5 hou s p io o analysis.
DNA cons uc s
pEGFP-C1 and –C2 ec o s we e pu chased om
Clon ech Inc. (Moun ain View, CA, USA). pEGFP-
C2-S a-1, and de i ed mu an cons uc s (i.e. WCA*,
A si e WCA*, D si e WCA*, A + D si e WCA*) we e
desc ibed p e iously [7] and co espond o he splice
a ian CYFIP1a, sequence AJ567911. mChe y-
agged S a-1 WCA* was gene a ed by swapping
EGFP wi h mChe y, kindly p o ided by D . Roge
Tsien (Uni e si y o Cali o nia a San Diego, La Jolla,
Cali o nia, USA) using NheI/Bs GI es ic ion si es.
pRK5-myc-Rac1L61 and pRK5-myc-RhoAV14 we e
kindly p o ided by Alan Hall and Lau a Machesky
(CRUK Bea son Ins i u e, Glashow, UK). Cdc42L61
(placen al iso o m) was syn hesized by Eu o ins
Genomics and cloned in o pRK5-myc and pEGFP-
C1 ec o s. pEGFP-C1-Rac1L61 and pRK5-myc-
RhoGL61 we e as desc ibed [6,7]. Fo gene a ion o
pEGFP-C1-RhoGL61, he co esponding DNA ag-
men immobilized om pRK5-myc-RhoGL61 wi h
BamHI/EcoRI was liga ed in o pEGFP-C1 ec o
diges ed wi h BglII/EcoRI. pRK5-myc-Rac1L61-
ΔCAAX was gene a ed by si e di ec ed mu agenesis
using 5ʹ-GAGGAAGAGAAAATGACTGCTGTTGTA
AGTC-3ʹas o wa d p ime . The ideli y o all con-
s uc s was e i ied by sequencing.
CRISPR/Cas9-media ed genome edi ing
B16-F1 cells lacking unc ional CYFIP1 andCYFIP2 genes,
as well as Rac1, Rac2 and Rac3 genes we e gene a ed by
ea ing S a-1/PIR121 KO cells (clone #3) wi h pSpCas9
(BB)-2A-Pu o (PX459) ec o s a ge ing Rac1, Rac2 and
Rac3 genes. Speci ically, cells we e co- ans ec ed wi h
plasmids a ge ing ATGCAGGCCATCAAGTGTG
(Rac1/2) and ATGCAGGCCATCAAGTGCG (Rac3)
genomic egions as desc ibed [7]. A e pu omycin selec-
ion o ans ec ed cells (3 days), cells we e ex ensi ely
dilu ed and a ew days la e , mac oscopically isible colo-
nies picked, o ob ain single cell-de i ed clones. De i ed
cell clones al eady lacking S a-1/PIR121 we e sc eened o
he addi ional absence o Rac exp ession by Wes e n
Blo ing (see Figu e 1).
Wes e n blo ing
P epa a ion o whole cell lysa es was pe o med essen-
ially as desc ibed [7]. Wes e n blo ing was ca ied ou
using s anda d echniques. P ima y an ibodies used we e
S a-1/PIR121 [15], Rac1/3 (23A8, Me ck), Rac2 [6],
GAPDH (6C5, Calbiochem) and GFP (clones 7.1 and
13.1, Roche). HRP-conjuga ed seconda y an ibodies
we e pu chased om In i ogen. Chemiluminescence
signals we e ob ained upon incuba ion wi h ECL™P ime
Wes e n Blo ing De ec ion Reagen (GE Heal hca e),
and we e eco ded wi h ECL Chemocam image (In as,
Goe ingen, Ge many).
Immunop ecipi a ion
Fo EGFP-immunop ecipi a ion expe imen s, S a-1/
PIR121 KO cells (clone #3) co-exp essing EGFP alone
o EGFP- agged a ian s o cons i u i ely ac i e (Q61L)
GTPases oge he wi h mChe y- agged S a-1 WCA*
we e lysed wi h lysis bu e (1% T i on X-100,
8M. SCHAKS ET AL.