A F ac al Na u e o Polyme ized Laminin
Camila Hochman-Mendez
1,2
, Ma co Can ini
3
, Da id Mo a al
4
, Manuel Salme on-Sanchez
3
,
Ta iana Coelho-Sampaio
2
*
1Ins i u e o Biomedical Sciences, Fede al Uni e si y o Rio de Janei o, Rio de Janei o, B azil, 2Ins i u e o Biophysics Ca los Chagas Filho, Fede al Uni e si y o Rio de
Janei o, Rio de Janei o, B azil, 3Di ision o Biomedical Enginee ing, School o Enginee ing, Uni e si y o Glasgow, Glasgow, Uni ed Kingdom, 4Cen e o Bioma e ials and
Tissue Enginee ing, Uni e si a Poli e
`cnica de Vale
`ncia, Vale
`ncia, Spain
Abs ac
Polylaminin (polyLM) is a non-co alen acid-induced nano- and mic o-s uc u ed polyme o he p o ein laminin displaying
dis inguished biological p ope ies. Polylaminin s imula es neu i ogenesis beyond he le els achie ed by o dina y laminin
and has been shown o p omo e axonal egene a ion in animal models o spinal co d inju y. He e we used con ocal
luo escence mic oscopy (CFM), scanning elec on mic oscopy (SEM) and a omic o ce mic oscopy (AFM) o cha ac e ize i s
h ee-dimensional s uc u e. Rende iza ion o con ocal op ical slices o immunos ained polyLM e ealed he aspec o a
loose loccula ed meshwo k, which was homogeneously s ained by he an ibody. On he o he hand, an o dina y ma ix
ob ained upon adso p ion o laminin in neu al pH (LM) was cons i u ed o bulky p o ein agg ega es whose in e io was no
accessible o he same an i-laminin an ibody. SEM and AFM analyses e ealed ha he seed uni o polyLM was a la
polygon o med in solu ion whe eas he seed s uc u e o LM was highly he e ogeneous, in e cala ing od-like, sphe ical
and hin sp ead lamella deposi s. As polyLM was isualized a p og essi ely inc easing magni ica ions, we obse ed ha
he mo phology o he polyme was alike independen ly o he magni ica ion used o he obse a ion. A sea ch o he
Hausdo dimension in images o he wo ma ices showed ha polyLM, bu no LM, p esen ed ac al dimensions o 1.55,
1.62 and 1.70 a e 1, 8 and 12 hou s o adso p ion, espec i ely. Da a in he p esen wo k sugges ha he in insic ac al
na u e o polyme ized laminin can be he s uc u al basis o he ac al-like o ganiza ion o basemen memb anes in he
neu ogenic niches o he cen al ne ous sys em.
Ci a ion: Hochman-Mendez C, Can ini M, Mo a al D, Salme on-Sanchez M, Coelho-Sampaio T (2014) A F ac al Na u e o Polyme ized Laminin. PLoS ONE 9(10):
e109388. doi:10.1371/jou nal.pone.0109388
Edi o : Heida -Ali Tajmi -Riahi, Uni e si y o Quebec a T ois-Ri ie es, Canada
Recei ed June 9, 2014; Accep ed Sep embe 2, 2014; Published Oc obe 8, 2014
Copy igh : ß2014 Hochman-Mendez 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.
Da a A ailabili y: The au ho s con i m ha all da a unde lying he indings a e ully a ailable wi hou es ic ion. All ele an da a a e wi hin he pape and i s
Suppo ing In o ma ion iles.
Funding: This wo k was suppo ed by a g an om he B azilian Na ional Resea ch Council (CNPq; 476772/2008-7) o TCS. MSS acknowledges suppo om he
Eu opean Resea ch Council h ough ERC - 306990. 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 au ho s ha e decla ed ha no compe ing in e es s exis .
* Email: [email p o ec ed]
In oduc ion
Laminin is he majo molecula componen o he basemen
memb ane, a specialized ype o ex acellula ma ix cha ac e ized
by a la shee -like geome y. Laminin egula es a a ie y o
biological phenomena, including he p o ision o bounda ies
be ween neighbo ing issues, he es ablishmen o molecula il e s
and he modula ion o cell beha io [1-3]. The qua e na y
s uc u e o laminin is gi en by h ee di e en polypep ide chains,
which associa e o o m a c oss-shaped he e o ime , exhibi ing
one long and h ee sho e a ms. In he h ee-dimensional space
laminin has he shape o a h ee-lea ed clo e , in which he lea es
co espond o he h ee sho a ms while he s em co esponds o
he long a m. This 3-D s uc u e is pa icula ly adequa e o a o
he assembly o a shee -like polyme , whe e he h ee sho a ms
simul aneously in e ac wi h each o he wi hin a single spa ial
plane, while he long a m is le a ailable o in e ac wi h he
su ace o con iguous cells [4].
As a consequence o i s s uc u al p ope ies, laminin can
spon aneously sel -polyme ize in a es ube, equi ing ei he a
minimal p o ein concen a ion [5] o a dec ease in he solu ion pH
[6,7]. Polyme s o med upon pH acidi ica ion, designa ed as
polylaminin (polyLM
1
), p esen speci ic signaling p ope ies and
ha e been shown o s imula e he ou g ow h o neu i es wi h a
leas wice he e iciency o o dina y laminin (LM), namely a
ma ix ob ained by adso bing he p o ein dilu ed in neu al pH
on o a glass co e slip [8]. PolyLM was also shown o e e se he
loss o mig a o y and neu i ogenic po en ials o co ical neu ons
and o p omo e he su i al and he p oli e a ion o axo omized
e inal ganglion cells, bo h isola ed om newbo n oden s [9].
Finally, i was demons a ed ha polyLM, bu no he laminin
p o ein dilu ed in neu al bu e , p omo ed axonal egene a ion
and unc ional eco e y a e spinal co d inju y in a s [10].
The mo phology o polyLM has p e iously been s udied bo h a
he mic o and a he nanome e scales. Using nega i e s aining
ollowed by ansmission elec on mic oscopy i was possible o
cha ac e ize i as a egula polygonal ne wo k displaying he same
ea u es o he na u al laminin ne wo ks assembled by li ing cells
[11,12]. In such polyme s he uni polygon was a hexagon o
app oxima ely 30 nm o side, which well co esponded o he size
o each sho a m in he laminin molecule. Cu iously, a polygonal
a ay o compa able ea u es was obse ed a a h ee o de s o
magni ude la ge scale when immunolabeled polyLM was
analyzed by luo escence mic oscopy [7,8,13].
PLOS ONE | www.plosone.o g 1 Oc obe 2014 | Volume 9 | Issue 10 | e109388
The ac ha polyLM obse ed a di e en magni ica ions
showed simila s uc u es sugges ed ha he polyme could be a
ac al s uc u e. Ne e heless, he lack o images ob ained a
in e media y magni ica ions, as well as he insu icien esolu ion
o p e ious epi luo escence pho omic og aphs, p e en ed he
sea ch o a ac al dimension o polyLM. In he p esen wo k
we used con ocal luo escence mic oscopy (CFM), scanning
elec on mic oscopy (SEM) and a omic o ce mic oscopy (AFM)
o ob ain a de ailed s uc u al cha ac e iza ion o polyLM ha
would pe mi assessmen o i s pu a i e ac al na u e. We showed
ha polyLM p esen ed a ac al dimension, which inc eased i s
complexi y upon accumula ion o he polyme on a la subs a e.
These indings may ha e impo an implica ions as hey can
p o ide an in insic molecula basis o he ac al-like o ganiza-
ion o he basemen memb anes p esen a he neu ogenic niches
in he adul cen al ne ous sys em.
Expe imen al P ocedu es
P epa a ion o laminin ma ices
PolyLM was p oduced by dilu ing EHS laminin (laminin 111;
In i ogen) o 50 mg/mL wi h 20 mM sodium ace a e (pH 4),
con aining 1 mM CaCl
2
. The laminin p o ein has p e iously been
shown o polyme ize in solu ion wi hin a ew minu es a e dilu ion
in acidic bu e , independen ly o i s concen a ion [6]. The
polyme s p oduced in solu ion (polyLM) we e adso bed o glass
co e slips o p oduce he ma ices used he e o mic oscopic
analyses. LM was p oduced by dilu ing EHS laminin o 50 mg/mL
wi h 20 mM T is-HCl (pH 7) con aining 1 mM CaCl
2
. This
concen a ion was below he c i ical p o ein concen a ion
necessa y o igge laminin polyme iza ion in solu ion a neu al
pH [5], so ha he LM ma ix was o med as he p o ein decan ed
and aised i s concen a ion a he glass su ace. In o de o a oid
unwan ed polyme iza ion in he highly concen a ed s ock solu ion
o EHS laminin (0.5–1.5 mg/mL), wo king aliquo s (2–10 mL)
we e s o ed ozen and indi idually hawed in ice immedia ely
be o e dilu ion. Unless o he wise indica ed, incuba ions wi h glass
co e slips we e ca ied ou a 37uC o 12 hou s, which is known
o be su icien o wa an ha a leas 60% o he p o ein would
decan and adso b ega dless o he solu ion pH [8].
Immunos aining and con ocal mic oscopy
Laminin ma ices adso bed on glass co e slips we e ixed wi h
pa a o madehyde 4% o 20 min and p epa ed o indi ec
immuno luo escence analysis. Co e slips we e washed 3 imes
o 5 min in PBS and incuba ed wi h bo ine se um albumin 5% in
PBS (PBS-BSA 5%) o 30 min. The p ima y an ibody was a
polyclonal abbi an i-laminin an ibody (1:30, Sigma-Ald ich,
no. L9393). A e o e nigh incuba ion a 4uC, co e slips we e
washed 3 imes o 5 min in PBS and incuba ed wi h an Alexa
Fluo 488 an i- abbi seconda y an ibody (1:300; Li e Technology,
no. A-11001) o 1 hou a oom empe a u e. They ecei ed
h ee 5-min washes wi h PBS and one wi h dis illed wa e be o e
being moun ed in n-p opyl galla e in 80% glyce ol (Sigma-
Ald ich). Con ocal images we e ob ained in a Leica TCS-SP5
con ocal lase scanning mic oscope using a HCX PL APO lambda
blue 63X objec i e o oil imme sion (1.4 o nume ical ape u e).
Images co espond o ende ized s acks o 74 op ical slices
ob ained wi h a zoom o 3.2 a each 21.4 mm ( o al wid h o
1530.8 mm).
Scanning elec on mic oscopy
Laminin ma ices a ached o co e slips we e ixed in
Ka nowsky eagen (4% PA and 0.5% glu a aldehyde in 0.1 M
cacodyla e bu e , pH 7.2) o 2 hou s, washed h ee imes wi h
sodium cacodyla e bu e 0.1 M, pH 7.2, dehyd a ed h ough
inc easing concen a ions o e hanol and d ied in E300 (Pola on,
Quo um Technologies L d, Laugh on, Uni ed Kingdom) c i ical
poin . The samples we e hen coa ed wi h a hin laye o gold
spu e (Leica EM MED020) and iewed unde a scanning
elec on mic oscope Jeol JSM6300.
T ansmission elec on mic oscopy
T ansmission elec on mic oscopy a e nega i e s aining was
ca ied ou as p e iously desc ibed [11]. B ie ly, 5 ml o laminin in
acidic bu e (polyLM) was deposi ed on a Fo m a -coa ed coppe
g id and a 5 ml d op o 2% u anyl ace a e was added o e i .
Samples we e isualized in a Zeiss EM 900 ansmission elec on
mic oscope ope a ed a 80 kV.
A omic o ce mic oscopy
Laminin ma ices we e ixed in 4% pa a o maldehyde o 20
minu es and d ied in c i ical poin d ye E300 (Pola on, Quo um
Technologies L d, Laugh on, Uni ed Kingdom). AFM analyses
we e pe o med using a Mul imode AFM equipped wi h a
NanoScope IIIa con olle (B uke ) ope a ing in apping mode
in ai ; he Nanoscope 5.30 2 so wa e e sion was used o image
p ocessing and analysis. Si-can ile e s MPP-21120 om B uke
we e used, wi h o ce cons an o 3 N.m
21
and esonance
equency o 75 kHz. The phase signal was se o ze o a a
equency 5–10% lowe han he esonance one. D i e ampli ude
was 200 mV and he ampli ude se -poin (A
sp
) was 1.4 V. The
a io be ween he ampli ude se -poin and he ee ampli ude (A
sp
/
A
0
) was kep equal o 0.7.
Quan i ica ion o laminin adso p ion
The amoun o adso bed laminin was measu ed using a Mic o
BCA P o ein Assay Ki (23235#, The mo Scien i ic Pie ce).
Following he s anda d p o ocol, he wo king eagen (WR) was
p epa ed om 25 pa s o MA (sodium ca bona e, sodium
bica bona e and sodium a a e in 0.2 N NaOH), 24 pa s o
MB (4% BCA in wa e ) and 1 pa o MC (4% cup ic sul a e,
pen ahyd a e in wa e ). As s anda ds, nine bo ine se um albumin
(BSA) solu ions wi h concen a ions anging om 0.0 o 200 mg/
mL we e p epa ed by dissol ing BSA in he bu e s used o
laminin adso p ion (acidic bu e and neu al bu e ). The amoun
o adso bed p o ein was calcula ed by measu ing he amoun o
p o ein emaining in he supe na an a each ime poin . The
samples and he s anda ds we e incuba ed wi h WR 1:1 a 37uC
o wo hou s be o e cooling o oom empe a u e. Then, he
abso bance a 562 nm was measu ed wi h he pla e eade Vic o 3
(Pe kinElme , Wal ham, Massachuse s). All he abso bance alues
we e co ec ed by he a e age 562 nm abso bance eading o he
blank s anda d eplica es. Each measu emen was pe o med in
duplica e.
Calculus o he ac al o he Hausdo dimension
All image p ocessing and analysis was done using an in-house
so wa e de eloped unde MATLAB R2006a (The Ma hWo ks,
Inc., Na ick, MA). The ac al dimension was de e mined using a
box-coun ing dimension es ima e o he Hausdo dimension,
which is a desc ip o o he complexi y o geome y o a gi en se
[14]. As he de ini ion o he Hausdo dimension does no o e
any guideline o an es ima e calcula ion, he box-coun ing
dimension has been used. The box-coun ing dimension is an
es ima e o he Hausdo dimension based on co e ing he
in es iga ed se wi h a ixed g id o size [15,16]. This
A F ac al Na u e o Polyme ized Laminin
PLOS ONE | www.plosone.o g 2 Oc obe 2014 | Volume 9 | Issue 10 | e109388
box-coun ing dimension can be calcula ed using he equa ion (1):
DH~
{Dlog k ðÞ½
Dlog ð1Þ
whe e k( ) is a numbe o g id boxes ha con ain any pa o he
in es iga ed se , and whe e he p ocess is epea ed wi h di e en
alues o he g id size. D
H
is known as he Hausdo dimension,
he Minkowski-Bouligand dimension, he Kolmogo o capaci y o
dimension, o simply he box-coun ing dimension.
Be o e applying he box-coun ing algo i hm, he g ayscale
image his og am was equalized and he esul ing image was
bina ized using he O su’s me hod [17]. Finally, he box-coun ing
es ima e was calcula ed on his image. All his p ocess was
pe o med on en di e en image c ops o he same size (100
pixels6100 pixels) o he o iginal images, and all hese image c ops
we e escaled en imes o ob ain an image big enough o apply he
box-coun ing algo i hm.
Resul s
Th ee-dimensional s uc u es o polyLM and LM assessed
by con ocal luo escence and scanning elec on
mic oscopy
Ma ices o adso bed laminin ob ained a acidic (polyLM) o
neu al pH (LM) we e ini ially analyzed by econs uc ing a se ies
o 74 con ocal op ical slices ende ized o e eal hei 3-D
s uc u e. PolyLM co esponded o a sponge-like ne wo k o an
appa en ly homogeneous densi y (Fig. 1A). Labeling was equally
dis ibu ed, indica ing ha he an ibody could e enly access
p o ein epi opes wi hin he polyme . On he o he hand, LM
p esen ed a b anched mo phology, esembling ha o a ma ine
co al, which p o uded om he glass su ace (Fig. 1B). The
an ibody did no pene a e he sphe ical p o ein clumps and only
hei con ou s we e b igh ly s ained. No ewo hy was he
obse a ion ha in LM a signi ican amoun o p o ein adso bed
di ec ly o he glass co e slip. This likely co esponds o he
p o ein no inco po a ed o he agg ega es. As a compa ison, he
backg ound o he image depic ing polyLM was da k, indica ing
ha i ually all laminin p o ein engaged in o ha polyme . The 3-
D s uc u es o he wo laminin ma ices can be be e app ecia ed
in he anima ion mo ies p esen ed as Mo ies S1 and S2.
The 3-D s uc u es o polyLM and LM we e addi ionally
in es iga ed by using SEM. While polyLM was again seen as a
homogeneous mesh, LM showed a leas wo s uc u al compo-
nen s (Fig. 1C and D). Besides he sphe ical agg ega es al eady
iden i ied by CFM, i was possible o de ise he p esence o od-
like s uc u es. In addi ion, he ips o hese ods possessed lamella
e mina ions, sugges ing he occu ence a hi d s uc u al compo-
nen o he LM ma ix (a ows in Fig. 1D).
Kine ics o o ma ion o laminin ma ices
The amoun o adso bed laminin was calcula ed by measu ing
he concen a ion o p o ein emaining in solu ion a 20, 40 and
60 min and a e 4, 8 and 12 hou s o adso p ion (Fig. 2A).
Laminin decan ed mo e quickly a neu al pH o o m he LM
ma ix. Mo e han 60% o he p o ein was abso bed a e 20 min
o incuba ion and such amoun sligh ly inc eased up o 80% a
12 hou s. On he o he hand, a acidic pH he kine ic o
adso p ion was mo e linea , whe eas 45% o he p o ein was
adso bed a 20 min and such p opo ion inc eased o 90% a
12 hou s. The mo phologies o he wo ma ices we e analyzed a
1, 8 and 12 hou s by SEM (Fig. 2B–G). Al eady a 1 hou polyLM
decan ed exhibi ing a s uc u ed 2-D mo phology consis en wi h
he o ma ion o he meshwo k obse ed a 12 hou s (Fig. 2B, D).
Cha ac e iza ion o he seed uni s o polyLM and LM
When he seed uni o polyLM was obse ed a highe
magni ica ion one could see ha i was i sel composed o polygons
in a plana o ganiza ion. Such seed uni was consis en ly p esen in
ma ices ob ained wi hin one hou o incuba ion (Fig. 3A–C). The
ex en o hei longes axes anged be ween 14 and 28 mm
(Fig. 3D). A e 8 and 12 hou s o incuba ion we could al eady
obse e he p esence o a mesh-like ne wo k whose mo phology
was compa ible wi h he o e lay o he seed uni s obse ed a one
hou ; hese uni s howe e could no longe be dis inguished wi hin
he meshwo k (Fig. 3E, F). In LM we could obse e lamella -like
deposi s adso bed di ec ly o he co e slips (pseudo colo ed g een
in Fig. 3). These lamella deposi s we e ei he loca ed a he end o
od-like s uc u es (yellow) o hey appea ed as indi idual pa ches
(a owheads in Fig. 3I). The seed uni was pseudo colo ed as o
e eal he h ee ypes o deposi s seen in LM, namely sphe es
(pink), ods (yellow) and lamella deposi s (g een) (Fig. 3G–I).
Th ee-dimensional s uc u es o polyLM and LM assessed
by a omic o ce mic oscopy
We nex examined he 3-D ea u es o polyLM and LM using
AFM. When a eas o 50650 mm we e scanned, he o e all
appea ances o polyLM and LM we e compa able o hose
isualized by CFM and SEM (Fig. 1). PolyLM displayed he
mo phology o a mul ilaye ed meshwo k con aining homogeneous
s u s (Fig. 4A), while LM exhibi ed ods and lamella deposi s
(Fig. 4B). The sphe ical agg ega es p e iously seen unde con ocal
luo escence (Fig 1B) and SEM (Fig 1D and 3G–I) we e no
Figu e 1. Th ee-dimensional s uc u e o laminin polyme s
unde con ocal luo escence mic oscopy and scanning elec on
mic oscopy. Laminin was incuba ed on glass co e slip o 12 hou s in
acidic (polyLM) o in neu al bu e (LM). (A, B) Indi ec immuno luo-
escence was pe o med using a polyclonal an ibody agains laminin.
The images depic z-s acks ob ained by he supe posi ion o 74
con ocal slices ende ized using he so wa e 7.2.3 (Bi -plane; ee ial).
(C, D). Scanning elec on mic og aphies (SEM) o he polyme s shown a
a simila magni ica ion. A ows in D poin o lamella deposi s o
laminin. The scale ba s apply o panels A–D and ep esen 10 mm.
doi:10.1371/jou nal.pone.0109388.g001
A F ac al Na u e o Polyme ized Laminin
PLOS ONE | www.plosone.o g 3 Oc obe 2014 | Volume 9 | Issue 10 | e109388
obse ed unde AFM due o hei la ge size, which was beyond he
Z scan ange o he AFM.
To u he cha ac e ize he homogenei y o he s u s in
polyLM, he ma ix was scanned a highe magni ica ions
(Fig. 5A, B). In ields o 0.560.5 mm, we measu ed he heigh s
o 10 indi idual s u s o he mesh, chosen as hose in di ec
con ac wi h he glass suppo , i.e., hose in he bo om laye o he
mesh. We ound alues anging be ween 50 and 73 nm, wi h a
mean heigh o 60.2561.764 nm (Fig. 5C, D). In he he e oge-
neous LM ma ix, we measu ed he heigh s o ods and lamella
deposi s, which displayed a e age heigh s o 12136134.6 nm
(Fig. 5E–H) and 125.1613.51 nm (Fig.5I–L), espec i ely.
E idence o a ac al na u e o polyLM
When polyLM was obse ed a a highe magni ica ion wi h
AFM i was possible o obse e he occu ence o igu es ha
ma ched he hexagon-like shape o polyme ized laminin a he
molecula le el (Fig. 6A–C). Such igu es we e s ill one o de o
magni ude la ge han he basic hexagons o med by he
associa ion o he sho a ms o indi idual laminin molecules
(Fig. 6G, H) [4,12]. In ha s uc u e, each side o he hexagon
possesses ,30 nm, esul ing om he in e ac ion o he laminin
sho a ms (35–50 nm long). The sides o he pu a i e hexagons
obse ed he e we e la ge and hei leng h was in he ange o a
ew hund eds o nanome e s. Ne e heless, hese polygons we e
made ou o small globules wi h a diame e and a spacing a ound
30–40 nm, compa ible wi h he cha ac e is ic leng h o laminin
polyme ized h ough in e ac ion be ween sho a ms (Fig. 6D–F).
These s uc u es could no be u he esol ed ia AFM.
The esul s ob ained up his poin sugges ed ha he hexagonal
ne wo k o med by he associa ion o indi idual laminin molecules
could ep oduce i sel a highe le els o o ganiza ion. In o de o
in es iga e his hypo hesis we compa ed images o polyLM
ob ained wi h SEM and wi h ansmission elec on mic oscopy
a e nega i e s aining (Fig. 7). Su p isingly, he mo phologies o
polyLM we e e y simila unde SEM (Fig. 7A) and ansmission
elec on mic oscopy (Fig. 7B) ega dless o a di e ence in
magni ica ion o 1,000 old. This obse a ion sugges s ha polyLM
p esen s a ac al na u e.
De e mina ion o he ac al dimension o polyLM
Based on e idence ha polyLM possessed a ac al s uc u e, we
analyzed images o polyLM ob ained a inc easing incuba ion
imes in sea ch o i s ac al dimension. F ac al o Hausdo
dimensions o inc easing complexi ies we e ound o polyLM as
he adso p ion ime inc eased om 1 o 12 h (Fig. 8). The
calcula ed alues we e 1.55, 1.62 and 1.70 a e 1, 8 and 12 hou s
o adso p ion, espec i ely. By con as , he LM ma ix did no
p esen a ac al s uc u e om which a ac al dimension could be
ob ained.
Figu e 2. Kine ics o adso p ion o polyLM and LM. (A) Laminin was incuba ed in acidic (polyLM) o neu al bu e (LM) and a kine ic o
adso p ion was ca ied ou by collec ing aliquo s o he supe na an a 10 minu es, 30 minu es, 1 hou , 4 hou s, 8 hou s and 12 hou s o
quan i ica ion o he p o ein con en emaining in solu ion. Open symbols ep esen polyLM and closed symbols ep esen he LM. (B–G) SEM images
show he polyme s ob ained in acidic (B–D) o neu al (E–G) bu e s a he indica ed imes. The a ows (B) poin o s uc u ed polyme s obse ed a
1 hou o incuba ion.
doi:10.1371/jou nal.pone.0109388.g002
A F ac al Na u e o Polyme ized Laminin
PLOS ONE | www.plosone.o g 4 Oc obe 2014 | Volume 9 | Issue 10 | e109388
Discussion
In he p esen wo k we desc ibed ha wo ma ices o laminin,
polyLM and LM, ob ained in di e en condi ions p esen ed highly
di e en s uc u es when obse ed a a wide ange o magni ica-
ions. In a p e ious s udy [11], we had al eady shown ha one o
hese polyme s, polyLM, displayed he same nanos uc u e
epo ed o laminin a ays sec e ed by cells [12]. Mo eo e , we
had desc ibed ha polyLM and LM p esen ed di e en mo phol-
ogies in he ange o ens o mic ome e s [8]. Ne e heless
in e media y magni ica ions be ween hese wo anges o sizes had
ne e been assessed be o e. Using con ocal, elec onic and a omic
o ce mic oscopy we illed in his gap and ound ha su p isingly
polyLM p esen ed simila s uc u es independen ly o he magni-
ica ion used o obse e i . Since his p ope y is a ea u e o
ac als, we sea ched o a possible ac al dimension and
con i med ha polyLM indeed co esponded o a ac al s uc u e.
Con e sely, he second polyme , LM, was mo e he e ogeneous
and did no p esen a ac al na u e.
Be o e add essing he biological signi icance o he p esen
indings i is impo an o ecapi ula e he ea u es o he polyme s
s udied he e. The e m o dina y laminin (LM) is used o e e o
Figu e 3. Cha ac e iza ion o polyme uni s in polyLM and LM. Laminin polyme s we e analyzed a high magni ica ion in o de o
cha ac e ize he mo phologies o he seed uni s o each polyme . (A–C) A 1 hou polyLM o ms s a -like 2D s uc u es as exempli ied in he h ee
panels. (D) The sizes o he longe axes in hese s uc u es we e quan i ied and shown o a e age a 20.8465.449 mm. (E, F) High magni ica ion
images o polyLM a 8 and 12 hou s show a meshwo k pa e n compa ible wi h he deposi ion o he s a -like s uc u es. (G–I) LM obse ed a high
magni ica ion e eals h ee ypes o seed s uc u es: ods (pseudocolo ed yellow), sphe es (pseudocolo ed pink) and lamellas (pesudocolo ed g een).
The scale ba in I applies o all panels and ep esen s 10 mm.
doi:10.1371/jou nal.pone.0109388.g003
Figu e 4. O e all mo phology o polyLM and LM unde AFM. A omic o ce mic oscopy images o polyLM (A) and LM (B) a e shown in heigh
mode a e c i ical-poin d ying o he samples. Bo h ma ices we e ob ained by incuba ing laminin wi h glass co e slips in he app op ia e bu e s o
12 hou s. The scanned a ea was 2500 mm2.
doi:10.1371/jou nal.pone.0109388.g004
A F ac al Na u e o Polyme ized Laminin
PLOS ONE | www.plosone.o g 5 Oc obe 2014 | Volume 9 | Issue 10 | e109388

Figu e 5. AFM analysis o polyLM and LM a inc easing magni ica ions. PolyLM (A, B) and LM (E, F, I, J) ob ained as desc ibed in Figu e 4
we e scanned in a eas o 225 mm2 (A, E, I) o 0.25 mm2 (B, F, J) and shown in heigh mode. In o de o de e mine he hickness o he s uc u al uni s
o ming each polyme , he heigh s o 10 s u s we e calcula ed in he ields depic ed in B (s u s o he polyLM mesh), F ( ods in LM) and J (lamellas in
LM). Conside ing ha bo h ma ices we e mul ilaye ed, each s uc u e selec ed o measu emen ollowed he c i e ia o being he closes possible o
he suppo (glass co e slip). Panels C, G and K depic examples o h ee measu emen s and panels D, H and L show he dis ibu ion o he alues
ob ained o each 10 s uc u es. The whi e squa e in I ep esen s an a ea a he edge o he lamella s uc u e used o he heigh measu emen . Panel
M shows he dis ibu ion o heigh s ob ained a each condi ion all oge he o compa ison.
doi:10.1371/jou nal.pone.0109388.g005
A F ac al Na u e o Polyme ized Laminin
PLOS ONE | www.plosone.o g 6 Oc obe 2014 | Volume 9 | Issue 10 | e109388
clus e s o laminin adso bed on o a glass su ace a a concen a ion
below he c i ical concen a ion o 60–100 mg/ml, p e iously
shown o induce solu ion polyme iza ion a pH 7 [5]. In his
condi ion he p o ein does no sel -assemble in solu ion bu i ends
o o m clus e s as i s concen a ion inc eases a he glass su ace
upon decan a ion/adso p ion. Since laminin is used as a coa ing
subs a e o cell a achmen a concen a ions below he c i ical
concen a ion ( ypically be ween 1 and 20 mg/ml), o dina y
Figu e 6. A omic o ce mic oscopy e eals he occu ence o hexagonal-like igu es in polyLM. AFM was pe o med on polyLM ma ices
ob ained as desc ibed in Figu e 4 and a eas o 1 (A, B) o 0.25 mm
2
(C) we e scanned in heigh mode. Hexagons-like igu es simila o hose occu ing
in na u al laminin polyme s [12] we e iden i ied. These hexagons we e isible a di e en magni ica ions (A–C) and p esen ed a iable side leng hs
(ske ched wi h whi e dashed lines), bu hey we e ne e as sho as 30 nm as hey should be o co espond o he sho a m o a laminin molecule.
The smalles dis inguishable s uc u es con ained wi hin he sides o he hexagons we e li le globules (D) whose size and spacing was measu ed in
images o 0.02 mm
2
(D). Panel E shows he dis ibu ion o spacing alues, which a e compa ible wi h he cha ac e is ic leng h (,30 nm) o laminin
polyme ized ia he sho a ms. Panel F depic s a h ee-dimensional econs uc ion o he same a ea shown in panel D, wi h supe posi ion o
compa ible loca ions o laminin molecules. Schemes o one indi idual laminin molecule (long a m dashed and sho a ms colo ed blue, g een and
o ange), wi h indica ion o i s cha ac e is ic dimensions (G) and o he hexagonal polyme gene a ed by he in e ac ion be ween indi idual laminin
molecules (H) a e also shown.
doi:10.1371/jou nal.pone.0109388.g006
A F ac al Na u e o Polyme ized Laminin
PLOS ONE | www.plosone.o g 7 Oc obe 2014 | Volume 9 | Issue 10 | e109388
laminin can be conside ed as he s anda d o m o he p o ein
e e ed o in he li e a u e. On he o he hand, polylaminin
(polyLM) is an a i icial polyme gene a ed upon pH acidi ica ion.
I is o med independen ly o he p o ein concen a ion and i is
no dis up ed a e inc easing he pH o 7 [8]. I was ini ially
desc ibed as a high molecula weigh en i y obse ed in solu ion by
moni o ing a spec oscopic pa ame e , namely s a ic ligh sca e -
ing [6]. A a gi en medium and a a ixed wa eleng h, he in ensi y
o ligh sca e ing is ela ed o he size o he pa icles in solu ion,
which allows o he use o his echnique o ollow he agg ega ion
s a e o p o eins in he p esence o no o ligands o o he
in e ac ing pa icles o biological in e es [18]. The e o e, he 40-
old inc ease in ligh -sca e ing in ensi y obse ed upon ans e -
ing laminin om acidic o neu al bu e e lec ed an inc ease in
he olume o p o ein pa icles in solu ion [6]. These pa icles we e
subsequen ly called ‘‘polyme s’’, ins ead o ‘‘agg ega es’’, which
would sugges ha hey co esponded o clus e s o dena u a ed
p o ein. The e m ‘‘polyme ’’ was employed due o e idence ha
1) he e ia y s uc u e o laminin was p ese ed wi hin he
clus e ed pa icles [6]; 2) decan ed/adso bed pa icles o med
biomime ic ma ices bo h a he nano [11] and a he mic ome e
scales o size [8], and 3) key signaling p ope ies o laminin we e
p ese ed and e en augmen ed a e he acid-induced assemblage,
which was demons a ed mainly o neu ons [8], bu also o o he
cells ypes as glial [9] and hy oid cells [19].
The concep ha he polyme ic s uc u e o p o eins can
in luence hei biological p ope ies has gained inc easing con i -
ma ion in ecen yea s. In pa icula , laminin, a p o ein ha occu s
in he polyme ic o m in na u al basemen memb anes, is known
o ha e hei signaling p ope ies dependen on he es ablishmen
o he polyme ic a ay [3,4]. The in e ac ion o laminin wi h o he
componen s o basemen memb anes, such as nidogens and/o he
p o eoglycan pe lecan, is also in luenced by polyme iza ion, which
is pos ula ed o c ea e new in e ac ing si es a he nanoscale ha
did no exis in he indi idual molecule [20]. These s udies
howe e conside ed polyme iza ion as ha ing only wo s a es (non-
polyme ized and polyme ized). In o he wo ds, he polyme ic s a e
co esponded o a single en i y, a sup amolecula a ay in which
he laminin ime s in e ac ed wi h each o he o o m a shee -like
polyme ancho ed o he plasma memb ane h ough cellula
ecep o s. Such shee -like polyme co esponds o he in e nal
laye o basemen memb anes. Howe e , he e is e idence ha
ce ain issues can p oduce o he ypes o laminin s uc u es. Fo
ins ance, skele al muscle ibe s in i o display memb ane-bound
deposi s o laminin o wo dis inc mo phologies [21], a e icula
and a ib illa one. These wo mo phologies we e assigned o esul
om in e ac ions wi h di e en cellula ecep o s, which, by
p esen ing dis inc egional dis ibu ions on he memb ane, would
lead o he o ma ion o each laminin deposi . One complemen-
a y explana ion, howe e , is ha he mo phologies o he wo
deposi s a he mic ome e scale would e lec speci ic molecula
in e ac ions a he nanome e ange and he e o e could in ol e
in e ac ion wi h he same ecep o . In he ne ous sys em, deposi s
o laminin appea wi h ou di e en shapes h oughou he
de elopmen o he b ain [22,23]. In consonance wi h hese in
i o indings, neu ons isola ed om la e al and medial egions o
he midb ain ha e been shown o sec e e ei he punc a e o
ib illa laminin ma ices, espec i ely [24]. Neu ons om
emb yonic and neona al b ain co ex ha e also been shown o
p oduce dis inc laminin ma ices, whe eas one emained associ-
a ed o he cell memb ane, he o he ex ended away om he cell
su ace, exhibi ing he appea ance o an a ay o angled h eads
[13]. Finally, laminin deposi s ound in he neu al s em cell niche,
p esen wo di e en mo phologies desc ibed as punc a al e na ed
wi h hin linea memb anous s uc u es ancho ed o blood essels
[25,26]. We p opose ha he wo laminin ma ices obse ed in he
p esen wo k e lec wo al e na i e manne s o p o ein assembly.
One impo an ques ion o add ess is how his ac al would be
gene a ed and wha would de e mine he sel -assembly o laminin
uni s in o polyLM, LM o any o he polyme . In a biological
se ing, he in e ac ion wi h in eg ins and o he laminin ecep o s
would guide he p ocess. As mos o hem bind o he long a m o
laminin, i can be p edic ed ha he simul aneous in e ac ion o
laminin ime s wi h memb ane ecep o s will in luence he
o ma ion o he ype o polyme iza ion. The ideal dis ance
be ween wo neighbo ing ecep o s o selec i ely a o in e ac ions
only among he sho a ms o laminin should co espond o he
dis ance be ween he cen e s o he laminin molecules in he
hexagonal ne wo k, i.e., app oxima ely 52 nm (Fig. 6). I he
dis ance is such o sho e a la polyme should be a o ed. On
he o he hand, as he spacing be ween ecep o s inc eased, o he
a angemen s would be allowed. In e es ingly, i has been shown
ha in ib oblas s and mesenchymal cells he ideal dis ance o
signaling h ough in eg ins was in he ange o 50–70 nm [27,28].
In he case o a polyme gene a ed in a cell- ee sys em as
polyLM he lack o cellula ecep o s demands an al e na i e
explana ion. By analyzing he dis ibu ion o su ace cha ges, we
ha e p e iously shown ha he pH acidi ica ion necessa y o
igge he o ma ion o polyLM ends he dis al po ion o he
long a m ( agmen s LG4 and LG5) comple ely posi i e [11]. We
p oposed ha his would be he de e minan o he p e en ion o
he in e ac ion be ween wo long a ms, which is he p edominan
laminin-laminin in e ac ion in he absence o o he molecules [5].
The e o e, polyLM could gi e ise o a hexagonal polyme
mimicking he shee -like polyme assembled on he cell su ace
e en be o e adso p ion o he co e slip. The p e ious epo ha
polyLM inc eased he ligh sca e ing o he solu ion con aining i ,
while LM in simila condi ions did no , suppo s his no ion [6].
The seed polyme al eady decan s as a ac al s uc u e wi hin one
hou o incuba ion and he complexi y o he ac al inc eases as
deposi ion p oceeds (Fig. 8D). The heigh o he lowes s uc u es
ound in polyLM and LM we e a ound 60 nm and 120 nm,
espec i ely (Fig. 5D, L). In e es ingly, he o me is he app ox-
ima e size o he ex ended long a m o laminin (Fig. 6G). Such an
ex ended con o ma ion o indi idual laminin molecules has been
p e iously de ec ed by using elec on mic oscopy a e o a o y
shadowing [5] and AFM [29]. Thus, i is likely ha polyLM
sedimen s as la 2-D polyme s in which only in e ac ions among
he sho a ms occu , as also sugges ed by he AFM measu emen s
(Fig. 6D–F). On he o he hand, in LM a leas wo laye s o la
polyme s media ed by in e ac ions be ween long a ms would be
Figu e 7. PolyLM displays simila mo phologies a bo h 200
and 200,000 old magni ica ions. Images o polyLM we e ob ained
using SEM (A) o ansmission elec on mic oscopy (B) a e nega i e
s aining. Unde SEM he magni ica ion was 200 old while unde TEM i
was 200,000 old. No e ha he obse ed pa e ns we e alike despi e
he 1000 old inc ease in magni ica ion.
doi:10.1371/jou nal.pone.0109388.g007
A F ac al Na u e o Polyme ized Laminin
PLOS ONE | www.plosone.o g 8 Oc obe 2014 | Volume 9 | Issue 10 | e109388
necessa y o accoun o he 120 nm heigh obse ed in he lowes
deposi s.
In he p esen wo k we demons a e ha he p o ein laminin
can gi e ise o a ac al s uc u e. The obse a ion ha he
sup amolecula o ganiza ion o a pu e p o ein (polyLM) is ac al
implies ha he in o ma ion con ained ul ima ely wi hin i s
p ima y sequence is su icien o de e mine he mo phology o
la ge s uc u es ha will spa ially o ganize issue compa men s.
This is pa icula ly in e es ing because i co ela es wi h he
‘‘ ac al-like’’ o ganiza ion o he niche o s em cells in he
sub en icula zone o he adul b ain. In his case, a laminin- ich
basemen memb ane, named ‘‘ ac one’’, has been p oposed o
o ien he binding o p o eoglycans, which, in u n, o ganize he
dis ibu ion o he g ow h ac o s con olling he main enance o
he s em cell niche [26,30], as shown o be he case o bFGF [31].
Al hough ac ones ha e been desc ibed only in he cen al
ne ous sys em, i is well possible ha a simila ac al-like
ex acellula ma ix is p esen in o he s em cell niches. Adul s em
Figu e 8. Calcula ion o he ac al dimension (Hausdo es ima e). (A, B) Image p ocessing in o de o p epa e he image o he box-
coun ing algo i hm o LM (A) and polyLM (B). 1, o iginal image; 2, o iginal image in which he his og am has been equalized; 3, bina ized image
using O su’s me hod). (C) F om images A.3 and B.3 he Hausdo dimension es ima es can be calcula ed supe imposing a g id o a iable size (C.1-
C.4, examples o he same image on which a g id o a iable size has been supe imposed). (D) Repea ing he p e ious p ocess o di e en alues o
g id size and compu ing he numbe o g id boxes ha con ain any pa o he in es iga ed se , he Hausdo dimension o simply he box-coun ing
dimension can be calcula ed. (E) F ac al dimension calcula ed o polyLM s uc u es as a unc ion o ime.
doi:10.1371/jou nal.pone.0109388.g008
A F ac al Na u e o Polyme ized Laminin
PLOS ONE | www.plosone.o g 9 Oc obe 2014 | Volume 9 | Issue 10 | e109388