A fractal nature for polymerized laminin

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