IC-Tagging and P o ein Reloca ion o ARV muNS
Inclusions: A Me hod o S udy P o ein-P o ein
In e ac ions in he Cy oplasm o Nucleus o Li ing Cells
Albe o B anda iz-Nun
˜ez, Rebeca Menaya-Va gas, Ja ie Bena en e, Jose Ma inez-Cos as*
Depa men o Biochemis y and Molecula Biology, Facul y o Pha macy and Cen e o Resea ch in Biological Chemis y and Molecula Ma e ials, Uni e si y o San iago
de Compos ela, San iago de Compos ela, Spain
Abs ac
Backg ound:
Cha ac e iza ion o p o ein-p o ein in e ac ions is essen ial o unde s anding cellula unc ions. Al hough
he e a e many published me hods o analyze p o ein-p o ein in e ac ions, mos o hem p esen se ious limi a ions. In a
di e en s udy we ha e cha ac e ized a no el a ian eo i us muNS-based p o ein agging and inclusion a ge ing me hod,
and demons a ed i s alidi y o pu i y ee an immobilized p o ein.
Me hodology/P incipal Findings:
He e we p esen a me hod o iden i y p o ein-p o ein in e ac ions inside li ing euka yo ic
cells ( es ed in p ima e and a ian cells). When p53 was agged wi h In e coil (IC; muNS esidues 477–542), i no only go
in eg a ed in o muNS cy oplasmic inclusions, bu also a ac ed i s known ligand SV40 la ge T an igen (TAg) o hese
s uc u es. We ha e also adap ed his sys em o wo k wi hin he cell nucleus, by c ea ing muNS- ela ed p o ein chime as
ha o m nuclea inclusions. We show ha nuclea muNS-de i ed inclusions a e as e icien as cy oplasmic ones in cap u ing
IC- agged p o eins, and ha he p o eins a ge ed o nuclea inclusions a e able o in e ac wi h hei known ligands.
Conclusions/Signi icance:
Ou p o ein edis ibu ion me hod does no p esen he a chi ec u al equi emen o e-
cons uc ing a ansc ip ion ac o as any o he wo-hyb id sys ems do. The me hod is simple and equi es only cell
ans ec ion and a luo escence mic oscope. Ou agging me hod can be used ei he in he cy oplasm o he nucleus o
li ing cells o es p o ein-p o ein in e ac ions o o pe o m unc ional s udies by p o ein ligand seques a ion.
Ci a ion: B anda iz-Nun
˜ez A, Menaya-Va gas R, Bena en e J, Ma inez-Cos as J (2010) IC-Tagging and P o ein Reloca ion o ARV muNS Inclusions: A Me hod o
S udy P o ein-P o ein In e ac ions in he Cy oplasm o Nucleus o Li ing Cells. PLoS ONE 5(11): e13785. doi:10.1371/jou nal.pone.0013785
Edi o : Anna Mi aki, Uni e si y o C e e, G eece
Recei ed June 23, 2010; Accep ed Oc obe 8, 2010; Published No embe 2, 2010
Copy igh : ß2010 B anda iz-Nun
˜ez e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s
un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed.
Funding: This wo k was suppo ed by g an s om he Eu opean Commission unde con ac s ERAS-CT-2003-980409 (as pa o he Eu opean Science
Founda ion EUROCORES P og amme Eu oSCOPE, web: h p://www.es .o g/eu oscope); he Spanish Minis e io de Ciencia y Tecnologı
´a (BFU2007-61330, BFU 205-
24982-E, web: h p://www.mi yc.es/) and Xun a de Galicia (08CSA009203PR, web: h p://www.conselle iaiei.o g/ga/dxidi/index.php). ABN was he ecipien o a
p edoc o al FPI ellowship om he Spanish Minis e io de Ciencia y Tecnologı
´a. The unde s had no ole in s udy design, da a collec ion and analysis, decision o
publish, o p epa a ion o he manusc ip .
Compe ing In e es s: The esul s p esen ed in his manusc ip a e pa en pending. Applica ion numbe : P201030204. In en o s: Albe o B anda iz-Nun
˜ez,
Rebeca Menaya Va gas, Ja ie Bena en e and Jose Ma inez-Cos as. Coun y: Spain. All he ma e ials desc ibed will be a ailable o esea ch pu poses. This does
no al e he au ho s’ adhe ence o all he PLoS ONE policies on sha ing da a and ma e ials.
* E-mail: jose.ma inez.cos [email protected]
In oduc ion
Vi oplasms, i al ac o ies o i us inclusion bodies a e di e en
names gi en o he cellula compa men s whe e mos i uses
ca y ou hei mo phogenesis. They a e usually gene a ed om
one o se e al i al p o eins ha ac as a sca old o ma ix,
nuclea ing he inclusion ha is o med by p o ein-p o ein
in e ac ions. The ma ix p o eins a ac and concen a e he i al
componen s, inc easing he o e all e iciency o he i al
eplica ion p ocess [1,2].
A ian eo i uses belong o he genus O ho eo i us, amily
Reo i idae [3,4] and cons i u e dange ous poul y pa hogens [5,6].
De ails on hei s uc u e and eplica ion cycle a e a ailable
elsewhe e [7,8,9]. Al hough hese i uses eplica e in he
cy oplasm o in ec ed cells, a leas wo i al p o eins ha e been
epo ed o display nuclea localiza ion [10,11]. In ecen yea s,
ou labo a o y has in es iga ed he mechanisms ha a ian
eo i uses use o p oduce i al ac o ies. The esul s e ealed ha
a ian eo i us non-s uc u al p o ein muNS is able o gene a e
ac o y-like inclusions when exp essed in di e en cell lines and
using di e en exp ession sys ems, sugges ing ha his p o ein
o ms he ma ix o he ac o ies in in ec ed cells [12]. Addi ionally,
muNS a ac s o he i al p o eins in a speci ically and empo ally
con olled way, hus con ibu ing o egula e he mo phogenesis o
he i al pa icle [13]. In a ecen s udy we ha e demons a ed ha
a ian eo i us inclusion o ma ion does no depend on he
cy oskele on, and ha a ian eo i us ac o ies and muNS-de i ed
inclusions a e no mic o ubule-associa ed [14]. An analysis o he
inclusion- o ming capabili y o muNS unca ions e ealed ha
he minimal muNS po ion able o gene a e in acellula
inclusions comp ises i s C- e minal one hi d ( esidues 448–635).
We designa ed i muNS-Mi, and cha ac e ized he ole ha i s
ou di e en domains (Coil1, Coil2, In e coil and C-Tail) play in
inclusion o ma ion. Mos no ably, we we e able o demons a e
ha Coil1 egion ( esidues 448 o 477) can be eplaced by a
dime iza ion domain, and ha he C-Tail domain ( esidues 605–
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635) o ien s muNS in e -monome con ac s o gene a e basal
oligome s ha dic a e he inclusion shape and inclusion- o ming
e iciency [14]. In he same s udy, we de eloped a simple p o ocol
o he pu i ica ion o he inclusions made by muNS in
baculo i us-in ec ed cells.
Based on he esul s ob ained, and in a di e en s udy
(manusc ip in p epa a ion), we de eloped a me hod o a ge
o eign p o eins o he muNS- ela ed inclusions in ecombinan
baculo i us-in ec ed insec cells. I is based on he s ong a ini y
be ween muNS-de i ed inclusions and he 66 esidue-long,
In e coil domain (IC, muNS esidues 477–542). Thus, agging
p o eins wi h IC caused hei e-localiza ion o he muNS-de i ed
inclusions. Using a me hod ha we had p e iously designed o he
pu i ica ion o muNS-de i ed inclusions [14], we de eloped a
p o ocol o pu i ica ion o o eign p o eins ha had been agged
wi h he IC domain. We demons a ed ha he inclusion- a ge ed
p o eins we e ac i e ei he when in eg a ed in he inclusions, o
a e hei solubiliza ion and sepa a ion om he muNS- ela ed
inclusions. Ou s udy also showed ha he inclusion-in eg a ed
p o eins we e ac i e bo h in i o and in i o [25].
In he p esen s udy we demons a e ha ou IC- agging and
inclusion- a ge ing me hod wo ks as well in ans ec ed cells o
a ian and mammalian o igin. We also show ha he cellula
p o ein p53, in spi e o being a nuclea p o ein, is eloca ed o
cy oplasmic muNS- ela ed inclusions by IC- agging. Addi ionally,
when p53 ge s eloca ed o he inclusions, i a ac s he SV40
La ge T an igen (TAg). We showed ou p o ocol o wo k in wo
di e en cell lines o di e en o igin: COS-7, a mammalian cell
line exp essing TAg endogenously, and CEF (a ian) in which we
exp essed TAg by plasmid ans ec ion. The me hod desc ibed in
his s udy can indi e en ly use any o he ou di e en muNS-
ela ed p o eins as he inclusion- o ming uni : muNS, muNS-Mi,
GFP-muNS and GFP-muNS-Mi, and all o hem e icien ly
cap u e he IC- agged p o eins. Fu he mo e, we ha e also
adap ed his highly e sa ile sys em o wo k wi hin he cell
nucleus, by c ea ing muNS p o ein chime as ha a e able o o m
nuclea inclusions. We showed ha nuclea inclusions a e as
e icien as cy oplasmic ones in cap u ing IC- agged p o eins, and
ha he p o eins a ge ed o nuclea inclusions a e pe ec ly able o
in e ac wi h hei known ligands. The nuclea inclusion
en i onmen ep esen s an ideal means o s udying he in e ac-
ions be ween p o eins ha no mally eside a he cell nucleus.
Also, i opens he possibili y o using hem o cap u ing o
seques e ing nuclea p o eins, and hus emo ing hem om hei
no mal in a-nuclea localiza ion o unc ional s udies.
Resul s
IC-di ec ed a ge ing o muNS- ela ed inclusions o
de ec ing in acellula p o ein-p o ein associa ions in
li ing cells
P e iously [25] we ha e demons a ed ha he a ian eo i us
muNS IC domain is a sui able ag o a ge ing used p o eins o
he inclusions o med by di e en muNS e sions: muNS-Mi,
GFP-muNS and GFP-muNS-Mi (Figu e 1A and manusc ip in
p epa a ion). In he p esen wo k we in es iga ed whe he IC-
agging could also be employed o de ec ing p o ein-p o ein
in e ac ions wi hin li ing cells, by using he s a egy ou lined in
Figu e 1. S a egy o de ec ing p o ein-p o ein in e ac ions based on muNS-de i ed inclusion a ge ing by IC agging. A. CEF cells
we e ans ec ed wi h plasmids exp essing he p o eins indica ed on op o he pic u es. The cells we e immunos ained o muNS (g een), excep
hose exp essing GFP ha we e isualized wi hou an ibodies. Nuclei we e coun e s ained blue wi h DAPI. A schema ic ep esen a ion o he muNS-
de i ed cons uc s is depic ed below each pic u e, whe e he p e iously desc ibed muNS-Mi domains (Coil 1 o C1; In e coil o IC; Coil2 o C2, and C-
Tail o CT, [14]) a e shown in boxes. The g een ba el ep esen s GFP. B. P inciple o he me hod. Plasmids exp essing a muNS-de i ed inclusion-
o ming p o ein and an IC- agged bai p o ein (B) a e co ans ec ed in o cells. The IC-B p o ein should be inco po a ed in o inclusions and should
ec ui a bai -in e ac ing ish p o ein (F) in o hese s uc u es. The F p o ein could be an endogenous p o ein o a plasmid-exp essed p o ein.
doi:10.1371/jou nal.pone.0013785.g001
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Figu e 1B. In b ie , a plasmid exp ession ec o ca ying he gene
encoding he muNS-de i ed p o ein is co- ans ec ed in o cells
wi h a plasmid exp essing a IC- agged p o ein B (bai ) ha should
be inco po a ed in o he inclusions. A hi d plasmid exp essing a
p o ein F ( ish) ha is hough o in e ac wi h he bai is also
ans ec ed and, i B and F in e ac , F would be also a ac ed o
he inclusions by i s in e ac ion wi h B. In con as , i B and F do
no associa e, F will e ain i s no mal in acellula dis ibu ion in
he ans ec ed cell. The localiza ion o bo h p o eins can be easily
isualized by immunos aining wi h speci ic an ibodies.
Abili y o he di e en inclusion-compe en muNS
e sions o ec ui p53-IC
To es he gene al u ili y o his echnology, we ook ad an age
o he well-de ined in e ac ion be ween he mammalian umo
supp esso p o ein p53 and he SV40 umo p o ein TAg [15].
Thus, we decided o ag p53 wi h IC and check i s abili y o e-
localize and ec ui he SV40 TAg o muNS- ela ed cy oplasmic
inclusions. We used a plasmid ha exp esses he IC- agged p53
p o ein [25] (manusc ip in p epa a ion) and i s analyzed i s
localiza ion in ans ec ed CEF cells by immuno luo escence. Bo h
he un agged (no shown) and IC- agged p53 (Figu e 2A) localized
p ima ily o he nucleus, showing ha IC- agging did no a ec he
no mal localiza ion o p53. Nex , we es ed he abili y o he IC ag
o a ge p53 o muNS-de i ed inclusions by co- ans ec ion. In
he p esence o muNS, un agged p53 localized p ima ily in he
nucleus wi h no isible co-localiza ion wi h muNS inclusions
(Figu e 2B, ow 1). This esul demons a es ha p53 does no
associa e wi h muNS inclusions on i s own, and also ha he p53-
speci ic an ibody used o immunos aining does no c oss- eac
wi h muNS inclusions. In con as , IC- agged p53 localized almos
exclusi ely o muNS inclusions in co- ans ec ed cells (Figu e 2B,
ow 2). These esul s demons a e ha inco po a ion o p53-IC
in o muNS inclusions did no dis up inclusion o ma ion and ha
ou inclusion- a ge ing sys em wo ks also wi h nuclea p o eins.
Exac ly he same esul s we e ob ained when using all he o he
inclusion- o ming p o eins desc ibed in Figu e 1 in he co-
ans ec ion expe imen s. Thus, he inclusions o med by GFP-
muNS (Figu e 2B, ow 3), muNS-Mi (Figu e 2B, ow 4) o GFP-
muNS-Mi (Figu e 2B, ow 5), all a ac ed he IC- agged p53 ha
was comple ely e-localized o he espec i e inclusions. In
con as , un agged p53 showed no co-localiza ion wi h any o
he inclusions and emained in he nucleus in he co- ans ec ion
expe imen s (Figu e 2B, ow 1 and da a no shown).
Figu e 2. In acellula dis ibu ion o p53 and p53-IC in he p esence o muNS-de i ed cy oplasmic inclusions. A. In acellula
dis ibu ion o p53-IC in single- ans ec ed CEF cells. p53 is immunos ained ed and nuclei a e coun e s ained blue wi h DAPI. B. CEF cells we e
co ans ec ed wi h he plasmids exp essing he p o eins indica ed on he le o he igu e. Cells we e immunos ained wi h abbi an i-muNS
an ibodies (muNS, g een) and mouse an i-p53 monoclonal an ibody (p53, ed). The cons uc s con aining GFP we e isualized wi hou an ibodies
(g een). Nuclei we e coun e s ained blue wi h DAPI.
doi:10.1371/jou nal.pone.0013785.g002
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In e es ingly, in mos cells co-exp essing muNS-Mi and p53-IC
we obse ed he p esence o bo h cy oplasmic and nuclea
inclusions (Figu e 2B, ow 4). Howe e , only cy oplasmic
inclusions we e de ec ed in cells co-exp essing GFP-muNS-Mi
and p53-IC (Figu e 2B, ow 5). We hypo hesized ha , due o i s
small size, a ac ion o he muNS-Mi p o ein is ansloca ed o he
nucleus in associa ion wi h p53-IC, and ha nuclea muNS-Mi is
s ill able o o m inclusions and o ec ui IC- agged p53.
Associa ion o p53 wi h endogenous SV40 T-An igen
(TAg) in he cy oplasm o COS-7 cells
We nex decided o s udy whe he p53-IC is able o ec ui TAg
o muNS-de i ed inclusions. Fi s , we used COS-7 cells, because
hese cells cons i u i ely exp ess endogenous TAg, which la gely
localizes o he nucleus [16,17]. As expec ed, and in ag eemen wi h
he esul s ob ained in CEF, in co- ans ec ed COS-7 cells
exp essing p53-IC and any o he muNS- ela ed inclusion-
compe en uni s (muNS, GFP-muNS, muNS-Mi and GFP-muNS-
Mi), agged p53 was localized almos exclusi ely in associa ion wi h
inclusions (Figu e 3A and da a no shown). In cells exp essing
muNS-Mi and p53-IC we again obse ed he o ma ion o nuclea
inclusions, as in CEF cells (Figu e 2B, ow 4 and da a no shown). In
con as , un agged p53 localized o he nucleus, showing no co-
localiza ion wi h inclusions (no shown). These esul s con i m ha
ou inclusion- a ge ing sys em wo ks well in di e en cells ypes
(CEF, COS-7 and S 9; [25].
We nex analyzed he in acellula dis ibu ion o TAg in COS-7
cells co-exp essing p53 and an inclusion- o ming p o ein. TAg
localized o he nucleus, and did no co-localize a all wi h
inclusions, when co-exp essed wi h un agged p53 (Figu e 3B, uppe
ow and da a no shown). These esul s indica ed ha TAg does no
independen ly associa e wi h inclusions and ha he TAg-speci ic
an ibody used o immunos aining does no c oss- eac wi h he
muNS and GFP moie ies o he inclusion- o ming p o ein.
Howe e , when we used IC- agged p53, a signi ican po ion o
TAg was ound in associa ion wi h cy oplasmic inclusions (see
Figu e 3B, middle ow). The same esul s we e ob ained wi h all
ou desc ibed inclusion- o ming p o eins (Figu e 3B, bo om ow
and da a no shown). We again obse ed some nuclea inclusions, in
addi ion o cy oplasmic ones, when using muNS-Mi as inclusion-
o ming uni (Figu e 3B, bo om ow). The nuclea inclusions
obse ed in COS-7 cells we e mo e nume ous han he ones
p e iously obse ed in CEF, p obably because he endogenous TAg
o COS-7 cells helps p53 in owing muNS-Mi o he nucleus.
Associa ion o p53 wi h ansien ly exp essed TAg in he
cy oplasm o CEF cells
So a , we ha e demons a ed he u ili y o ou sys em o
de ec ing he in e ac ion be ween plasmid-exp essed p53 and
endogenous TAg in double- ans ec ed COS-7 cells. Nex , we
wan ed o demons a e he alidi y o ou me hod o de ec ing he
in e ac ion be ween wo plasmid-exp essed p o eins in iple-
ans ec ed cells. Fi s , o check ha he TAg speci ic an ibody
does no c oss- eac wi h p53, we exp essed p53-IC and examined
by immuno luo escence whe he i was ecognized by an ibodies
agains muNS and TAg. As expec ed, p53-IC could no be
de ec ed using he TAg-speci ic an ibody, bu was isualized in he
nucleus o he ans ec ed cells by eac ion wi h muNS speci ic
an ise um (Figu e 4A). Nex , we ocused in localizing he
in acellula dis ibu ion o TAg in iple- ans ec ed CEF. TAg
was localized exclusi ely in he nucleus o CEF cells co-exp essing
un agged p53 and an inclusion- o ming p o ein (Figu e 4B, uppe
Figu e 3. In acellula dis ibu ion o endogenous TAg in COS-7 cells exp essing inclusion-compe en p o eins and p53/p53-IC. A.
In acellula localiza ion o muNS (g een) and p53-IC ( ed) in co ans ec ed COS-7 cells. Nuclei we e coun e s ained blue wi h DAPI. B. COS-7 cells
we e co- ans ec ed wi h plasmids exp essing he p o eins indica ed a he le o he igu e. The cells we e immunos ained wi h abbi an i-muNS
an ibodies (muNS, g een) and mouse an i-TAg (TAg, ed). Nuclei we e coun e s ained blue wi h DAPI.
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ow, and da a no shown). Howe e , when co-exp essed wi h p53-
IC, TAg localized mos ly in cy oplasmic muNS inclusions,
showing ha ou inclusion- a ge ing sys em wo ks pe ec ly in
di e en cell ypes and wi h di e en exp ession sys ems (Figu e 4B,
middle ow). When muNS-Mi was used as inclusion- o ming
p o ein, again some nuclea inclusions we e e iden in he
ans ec ed cells, and hey success ully a ac ed bo h agged p53
and TAg (Figu e 4B, bo om ow).
Enginee ing muNS- ela ed p o eins o o m p o ein
inclusions in he nucleus
Ou inding ha nuclea inclusions a e o med in cells co-
exp essing p53-IC and muNS-Mi p omp ed us o in es iga e he
possibili y o adap ing ou sys em o de ec ing p o ein-p o ein
in e ac ions wi hin he cell nucleus, which would make ou sys em
mo e sui able o de ec ing associa ions be ween nuclea p o eins.
Fo his, we ied o gene a e nuclea -inclusion- o ming p o eins by
using nuclea localiza ion sequences (NLS) o muNS- ela ed
p o eins in o de o see i hey could each he nucleus and gene a e
inclusions he e. We chose he TAg nuclea localiza ion sequence
PKKKKKV [17] as a sho , p e iously cha ac e ized NLS o be
added o he N e minus o he di e en inclusion- o ming p o eins.
Addi ionally, we also conside ed using a bigge domain o
p o iding a unc ional NLS ins ead o he sho sequences ha
could be no p ope ly olded in he chime ic p o eins due o hei
small size. Thus, we used he VP16 He pes i us domain wi h a
used TAg NLS, ha is pa o he Mammalian Ma chmake Two-
Hyb id Assay Ki (Clon ech, Sain Ge main en Laye, F ance), and
used i o he N- e minus o he ou di e en muNS a ian s.
Fluo escence mic oscopy analysis o he in acellula dis ibu ion o
he used cons uc s e ealed ha muNS, GFP-muNS and GFP-
muNS-Mi p oduced nuclea inclusions when used o ei he TAg
NLS o VP16 (Figu e 5A, columns 1, 3 and 4). S ikingly, he
chime as con aining used muNS-Mi did no gene a e inclusions
(Figu e 5A, column 2), bu we e dis ibu ed di usely ei he
h oughou he cy oplasm (when used o TAg NLS), o h oughou
he nucleus (when used o VP16).
To ule ou he possibili y ha any o he nuclea -inclusion-
o ming chime as we e subs an ially mis olded and/o a ge ed o
deg ada ion by he ubiqui in-p o easome sys em, he cells
exp essing he nuclea inclusion-compe en cons uc s we e
immunos ained wi h MAB FK2, which ecognizes conjuga ed
ubiqui in [18]. None o he cons uc s co-localized wi h conjuga ed
ubiqui in, sugges ing ha hey we e o ming inclusions by speci ic
in e ac ions be ween monome s and no by simple agg ega ion
(Figu e 5B, and da a no shown).
IC- agged p o eins a e a ge ed o nuclea inclusions
To demons a e ha muNS-de i ed nuclea inclusions a e able
o ec ui IC- agged p o eins, we cons uc ed plasmids ha exp ess
IC used bo h o he C e minus o he nuclea p o ein GAL4
DNA binding domain, and o a p o ein ha dis ibu es uni o mly
h oughou he whole cell (GFP). Fluo escence mic oscope analysis
o single- ans ec ed CEF cells e ealed ha IC- agging does no
change he no mal in acellula dis ibu ion o any o he wo
p o eins; GAL4-IC was e enly dis ibu ed h oughou he nucleus,
whe eas GFP-IC emained dis ibu ed h oughou he whole cell
(Figu e 6A). The dis ibu ion o he wo un agged p o eins did no
change upon co-exp ession wi h muNS-de i ed nuclea -inclusion-
o ming p o eins (Figu e 6B, ows 1 and 3), indica ing ha nei he
o hese p o eins in e ac wi h inclusions and ha he an ibody
agains GAL4 does no c oss- eac wi h he inclusion- o ming
p o eins. The same was ue o p53-IC ha was used on he
expe imen s shown in Figu es 3 and 4 (Figu e 6B, ow 5). In
Figu e 4. In acellula dis ibu ion o plasmid-exp essed TAg in CEF cells co-exp esing inclusion-compe en p o eins and p53/p53-
IC. A. CEF cells we e ans ec ed wi h he plasmid exp essing p53-IC and hey we e subsequen ly immunos ained wi h an ibodies agains muNS
(g een) and TAg. Nuclei we e coun e s ained blue wi h DAPI. B. CEF cells we e co- ans ec ed wi h plasmids exp essing he p o eins indica ed a he
le o he igu e. The cells we e immunos ained wi h an ibodies agains muNS (g een) and an i-TAg ( ed). Nuclei we e coun e s ained blue wi h DAPI.
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con as , he h ee p o eins we e ound o collec in o muNS-
de i ed nuclea inclusions when agged wi h IC (Figu e 6B, ows
2, 4 and 6), showing ha ou IC- agging and inclusion associa ion
me hod is also able o a ge nuclea and nonnuclea p o eins o
nuclea inclusions. The same esul s we e ep oduced wi h all he
nuclea -inclusion- o ming p o eins shown in Figu e 5 (no shown),
including hose ha p oduce luo escen inclusions. These esul s
u he showed ha he inco po a ion o IC- agged p o eins in o
muNS-de i ed nuclea inclusions does no dis up inclusion
o ma ion. Finally, inclusion-associa ed GFP is p ope ly olded
since i con inues o emi luo escence.
Associa ion o p53 wi h SV40 TAg wi hin he nucleus
Once demons a ed ha nuclea muNS-de i ed inclusions a e
able o ec ui IC- agged p o eins, we assessed he alidi y o ou
sys em o de ec p o ein-p o ein in e ac ions wi hin he nucleus.
Fo his, we examined again he in e ac ion o p53 wi h SV40 TAg
in ei he COS-7 cells (endogenous TAg) o CEF cells (plasmid-
d i en exp ession o TAg). Fi s , we e i ied ha p53-IC is
a ge ed o nuclea inclusions in COS-7 cells as we ha e jus
shown o CEF (no shown). We nex in es iga ed he in acellula
dis ibu ion o endogenous TAg in each o he wo co- ans ec ed
cells. TAg was di usely dis ibu ed h oughou he nucleus o
COS-7 cells co-exp essing p53 and VP16-muNS (Figu e 7A,
uppe ow), indica ing ha TAg does no associa e wi h nuclea
inclusions and ha he TAg-speci ic an ibody does no c oss- eac
wi h muNS-de i ed inclusions. In con as , TAg e-localized o
nuclea inclusions in cells exp essing p53-IC (Figu e 7A, bo om
ow), demons a ing ha ou me hod is able o de ec he
in e ac ion o plasmid-exp essed p53 wi h endogenous TAg in
he nucleus o COS-7 cells.
The same esul s we e ob ained in iple- ans ec ed CEF cells,
when TAg was p o ided by plasmid exp ession. As in COS-7 cells,
TAg does no associa e wi h muNS-de i ed nuclea inclusions in
CEF (Figu e 7B, uppe ow). Only in cells exp essing p53-IC, bu
no in hose exp essing un agged p53, plasmid-exp essed TAg
localized o nuclea inclusions (Figu e 7B, bo om ow), indica ing
ha ou me hod is also able o de ec he in e ac ion wi hin he
nucleus o wo ansien ly exp essed p o eins.
The in e ac ion p53-TAg in he wo cell ypes was success ully
de ec ed when using each o he di e en nuclea -inclusion-
compe en muNS chime as shown in Figu e 5 (da a no shown),
indica ing ha se e al di e en inclusion- o ming p o eins,
including luo escen p o eins, can be used in ou sys em.
Simul aneous de ec ion o p53 and TAg in muNS-de i ed
inclusions
To conclusi ely demons a e he p esence o bo h in e ac ing
p o eins inside muNS-de i ed inclusions, we pe o med double
labeling expe imen s using speci ic an ibodies agains p53 and TAg.
Figu e 5. Subcellula localiza ion o muNS-de i ed p o eins con aining nuclea localiza ion sequences. A. CEF cells we e ans ec ed
wi h plasmids exp essing he chime as o med by using he p o eins indica ed on op o he igu e wi h he ags indica ed on he le . The cells we e
immunos ained o muNS (g een), excep hose con aining GFP ha we e isualized wi hou an ibodies (g een). Nuclei we e coun e s ained blue
wi h DAPI. B. CEF cells we e ans ec ed wi h plasmids exp essing he p o eins indica ed a he le o he igu e and immunos ained wi h an ibodies
agains muNS (g een) and conjuga ed ubiqui in ( ed). The cons uc con aining GFP was isualized wi hou an ibodies (g een). Nuclei we e
coun e s ained blue wi h DAPI.
doi:10.1371/jou nal.pone.0013785.g005
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Once e i ied ha he e is no c oss- eac ion be ween bo h an ibodies
(Figu e 4A and Figu e 8, lane 1), we we e able o simul aneously de ec
IC- agged p53 and TAg inside cy oplasmic and nuclea muNS-
de i ed inclusions in COS-7 (Figu e 8, lanes 2 and 4) and CEF cells
(Figu e 8, lanes 3 and 5). Simila esul s we e ob ained wi h all o he
inclusion- o ming cons uc s desc ibed in his s udy (no shown).
Discussion
In a p e ious s udy we ha e cha ac e ized a simple and
inexpensi e me hod o pu i ying p o eins exp essed in baculo-
i us-in ec ed cells. The me hod is based on: i) he abili y o
di e en a ian eo i us muNS e sions o o m cy oplasmic
inclusions ha can be easily pu i ied; and ii) he abili y o hese
inclusions o ec ui p o eins agged wi h muNS domains, wi h IC
being he mos e ec i e domain [25]. In his s udy we ha e
ex ended he applicabili y o his me hod o de ec ing p o ein-
p o ein in e ac ions wi hin euka yo ic cells o a ian and mamma-
lian o igin.
Ou me hod is e y simple and inexpensi e, since only equi es
ansien exp ession o he es p o eins and a egula luo escence
mic oscope. I is also highly e sa ile, because i wo ks success ully in
di e en cell lines (a ian and mammalian), can be used o de ec
p o ein-p o ein in e ac ions in he nucleus o he cy oplasm, and can
use se e al di e en inclusion- o ming p o eins, some o which a e
au o- luo escen he eby acili a ing inclusion de ec ion. The e sa ili y
o ou me hod also pe mi s de ec ion o in e ac ions be ween nuclea
and non-nuclea p o eins in ei he he nucleus o he cy oplasm, as well
as he in e ac ion be ween wo plasmid-exp essed p o eins o be ween
a plasmid-exp essed p o ein and an endogenous p o ein. I s main
limi a ion esides on he a ailabili y o speci ic an ibodies, bu his can
also be ci cum en ed wi h he use o agged p oblem p o eins.
Figu e 6. In acellula dis ibu ion o GFP/GFP-IC, GAL4/GAL4-IC and p53/p53-IC in cells exp essing nuclea inclusion-compe en
p o eins. A. CEF cells we e ans ec ed wi h he plasmids exp essing he p o eins indica ed on op o he igu e and immunos ained o GAL4 ( ed)
excep hose con aining GFP ha we e isualized wi hou an ibodies (g een). Nuclei we e s ained wi h DAPI (blue). B. CEF cells we e co- ans ec ed
wi h plasmids exp essing he p o eins indica ed on he le o he igu e and nuclea inclusion-compe en , muNS-de i ed p o eins. The a ge ed
p o eins we e isualized in ed wi h he excep ion o GFP (g een) and he inclusions in g een, wi h he excep ion o hose in cells co-exp essing GFP,
whe e hey we e s ained in ed. Nuclei we e coun e s ained wi h DAPI (blue).
doi:10.1371/jou nal.pone.0013785.g006
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Figu e 7. In acellula dis ibu ion o TAg in cells exp esing VP16-muNS and p53 o p53-IC. Cells we e ans ec ed wi h he plasmids
exp essing he p o eins indica ed on he le o he igu e and immunos ained wi h an i-muNS an ibodies (g een) and an i-TAg ( ed). Nuclei we e
s ained wi h DAPI (blue). A. COS-7 cells. B. CEF cells.
doi:10.1371/jou nal.pone.0013785.g007
Figu e 8. In acellula dis ibu ion o TAg and p53-IC in he p esence o cy oplasmic and nuclea inclusions. Cells we e ans ec ed wi h
he plasmids exp essing he p o eins indica ed on he le o he igu e and immunos ained wi h an i-p53 (g een) and an i-TAg ( ed) an ibodies.
Nuclei we e s ained wi h DAPI (blue). Cell ypes a e indica ed in b acke s on he igh o he igu e.
doi:10.1371/jou nal.pone.0013785.g008
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Ou inding ha an endogenous nuclea p o ein like COS7
TAg can be eloca ed o p53-IC-con aining cy oplasmic inclusions
sugges s ha ou me hod migh also be used o emo ing a
p o ein om i s no mal nuclea localiza ion, which would allow
s udying he e ec ha i s loss o nuclea unc ion has on cellula
p ocesses. On he o he hand, o acili a e he de ec ion o he
in e ac ion be ween nuclea p o eins, we ha e adap ed ou
me hod o de ec ing in e ac ions inside he nucleus o euka yo ic
cells by adding nuclea a ge ing sequences o he muNS-de i ed
inclusion- o ming p o eins. Nuclea inclusions we e e icien o
bo h cap u ing IC- agged p o eins and de ec ing he in e ac ion
be ween p53-IC and TAg wi hin he nucleus, hei na u al
en i onmen .
The e a e se e al published me hods aimed a s udying
p o ein-p o ein in e ac ions inside li ing cells o highe euka y-
o es [19,20], being he mammalian wo-hyb id sys em he mos
widely used. Howe e , we belie e ha he wo-hyb id sys em has
se e aldisad an agesincompa isonwi hou me hod.The i s
limi a ion o he wo-hyb id sys em is ha i can only be used o
de ec in e ac ions be ween wo plasmid-exp essed p o eins, bu
no he associa ion o a plasmid-exp essed p o ein wi h an
endogenous p o ein. The second limi a ion is ha he wo-hyb id
sys em can only de ec in e ac ions be ween wo agged p o eins,
whe eas ou me hod only equi es he exp ession o one agged
p o ein. Fu he mo e, he wo-hyb id sys em can only de ec
p o ein-p o ein in e ac ions wi hin he nucleus, and his may no
wo k ou well wi h cy oplasmic p o eins. Finally, in he wo-
hyb id sys em he associa ion o a p o ein A-con aining
ac i a ing domain wi h a p o ein B-con aining DNA-binding
domain has o econs i u e a unc ional ansc ip ion ac o o
success ully de ec ing he A-B in e ac ion. This s a egy has an
ob ious a chi ec u al disad an age, since a posi i e esul
depends on he geome y o he associa ion be ween wo agged
p o eins, because A-B associa ion could occlude/inac i a e he
ac i a ing and/o he DNA-binding domains. Howe e , in ou
me hod he geome y o he A-B associa ion is i ele an o i s
de ec ion.
Recen ly, a mammalian eo i us muNS-de i ed pla o m o
isualizing p o ein associa ions inside cells was de eloped and
e med PIP (p o ein in e ac ion pla o m; [21]). Using he
mammalian eo i us muNS ins ead o he a ian was no he only
di e ence wi h ou me hod, a he he whole concep is di e en .
PIP does no a ge agged p o eins o an inclusion o med by a
di e en polypep ide, bu use oge he he bai p o ein and he
minimal inclusion- o ming po ion o muNS, and wai o he ish
polypep ide o be ec ui ed o he bai -con aining inclusion. This
pla o m was subsequen ly adap ed o he isualiza ion o
in e ac ing p o eins in yeas [22]. The au ho s claim ha PIP is
be e and mo e e icien in yeas han he wo-hyb id sys em,
because i is able o de ec mo e chape one-e ec o in e ac ions
[22]. This is p obably due o he lack o geome ic es ic ions o
PIP ela i e o he wo-hyb id sys em.
Howe e , he mammalian eo i us muNS-de i ed PIP me hod
p esen s some disad an ages ha he au ho s acknowledge, and
ha a e sol ed o a big ex en wi h ou inclusion- a ge ing
p o ocol. Fo ins ance, PIP elies on he inclusion- o ming abili y
o a bai -muNS usion, and ha usion could al e he olding and/
o ac i i y o he used pa ne s. The used bai may abla e he
inclusion- o ming abili y o he eo i al p o ein, so ha he bai -
muNS usion does no longe o m inclusions. In suppo o his, we
ha e ound ha mino al e a ions in he sequence o some
inclusion-compe en muNS e sions leads o ei he ab oga ion o
inclusions o he gene a ion o amo phous agg ega es ins ead o
egula inclusions (Figu e 5; [14]). On he o he hand, he used
mul ime ic eo i al p o ein, which in e wines o c ea e a big
p o ein inclusion, migh al e he p ope olding o he bai
pa ne . Ou inclusion a ge ing p o ocol is less likely o ha e his
p oblem. Fi s o all, he small size o he IC ag o which he bai
p o ein is used should no signi ican ly modi y he na i e
con o ma ion o he bai . Second, he bai p o ein does no
in e ac di ec ly wi h inclusions, bu h ough he IC ag, and we
ha e shown ha such associa ion does no dis up he capaci y o
ou muNS-de i ed p o eins o o m inclusions. Fu he mo e, ou
me hod should be e sa ile enough o sol e olding p oblems, since
i can use di e en muNS-de i ed inclusion- o ming uni s. An
addi ional ad an age o ou me hod is ha i has been adap ed o
isualize p o ein associa ions inside he nucleus, which should be
e y use ul o de ec ing in e ac ions be ween nuclea p o eins in
hei no mal en i onmen , inc easing he p obabili ies o success.
The gene a ion o nuclea compa men s whe e pa icula
p o eins could be ec ui ed o unc ional s udies is o he possible
ad an age o ou sys em.
We a e awa e ha ou me hod p esen s he limi a ions o any
o he agging me hod, since ag usion could in e e e wi h he
p ope olding o he bai p o ein. Howe e , he ex emely posi i e
and eliable esul s ob ained wi h he IC ag in he ec ui ing
expe imen s desc ibed he e and in a p e ious s udy [25], sugges
ha IC p obably has a e y compac and independen olding.
Addi ionally, i s small size makes i less likely o in e e e wi h he
bai p o ein olding. In he p esen and an addi ional s udy [25] we
show examples whe e he IC- agging and inclusion- a ge ing
sys em success ully wo ks wi h many di e en p o eins, wi h
di e en in acellula localiza ions, exp essed in cells o di e en
o igins and wi h di e en exp ession sys ems. In all shown
examples, he agged p o ein emained ac i e and/o associa ed
wi h i s known in e ac ing pa ne .
Ma e ials and Me hods
Cells and an ibodies
P ima y cul u es o chicken emb yo ib oblas s (CEF) we e
p epa ed om 9- o 10-day-old chicken emb yos [23] and g own
in monolaye s in medium 199 (In i ogen, Ba celona, Spain)
supplemen ed wi h 10% (w/ ) yp ose-phospha e b o h and 5%
( / ) cal se um. COS-7 cells [16] we e g own in monolaye s in
medium D-MEM supplemen ed wi h 10% e al bo ine se um.
Rabbi polyclonal an ise um agains a ian eo i us S1133 muNS
p o ein was aised in ou labo a o y [12]. Monoclonal an ibody
PAB40 speci ic o p53 was ob ained om Sigma-Ald ich (Mad id,
Spain). Monoclonal an ibody PAb101 speci ic o SV40 TAg was
ob ained om BD biosciences (Mad id, Spain). Monoclonal
an ibody speci ic o GAL4 DNA binding domain was ob ained
om Clon ech (Sain Ge main en Laye, F ancia). Polyclonal
an ibody agains p53 was ob ained om San a C uz Bio echnol-
ogy (San a C uz, Cali o nia). The mouse monoclonal an ibody
(Mab) FK2 agains conjuga ed ubiqui in [18] was om Biomol
In e na ional L.P. (Exe e , Uni ed Kingdom). The ollowing
seconda y an ibodies we e used as app op ia e o di e en
expe imen s: Alexa 594 and Alexa 488 conjuga ed an ibodies
agains mouse and abbi IgG, espec i ely (Sigma-Ald ich,
Mad id, Spain).
T ans ec ions and IF mic oscopy
T ans ec ions o cell monolaye s we e done wi h he Lipo ec a-
mine Plus eagen (In i ogen, Ba celona, Spain), acco ding o he
manu ac u e ’s ins uc ions. T ans ec ed cells we e incuba ed a
37uC o 18 h, unless o he wise s a ed.
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