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Etimologie van vimentin

Etimologie van vimentin


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Wat is die etimologie van die intermediêre filament, Vimentin?


Volgens hierdie koerant:

Van die Latynse woord vimentum, wat gebruik word om skikkings van buigsame stawe te beskryf, beide geordende (bv. Latices, filigres en rietwerk) en nie-geordende (bv. Kwashout).

Vimentien:

Filigraan kuns:


Vimentin

Vimentin

Vimentin, ook bekend as fibroblast intermediêre filament, is die belangrikste intermediêre filament wat in nie -spierselle voorkom (Colvin et al., 1995). Hierdie seltipes sluit in fibroblast, endoteelselle, makrofage, melanosiete, Schwann -selle en limfosiete. Dit is in teenstelling met keratien, wat die intermediêre filament is wat in epiteelselle voorkom. Vimentin bestaan ​​uit 'n enkele subeenheid en het 'n molekulêre gewig van ongeveer 57 kD. In vivo, vimentien is gewoonlik nie in normale epiteelselle teenwoordig nie, maar dit is gekweek in vitro in epiteelselle en kan ook uitdrukking toon in tumorselle van epiteeloorsprong, gewoonlik in paraffienweefsel na antigeenherwinning. Mesenchymale en endoteelselle vlek gewoonlik vimentien positief en dien dus as 'n maatstaf vir interne kwaliteitskontrole in immunoreaktiwiteit. Afwesigheid van immunokleuring in hierdie gebiede dui dikwels daarop dat daar aansienlike skade aan weefselantigene en verlies aan strukturele argitektuur is.

Die goedaardige epiteel van die eierstokoppervlak is 'n enkele laag epiteelselle wat van 'n gespesialiseerde mesodermale oorsprong in die eierstok is. Dit is baie kompleks en het blykbaar kenmerke wat tipies is vir mesenchymale selle in vivo en wanneer dit in kultuur gegroei word. Hierdie selle druk dikwels vimentien uit, en tot 40% van die eierstokkankergevalle is vimentien positief. Daar is egter geen kliniese korrelasie tussen vimentienvlekke en die graad van tumordifferensiasie of die teenwoordigheid van nodale siekte nie. Die gebruik van vimentien word aansienlik beperk deur die gebrek aan spesifisiteit daarvan, wat die gevolg is van die wye verspreiding daarvan in 'n verskeidenheid seltipes in vroeë embrionale lewe voordat hulle later in ontwikkeling deur meer spesifieke intermediêre filamente vervang word.

Deur immunologiese metodes te gebruik, kan vimentien maklik gedemonstreer word in bevrore gedeeltes sowel as paraffienafdelings. In formalien-vaste weefsel kan daar egter vimentienkleuring wees, en die resultate kan inkonsekwent wees. Sommige van hierdie inkonsekwente uitdrukking kan verminder word met behulp van antigeen-herwinningsmetodes. Oor die algemeen is primêre OC's meer gereeld vimentien positief in vergelyking met primêre pulmonêre adenokarsinoom. Daarom sal 'n gebrek aan vimentien -vlekke in hierdie geval die voorkeur gee aan 'n long primêr bo 'n primêre eierstokkarsinoom.


Selbiologie van glioblastoma multiforme: van basiese wetenskap tot diagnose en behandeling

Eerste beskryf in die 1800's, glioblastoma multiforme (GBM), 'n klas IV neoplasma met astrocytiese differensiasie, volgens die hersiene 2016 Wêreldgesondheidsorganisasie klassifikasie van tumore van die sentrale senuweestelsel (SSS) is die mees algemene kwaadaardige gewas van die SSS. GBM het 'n uiters wye reeks veranderings, beide geneties en epigeneties, wat 'n groot aantal mutasie -subgroepe oplewer, waarvan sommige 'n gevestigde rol speel in die onafhanklike oorlewing en behandeling van die pasiënt. Al hierdie komponente verteenwoordig nie net 'n geslote siklus nie, maar is ook relevant vir die gewas biologiese gedrag en weerstand teen behandeling aangesien hulle die patobiologiese gedrag en kliniese verloop vorm. Die teenwoordigheid van verskillende aanvangsmutasies op die agtergrond van die teenwoordigheid van sleutelmutasies in die GBM -stamselle (GBMsc), skei GBM verder as primêre ontstaan ​​de novo van neurale stamselvoorlopers wat ontwikkel tot GBMsc en sekondêr, deur middel van geaggregeerde mutasies. Sommige van die veranderinge in sellulêre biologie in GBM kan waargeneem word deur middel van ligmikroskoop, aangesien dit die sellulêre en weefselkenmerke van die toestand vorm. Veranderinge in genetiese inligting, wat lei tot verandering, onderdrukking en uitdrukking van gene in vergelyking met hul fisiologiese vlakke in gesonde astrosiete, lei nie net tot sellulêre nie, maar ook na ekstrasellulêre matriksherorganisasie. Hierdie veranderinge lei tot 'n veelvuldige aantal mikromorfologiese en suiwer immunologiese/biochemiese vorms. Daarom het die term multiforme in die een-en-twintigste eeu nuwe gewildheid gekry op die agtergrond van genotipiese diversiteit in hierdie neoplastiese inskrywing.

Dit is 'n voorsmakie van intekeninginhoud, toegang via u instelling.


Die oorsprong van vimentine -uitdrukking by indringende borskanker: epiteel -mesenchymale oorgang, myoepitheliale histogenese of histogenese van stamvader selle met bilinaire differensiasie potensiaal?

Vimentien-uitdrukking is 'n taamlik seldsame bevinding in indringende borskanker, en word geassosieer met hoë tumorindringendheid en chemoreweerstand. Dit word tans verduidelik deur twee verskillende biologiese teorieë: direkte histogenetiese afleiding van mioepiteelselle, en epiteel-mesenchimale oorgang (EMT) wat die eindstadium van borskanker-dedifferensiasie weerspieël. In hierdie studie het ons ten doel gehad om verdere insigte te verkry oor die biologiese kenmerke van hierdie vimentien-uitdrukkende borskanker. Ons het immunohistochemie vir vimentien en 15 ander differensiasiemerkers toegepas op 'n reeks van 364 indringende borskankergevalle, met behulp van weefselmikroskikking-tegnologie. 7,7% van alle gewasse het vimentien uitgedruk. Byna al hierdie gevalle (19/21) was graad 3 indringende ductale karsinome, en die meerderheid (13/21) hiervan was geassosieer met 'n ductale in situ komponent. Vimentien uitdrukking is ook gesien in die onderskeie in situ komponente en korreleer positief met die uitdrukking van SMA, CD10, CK 5, p53, Mib-1 en EGFR. 'N Negatiewe korrelasie is gesien vir die uitdrukking van CK 8/18 en die estrogeenreseptor. Karsinoom wat deur Vimentin tot uitdrukking kom, het 'n aansienlik hoër gemiddelde absolute aantal sitogenetiese veranderinge per geval geopenbaar, maar 'n aansienlik laer frekwensie van chromosoom 16q verliese in vergelyking met vimentien-negatiewe gevalle. Ons huidige resultate toon aan dat, ondanks analogieë tussen vimentien-positiewe borskankers en myoepiteelselle in hul uitdrukking van differensiasieverwante proteïene, nie myoepitheliale histogenese of EMT die biologie van hierdie afsonderlike gewasse uitsluitlik kan verklaar nie. Dit word hoofsaaklik ondersteun deur die aansienlik hoër voorkoms van vimentien-uitdrukkende borskanker in vergelyking met enige ander mioepiteel-borsgewasse en die feit dat vimentien reeds in ductale waargeneem word. in situ komponente. Ons stel dus die alternatiewe hipotese voor dat vimentien-uitdrukkende borskarsinoom kan voortspruit uit borsvader selle met bilinêre (klier- en myoepitheliale) differensiasie potensiaal. Kopiereg © 2005 Pathological Society of Great Britain and Ireland. Uitgegee deur John Wiley & Sons, Ltd.


BESPREKING

VIF belemmer die vorming van lamellipodia

Ons resultate toon dat daar gradiënte van die verskillende samestellingstoestande van vimentien in migrerende fibroblaste is, soos aangedui deur 'n hoë konsentrasie VIF binne die stert- en perinukleêre streke, 'n relatiewe afname in lang VIF en toename in kronkels (kort IF) en nie-filamentagtige deeltjies binne die lamella, en 'n verryking van deeltjies binne die lamellipodium. Die verspreidings van die verskillende samestellingstoestande van vimentien is dus gekorreleer met die vorm en polariteit van voortbewegingselle. Ter ondersteuning van hierdie waarnemings is voorheen aangetoon dat vimentien 'n dominante rol speel in die vormoorgange en verhoogde beweeglikheid van selle wat die EMT ondergaan (Hendrix et al., 1997 Mendez et al., 2010). Ander bewyse is verkry uit die mikro -inspuiting van die mimetiese vimentin 1A helix -aanvangspeptied, wat VIF hoofsaaklik in monomere en dimere demonteer, wat veroorsaak dat fibroblaste heeltemal afrond en hul substraataanhegsels verloor (Goldman et al., 1996). Daarbenewens veroorsaak stillegging van ander tipe III IF-proteïene soos gliale fibrillêre suurproteïen en periferien beduidende vormveranderinge in onderskeidelik astrositoom- en PC12-selle (Weinstein et al., 1991 Helfand et al., 2003).

In hierdie studie toon ons aan dat die mimetiese vimentinepeptied, 2B2, VIF in ULF demonteer, eerder as die laer orde strukture as gevolg van die 1A peptied eksperimente (Goldman et al., 1996). Lae konsentrasies van 2B2 mikro-inspuiting in serum-gehongerde fibroblaste het dikwels veroorsaak dat VIF uitmekaar gehaal en teruggetrek het van die rand van die sel naby die inspuitplekke waar lamellipodia gevorm het. By hoër konsentrasies het die 2B2 -peptied gereeld meer uitgebreide VIF -demontage en terugtrekking veroorsaak, en dit veroorsaak dat membraan rondom die hele seloppervlak ruk. In soortgelyke eksperimente, is perifere nie-ruppelende streke van fibroblaste wat in 2% serum gekweek is, deur 2B2-mikro-inspuiting veroorsaak om te ruk, en daarna het hierdie selle hul rigting van translokasie verander. Hierdie resultate demonstreer dat die geteikende demontage en terugtrekking van die VIF-netwerk vanaf die selomtrek membraan-ruppeling kan veroorsaak en selbeweging kan verander.

Ons resultate toon ook aan dat beweging in vim-/-selle, selle wat deur vimentien stilgemaak word, en selle wat die dominante-negatiewe vimentien uitdruk, belemmer word(1–138)alhoewel selle onder elk van hierdie eksperimentele toestande grootliks om hul hele omtrek rits, wat daarop dui dat VIF 'n rol speel in selmotiliteit. Hierdie inhibisie van motiliteit in die afwesigheid van normaal georganiseerde VIF kan verband hou met die onvermoë van die geaffekteerde fibroblaste om die polariteit wat benodig word vir motiliteit vas te stel. Die regulering van die demontage van VIF kan dus optree as 'n molekulêre koppelaar wat die aktien-gebaseerde masjinerie wat verantwoordelik is vir die beweging van selle moduleer. In teenstelling hiermee, wanneer VIF onderhewig aan die seloppervlak gepolimeriseer word, dien hulle as 'n rem en 'n meganiese stabiliseerder om die vorming van lamellipodia te inhibeer.

IF's as meganiese stabiliseerders in die regulering van selbeweging

Ter ondersteuning van hul rol as meganiese stabiliseerders van die seloppervlak, is bewys dat VIF aansienlik groter meganiese spanning in vitro weerstaan ​​as mikrotubules of mikrofilamente, en dit vertoon ook rekverhardende eienskappe en kan tot ×3 × hul lengte gestrek word (Janmey et al., 1991 Kreplak et al., 2005, 2008). Hierdie eienskappe is inherent aan alle soorte sitoplasmiese IF -proteïene (Lin et al., 2010). In vivo-ondersteuning vir hul meganiese rolle word verkry uit die feit dat mutasies in keratien-IF-proteïene geassosieer is met blaassiektes van die vel, waarin keratienosiete broos word en geneig is om te skeur as gevolg van ligte fisiese stres (Chan) et al., 1994). Dit is ook gedemonstreer dat die organisasie van IF-netwerke vinnig verander in reaksie op skuifkragte (Helmke et al., 2000 Sivaramakrishnan et al., 2008). Skerp veroorsaak byvoorbeeld veranderinge in die maasgrootte van die keratien IF -netwerk, wat 'n toename in sy styfheid veroorsaak, wat verder aantoon dat die IF -netwerk selle 'n meganisme bied om eksterne meganiese kragte te weerstaan ​​(Sivaramakrishnan et al., 2008). Dit is waarskynlik dat die buigsaamheid en vervorming-verhardingseienskappe van IF die styfheid van streke van 'n sel plaaslik kan beheer, en daardeur optree as determinante van die ligging en posisionering van lamellipodia.

Die plaaslike regulering van selstyfheid deur VIF kan help om die gedrag van lokomotiewe fibroblaste te verduidelik. In die oorspronklike beskrywings van Abercrombie (1961) van die eienskappe van geplooide membrane in bewegende fibroblaste, het hy opgemerk dat 'n toonaangewende lamellipodium gereeld ophou ruffel terwyl 'n nuwe ruffel elders op die seloppervlak vorm, waardeur die sel in 'n ander rigting beweeg. Min is bekend oor die faktore wat die plek van die vorming van 'n lamellipodium bepaal. Dit behels beslis 'n reeks komplekse interaksies tussen eksterne stimuli (bv. Groeifaktore), seloppervlakreseptore, interne seinweë (bv. Rho en Rac) en die herorganisasie van die sitoskeletale stelsels en hul gepaardgaande proteïene. Op grond van ons resultate veronderstel ons dat die plaaslike organisasie van VIF deelneem aan die bepaling van hierdie webwerwe. Ter ondersteuning hiervan het ons gevind dat gebiede wat relatief min VIF bevat, veroorsaak kan word deur aansienlik minder PA-Rac1-aktivering as wat nodig was in streke wat meer VIF bevat. Hierdie resultate kan die vorige waarneming verduidelik dat sommige sellulêre streke veel groter bestraling benodig as ander om lamellipodia te begin na PA-Rac1-aktivering (Wu et al., 2009).

Vimentienfosforilering en die regulering van IF-samestelling en organisasie

Die funksionele betekenis van die bevinding dat daar 'n verryking van nie-filamenteuse vimentiendeeltjies binne nuutgevormde lamellipodia is, is onbekend. Daar is getoon dat hierdie strukture voorlopers is in die samestelling van kort IF (krummels Prahlad et al., 1998). Soortgelyke voorlopers vir keratien IF is aangemeld in die perifere streke van epiteelselle (Windoffer et al., 2006). Alhoewel die presiese oorsprong van die vimentiendeeltjies nog bepaal moet word, is dit waarskynlik dat ten minste sommige van hierdie strukture voortspruit uit die demontage van voorafbestaande VIF. Hierdie demontage word waarskynlik gereguleer deur proteïenkinases (bv. Lam et al., 1989), aangesien aangetoon is dat fosforilering 'n kritieke rol speel in die regulering van die samestellingstoestande van VIF. Byvoorbeeld, fosforilering van Ser-55 in die nie-a-helikale N-terminale domein van vimentien deur Cdk1 is verantwoordelik vir VIF demontage in nie-filamentagtige deeltjies soos selle afrond om mitose te betree (Chou) et al., 1990). Die bepaling van die relevansie van fosforilering tydens die induksie van rommel is egter kompleks, aangesien vimentien 53 bekende en vermeende fosforileringsplekke het (Li) et al., 2002 -konsortium, 2010). Hiervan woon 19 plekke in die N-terminus, 'n domein waarvan bekend is dat dit 'n belangrike rol speel in die polimerisasie van IF (Chou et al., 1991, 1996 Herrmann et al., 1996). Baie van hierdie plekke is teikens van kinases wat betrokke is by die induksie van lamellipodia en selmotiliteit. Dit sluit in PI3Kγ (Barberis et al., 2009), ROKα (Sin et al., 1998), PKC (Inagaki et al., 1987), Raf-1 (Janosch et al., 2000), proteïenkinase A (Howe, 2004), PAK (Goto et al., 2002) en AKT1 (Zhu et al., 2011). Vimentin Ser-38 is 'n besonder interessante webwerf omdat dit deur ten minste sewe kinases geteiken word, waaronder PAK, PKCε en AKT (Ando et al., 1989 Izawa en Inagaki, 2006 Zhu et al., 2011). Dit is bekend dat laasgenoemde drie geaktiveer word deur Rac1 of serumbyvoeging. Deur 'n teenliggaam teen vimentin pSer-38 te gebruik, toon ons dat daar 'n toename van 50350–400% in fosforilering op hierdie plek is, wat saamval met die induksie van membraanroes. Dit gaan gepaard met beide die plaaslike demontage van VIF naby die seloppervlak en 'n toename in die tempo van die uitruil van subeenhede langs die oorblywende VIF, soos aangedui deur FRAP -analise. Interessant genoeg word die plaaslike fotoaktivering van PA-Rac1 binne 'n ~10-μm-deursnee-vlek geassosieer met 'n onmiddellike toename in Ser-38-fosforilering slegs binne die verligte area. Hierdie plaaslike fosforilering word vinnig langs die VIF-netwerk regdeur die sel gepropageer in <1 min. Tog kan ons onder hierdie eksperimentele toestande die demontage en terugtrekking van VIF langs die seloppervlak slegs naby die aanvanklike plek van PA-Rac1-aktivering opspoor.

'N Moontlike verklaring vir die plaaslike demontage van VIF tydens die vorming van lamellipodia kan verband hou met die vlakke van vimentienfosforylering. Byvoorbeeld, die omskakeling van VIF in deeltjies en kronkels wat onder ruffles gesien kan word, kan 'n hoër fosforyleringvlak vereis as wat nodig is om die omset van subeenhede in die res van die VIF -netwerk te veroorsaak. Ons FRAP -gegewens na die toevoeging van serum dui daarop dat dit die geval kan wees, aangesien ons 'n tweeledige toename in die uitruil van subeenheid in die VIF -netwerke toon in streke wat nie met die geplooide membraan verband hou nie. Ander faktore wat ons begrip van die regulering van VIF-samestellingstoestande bemoeilik, is die groot aantal kinases wat bekend is om met vimentien in wisselwerking te tree, die regulering van die talle kinase/fosfatase-ewewigte wat betrokke is by die regulering van die dinamiese eienskappe van VIF, en die aantal posttranslasioneel gemodifiseerde residue in die vimentien-proteïenketting. Dit is ook moontlik dat, na die fosforilering van een terrein, addisionele terreine meer toeganklik word en dus meer geneig is om gefosforileer te word. Op hierdie manier is dit waarskynlik dat verskeie vlakke van regulering bydra tot die toestand van vimentiensamestelling. Om te begin om die rol van spesifieke fosforileringsplekke te bepaal, het ons voorlopige eksperimente begin oor die effek van serumtoevoeging tot serum-gehongerde, vimentien-nul selle wat netwerke van 'n nie-fosforileerbare mutante vimentien (S38A) uitdruk. Tot op hede het ons nie 'n duidelike verandering in die algehele reaksie op serumbyvoeging relatief tot kontroles opgespoor nie (data nie gewys nie). Hierdie voorlopige resultaat dui daarop dat toekomstige studies met behulp van die mutagenese van verskeie terreine nodig sal wees om die rol van fosforilering by die regulering van die dinamiese eienskappe van VIF, die demontage en terugtrekking daarvan tydens die vorming van lamellipodia, te bepaal.

Daar moet kennis geneem word dat sommige deeltjies teenwoordig binne lamellipodia nuut gesintetiseerde proteïen kan verteenwoordig. Ondersteuning vir hierdie moontlikheid kom van die teenwoordigheid van vimentien-mRNA binne die voorste rand van selle (Lawrence en Singer, 1986) sowel as die bevinding dat vimentiendeeltjies gereeld geassosieer word met mRNA wat betrokke is by die proses van "dinamiese ko-translasiesamestelling" (Chang) et al., 2006). Verder kan hierdie nie -filamentagtige deeltjies funksies hê wat verskil van dié van volledig gepolymeriseerde VIF. Dit is byvoorbeeld tydens aksonale regenerasie aangetoon dat nuut gesintetiseerde, ongepolimeriseerde vimentien 'n kompleks vorm met geaktiveerde MAP-kinase, importin-ß en sitoplasmiese dineïen voor vervoer na die kern en die daaropvolgende modulasie van geenuitdrukking (Perlson) et al., 2005). Dit is ook moontlik dat die verskillende vimentienstrukture wat in die lamella/lamellipodiale streke voorkom, betrokke kan wees by die modulasie van fokale adhesies tydens selmotiliteit. Ter ondersteuning hiervan is vimentien geassosieer met die verspreiding van β3-integrienbevattende fokuskomplekse (Bhattacharya). et al., 2009), die regulering van β1 integriene (Kim et al., 2010), en die omsetkoers van paxillien, 'n 'adapterproteïen' wat algemeen is vir alle fokale adhesies (Mendez et al., 2010). Ten slotte veronderstel ons dat ten minste 'n subbevolking van hierdie deeltjies 'n stoorvorm verteenwoordig vir die samestelling van VIF -netwerke wat die verspreiding van sitoplasma meganies stabiliseer terwyl 'n fibroblast vorentoe beweeg in die rigting van die voorste lamellipodium.

Gevolgtrekkings

Hierdie studie definieer 'n rol vir vimentien in die regulering van lamellipodiumvorming en selmotiliteit. Spesifiek, wanneer netwerke van VIF saamgestel word en met die seloppervlak geassosieer word, word membraan-ruppeling en die vorming van lamellipodia geïnhibeer. Omgekeerd kan lamellipodia gevorm word deur die gereguleerde demontage van VIF in hul strukturele boustene. Deur die vorming van lamellipodia te moduleer, neem VIF deel aan die regulering van selpolariteit en motiliteit.


Metodes

Reagense

HNE, 15-deoxy-Δ 12,14 -prostaglandien J2 (15d-PGJ2), prostaglandien A1 (PGA1) en hul gebiotinyleerde derivate was van Cayman Chemical. Diamide, DBB, Iac-B, TPEN, Z-LLL of MG132, Zinquin acid and Zinquin ethyl ester, anti-actin, anti-tubulin and anti-vimentin antibodies were received from Sigma. Monoklonale anti-Hsp90 (sc-7947), anti-Lamp1 (sc-20011) anti-vimentin V9 teenliggaam (sc-6260) en sy Alexa488, Alexa405 en agarose-konjugate is verkry van Santa Cruz Biotechnology. Gesuiwerde rekombinante Siriese hamster-vimentien (toegangsnommer AH001833) was van Cytoskeleton, Inc. Anti-HNE Michael-addukte is van Calbiochem verkry. Die monoklonale anti-tubulien P1C3-teenliggaam was die vrygewige geskenk van dr Isabel Barasoaín (Centro de Investigaciones Biológicas, CSIC, Madrid, Spanje). LTR, phalloidin-Alexa468 en phalloidin-Alexa568 was afkomstig van Molecular Probes. Pefablock protease -remmer was van Roche.

Selkultuur en behandelings

Die volgende selkulture is verkry vanaf die Nasionale Instituut vir Algemene Mediese Wetenskappe (NIGMS) Menslike Genetiese Sel Bewaarplek by die Coriell Instituut vir Mediese Navorsing (Candem, NJ): Menslike primêre fibroblaste van kontrolepersone (AG10803) en van pasiënte met AE (ONIM) entry 201100, cell line GM02814A), en is gemanipuleer volgens die instruksies van die verskaffer. Die studie is uitgevoer volgens die Declaration of Helsinki beginsels en is goedgekeur deur die Commission of Bioethics and Biosafety of Centro de Investigaciones Biológicas en deur die Bioethics Committee of Consejo Superior de Investigaciones Científicas (Spanje). SW13/cl.2 vimentien-tekort selle was die ruim geskenk van Dr A. Sarria 43 en is gekweek in DMEM aangevul met 10% (vol/vol) fetale bees serum en antibiotika (100 U ml −1 penisillien en 100 μg ml − 1 streptomisien). Mesangiale selle van rotte is verkry uit rotniere met behulp van 'n tegniek van gegradeerde sif 40 en gekweek in RPMI1640 met aanvullings soos hierbo. HeLa-selle was van die American Type Cell Culture Collection. Vir behandelings is selle verbou in die afwesigheid van serum. Behandelings met Z-LLL vir proteasoom inhibisie is uitgevoer by 200 nM vir 20 uur. Diamied is bygevoeg vir 20 min by 1 mM, waar aangedui. TPEN is gebruik vir sinkuitputting in selle by 10 μM vir die aangeduide tye.

Plasmiede en transfeksies

Lamp1-GFP was die vrygewige geskenk van professor J. Lippincott-Schwartz (National Institutes of Health). Die GFP-vimentin wt en GFP-vimentin C328S konstrukte is voorheen beskryf 40. Van hierdie plasmiede is vimentin wt en C328S gesubkloneer in die EcoRI- en BamHI-terreine van pIRES2-AcGFP1 en pIRES2 DsRed-Express2 bicistroniese plasmiede, verkry uit Clontech, om konstrukte te lewer wat die fluorescerende proteïen en die ongemerkte vimentien tot afsonderlike proteïene uitdruk. Hierdie konstrukte sal regdeur die manuskrip GFP // vimentin wt of C328S en RFP // vimentin wt of C328S genoem word. Hierdie strategie verseker die monitering van selle wat getransfekteerde vimentien (onderskeidelik GFP of RFP positief) uitdruk om hulle te onderskei van die paar SW13 selle wat spontane terugkeer van vimentien uitdrukking kan ondergaan. Die plasmiede GFP-vimentin C328A en RFP//vimentin C328A is gegenereer uit GFP-vimentin en RFP//vimentin wt, onderskeidelik, deur die Quickchange XL-mutagenese-stel van Stratagene met oligonukleotiede: 5'-GGTGCAGTCAGTCGATCGCT-reverse en sy komplimentêre . Vir verbygaande transfeksies is selle in p35 -plate met 1 μg DNA getransfekteer met behulp van Lipofectamine 2,000 (Invitrogen). Vir medetransfeksiebepalings is 1 μg bicistroniese plasmiede wat kodeer vir ongemerkte vimentien wt of C328S en RFP vir die identifisering van getransfekteerde selle (RFP // vimentin), saam met 0,2 μg van die GFP-vimentin fusiekonstruksies gebruik. Vir die opwekking van stabiel getransfekteerde selle, is seleksie uitgevoer deur kweek in die teenwoordigheid van 500 μg ml −1 G-418 (Gibco) vir ten minste 3 weke.

In vitro toetse vir vimentienmodifikasie en polimerisasie

Vir modifikasie deur verskeie elektrofiele, is vimentien by 5 μM in 5 mM PYPE (pH 7.0) en 0.1 mM dithiothreitol (DTT) geïnkubeer in die teenwoordigheid van die aangeduide verbindings opgelos in dimetielsulfoksied. In hierdie toetse is óf oplosbare óf gepolymeriseerde vimentien gebruik. In die geval van inkorporering van gebiotinileerde reagense, is biotienopsporing bereik deur inkubasie van vlekke met peperwortelperoksidase (HRP)-streptavidien en verbeterde chemiluminisensie (ECL). HNE-inkorporering is opgespoor met 'n teen-HNE Michael adducts teenliggaam.

Vir polimerisasie is vimentien geïnkubeer in die teenwoordigheid van 5 mM PIPES (pH 7,0) en 150 mM NaCl, vir 5–20 minute by 37 ° C. In ander toetse is polimerisasie veroorsaak deur die toevoeging van verskillende soute van tweewaardige katione, soos aangedui. Die effek van imidasool op ZnCl2-geïnduseerde polimerisasie is geassesseer deur ko-inkubasie met 1 M imidasool. Vir omkering van ZnCl2geïnduseerde polimerisasie, vimentien gepolymeriseer deur inkubasie met 500 μM ZnCl2 vir 1 uur is daarna vir 30 minute met 1 mM TPEN geïnkubeer. In alle gevalle is oplosbare (S) en gepolymeriseerde (P) vimentien geskei deur ultrasentrifugering by 100,000g vir 30 minute by 4 ° C, waarna hoeveelhede van die supernatant, wat oplosbare vimentien bevat, en van die korrels, wat in Laemmli -buffer gesuspendeer is, deur SDS – PAGE en WB geanaliseer is.

Kruisbindingstoetse is uitgevoer met behulp van vimentien wat vooraf geïnkubeer is in die afwesigheid of teenwoordigheid van 150 mM NaCl, by 3 μM, deur inkubasie in die teenwoordigheid van 20 μM DBB vir 1 uur by 37 °C. Alikvote van die inkubasies is geskei op 7,5% SDS -poliakrylamied gels en geanaliseer deur WB.

Vir die bepaling van sinkbinding het ons 'n wysiging gebruik van 'n onlangs beskryfde prosedure vir die opsporing van sinkbindende proteïene, wat gebaseer is op die vermindering van die inkorporering van 'n sistienreaktiewe sonde in die teenwoordigheid van hierdie metaal 66. Kortliks is gesuiwerde vimentien 10 minute lank by 37 ° C met 150 mM NaCl gepolymeriseer voor 1 uur by kamertemperatuur. met 10 mM EDTA of 500 μM ZnCl2, CaCl2 of MgCl2. Daarna is 10 μM Iac-B bygevoeg vir ekstra 30 minute. Inkorpasie van Iac-B is beoordeel deur SDS-PAGE en vlek met HRP-streptavidien en vimentien hoeveelheid deur WB. In hierdie toets dien inkubasie met EDTA as 'n kontrole om potensiële tweewaardige katione wat aan die proteïen gebind is, te verwyder. Aangesien vimentien in hierdie geval onder sterk denatureringstoestande gesuiwer word, word dit nie verwag dat die proteïen gekonjugeerde metale behou nie.

Visualisering van vimentinstrukture deur optiese mikroskopie

'N Alikwot van 5 ul vimentien teen 10 μM in 5 mM PYPE (pH 7,0) en 0,1 mM DTT is op 'n glasplaat geplaas en 5 μl 5 mM ZnCl2 oplossing is bygevoeg en met die pipetpunt gemeng, waarna 'n dekglappie bo -op die oplossing geplaas is en die monster op 'n SP2 -konfokale mikroskoop gevisualiseer word met behulp van die differensiële interferensie -kontrasmodus vir beeldverwerwing. Bepalings is ook uitgevoer teen 500 μM sink met soortgelyke resultate. Vir die bereiding van fluorescerende vimentien is vimentien teen 17 μM vir 15 minute by r.t. in die teenwoordigheid van 'n 10-voudige molêre oormaat FITC, waarna oortollige FITC verwyder is deur gelfiltrering op Zeba-ontsoutende mikrospinkolomme (Thermo Scientific) volgens die instruksies van die vervaardiger. FITC – vimentin is gemeng met onveranderde vimentien in 'n verhouding van 1: 4 en verwerk soos hierbo vir visualisering deur optiese mikroskopie. Vir Zinquin -kleuring is 1 mM Zinquin -suur by die vimentin -ZnCl gevoeg2 mengsel voor die dekstrokie geplaas word. In sommige toetse word die mengsel van vimentien-ZnCl2, geïnkubeer vir polimerisasie soos hierbo, is daarna behandel met 2 mM TPEN voor byvoeging van Zinquin. Sinkin fluoressensie is opgespoor deur eksitasie met 'n ultraviolet laser by 351 en 364 nm, en emissie tussen 450 en 520 nm is verkry.

Elektronmikroskopie

Vimentin by 3 μM is geïnkubeer in die teenwoordigheid van verskillende soute by r.t. Die broeimengsels is onmiddellik voor toediening op koolstofbedekte koper-palladiumroosters met 0,25% glutaraldehied vasgemaak. Nadat oortollige monsters verwyder is, is die roosters met polimerisasiebuffer gewas en 90 sekondes negatief met 2% uranielasetaat gekleur. Monsters is waargeneem in 'n JEOL JEM-1230 elektronmikroskoop wat werk teen 100 kV toegerus met 'n CMOS TVIPS TemCam-F416 digitale kamera en verkry teen 50 K vergroting.

Immunofluoressensie

Na die verskillende behandelings is selle gefixeer met 4% paraformaldehied vir 25 min by r.t. Selle is deurlaatbaar met 0.1% Triton X-100 in PBS, geblokkeer deur inkubasie in 1% beeserumalbumien in PBS (blokkeeroplossing) en geïnkubeer met anti-vimentin-Alexa488 of die aangeduide primêre teenliggaampies by 1: 200-verdunning in dieselfde oplossing . Wanneer nodig, is sekondêre teenliggaampies gebruik teen 1:200 verdunning in blokkeeroplossing. Selle is teengekleur met 4,6-diamidino-2-fenilindool by 3 μg ml -1 in PBS vir 15 min. Vir aktinevisualisering is selle gevlek met Phalloidin-Alexa488 of Alexa568, volgens die instruksies van die vervaardiger. Lisosome is gekleur met LTR deur selle te inkubeer met 25 nM LTR vir 30 min by 37 °C (verwys. 67). Toe dekglase gebruik is, is dit met Fluorsave van Calbiochem gemonteer.

Konfokale mikroskopie

Selle is gevisualiseer op Leica SP2- of SP5 -mikroskope. Individuele snitte is elke 0.5 μm geneem met 'n numeriese opening van × 63, en algehele projeksies word getoon, tensy anders vermeld.

Vir FRAP-toetse is die Leica SP5-konfokale mikroskoop toegerus met 'n termostatiese kamer by 37 °C gebruik. Kortliks is 'n voorbleikbeeld geneem, waarna drie pulse van 488 nm laserkrag toegepas is om 'n area van 18 × 1,5 μm te bleik. Na-bleikbeelde is elke 3 sekondes vir 10 minute verkry met behulp van sagteware van Leica. Vir ontleding van fluoressensieherwinning, is die intensiteit in gedefinieerde punte van gebleikte filamente gemeet op die verskillende tydpunte met Fiji ImageJ sagteware 68 en uitgedruk in arbitrêre eenhede om herstelgrafieke te plot. 'n Totaal van 20 FRAP-toetse is uitgevoer per eksperimentele toestand 26 wt en 48 C328S filamente is in totaal gemonitor.

Vir die bepaling van die dikte van die vimentien-filament, is deursnee van enkele konfokale mikroskopiese vliegtuie van fibroblaste wat deur IF gekleur is, opgestel om profiele te verkry met behulp van Fiji ImageJ sagteware 68. Die breedte van pieke met intensiteit en maksimum intensiteit van 50% is individueel gemeet, sowel as die aantal filamente per selafdeling.

Vir visualisering van intrasellulêre sink, is selle wat op glasbodemskottels gekweek is, geïnkubeer met 25 μM Zinquin ethyl ester in Hank's Balanced Salt Solution (HBSS) en waargeneem op 'n Leica SP2 mikroskoop met behulp van 'n termostatiese kamer by 37 °C. Zinquin -fluoressensie is opgespoor met die eksitasie- en emissieparameters wat hierbo gespesifiseer is.

Vimentin netwerk uitbreiding toets

SW13 -selle wat stabiel getransfekteer is met RFP // vimentin wt of C328S, is getrypsineer en op glasbedekkings geplateer. Op die aangeduide tydstip na platering is selle vasgemaak en verwerk vir IF met anti-vimentien teenliggaam. Die posisie van kerne is verkry deur 4,6-diamidino-2-fenilindoolkleuring en die area wat deur die sitoplasma beset is, is deur RFP-fluoressensie geopenbaar. Die patroon van die vimentiennetwerk is blindelings deur onafhanklike waarnemers beoordeel om die verhouding van selle met vimentienfilamente wat omskryf is tot die perinukleêre gebied of na die periferie van die selle te evalueer. 'N Minimum van 100 selle per toets is gemonitor.

Vimentin -oplosbaarheidstoetse

Die verhouding van oplosbare en onoplosbare vimentien in selle is bepaal deur selekstraksie met 'n hoë sout, skoonmaakmiddelbuffer (20 mM Tris-HCl, pH 7.4, 600 mM NaCl, 0.5% Triton X-100, 0.1 mM natriumortovanadaat en protease-inhibeerders ( 2 μg ml -1 elk van leupeptien, aprotinien en tripsien inhibeerder, en 1,3 mM Pefablock) en skeiding deur sentrifugering by 12 000g vir 10 min by 4 °C (verw. 69). Gelyke hoeveelhede van oplosbare en onoplosbare breuke is geanaliseer deur SDS – PAGE en WB, en verskille in proteïenbelading is gemonitor deur WB anti-aktien. Onversadigde blootstellings van drie onafhanklike eksperimente is geanaliseer deur beeldskandering, en die verhouding oplosbare vimentien is bereken met betrekking tot totale vimentien.

WB en immunopresipitasie

For SDS–PAGE, cells were lysed in 20 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 0.1 mM EGTA, 0.1 mM β-mercaptoethanol, containing 0.5% SDS, 0.1 mM sodium orthovanadate and protease inhibitors (2 μg ml −1 each of leupeptin, aprotinin and trypsin inhibitor, and 1.3 mM Pefablock). Aliquots containing 20 μg of protein were separated on SDS–polyacrylamide gels of the adequate percentage of polyacrylamide, as indicated. Gels were transferred to Immobilon-P membranes (Millipore) using the three buffer system recommended by the manufacturer. Blots were incubated with the antibodies against the proteins of interest (typically at 1:500 dilution) followed by secondary antibodies (HRP conjugated) at 1:2,000 dilution, and immunoreactive bands were detected with ECL (GE Healthcare). In some instances, lanes from the same gel have been cropped for presentation. This is indicated by dotted lines in figures and uncropped scans of blots are presented in Supplementary Fig. 6.

For vimentin immunoprecipitation, cells were lysed in 50 mM Tris-HCl, pH 7.5, 0.1 mM DTT and 0.5% SDS containing protease inhibitors. Lysates were diluted 1:5 in the same buffer in which SDS was substituted by 1% NP-40 and incubated overnight with anti-vimentin V9 agarose-conjugated antibody. Immunoprecipitates were washed four times with PBS and eluted by incubation at 95 °C for 5 min with Laemmli buffer.

Proteomiese analise

Immunoprecipitates obtained from 400 μg of lysates of DBB-treated HeLa cells with the anti-vimentin agarose-conjugated antibody were separated by SDS–PAGE. Gels were stained with Sypro Ruby and were visualized under ultraviolet light. The region of the gel corresponding to the DBB-induced oligomers detected by WB was excised and destained using 50 mM ammonium bicarbonate/50% acetonitrile (ACN), reduced with 10 mM DTT for 30 min at r.t., alkylated with 55 mM iodoacetic acid in the dark for 30 min at r.t. and digested with 12.5 ng μl −1 trypsin in 50 mM ammonium bicarbonate, overnight (o/n) at 30 °C. Peptides were extracted with ACN and 5% trifluoroacetic acid (TFA) and cleaned using ZipTips (0.6 μl C18 resin, Millipore).

Peptides were resuspended in 0.1% formic acid/2% ACN (buffer A) and analysed in an LTQ Orbitrap Velos (Thermo Scientific) in the positive ion mode, coupled to a nanoEasy HPLC (Proxeon). Peptides were first trapped onto a C18-A1 ASY-Column 2 cm precolumn (Thermo Scientific), and then eluted onto a Biosphere C18 column (C18, inner diameter 75 μm, 15 cm long, 3 μm particle size NanoSeparations) and separated using a 70-min gradient from 0 to 35% buffer B (buffer B: 0.1% formic acid in ACN) at a flow rate of 250 nl min −1 . Full-scan mass spectra (m/z 300–1,700) were acquired in the Orbitrap with a target value of 1,000,000 at a resolution of 30,000 at m/z 400, and the 15 most intense ions were selected for collision-induced dissociation fragmentation in the LTQ with a target value of 10,000 and normalized collision energy of 35%. Acquired spectra were searched against human UniProt database (090513) using Sequest search engine through Proteome Discoverer (version 1.4.1.14, Thermo). As for the search parameters, precursor and fragment mass tolerance were set to 10 p.p.m. and 0.8 Da, respectively. Carbamidomethylation of cysteines was set as a fixed modification, and oxidation of methionines was set as a dynamic modification. Two missed cleavages were allowed. Identified peptides were validated using Percolator algorithm with a q value threshold of 0.01 and those with a high confidence level were accepted. The number of peptides from any given identified protein matching these criteria is referred to as peptide spectrum match.

PAR-competition assays

Assays for the competition between PAR and vimentin were based in the procedure described by Koch et al. 51 , with the following modifications: PAR was used at 100 μM final concentration and vimentin at 1 μM. The assay was carried out in 5 mM PIPES, pH 7.0, at r.t. Assays were started by the addition of ZnCl2 solution to give final concentrations between 1 and 100 μM. The formation of the ZnPAR2 complex (Kd of ZnPAR2, 3 × 10 −13 ref. 51) was determined photometrically at 492 nm. No significant changes in absorbance were observed during the time of the assay (typically 10 min). In some assays, vimentin was preincubated with 20 mM MgCl2 for 50 min, after which ZnCl2 was added. In this case, changes in absorbance with time were followed. PAR samples received an equivalent amount of MgCl2, which did not affect colour development.

Statistiese analise

All experiments were repeated at least three times. For experiments involving visual inspection of morphological features, preparations were evaluated by two independent observers in a blind fashion, and, unless otherwise stated, at least 50 cells were monitored per experimental condition. All results are presented as average values±s.e.m. Statistical differences were evaluated by the Student’s t-test and were considered significant when P<0.05, which is denoted in graphs by an asterisk.


Erkennings

We thank Nai He Jin (Institute of Biochemistry and Cell Biology, Chinese Academy of Science, Shanghai, China) for assistance with nestin staining. We thank Xiao Yan Ding (Institute of Biochemistry and Cell Biology, Chinese Academy of Science, Shanghai) and Bing Liu (Beijing Institute of Basic Medical Sciences, Beijing) for their critical reading of the manuscript. We would also like to thank all the members of our laboratory for their help in this study. This work was supported by the National Basic Research Program of China (G1999054300, 2005CB522705), Shanghai Science and Technology Development Foundation.


Etymology of vimentin - Biology

a Institute for X-Ray Physics, University of Göttingen, 37077 Göttingen, Germany
E-pos: [email protected]

b Cluster of Excellence “Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells” (MBExC), University of Göttingen, Germany

Abstrak

Despite the importance for cellular processes, the dynamics of molecular assembly, especially on fast time scales, is not yet fully understood. To this end, we present a multi-layer microfluidic device and combine it with fluorescence fluctuation spectroscopy. We apply this innovative combination of methods to investigate the early steps in assembly of vimentin intermediate filaments (IFs). These filaments, together with actin filaments and microtubules, constitute the cytoskeleton of cells of mesenchymal origin and greatly influence their mechanical properties. We are able to directly follow the two-step assembly process of vimentin IFs and quantify the time scale of the first lateral step to tens of ms with a lag time of below 3 ms. Although demonstrated for a specific biomolecular system here, our method may potentially be employed for a wide range of fast molecular reactions in biological or, more generally, soft matter systems, as it allows for a precise quantification of the kinetics underlying the aggregation and assembly.


<p>Hierdie afdeling verskaf inligting oor die kwaternêre struktuur van 'n proteïen en oor interaksie(s) met ander proteïene of proteïenkomplekse.<p><a href='/help/interaction_section' target='_top'>Meer. </a></p> Interaksie i

<p>This subsection of the <a href="http://www.uniprot.org/help/interaction%5Fsection">'Interaction'</a> section provides information about the protein quaternary structure and interaction(s) with other proteins or protein complexes (with the exception of physiological receptor-ligand interactions which are annotated in the <a href="http://www.uniprot.org/help/function%5Fsection">'Function'</a> section).<p><a href='/help/subunit_structure' target='_top'>More. </a></p> Subunit structure i

Homomer assembled from elementary dimers (By similarity). Identified in complexes that contain VIM, EZR, AHNAK, BFSP1, BFSP2, ANK2, PLEC, PRX and spectrin (PubMed:21745462).

Interacts with BCAS3 (By similarity).

Interacts with LGSN (PubMed:18178558).

Interacts with SYNM (PubMed:17356066).

Interacts (via rod region) with PLEC (via CH 1 domain) (PubMed:15128297).

Interacts with STK33 (By similarity).

Interacts with LARP6 (By similarity).

Interacts with RAB8B (By similarity).

Interacts with TOR1A the interaction associates TOR1A with the cytoskeleton (PubMed:18827015).

Interacts with TOR1AIP1 (By similarity).

Interacts with DIAPH1 (By similarity).

Interacts with EPPK1 interaction is dependent of higher-order structure of intermediate filament (By similarity).

Interacts with the non-receptor tyrosine kinase SRMS the interaction leads to phosphorylation of VIM (By similarity).

Interacts with NOD2 (By similarity).

Interacts (via head region) with CORO1C (PubMed:27178841).

Interacts with HDGF (By similarity).

Interacts with PRKCE (via phorbol-ester/DAG-type 2 domain) (By similarity).

Interacts with BFSP2 (PubMed:19029034).

Interacts with PPL (PubMed:19029034).

Handmatige bewering afgelei uit volgorde-ooreenkoms met i

Handmatige bewering gebaseer op eksperiment in i

& ltp> Hierdie onderafdeling van die '& lta href = "http://www.uniprot.org/help/interaction%5Fsection"> Interaksie & lt/a>' afdeling bied inligting oor binêre proteïen-proteïen interaksies. Die data wat in hierdie afdeling aangebied word, is 'n kwaliteit gefiltreerde deelversameling van binêre interaksies wat outomaties afgelei is van die & lta href = "https://www.ebi.ac.uk/intact/"> IntAct databasis & lt/a>. Dit word by elke <a href="http://www.uniprot.org/help/synchronization">UniProt-vrystelling</a> bygewerk.<p><a href='/help/binary_interactions' target='_top'>Meer. & lt/a> & lt/p> Binêre interaksies i

P20152

GO - Molecular function i

Proteïen-proteïen interaksie databasisse

Die Biologiese Algemene Bewaarplek vir Interaksie Datastelle (BioGRID)

CORUM comprehensive resource of mammalian protein complexes

Database of interacting proteins

Proteïeninteraksie databasis en analise stelsel

Molekulêre INTERAKSIE databasis

STRING: funksionele proteïenassosiasienetwerke

Diverse databasisse

RNAct, proteïen-RNA interaksievoorspellings vir modelorganismes.


Resultate

Loss of Vimentin Leads to Deficient Wound Healing.

Burns or excisional skin wounds in mice are common experimental approaches to assess molecular, cellular, and tissue movements associated with repair. To determine whether vimentin is an important determinant in wound healing, full-thickness standardized burn wounds (1 cm in diameter) were induced in 8- to 10-wk-old VIM −/− mice and their WT littermates. A histological analysis of wound morphology 9 d after injury revealed epidermal healing of only 17% reepithelialization of the wounds in VIM −/− mice compared with 32% in the WT group (Fig. 1 A en B). At day 15 postinjury the wounds in WT mice were reepithelialized completely, with a prominent keratinized layer (Fig. 1 A en B). In contrast, the epithelium layer in VIM −/− mice remained open (∼56% reepithelialization) and in many mice was still covered by a large scab (Fig. 1 A en B). Furthermore, the wound-closure area, quantified at different time points following injury, showed that from day 4 postinjury healing was significantly slower in the VIM −/− group than in the WT group (Fig. 1 C en D). As shown in Fig. 1E, the time to healing (i.e., when the scab falls off) was ∼3 d longer in the VIM −/− group than in WT group. As with burn wounds, the healing of excisional wounds was severely impaired in VIM −/− mice (Fig. S1). To examine the possible influence of basal skin conditions on wound healing, we analyzed the overall features of uninjured control skin of prenatal mice at embryonic day 14 (E14), preweaning (18-d-old) mice, and adult mice (age 10 wk old up to 10 mo) (Fig. S2). In this examination of the uninjured skin, no striking differences in the organization of epidermis, dermis, and cell components could be observed between WT and VIM −/− skins (Fig. S2 AD). The results demonstrate that loss of vimentin inhibits normal wound healing, resulting in a slow and incomplete recovery of the tissue.

VIM −/− mice display wound-healing defect in a burn wound model. (A) Representative pictures showing immunohistochemical labeling of pan-keratin in WT and VIM −/− wounds on days 9 (D9) and 15 (D15) postinjury. (Scale bar, 100 μm.) (B) Quantification of the percentage of wound reepithelialization at different time points after wounding in VIM −/− and WT wounds. Data are shown as means ± SEM n = 6. (C) Representative wound pictures from VIM −/− and WT mice during the 15-d wound-healing period. (D) Quantification of the remaining wound area at different time points after wounding in WT and VIM −/− groups. Data are shown as means ± SEM n = 6–12. (E) Comparison of the healing times (scab falling off) in the days after wounding. Data are shown as means ± SEM n = 12. *P & lt 0,05 **P & lt 0.01 ***P & lt 0,001.

VIM −/− mice have slower wound healing in an excisional wound model (related to Fig. 1). (A) Representative pictures of immunohistochemical labeling of pan-keratin in wounds in VIM −/− and WT mice on days 9 (D9) and 15 (D15) postinjury. (B) Representative pictures of wounds in WT and VIM −/− mice 15 d after excisional injury. (C) Quantification of the wound area remaining at different time points after wounding in the WT and VIM −/− groups. Data are shown as means ± SEM n = 4. *P & lt 0,05 **P < 0.01 ns, not significant.

Vimentin control mice have normal skin composition and collagen accumulation (related to Figs. 2 and 5). (A) Representative pictures showing immunohistochemical labeling of pan-keratin in uninjured skin from control mice on E14 and from 18-d-old, 10-wk-old, and 10-mo-old mice. (B) Representative confocal pictures of immunofluorescent labeling of N-cadherin (red) and keratin 5 (K5, green) in skins of uninjured control mice. (C en D) Quantitation of K5 + DAPI + cells (C) and N-cadherin + DAPI + cells (D) in the epidermal and dermal region. (E) Representative pictures of Picro-Sirius Red staining of collagen in the corresponding sections of skins from VIM −/− and WT control mice. (F) The quantitation of collagen accumulation (Picro-Sirius Red-positive areas) in the mesenchymal/dermal regions of wounds. In A, B, en E, D, dermis region E, epidermis region. (Scale bars, 100 μm.) In C, D, en F, data are shown as means ± SD n = 2–4.

The Defect in Reepithelialization Is Associated with an Inactive EMT Program in VIM −/− Wounds.

To evaluate skin reepithelialization during wound healing further, we analyzed the keratinocytes at wound margins, which form a coordinated cell sheet with de novo production of injury-specific keratin proteins, such as keratin 6, 14, and 16 (25, 26). WT wounds had higher keratin 6 intensity than VIM −/− wounds at days 9 and 15 postinjury (Fig. 2A), and a similar tendency was observed for keratin 14 and with labeling by a pan-keratin antibody (Fig. 2 B en C). By day 15 postinjury, keratinocyte colonies had fused successfully with the newly formed stratified mature epidermis in WT wounds. In contrast, reepithelialized VIM −/− wound regions displayed a very thin epidermal layer with a large scab (right images in Fig. 2 A en B) the average thickness of pan-keratin + epidermis was barely 30.5% of that in the WT group (Fig. 2D). Furthermore, certain regions of keratinocyte colonies in the epidermis of VIM −/− wounds were poorly organized (Fig. 2C). These observations imply that migration, maturation, and stratification of the epidermis, all prerequisites for fast, spontaneous wound healing, are severely compromised in the VIM −/− group.

Compromised reepithelialization and EMT differentiation in VIM −/− wounds. (A en B) Representative pictures of confocal images of keratin 6 (green) and DAPI (red) (A) and keratin 14 (green) and DAPI (red) (B) in VIM −/− and WT wounds on day 9 and 15 after skin burn injury. D, dermis region S, scab region. (Skaalstawe, 200 μm.) (C) Representative confocal images of keratin expression as visualized by a pan-keratin antibody (green), vimentin (red), and DAPI (blue) in VIM −/− and WT wounds on day 15 postinjury. White arrows indicate the region of thin and poor keratinization in VIM −/− wounds. (D) Quantification of average epidermis thickness on day 15 after burn injury. Bars indicate the mean fold changes relative to WT ± SEM n = 3. (E) qRT-PCR analysis of mRNA transcripts for Slug, N-cadherin (Cdh2), FSP-1 (S100a4), and fibronectin (Fn1) in isolated epidermal regions of VIM −/− and WT wounds on days 0, 3, 9, and 15 after burn injury. Bars indicate the mean fold changes ± SEM relative to day 0 WT **P < 0.01 n = 3.

EMT or an EMT-like transdifferentiation program induces the transient transition of secondary epithelial cells to a more migratory phenotype, which is critical for rapid reepithelialization of injured epithelium (27, 28). To address the role of vimentin in this process, we analyzed whether the absence of vimentin affects EMT during wound repair in injured epidermis. We found that during normal WT wound closure, the expression of a major EMT initiator, the transcription factor Slug (also termed Snai2), remained at the same levels found in samples of day 0 uninjured WT skin until day 3 (Fig. 2E). In the WT injured skin samples, the expression of Slug mRNA increased dramatically from day 0 until day 9 and then ceased by day 15 (Fig. 2E). In comparison, skin samples from VIM −/− mice showed significantly reduced mRNA expression of Slug during wound healing we observed a 4.7-fold reduction on day 3 and a prominent 11.5-fold reduction on day 9 in the wounds of VIM −/− mice as compared with their WT littermates (Fig. 2E). On day 15, Slak expression returned to similar levels in WT and VIM −/− wounds (Fig. 2E), in accordance with previously observed kinetics of transient Slug induction in several skin-injury studies (3, 29). Correspondingly, the mRNA expression of EMT-dependent markers downstream of Slug, including N-cadherin (Cdh2), fibroblast-specific protein 1 (S100a4), and fibronectin (Fn1) (30), increased until day 9 and decreased by day 15 during normal WT wound closure (Fig. 2E). The baseline expression of these genes in skin samples from VIM −/− mice were at the same levels as in day 0 WT skin samples but dropped drastically during wound healing, especially at day 9 (Fig. 2E), further supporting the vimentin dependence of the Slug–EMT response during wound repair.

We also found keratin + cell colonies at the WT epidermis–dermis interface that were absent in VIM −/− wounds at day 9 postinjury (Fig. S3 A en B). Additionally a significant portion of cells in the dermal regions of WT wounds coexpressed keratin and the mesenchymal marker α-SMA (Fig. S3C) at day 9 postinjury. Another mesenchymal marker, N-cadherin, was coexpressed with keratin 5 in WT epidermal cells on day 15 postinjury (Fig. S3 E en F). Such cells, which may be at the intermediate stages of EMT, with continued expression of epithelial markers but acquiring the expression of mesenchymal markers, were much less abundant in the healing VIM −/− skin (Fig. S3 CF). Therefore, in the absence of vimentin, the consequent dysfunction of the EMT program is likely to be a primary reason for the inhibited reepithelialization during wound repair in our models.

Compromised reepithelialization and keratinocyte transdifferentiation in VIM −/− wounds (related to Fig. 2). (A) Representative confocal images of the expression of pan-keratin (green) and DAPI (blue) in wounds of VIM −/− and WT mice on day 9 postinjury (D9). The white arrowheads indicate examples of migrating pan-keratin + cell colonies locating in the dermis–epidermis interface. (Scale bar, 100 μm.) (B) Quantitation of pan-keratin + cell colony numbers in dermis–epidermis interface regions on day 9 postinjury. (C) Representative confocal images of the expression of α-SMA (red) and pan-keratin (green) in VIM −/− and WT wounds on day 9 postinjury. The white arrowheads indicate examples of pan-keratin + α-SMA + cells in the dermal region of WT wounds on day 9 postinjury. (Scale bars, 20 μm.) (D) Quantitation of pan-keratin + α-SMA + cells in wounds in VIM −/− and WT mice on day 9 postinjury. (E) Representative pictures of immunofluorescent labeling of keratin 5 (green) and N-cadherin (red) in wounds of VIM −/− and WT mice on day 15 postinjury (D15). (Scale bar, 100 μm.) (F) Quantitation of N-cadherin + K5 + cells in epidermis in day 15 wounds. In A en E, E: epidermis region D: dermis region. In B, D, en F, data are shown as means ± SEM n = 3. **P & lt 0,01.

TGF-β Derived from Dermal Fibroblasts Triggers the Slug–EMT Program and Epithelial Cell Migration.

An EMT-like keratinocyte transdifferentiation program can be modulated rapidly by a variety of factors in the wound environment (31, 32). Therefore we explored the possible involvement of these signaling factors by gene-expression profiling and found that TGF-β1 (Tgfb1), a primary inducer of EMT, had a significantly lower expression at day 3 and day 9 postinjury in VIM −/− wound tissues than in their WT counterparts (Fig. 3A). Recombinant TGF-β1 potently inhibited the expression of the epithelial markers desmoplakin and E-cadherin and induced the expression of Slug, N-cadherin, and vimentin (all hallmarks of an EMT-like transdifferentiation process) in mouse keratinocytes following cytokine stimulation (Fig. 3 BD). To examine the involvement of Slug in wound healing-mediated EMT, we silenced Slug expression in keratinocytes using Slug siRNA in the absence or presence of TGF-β1 (Fig. 3 E en F). Keratinocytes transfected with Slug siRNA had reduced expression of N-cadherin and vimentin as well as a slight induction of desmoplakin upon TGF-β stimulation (Fig. 3 EG), indicating that Slug is needed to induce EMT downstream of TGF-β. Taken together, these data suggest that TGF-β1 produced in WT wounds is capable of driving an active EMT transdifferentiation program via Slug.

TGF-β1–Slug signaling promotes keratinocyte differentiation and migration. (A) qRT-PCR analysis of transcripts for TGF-β1 (Tgfb1) in VIM −/− and WT wounds on days 3, 9, and 15 after wounding. Bars show mean fold changes ± SEM relative to WT n = 3. (B) Mouse keratinocytes were stimulated with 5–10 ng/mL TGF-β1 for 0, 3, or 5 d. Cell lysates were collected and blotted with antibodies against desmoplakin, E-cadherin, N-cadherin, vimentin, and Hsc-70 as loading control. (C en D) Quantification of Slug (C) and N-cadherin (D) intensity in B equalized to Hsc-70. Bars show mean fold changes ± SEM relative to day 3 control mice n = 3. (E) Mouse keratinocytes were transfected with scramble siRNA (si Scra) or Slug siRNA (si Slug) oligos for 2 d and then were stimulated with 5 ng/mL TGF-β1 for 3 d. Cell lysates were collected for Western blotting analysis of Slug, N-cadherin, vimentin, desmoplakin, and loading control Hsc-70. (F en G) Quantification of Slug (F) and N-cadherin (G) intensity in E equalized to Hsc-70. Bars show the mean fold changes ± SEM relative to mock transfections (Ctrl) n = 3 *P & lt 0,05 **P & lt 0.01 ***P < 0.001 ns, not significant.

Because vimentin as a mesenchymal marker is expressed mainly in dermal compartments, we postulated that activated dermal fibroblasts would be responsible for the TGF-β production observed upon injury, thereby triggering epidermal keratinocyte transdifferentiation. In support of this hypothesis, we found that mouse dermal fibroblasts (MDFs) isolated from VIM −/− mice have lower TGF-β1 gene expression than MDFs from WT mice (Fig. 4A). Correspondingly, the levels of active TGF-β1 in the supernatants of VIM −/− MDFs were significantly lower than in the supernatants of WT MDFs (Fig. 4B). Cell-culture supernatants of WT MDFs containing greater amounts of TGF-β1 induced a stronger EMT program in the cultivated mouse keratinocytes than did supernatants from VIM −/− MDFs (Fig. 4C). Consistently, in a scrape-wound assay, mouse keratinocytes closed the scrape wounds considerably faster in the presence of conditioned medium from WT MDFs than in conditioned medium from VIM −/− MDFs (Fig. 4 D en E). To exclude the involvement of other cellular effectors of inflammation that might be relevant to the keratinocyte phenotype, we assessed the expression of key cytokines and the presence of inflammatory cells during wound repair. We found that the expression of the mRNA of proinflammatory cytokines IL-6 (Il6), IL-1α (Il1a), IL-1β (Il1b), IL-23 (Il23), and TNF-α (Tnf) remained at similar levels (less than 1.5-fold changes) between WT and VIM −/− wounds on day 3 and day 9 postinjury (Fig. S4A). Although on day 9 postinjury, 1.5-fold fewer neutrophil cells had infiltrated to the edges of WT wounds than to the edges of VIM −/− wounds (Fig. S4 B en C), there was no significant change in the myeloperoxidase activity of neutrophils (MPO + ) in WT and VIM −/− wounds at day 9 postinjury (Fig. S4 D en E). In addition, the presence of macrophages (CD11b + ) and activated T cells (Zap-70 + ) was comparable between WT and VIM −/− wounds on day 9 of wound repair (Fig. S4 D en E). Cell-culture supernatants of WT and VIM −/− macrophages also contained equal levels of TGF-β1 (Fig. S4F), demonstrating that the loss of vimentin in the regional fibroblasts, but not the infiltrated inflammatory cells, is the key effector of insufficient TGF-β production and signaling.

Vimentin promotes TGF-β production from fibroblasts driving EMT and migration of keratinocytes. (A) qRT-PCR analysis of transcripts for TGF-β1 (Tgfb1) in VIM −/− and WT MDFs. Bars show mean fold changes ± SEM relative to WT n = 6. (B) Level of active and latent forms of TGF-β1 in the supernatants of 6-d MDF cell cultures were analyzed by ELISA. Data are shown as mean ± SEM n = 3. (C) VIM −/− and WT MDF cell-culture media were extracted on 0, 3, and 6 d after cell growth. The growth medium of mouse keratinocytes was replaced with the MDF-conditioned medium for 5 d. Cell lysates were collected and blotted with antibodies against desmoplakin, E-cadherin, N-cadherin, vimentin, Slug, and loading control GAPDH. (D en E) In vitro wound-healing assay of mouse keratinocytes grown in 6-d conditioned medium from VIM −/− MDFs and WT MDFs. The cell gap was monitored over 24 h, and the wound areas were measured and plotted against the time point. At least four wound scratches were analyzed per experiment. Data are shown as means ± SEM n = 3. (F) Mouse keratinocytes grown in 6-d conditioned medium from the VIM −/− and WT MDFs were treated with control IgG (Ctrl IgG, 10 μg/mL), pan–TGF-β neutralizing antibody 1D11 (1D1 Ab, 10 μg/mL), two chemical inhibitors of TGF-β receptors [LY2109761 (2 μM) and SB431542 (2 μM)] or were grown in the corresponding DMSO control (DMSO, 2 μM) for 3 d. The cell lysates from this experiment were blotted with antibodies against pSmad2/3, total Smad2/3, Slug, N-cadherin, and loading control GAPDH. (G en H.) Quantification of Slug and N-cadherin intensity in F equalized to GAPDH bars show the mean fold changes relative to WT Ctrl IgG ± SEM n = 3. (Ek) In vitro wound-healing assay of mouse keratinocytes (at 16 h of wound healing) in the treatments in F. (J) Die y axis shows the percentage of area covered (at 16 h of wound healing) by keratinocytes transfected with mock, scramble siRNA (si Scra), or Slug siRNA (si Slug) oligos for 2 d and incubated in 6-d WT or VIM −/− MDF-conditioned medium. In Ek en J, at least four wound scratches were analyzed per experiment. Data are shown as means ± SEM n = 3 *P & lt 0,05 **P & lt 0.01 ***P & lt 0,001.

Inflammatory cytokines and infiltrated inflammatory cells in wounds in WT and VIM −/− mice (related to Fig. 4). (A) qRT-PCR analysis of transcripts for IL-6 (Il6), IL-1α (Il1a), IL-1β (Il1b), IL-23 (Il23), and TNF-α (Tnf) in wounds in VIM −/− and WT mice on days 3 (D3), 9 (D9), and 15 (D15) after wounding. Data are shown as means ± SEM n = 3. (B) Representative pictures of immunohistochemical labeling of mouse neutrophils in wounds in VIM −/− and WT mice on days 3 and 9 postinjury. The inflamed tissue is indicated by black dashed lines. (Scale bars, 20 mm.) (C) Comparison of the temporal regulation of inflammation (the wound-edge regions infiltrated with neutrophils) in wounds in VIM −/− and WT mice. Data are shown as means ± SEM n = 3. (D en E) Representative pictures (D) and quantitative comparison (E) of the myeloperoxidase activity of neutrophils (MPO), monocyte/macrophage marker CD11b, and T-cell marker Zap-70 in the wound-edge regions of wounds in VIM −/− and WT mice 9 d after injury. (Scale bar, 100 μm.) Data in E are shown as mean ± SEM n = 3. (F) Level of active and latent forms of TGF-β1 in the supernatants after 6 d of WT and VIM −/− macrophage cell culture were analyzed by ELISA. Data are shown as means ± SEM n = 5. *P & lt 0,05 **P & lt 0.01 ***P < 0.001 ns, not significant.

To test further whether TGF-β1 produced from MDFs is required to drive EMT-like changes in keratinocytes, a pan–TGF-β neutralizing antibody, 1D11, and two chemical inhibitors of TGF-β receptors, LY2109761 and SB431542, were added to MDF-conditioned medium all effectively inhibited the phosphorylation of Smad2/3 (Fig. 4F), downstream effectors of the TGF-β signaling pathway. Further corroborating the involvement TGF-β signaling were the results showing that when TGF-β signaling was blocked, the expression of EMT markers (Slug and N-cadherin) in keratinocytes was reduced (Fig. 4 G en H.), and cell migration was inhibited (Fig. 4Ek). This effect was prominent in cells incubated in the presence of WT MDF supernatants (Fig. 4 GEk), which contained higher levels of active TGF-β1 than VIM −/− MDF supernatants (Fig. 4B), reinforcing the notion that TGF-β signaling is a major driver in keratinocyte transdifferentiation and migration induced by fibroblast vimentin. Inhibition of Slug expression by siRNA in keratinocytes also inhibited cell migration (Fig. 4J), further demonstrating that vimentin regulates keratinocyte migration through TGF-β –Slug signaling.

Vimentin Promotes Fibroblast Proliferation, Collagen Accumulation, and Paracrine EMT Signals.

At later stages of tissue repair, we found that the VIM −/− wounds displayed a striking reduction in s.c. proliferating fibroblasts, as indicated by the loss of cells positive for the proliferation marker nuclear antigen ki67 (Fig. 5 A en B). To assess whether this fibroblast deficiency would lead to a defect in ECM synthesis and buildup, we analyzed the accumulation of dermal collagen using Picro-Sirius Red staining (33). The normal granulation tissue with thick collagen fibers that was observed in WT wounds could not be observed in VIM −/− wounds, which instead showed either no fibers or at best a few scattered thin fibers (Fig. 5C). At 15 d postinjury, a striking difference was observed between WT and VIM −/− wounds: Collagen accumulation was largely absent in the dermal regions of VIM −/− wounds, whereas the majority (∼95%) of WT dermis was occupied by collagen fibers (Fig. 5D). There was no striking difference in collagen accumulation between normal, uninjured WT and VIM −/− adult skins (Fig. S2 E en F, 10 wk), excluding the possibility of a problem in s.c. collagen distribution in uninjured basal skin. Therefore the collagen defect can be attributed entirely to events during wound healing, and the observed absence in reepithelialization in VIM −/− wounds is closely associated with the inhibition of fibroblast proliferation and collagen accumulation.

Vimentin promotes mesenchymal cell proliferation and collagen accumulation in vivo. (A) Representative confocal images of the expression of Ki67 (punctate green signal in the nucleus) and vimentin (red) in VIM −/− and WT wounds on days 9 (D9) and 15 (D15) after burn wounding. Nuclei were counterstained with DAPI (blue). (Scale bars, 20 μm.) The white arrowheads indicate examples of ki67 + cells in dermal regions. D, dermis region E, epidermis region. (B) Quantitation of ki67 + cells in mesenchymal/dermal regions of wounds. (C) Representative pictures of Picro-Sirius Red staining of collagen (Boonste) and the immunohistochemical labeling of pan-keratin (Laer) in the corresponding sections of VIM −/− and WT wounds on day 15 postinjury. The right lower corner of each panel shows an enlarged image of the area in the white box. (Scale bars, 100 μm.) (D) The quantitation of collagen accumulation (Picro-Sirius Red-positive areas) in mesenchymal/dermal regions of wounds. In B en D data are shown as means ± SEM n = 3 *P & lt 0,05 **P & lt 0,01.

To investigate further whether vimentin plays a direct role in dermal fibroblast proliferation, we compared the proliferative potential of VIM −/− and WT MDFs. The results showed that VIM −/− MDFs grew much more slowly than WT MDFs (Fig. 6A), although the apoptotic rates in VIM −/− and WT MDFs were similar (Fig. S5A). To determine whether vimentin expression is required for the growth advantage, exogenous vimentin construct was titrated into VIM −/− MDFs. We found that reconstitution of vimentin in VIM −/− MDFs restored the cell proliferation capacity in a dose-dependent manner (Fig. 6A) that was associated with the reactivation of ERK1/2 signaling (Fig. 6B). Furthermore, the conditioned medium from these VIM −/− MDFs reexpressing increasing levels of vimentin gradually rescued the EMT and migration defect in keratinocytes (Fig. 6 C en D). These results suggest that the presence of vimentin in fibroblasts not only directly regulates the proliferation of these cells but also drives the transdifferentiation of keratinocytes by TGF-β1–mediated paracrine mechanisms.

Vimentin promotes mesenchymal cell proliferation and paracrine EMT signaling in vitro. (A) The growth curve of VIM −/− and WT MDF cells after transfected with different amounts of vectors encoding full-length vimentin (the total DNA per transfection was equalized with the control vector). Data are shown as mean ± SEM n = 3. (B) Immunoblotting of vimentin, pERK1/2, total ERK1/2, and GAPDH expression of the cell lysates in A. (C en D) Western blotting of individual markers (C) and migration (D) of mouse keratinocytes growing in 6-d conditioned medium (D6) from VIM −/− and WT MDFs transfected with the indicated amount of vimentin plasmids. Data are shown as means ± SEM n = 3. (E) Scheme showing the working model. Vimentin has a profound effect on fibroblast proliferation, which activates both collagen production and TGF-β secretion. The active fibroblast TGF-β induces Slug–EMT signaling in keratinocytes, promoting EMT-like transdifferentiation and keratinocyte migration.

Vimentin does not influence fibroblast survival (related to Fig. 6). (A) The cell-survival curve of VIM −/− and WT MDFs after transfection with different amounts of vectors encoding full-length vimentin (the total DNA per transfection was equalized with the control vector). Data are shown as means ± SEM n = 3. (B) Representative confocal pictures of immunofluorescent labeling of DAPI (blue), keratin 5 (green), and vimentin (red) (Links) or keratin 14 (green) and N-cadherin (red) (Reg) from isolated mouse keratinocytes. (Scale bars, 20 μm.)

Taking these results together, we have been able to validate the conceptual framework derived from in vivo observations by in vitro experiments with dermal fibroblasts and keratinocytes isolated from VIM −/− and WT mice. Our working model summarized in Fig. 6E proposes that vimentin orchestrates wound healing by regulating fibroblast proliferation and thereby both collagen accumulation and the TGF-β1–mediated Slug–EMT switch in keratinocytes.


Materiaal en metodes

Selkultuur.

MDA-MB-231 human breast cancer cells and MDA-MB-453 (American Type Culture Collection) were cultured in DMEM/Ham’s F-12 50/50 mix (Corning). SKBR-3 was cultured in McCoy’s 5A (modified) medium. The cell culture medium was further supplemented with 10% FBS (Atlanta Biologicals) as well as 1% penicillin-streptomycin (Corning). Cells were maintained at 37 °C in 5% CO2 until the time of experiment/fixation.

QRT-PCR Analysis.

mRNA for genetic analysis was extracted from MDA-MB-231 PGCCs and non-PGCCs using PureZOL RNA isolation reagent (Bio-Rad) according to the manufacturer’s recommendations. RNA integrity and purity were confirmed by spectrophotometry (Nanodrop 1000 Thermo Fisher Scientific). gDNA removal and cDNA synthesis were performed using the iScript gDNA Clear cDNA Synthesis Kit (Bio-Rad) according to the manufacturer’s protocol. Here 1 μg of RNA was converted into cDNA for real-time PCR analysis of EMT markers. The primers used are listed in SI Bylae, Table S1.

Scratch Wound Assay.

MDA-MB-231 cells were seeded at high concentration and allowed to grow to 100% confluency in a 24-well plate (Corning) (SI Bylae, Fig. S2). The well plates were pretreated with 20 μg/mL type I rat tail collagen (Corning) for 1 h before seeding. Before imaging, confluent monolayers were scratched vertically and horizontally in a cross fashion with a 10-μL pipette tip. The well was then washed three times with PBS and fed using medium with Hoechst 33342 (1 μg/mL) for an additional 30 min. Hoechst-stained cells were then imaged with a Nikon Eclipse Ti inverted fluorescent microscope equipped with an environmental chamber (37 °C and 5% CO2). Edges of scratch wounds were selected and imaged periodically every 10 min for 12 h. Time-lapse videos were then exported, and individual images were fed into CellProfiler (Broad Institute). Using a custom pipeline, individual cells were identified within migrated regions. In brief, we used object segmentation after thresholding at 0 h to identify individual nuclei. Identified nuclei were then dilated to estimate the region of the initial monolayer (i.e., the initial coverage area). We repeated this process at different intervals to establish a time course, allowing us to determine the coverage ratio as well as to isolate migrated populations. PGCCs were subsequently identified by measuring the area of segmented nuclei.

Immunofluorescence Staining.

Cells designated for imaging at high magnification or by confocal microscopy were seeded in 24-well plates containing 12-mm, 1.5-μm-thick glass coverslips (Electron Microscopy Sciences). Scratch wounding in a cross formation was performed using a 10-μL pipette tip, and cells were allowed to migrate before fixation for predetermined times. Cells were fixed using 4% formaldehyde and 1% Triton X-100, then stained for DNA, actin, and vimentin cytoskeletal networks as described previously (1 ⇓ –3). In brief, a 1:100 dilution of anti-rabbit vimentin antibody (Cell Signaling Technology) was added to individual coverslips for 1 h. After incubation with primary antibody, coverslips were returned to well plates and then washed twice with PBS. Next, a 1:1,000 anti-rabbit Alexa Fluor 488 secondary antibody (Invitrogen) was used in conjunction with rhodamine-phalloidin (0.2 uM) and DAPI (5 ug/mL) for 1 h. Coverslips were washed three times with PBS and then mounted on microscope slides using Fluoromount-G (SouthernBiotech).

Images of the scratch wound were divided into migrated, unjammed, and jammed regions, defined as follows. The migrated region includes cells that have migrated and moved into the initial scratch area. The unjammed region represents cells located near the edge of the scratch wound, which are uncaged/unjammed by their neighbors and can migrate freely. Finally, the jammed region includes areas toward the middle of the monolayer, where cells are caged/jammed by their neighbors and cannot move freely. Images of fluorescently labeled cells were analyzed for vimentin polarity. In brief, the direction of cell migration was determined primarily by the position of the cell with respect to the scratch wound and overall cell morphology (i.e., identification of axis of elongation and lamellipodia structures). Vimentin polarity was then assessed with respect to the direction of cell migration (SI Bylae, Fig. S3).

Fluorescence Imaging and Volume Analysis.

Mounted slides were imaged using a Nikon Eclipse Ti inverted fluorescent microscope with a 60× objective or an Olympus 3000 confocal microscope (Z-stacks for 3D volume reconstructions). Beelde van konfokale mikroskopie is ontleed met ImageJ met behulp van die Voxel Counter plugin na drempel en gaping sluiting om selvolume te bepaal. Kortliks is die konfokale Z-stapels van falloïdienbevlekte aktien deur 'n skyf gedompel, daarna is 'n gapingsfunksie gebruik om gate binne die drumpelarea te vul (om seloppervlakte per snit voor te stel). Laastens is die Voxel Counter-inprop gebruik om die aantal voxels wat in die drempelarea ingesluit is, te bereken om algehele selvolume te meet. 'N Soortgelyke benadering is toegepas op fluorescent gekleurde vimentien- en Hoechst-bevlekte kerne, en relatiewe vimentien/kerne-volume is bepaal in verhouding tot die totale volume van die sel. 3D -projeksies is gegenereer met behulp van die 3D viewer -funksie in Fidji.

SiRNA -behandeling.

MDA-MB-231 vimentin is stilgemaak met behulp van 'n poel van drie siRNA-dupleks wat verkry is met behulp van die TriFECTa DsiRNA Kit vir vimentin (Integrated DNA Technologies). Selle is getransfekteer met elke dupleks of met kontroles met behulp van Lipofectamine-3000 (Invitrogen) volgens die vervaardiger se aanbevelings. Knockdown-doeltreffendheid is bekragtig deur gebruik te maak van qRT-PCR-uitdrukkingsanalise van selle wat met die stilmaak-dupleks getransfekteer is in vergelyking met die verskafde scramble-kontrole. Die afwesigheid van effekte buite die teiken is bevestig met behulp van die siRNA-dupleks en die ekspressievlak van HPRT1 (kontrolegen wat nie deur die dupleks geteiken word nie) in vergelyking met HPRT1 knockdown dupleksbeheer.


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Kommentaar:

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