Este trabalho foi financiado por Lawrence Livermore National Lab LDRD 18-ERD-062 (para C.R.). Obrigado a John Muschler por seu gentil presente das células DgKO e DgKI. Obrigado à equipe do Laboratório de Microscópio Eletrônico da Universidade da Califórnia em Berkeley pelo aconselhamento e assistência na preparação de amostras de microscopia eletrônica e coleta de dados.

1. Descongelar e alíquota laminina-111
NOTA: A laminina-111 purificada está disponível comercialmente (normalmente purificada a partir da matriz de EHS vendida como Matrigel), mas nem todas as fontes fornecem proteína intacta e não degradada.
<ol><li>Confirmar que Ln1 não é degradado por fornecimento de Ln1 e cromatografia de exclusão de tamanho de corrida26 ou eletroforese em gel de poliacrilamida26. Ln1 desnaturado deve ter bandas em 337 kDa, 197 kDa e 177 kDa, e Ln1 nativo deve ter uma única banda em 711 kDa.</li>
	<li>Descongelar Ln1 no gelo em geladeira durante a noite e alíquota em tubos de microcentrífuga de baixa ligação a proteínas (tipicamente alíquota a 100 μg/tubo para sobreposições de cultura, ou 10 μg para revestimentos) usando pontas de baixa ligação a proteínas e técnica estéril27. Congelar alíquotas e conservar a -80 °C.</li>
</ol>2. Polimerização da laminina usando tampões de pH baixo

<ol><li>Faça tampão de polimerização poliLM: Pesar e misturar 1 mM de CaCl2 e 20 mM de acetato de sódio em água ultrapura. Em seguida, ajuste o pH para 4,0, usando HCl ou ácido acético. Filtrar estéril através de um filtro de 0,2 μm e conservar a 4 °C.</li>
	<li>Faça o controle do tampão de polimerização: Misture 1 mM CalCl2 e 20 mM Tris e ajuste o pH para 7,0, usando 10 N HCl ou 12 N NaOH conforme necessário. Filtrar estéril através de um filtro de 0,2 μm e conservar a 4 °C. NOTA: O protocolo pode ser pausado aqui.</li>
	<li>Descongele o número necessário de alíquotas de Ln-1 em uma geladeira durante a noite.</li>
	<li>Usando a técnica de cultura de células estéreis, adicione o tampão apropriado (tampão de polimerização poliLM da etapa 2.1 ou tampão de polimerização de controle da etapa 2.2) às alíquotas de laminina na concentração final de 100 μg/mL (ou seja, adicione 0,5 mL a 50 μg de alíquotas) e misture suavemente por pipetagem.</li>
	<li>Se utilizar laminina fluorescentemente marcada para visualização, reconstitua e adicione na proporção 1:50 m/peso (por exemplo, 2 μg/100 μg) e misture suavemente por pipetagem. Observação : se usando laminins como um substrato para a fixação celular, execute as etapas 2.6-2.8:</li>
	<li>Imediatamente após a diluição em tampão apropriado, transferir lamininas para plástico de cultura ou vidraria e incubar durante a noite em uma incubadora de cultura celular a 37 °C. Observe que, ao cobrir o revestimento, adicione um volume suficientemente grande para produzir uma queda equilibrada (normalmente, use 50 μL para uma lamínula redonda de 13 mm) e mantenha em uma câmara umidificada durante a noite a 37 °C.</li>
	<li>Utilizando técnica estéril27, lavar com solução salina tamponada com fosfato (PBS) 1x.</li>
	<li>Retire o líquido com cuidado. Não deixe a superfície completamente seca. NOTA: Se estiver usando lamininas para cobrir as células para indução de proteína do leite, siga as etapas 2.9-2.12:</li>
	<li>Cultura de células epiteliais mamárias. Cultura MepG dystroglycan knock in (DgKI) ou células MepG dystroglycan knockout cells (DgKO) em DMEM-F12 suplementado com 2% FBS, 0,01 mg/mL insulina, 0,005 μg/mL EGF, 0,05 mg/mL gentamicina e 0,05 g/mL normocina.</li>
	<li>Células DgKO e DgKI de sementes para estimulação de laminina a 10.500 células/cm2 em placas de poço ou placas de fundo de lamínula</li>
	<li>Incubar lamininas diluídas em uma incubadora de cultura celular a 37 °C por 30 min.</li>
	<li>Centrifugar alíquotas de poliLM a 2.000 x g por 20 minutos, mas centrifugar LM em pH neutro a 20.000 x g por 20 minutos devido ao menor tamanho das entidades proteicas e necessidade de velocidades mais altas para coletar efetivamente.</li>
	<li>Por técnica estéril27, retirar suavemente o sobrenadante e ressuspender em volume apropriado de meio de indução DMEM-F12 suplementado com 3 μg/mL de prolactina, 2,8 μM de hidrocortisona e 5 μg/mL de insulina</li>
	<li>Transferir laminina em meio de cultura celular imediatamente para estimular as células. Estimulamos células com 50-800 μg/mL Ln1</li>
</ol>3. Polimerização da laminina utilizando glicoproteínas isoladas
<ol><li>Descongele nidogênio-1 recombinante e Ln1 suavemente no gelo na geladeira durante a noite.</li>
	<li>Misturar Ln1 com nidogênio-1 na proporção 1:1 em peso em meio de cultura celular usando pontas e tubos de baixa ligação a proteínas e usando técnica estéril. Utilizar Ln1 puro em meio de cultura celular como controle.</li>
	<li>Se usar laminina fluorescentemente marcada para visualização, reconstitua e adicione na proporção 1:50 m/peso (por exemplo, 2 μg/100 μg). Misture suavemente por pipetagem.</li>
	<li>Incubar Ln1-nidogênio ou controlar Ln1 em incubadora de cultura celular a 37 °C por 30 min.</li>
	<li>Centrífuga de copolímeros de Ln1-nidogênio a 2.000 x g por 30 min e controle Ln1 a 20.000 x g por 30 min.</li>
	<li>Remover suavemente o sobrenadante e ressuspender em meio de cultura celular até uma concentração final de 100-800 μg de proteína total/mL.</li>
	<li>Use imediatamente para estimular as células, ou transfira para lamínulas e deixe aderir durante a noite a 37 °C em incubadoras de cultura de células umedecidas.</li>
	<li>Remova o meio de cultura das lamínulas, lave as lamínulas com 1x PBS e, em seguida, fixe com formalina a 10% (ou seja, formaldeído a 4% em PBS) por 15 minutos à temperatura ambiente.</li>
</ol>4. Polimerização de laminina utilizando células que expressam distroglicanos
<ol><li>Cultura de células epiteliais mamárias. Cultura MepG dystroglycan knock in (DgKI) ou células MepG dystroglycan knockout cells (DgKO) em DMEM-F12 suplementado com 2% FBS, 0,01 mg/mL insulina, 0,005 μg/mL EGF, 0,05 mg/mL gentamicina e 0,05 g/mL normocina.</li>
	<li>Sementes de células DgKI a 5.000 células/cm2 e células DgKO de sementes a 8.000 células/cm2 devido a diferenças nas taxas de crescimento. Cultura a 37 °C em incubadoras umedecidas com trocas de meios a cada 2-3 dias e passagens a cada 4-5 dias usando uma técnica estéril rigorosa. Contar as células cuidadosamente com um hemocitômetro ou contador automático de células27 para garantir taxas de crescimento adequadas.</li>
	<li>Células de sementes para estimulação de laminina a 10.500 células/cm2 em placas de poço ou placas de fundo de cobertura e cultura por 2 dias, usando células DgKI para gerar células fractais Ln1 e DgKO como controles negativos. Descongelar Ln-1 suavemente durante a noite em gelo em um refrigerador e, em seguida, misturar com meio de indução de DMEM-F12 suplementado com 3 μg/mL de prolactina, 2,8 μM de hidrocortisona e 5 μg/mL de insulina. Para uma placa de 35 mm, use 1 mL de meio de indução com 50-800 μg de Ln1.</li>
	<li>Se estiver usando Ln1 fluorescente, misture na proporção 50:1 em peso/mwt.</li>
	<li>Remover o meio das células e cobrir com laminina e meio de indução de DMEM-F12 suplementado com 3 μg/mL de prolactina, 2,8 μM de hidrocortisona e 5 μg/mL de insulina</li>
	<li>Cultivar células por 2 dias e, em seguida, fixar com formalina a 10%.</li>
</ol>5. Caracterização da microestrutura da laminina via microscopia óptica
<ol><li>Coletar amostras fixas e lavar 3 vezes com 1x PBS à temperatura ambiente por 5 min.</li>
	<li>Bloquear e permeabilizar amostras em 1x PBS com Triton X-100 a 0,5%, albumina de soro bovino a 3%, soro caprino a 10% e fragmento de anticorpo bloqueador de camundongo a 40 μg/mL por 1 h à temperatura ambiente em câmara umedecida. NOTA: Triton X-100 é perigoso. Manuseie com luvas e proteção ocular e descarte adequadamente.</li>
	<li>Misture o anticorpo antilaminina numa diluição de 1:100 com 1x PBS com Triton X-100 a 0,5% e BSA a 3% e adicione às amostras durante 2 h à temperatura ambiente ou durante a noite a 4 °C numa câmara humidificada (normalmente utilize caixas de ponta com um lenço de laboratório molhado no fundo).</li>
	<li>Remover o anticorpo primário e lavar 3 vezes com 1x PBS com Triton X-100 a 0,5% e BSA a 3%.</li>
	<li>Adicionar um anticorpo secundário adequado numa diluição de 1:500 às amostras em 1x PBS com Triton X-100 a 0,5% e BSA a 3% e incubar à temperatura ambiente no escuro numa câmara umedecida.</li>
	<li>Remover o anticorpo secundário e lavar 3 vezes com 1x PBS com Triton X-100 a 0,5% e BSA a 3%.</li>
	<li>Para amostras contendo células, adicionar 6 μM de faloidina por 45 min, seguido de 3 lavagens com PBS com Triton X-100 a 0,5% e BSA 3%, e seguidas de 3 lavagens com PBS. Em seguida, corar com 0,1 μg/mL DAPI em PBS por 5 minutos.</li>
	<li>Monte amostras, se for o caso.</li>
	<li>Estrutura da imagem ln-1 utilizando microscopia confocal de alta resolução. Use um microscópio de varredura a laser equipado com objetivas de imersão (Plano de imersão em água Apochromat 40X/1.1NA ou Plano de imersão em óleo Apochromat 63X/1.4NA).</li>
</ol>6. Caracterização de construções de laminina via dimensão de contagem de caixas
<ol><li>Analise as propriedades fractais usando algoritmos de dimensão de contagem de caixas disponíveis nas caixas de ferramentas do Matlab ou no ImageJ. Esses métodos limiares imagens de laminina e plotam em um log-log plotar o número de caixas contendo Ln1 em comparação com o tamanho das caixas. A inclinação da relação entre esses parâmetros é a dimensão de contagem de caixas: geometrias não-fractais terão uma dimensão de 0, 1 ou 2, enquanto geometrias fractais terão uma dimensão não-inteira28. NOTA: Ln1 tratado com ácido, glicoproteína e ligado a células DgKI tem uma dimensão fractal de contagem de caixa de ~1,7, enquanto Ln1 de controle tem uma dimensão fractal de 2,0.</li>
</ol>7. Caracterização de construções de laminina via microscopia eletrônica
<ol><li>Ressuspender as lamininas em meios de cultura celular e deixar adsorver wafers de sílica por 90 min em um ambiente úmido de 37 °C.</li>
	<li>Matrizes de fixação em glutaraldeído 2,5% em tampão cacodilato de sódio 0,1 M pH 7,2. NOTA: O glutaraldeído é perigoso e deve ser manuseado em uma capela com luvas e proteção ocular e descartado como resíduo perigoso.</li>
	<li>Matrizes pós-fixas em tetróxido de ósmio a 1% em tampão cacodilato de sódio 0,1 M pH 7,2. NOTA: O tetróxido de ósmio é perigoso e deve ser manuseado em um exaustor com luvas e proteção ocular e descartado adequadamente. O cacodilato é perigoso e deve ser manuseado em um exaustor com luvas e proteção ocular e descartado adequadamente.</li>
	<li>Lavar 3 vezes em tampão cacodilato de sódio a pH 7,2.</li>
	<li>Desidratar com concentrações crescentes de etanol.</li>
	<li>Sputter reveste matrizes com uma fina camada de ouro.</li>
	<li>Imagem utilizando microscopia eletrônica de varredura.</li>
</ol>

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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of Animal Cells: A Manual of Basic Techniques. 7th edition. , (2016).</li><li>Mandelbrot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Fractal Geometry of Nature. , (1982).</li><li>Schittny, J. C., Yurchenco, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terminal+short+arm+domains+of+basement+membrane+laminin+are+critical+for+its+self-assembly.">Terminal short arm domains of basement membrane laminin are critical for its self-assembly.</a> Journal of Cell Biology. 110 (3), 825-832 (1990).</li><li>Schuger, L., Yurchenco, P., Relan, N. K., Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+fragment+E4+inhibition+studies:+basement+membrane+assembly+and+embryonic+lung+epithelial+cell+polarization+requires+laminin+polymerization.">Laminin fragment E4 inhibition studies: basement membrane assembly and embryonic lung epithelial cell polarization requires laminin polymerization.</a> International Journal of Developmental Biology. 42 (2), 217-220 (1998).</li><li>Colognato, H., Yurchenco, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Form+and+function:+the+laminin+family+of+heterotrimers.">Form and function: the laminin family of heterotrimers.</a> Developmental Dynamics. 218 (2), 213-234 (2000).</li><li>Kabaeva, Z., Meekhof, K. E., Michele, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sarcolemma+instability+during+mechanical+activity+in+Large+myd+cardiac+myocytes+with+loss+of+dystroglycan+extracellular+matrix+receptor+function.">Sarcolemma instability during mechanical activity in Large myd cardiac myocytes with loss of dystroglycan extracellular matrix receptor function.</a> Human Molecular Genetics. 20 (17), 3346-3355 (2011).</li></ol>

Este trabalho foi realizado sob os auspícios do Departamento de Energia dos EUA pelo Lawrence Livermore National Laboratory sob o Contrato DE-AC52-07NA27344. Lançamento de IM LLNL-JRNL-799443.

As lamininas representam um elemento-chave da MEC em sistemas epiteliais, musculares e de órgãos neurais, e têm demonstrado in vitro desempenhar papéis essenciais na regulação da diferenciação funcional das células. Evidências crescentes indicam que a estrutura da laminina exibida às células regula sua sinalização e o fenótipo celular resultante 15,16, portanto, métodos para controlar a microestrutura de Ln1 devem ser de interesse para biólogos b...

Ao contrário dos fatores de crescimento ou citocinas, as proteínas da matriz extracelular (MEC) podem se agrupar em redes estruturais. Como a disfunção da MEC é um elemento-chave de muitas condições de doença, os mecanismos pelos quais as células detectam e transduzem sinais da composição, microestrutura e biomecânica da MEC são alvos atraentes para o desenvolvimento terapêutico. Evidências crescentes sugerem que a microestrutura dessas redes desempenha um papel importante em como essas proteínas sinalizam para as células. Até o momento, essa ligação entre a microestrutura da MEC e a função celular tem sido demonstrada para colágeno I1, fibronectina2 e fibrina3.
A laminina-111 (Ln1) é uma proteína trimérica, em forma de cruz, que é um componente estrutural chave da matriz extracelular que entra em contato com células epiteliais4, células musculares5 e células neurais6. Na glândula mamária, o Ln1 é necessário para a diferenciação funcional das células epiteliais da glândula mamária em células produtoras de leite7,8,9, e o Ln1 é crucial para a indução da reversão tumoral 10,11. Ln1 e outras lamininas são necessárias para o desenvolvimento de redes corticais de actina e organização do sarcolema no músculo12,13, e para migração de células neurais e crescimento de neuritos13 . Assim, compreender os mecanismos pelos quais a laminina sinaliza para as células é uma área ativa de pesquisa.
Ln1 contém múltiplos domínios adesivos para uma ampla gama de receptores, incluindo integrinas, sindecanos, distroglicanos, LBP-110 e LamR (Figura 1A)4,14. O fragmento E8, que contém os domínios globulares c-terminais da laminina α1, é necessário para a ligação de distroglicanos e integrinas α6β1 e α3β115, sendo necessário para a diferenciação funcional das células epiteliais 15,16. Em contraste, os braços curtos apresentam atividade biológica diferente17, o que significa que a disponibilidade para o reconhecimento desses diferentes domínios Ln1 após a formação da rede poderia alterar o comportamento celular.
Recentemente, demonstramos que Ln1 arranjado em uma rede polimérica exibe propriedades fractais (isto é, uma estrutura que é auto-similar em múltiplas escalas de comprimento)18. As redes fractais sinalizam diferentemente às células epiteliais em relação ao Ln1, que não possui esse arranjo19. Essa rede fractal indica uma estrutura particular de montagem, onde o braço longo é preferencialmente exposto às células18. Como resultado dessa exibição de braço longo diferente, as células epiteliais sofrem diferenciação funcional em células produtoras de leite com tight junctions bem organizadas e supressão das fibras de estresse de actina19.
Descrevemos 3 métodos para gerar redes Ln1 a partir de interações de ligação braço curto-braço curto, que mostram uma alta exibição do braço longo de Ln1: 1) por montagem livre de células com tampões ácidos, 2) por co-incubação com glicoproteínas, ou 3) por interação com distroglicanos de superfície celular. Estes três métodos geram microestruturas de laminina semelhantes através de três mecanismos muito diferentes, permitindo flexibilidade no planejamento experimental. Trabalhos anteriores sugerem que esses três métodos podem representar robustez biológica: no epitélio da glândula mamária, tanto distroglicanos de superfície celular, glicoproteínas ou laminina estruturada artificialmente podem induzir diferenciação funcional19. Assim, os pesquisadores podem escolher entre esses métodos com base no objeto de estudo: células que expressam distroglicanos não apresentam sensibilidade à microestrutura de Ln-1, pois o distroglicano na membrana celular induz a polimerização correta em uma rede fractal19,20. Matrigel, uma mistura de lamininas e glicoproteínas21, representa a fonte mais econômica de Ln-1, e oferece a microestrutura necessária para induzir células mamárias knockout distroglicanos a diferenciar20, mas contém uma mistura complexa de proteínas com variabilidade lote a lote. O PolyLM representa o sistema mais limpo e reducionista que proporciona essa função, porém com maior custo e menor eficácia para induzir a diferenciação das células mamárias em relação ao Matrigel19.
Esses três métodos apresentam estrutura de rede fractal característica de agregação limitada àdifusão18,19 e apresentam atividade biológica alterada em relação à laminina agregada em múltiplos sistemas experimentais22,23,24,25. Estudos futuros utilizando esses métodos poderiam identificar os receptores e a sinalização necessários para a diferenciação epitelial e determinar a restauração de interações normais célula-matriz em células em microambientesanormais20, como tumores.

<table><tbody><tr><td>10% formalin</td><td>Sigma Aldrich</td><td>HT501128</td><td></td></tr><tr><td>Anti-Laminin primary antibody</td><td>Sigma Aldrich</td><td>L9393</td><td></td></tr><tr><td>Anti-Rabbit Alexafluor 488 secondary antibody</td><td>Thermo</td><td>A32731</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma Aldrich</td><td>A9418</td><td></td></tr><tr><td>CaCl2</td><td>Sigma Aldrich</td><td>C4901 or similar</td><td></td></tr><tr><td>Cell Culture Incubator</td><td>Thermo</td><td>Heracell 150 or similar</td><td></td></tr><tr><td>Centrifuge for microcentrifuge tubes</td><td>Eppendorf</td><td>5418 or similar</td><td></td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>LSM 710 or equivalent</td><td></td></tr><tr><td>Coverslips</td><td>Thermo</td><td>25CIR-1 or similar</td><td></td></tr><tr><td>DMEM-F12, Liquid, High Glucose, +HEPES, L-glutamine, +Phenol red</td><td>Thermo</td><td>11330107</td><td></td></tr><tr><td>EGF (Epidermal Growth Factor)</td><td>Sigma Aldrich</td><td>11376454001</td><td></td></tr><tr><td>Fluorescently Labeled Laminin</td><td>Cytoskeleton Inc</td><td>LMN02</td><td></td></tr><tr><td>Gentamicin</td><td>VWR</td><td>VWRV0304-10G</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma Aldrich</td><td>G5882</td><td></td></tr><tr><td>HCl</td><td>Sigma Aldrich</td><td>320331 or similar</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H0888-5G</td><td></td></tr><tr><td>Insulin</td><td>Sigma Aldrich</td><td>16634-50mg</td><td></td></tr><tr><td>MepG Dystroglycan Knockout Cells</td><td>LBNL</td><td>N/A</td><td></td></tr><tr><td>NaOH</td><td>Sigma Aldrich</td><td>S8045 or similar</td><td></td></tr><tr><td>Normocin</td><td>Invivogen</td><td>ant-nr-1</td><td></td></tr><tr><td>Osmium Tetroxide</td><td>Sigma Aldrich</td><td>75633</td><td></td></tr><tr><td>Ovine Prolactin</td><td>Los Angeles Biomedical Research Institute</td><td>Ovine Pituitary Prolactin</td><td></td></tr><tr><td>Phalloidin</td><td>Thermo</td><td>A22287</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Thermo</td><td>10010023 or similar</td><td></td></tr><tr><td>Purified Laminin-111</td><td>Sigma Aldrich</td><td>L2020-1MG</td><td></td></tr><tr><td>Recombinant human Nidogen-1, carrier free</td><td>R&amp;amp;D systems</td><td>2570-ND-050</td><td></td></tr><tr><td>Sodium Acetate</td><td>Sigma Aldrich</td><td>S2889 or similar</td><td></td></tr><tr><td>Sodium Cacodylate</td><td>Sigma Aldrich</td><td>C0250</td><td></td></tr><tr><td>Sterile filters</td><td>Millipore</td><td>SLGP033RS or similar</td><td></td></tr><tr><td>Tris</td><td>Sigma Aldrich</td><td>252859 or similar</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Ultra-low protein binding tubes</td><td>VWR</td><td>76322-516, 76322-522 or similar</td><td></td></tr><tr><td>Ultrapure water</td><td>Thermo</td><td>15230253 or similar</td><td></td></tr></tbody></table>

generating a fractal microstructure of laminin-111 to signal to cells

A laminina-111 (Ln1) é parte essencial da matriz extracelular nos epitélios, sistemas muscular e neural. Nós demonstramos anteriormente que a microestrutura de Ln1 altera a maneira como ele sinaliza para as células, possivelmente porque a montagem de Ln1 em redes expõe diferentes domínios adesivos. Neste protocolo, descrevemos três métodos para gerar Ln1 polimerizado.

A laminina-111 é uma proteína trimérica com três domínios automontáveis no final de seus braços curtos (Figura 1A). Quando tratados adequadamente, os braços curtos podem polimerizar-se em uma rede hexagonal (Figura 1B,1C), que exibe propriedades fractais de agregados limitadas à difusão em escalas espaciais maiores (Figura 2). A laminina incubada em tampões de pH neutro contendo cálcio forma pequenos agr...

Watch this Scientific Journal Video about Generating a Fractal Microstructure of Laminin-111 to Signal to Cells at JoVE.com

Generating a Fractal Microstructure of Laminin-111 to Signal to Cells

Descrevemos três métodos para gerar polímeros Ln1 com propriedades fractais que sinalizam para as células de forma diferente em comparação com Ln1 não polimerizado.

Gerando uma Microestrutura Fractal de Laminina-111 para Sinalizar para Células

Laminin-111 (Ln1) is an essential part of the extracellular matrix in epithelia, muscle and neural systems. We have previously demonstrated that the ...

generating-a-fractal-microstructure-of-laminin-111-to-signal-to-cells

Research

JoVE Journal

Bioengineering

924 visualizações.  Texas Heart Institute. Descrevemos três métodos para gerar polímeros Ln1 com propriedades fractais que sinalizam para as células de forma diferente em comparação com Ln1 não polimerizado.

Vídeo: Generating a Fractal Microstructure of Laminin-111 to Signal to Cells

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Criando Two-Dimensional Substratos modelado para Confinamento de proteína e celular

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Bioengenharia

Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attaches to laminin &#947;-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.

Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution.

Estudos estruturais de macromoléculas em solução usando o pequeno ângulo de espalhamento de raios-x

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and ...

Bioquímica

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

Nanofibras ECM proteínas e Nanoestruturas Engineered Usando Assembléia iniciou-Surface

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microambientes sanduíche-como aproveitarar Telemóveis / interações materiais

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Preparação de 3D colágeno Géis e microcanais para o estudo das interações 3D<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Engenharia tridimensionais tecidos epiteliais incorporado dentro Matriz Extracelular

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

Matrizes ligando Nano-fragmentação em uma bicamada lipídica Compatível

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

Geração de Padrões para Microscopia de Tração de Micropattern

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the cells themselves. Here, we present the method of Light-Induced Molecular Adsorption of Proteins (LIMAP) using the PRIMO system as a patterning technique to produce micro-patterned extracellular matrix (ECM) substrates using a single or combination of proteins. The method enables printing of ECM patterns in micron resolution with excellent reproducibility. We provide a step-by-step protocol and demonstrate how this can be applied to study the processes of neuronal pathfinding. LIMAP has significant advantages over existing micro-printing methods in terms of the ease of patterning more than one component and the ability to generate a pattern with any geometry or gradient. The protocol can easily be adapted to study the contribution of almost any chemical component towards cell fate and cell behavior. Finally, we discuss common issues that can arise and how these can be avoided.

Our overall aim is to understand how cells sense extracellular cues that lead to directed axonal growth. Here, we describe the methodology of Light-Induced Molecular Adsorption of Proteins, used to produce defined micro-patterns of extracellular matrix components in order to study specific events that govern axon outgrowth and pathfinding.

Adsorção molecular induzida por luz de proteínas usando o sistema PRIMO para Micropadronização para estudar as respostas celulares às proteínas da matriz extracelular

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the ...

Neurociência

Fabricating Engineered Nanofibrillar Collagen with Directed Patterning

Production of Nanofibrillar Patterned Collagen for Tissue Engineering

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are designed to emulate the biophysical properties of native tissue, providing cellular phenotypic and morphological guidance that contributes to the restoration of the regenerative tissue niche. Collagen, a prevalent extracellular matrix protein, is a common component of these regenerative biomaterials due to its biocompatibility and other favorable properties. The current study describes a novel and straightforward method for the fabrication of engineered nanofibrillar collagen with directed fibril patterning. Through the manipulation of shear stress, temperature, and pH, collagen fibrillogenesis and alignment are precisely controlled without requiring specialized equipment. This approach allows for the creation of nanofibrillar collagen biomaterials that mimic the native structure of tissues exhibiting either anisotropic or isotropic characteristics. The flexibility in collagen nanofibril patterning not only facilitates the study of nanoscale patterning on cell behavior but also offers diverse possibilities for patterned tissue engineering applications.

The following protocol presents a shear-based extrusion method for the fabrication of collagen hydrogels with nanoscale patterned fibrils, which can be applied to a broad range of tissue engineering applications.

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are ...