CG é apoiada por doações da Agence National de la Recherche (ANR-13-JSV1-0008), a partir do Sétimo Programa-Quadro da União Europeia (Marie Curie Integração Carreira n˚8304162) e do Conselho Europeu de Investigação (ERC) no âmbito do Horizonte da União Europeia 2020 programa de inovação (ERC Starting Grant n˚639300) pesquisa e.

1. Conjugação de ligando a esferas magnéticas Nota: a conjugação ligando é realizada utilizando pérolas activadas por tosilo superparamagnéticas com um diâmetro de 2,8 uM (concentração da solução de estoque 10 8 esferas / mL, 30 mg de esferas / ml). O protocolo seguinte baseia-se amostras de cerca de 2 x 10 5 células, que correspondem a MRC 5-células cultivadas a 80% de confluência numa placa de cultura de tecidos de 60 mm. Ajuste o volume de contas e reagentes de acordo, se utilizando placas de diferentes tamanhos ou células em diferentes confluências. Usar uma quantidade de grânulos superparamagnéticas, a fim de ter 2 contas por célula. Portanto, 4 x 10 5 esferas são necessários para uma placa de 60 mm. <ol><li> Completamente ressuspender as contas dentro do recipiente original em vortex pelo menos 30 s. Aliquota de 40 ul das esferas superparamagnéticas ressuspenso (correspondendo a 4 x 10 6 grânulos) a 1,5 mL tubo de microcentrífuga. </li><li> Colocar o tubo em um separati magnéticano suporte, a fim de separar as esferas magnéticas a partir da solução. Descartar o sobrenadante, remover o suporte do tubo de separação magnética e voltar a suspender as pérolas em 1 ml de 0,1 M fosfato de Na pH 7,4. <ol><li> Repetir o passo de lavagem duas vezes com 1 mL de 0,1 M fosfato de Na pH 7,4. </li></ol></li><li> Após a lavagem final, voltar a suspender as pérolas em 1 ml de 0,1 M de Na-fosfato pH 7,4. </li><li> Combinar 100 ug Bovina A fibronectina (FN) ou qualquer outro ligando de receptor de adesão de célula a 1 mL de 0,1 M fosfato de Na pH 7,4 contendo esferas e misturar por pipetagem. </li><li> Repetir os passos anteriores, substituindo FN ou qualquer outro ligando específico com BSA, poli-D-lisina ou apo-transferrina para controlos negativos. </li><li> Opcionalmente, para análise em gel, ter uma alíquota de 10 ul de solução de ligando para análise de eficiência de ligação cruzada e misturar-se com um volume adequado de tampão de amostra de Laemmli concentrado. </li><li> Incubar grânulos com a solução contendo ligando durante 12-24 h a 37 ° C num rotor. </li><li> Se agregados talão aparecer após a reação ocorreu, sonicate (poder 39 W contínua) para não mais de 10-20 s. </li><li> Isolar as contas usando suporte de separação magnética, aspirar a solução restante e adicionar 1 ml de PBS / BSA a 0,2% pH 7,6 solução. Incubar durante 1 h no rotor a 37 ° C. </li><li> Lavar os grânulos utilizando um magnete duas vezes com 1 mL de PBS / BSA a 0,2%, pH 7,6. Ressuspender as pérolas em 1 ml PBS / BSA a 0,2%, pH 7,6. </li><li> grânulos sonicate se agregados aparecer para não mais do que 20-30 s. </li><li> Opcionalmente, remover uma alíquota de 10 uL para analisar a eficiência de acoplamento por Western blot (misturar com um volume adequado de tampão de amostra de Laemmli concentrado). </li><li> Prossiga para a célula ensaios ou loja de esferas, a 4 ° C por até 1 mês. </li><li> Opcionalmente, correr um gel de SDS-PAGE e mancha com azul de Coomassie para analisar FN reticulação às pérolas magnéticas. </li></ol> 2. Aplicação de forças de tensão sobre as esferas revestidas com ligando ligado à aderência receptores na dorsalSuperfície das células <ol><li> As células aderentes de cultura na placa de cultura de tecido de 60 mm de meio de crescimento adequado (geralmente DMEM 4,5 g / L de D-glucose suplementado com FCS a 10%) até atingir 80% de confluência. </li><li> Preparar tampão de lise não desnaturante não-iónico (20 mM Tris-HCl pH 7,6, NaCl 150 mM, MgCl2 2 mM, 0,1% de NP-40) e tubos de microcentrífuga frio. </li><li> Agregar as esferas revestidas com ligandos de secção 1.10 e ressuspender em 1 ml de PBS / 0,2% BSA a pH 7,6. Adicionar 100 ul da solução de pérolas revestidas com ligandos para 5 mL de meio de cultura quente e vortex. </li><li> médio aspirado na placa de cultura 60 mm e adicionar 5 ml de meio de crescimento quente suplementado com grânulos. </li><li> Incubar durante 20 min sob as condições de cultura de células (37 ° C e 5% de CO 2) para permitir que as esferas para sedimentar e aderir às células. É crucial para não exceder 20 min desde pérolas podem ser internalizados por fagocitose. </li><li> Opcionalmente, observe as contas sob um microscópio de luz usando um 10X ou 20X objective e verificar a aderência do grânulo agitando levemente o prato para discriminar entre esferas ligadas e flutuantes. </li><li> Enquanto se mantinha a placa de cultura na incubadora, trocar a tampa normal de prato para uma tampa com um íman de neodímio 38 milímetros rodada ligado na face superior. O íman está mantido no lugar por dois pequenos ímans de neodímio 13 mm posicionado na face inferior da tampa. Uma vez que os ímãs são muito poderosos, manipulá-los com cuidado. </li><li> Incubar as células submetidas a tensão para os pontos de tempo desejados em condições de cultura de células (37 ° C e 5% CO 2). </li><li> Após o tratamento, prato colocar em gelo e cuidadosamente aspirar todo o meio a partir do prato. </li><li> Adicionar 300 ul de tampão de lise de células, e incuba-se durante 10 min em gelo. Recolhe-se o lisado usando um raspador de células e transferir para um tubo de microcentrífuga de 1,5 ml pré-arrefecido. </li><li> Agregar as esferas magnéticas, utilizando o suporte de separação magnética e transferir o lisado celular total a um novo pré-frioed 1,5 mL tubo de microcentrífuga. Conserva-se a fracção de -20 ° C para posterior análise. </li><li> Lavam-se as esferas 3 vezes com 1 mL de tampão de lise arrefecido com gelo. Adicionar 50 ul de tampão de amostra de Laemmli ao sedimento de grânulo, misturar por pipetagem e em ebulição a 95 ° C durante 5 min, utilizando um aquecedor de bloco seco. Esta fracção contém os complexos de adesão isoladas. Proceder à análise bioquímica ou armazenar a -20 ° C. </li><li> A partir do lisado celular total obtida após separação de grânulos (cerca de 300 mL), remover uma alíquota de 50 uL para análise de mancha ocidental. NOTA: Os 250 uL esquerda pode ser utilizado em estudos bioquímicos, tais como estudos de interacção proteína-proteína, ensaios de imunoprecipitação (GST pull-down) ou ensaios de actividade de GTPase (mudando o tampão de lise pode ser necessário, dependendo da GTPase de interesse). </li></ol>

<ol>
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Os autores declaram não haver interesses financeiros concorrentes.

O método aqui descrito constitui uma abordagem simples para aplicar a tensão de receptores de adesão da superfície celular e permitir a sua purificação posterior. No entanto, alguns passos são essenciais para realizar a purificação eficiente de adesão e optimização de potencial pode ser feito em função dos receptores de adesão segmentados. Nós apresentamos os possíveis problemas que o usuário pode encontrar abaixo. Utilizou-se 2,8 um de diâmetro, mas as esferas magnéticas...

Em metazoa, tensão mecânica dirige o desenvolvimento do tecido e da homeostase através da regulação de uma miríade de processos celulares, como a proliferação, diferenciação e sobrevivência 1, 2. tensão mecânica pode surgir a partir da matriz extracelular ou podem ser gerados por células aderentes, que amostra o ambiente extracelular através da maquinaria contrátil actomiosina que puxa na matriz extracelular e as sondas a sua rigidez por meio de moléculas sensíveis à tensão. Em resposta à tensão, proteínas de adesão mechanosensitive sofrer alterações conformacionais que desencadeiam cascatas de sinalização complexas. Por sua vez, estas vias de sinalização orquestrar uma mechanoresponse englobando a proliferação, diferenciação e sobrevivência que ajusta o comportamento celular para o ambiente extracelular. Tais processos podem ser resolvidos em um período de curto prazo (segundos a minutos) para alimentar rapidamente de volta para o loop de mecha<html> notransduction modificando as estruturas mechanosensitive. Por exemplo, as adesões à base de integrina reforçar em resposta a tensão através de Rho GTPase mediada por remodelação do citoesqueleto 3, 4, 5. Em paralelo, outras vias de sinalização são ativados, durante horas e dias para controlar programas genéticos que, eventualmente, impactar o destino celular 6. Considerando que, muitos estudos têm destacado o efeito da rigidez da matriz no determinismo celular e desenvolvimento da doença 1, 2, os mecanismos moleculares precisos de mecanotransdução mediada por adesão continuam a ser evasivo. Várias abordagens foram desenvolvidas para estudar os efeitos de forças gerado-celulares ou as forças externas sobre o comportamento das células, incluindo sistemas de fluxo, de transferência de energia de ressonância de fluorescência (FRET) -tension sensores 7,lass = &quot;xref&quot;&gt; 8, 9 substratos compatíveis, pinças magnéticos, pinças ópticas 10 e de microscopia de força atômica (AFM) 11. Aqui apresenta-se um protocolo utilizando esferas superparamagnéticas para caracterizar vias mecanotransdução em resposta a forças de tensão aplicadas aos receptores de adesão específicas. superparamagnéticas grânulos são partículas que magnetizam reversivelmente, quando colocado num campo magnético. Uma vez revestida com um ligando para um receptor específico, estes grânulos de fornecer uma ferramenta poderosa para estudar os efeitos da aplicação de força extracelular. Este método foi validado por diversos estudos 3, 5, 12 - 17 e apresentam a vantagem de facilitar, em grande parte análises bioquímicas nas células aderentes. Usando esferas magnéticas revestidas com colagénio semelhante, seguido de análise bioquímica, os primeiros trabalhos relatados um aumento doa fosforilação da proteína tirosina e a activação de RhoA, em resposta à tensão 5, 18, 19. O método descrito abaixo foi também usado com fibronectina (FN) grânulos -Revestido para caracterizar as vias de sinalização a jusante da tensão aplicada às integrinas 3. Neste estudo, Guilluy et ai. mostrou que a tensão ativa RhoA através do recrutamento dos dois fatores de câmbio nucleótido guanina (GEFs), Larg e GEF-H1, a complexos de adesão integrina. Desde que, outros estudos mostraram que GEF-H1 é recrutado para complexos de adesão em resposta a tensão gerada por células utilizando diferentes métodos de 20, 21, demonstrando a robustez da metodologia aqui descrita. Como resultado, RhoA activada foi mostrado para promover a adesão de reforço, por meio de remodelação do citoesqueleto. Este sistema também foi utilizado para explorar a tensão aplicada tO receptores de adesão / célula a célula. A aplicação de forças sobre esferas magnéticas revestidas com o domínio extracelular da caderina-E induziu um aumento no recrutamento vinculina semelhante a integrina associada complexos de adesão 12. Collins e colegas observaram que a aplicação de tensão para PECAM-1 promove a integrina e ativação RhoA 13. Outra abordagem experimental utilizando esferas magnéticas é o estudo da tensão aplicada aos núcleos isolados. Usando esferas revestidas com anticorpos contra a proteína do envelope nuclear nesprin-1, os complexos de envelope nuclear foram purificados para mostrar que eles são regulados dinamicamente em resposta a tensão mecânica 22. Estes resultados suportam a powerfulness deste método no estudo das vias mecanotransdução. Além disso, enquanto que os sistemas de fluxo de força de tracção ou estimular os processos celulares gerais, esferas magnéticas visar especificamente um receptor de adesão de células utilizando um ligandos de receptores 13, 15. Outra vantagem deste método é o isolamento de complexos de adesão por meio de um procedimento de purificação por afinidade de ligando simples. É bem sabido que a adição de contas revestidas com ligando se liga a receptores de adesão de células e induz o recrutamento de várias proteínas de adesão 23. Além disso a aplicação de forças de esferas magnéticas revestidas com ligando transforma estes complexos de adesão em plataformas macromoleculares que medeia várias vias de sinalização dependentes de tensão 4, 24. A lise celular seguido de concentração grânulo utilizando um íman permite o isolamento das plataformas de adesão. Outros métodos utilizados para purificar complexos de adesão já têm sido utilizados em células aderentes. Eles combinam reticulação química para conservar as interacções proteína-proteínae um passo de lise celular por detergente e sonicação ou fluxo de cisalhamento 20, 21, 25, 26, 27, 28. O último passo é a coleta das membranas plasmáticas ventral resultantes contendo os complexos de adesão. Ao contrário destes métodos, esferas magnéticas permitir um nível de purificação maior de complexos de adesão celular por segmentação selectivamente uma família específica de receptores de adesão. Esférulas magnéticas já têm sido utilizadas para purificar complexos de adesão em células não-aderentes ligadas a microesferas revestidas com ligando, 29 30. O método descrito abaixo imita situações biológicas, onde a força é aplicada por um curto período sustentado (segundos a minutos). Portanto, ele fornece uma ferramenta poderosa para investigar tanto a composição molecular de complexos de adesão purificada eas vias a jusante mechanosensitive de sinalização. Aqui apresentamos um protocolo experimental detalhado para usando esferas magnéticas para aplicar forças de tracção às proteínas de superfície de adesão. Um íman de neodímio permanente é colocada no topo da superfície do prato de cultura. A face do polo do íman é colocado a uma altura de 6 mm, de modo que a força sobre uma única 2,8 uM esférulas magnéticas é constante (cerca de 30-40 PN) 31. A duração da estimulação tensão é determinado pelo operador, dependendo da molécula de interesse e o seu tempo de activação escala. As células são lisadas, finalmente, complexos de adesão são purificados por separação de grânulos utilizando um íman e análises bioquímicas são processados. Este protocolo inclui a preparação de grânulos superparamagnéticas recobertas pelo ligante, bem como a aplicação de tensão através do ímã seguido por análises bioquímicas. Além disso, nós fornecemos uma amostra representativa de dados que demonstram que a tensão aplicada à base ADH-integrinaesions induz adesão remodelação e altera a fosforilação da proteína tirosina.

<table><tbody><tr><td>Neodymium magnets (on the upper face of 60 mm dish)</td><td>K&amp;amp;J Magnetics, Inc</td><td>DX88-N52</td><td>grade N52 dimension: 1 1/2&quot; dia. x 1/2&quot; thick</td></tr><tr><td>Neodymium magnets (on the lower face of 60 mm dish)</td><td>K&amp;amp;J Magnetics, Inc</td><td>D84PC-BLK</td><td>grade N42 dimension: 1/2&quot; dia. x 1/4&quot; thick Black Plastic Coated&amp;nbsp;</td></tr><tr><td>Dynabeads M280 Tosylactivated</td><td>Thermofisher</td><td>14203</td><td>superparamagnetic beads&amp;nbsp;</td></tr><tr><td>DynaMag-2 Magnet</td><td>Thermofisher</td><td>12321D</td><td></td></tr><tr><td>Fibronectin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F1141-5MG</td><td>Fibronectin from bovine plasma</td></tr><tr><td>Poly-D-Lysine</td><td>Sigma-Aldrich</td><td>P7280-5MG</td><td></td></tr><tr><td>Apo-Transferrin</td><td>Sigma-Aldrich</td><td>T1428-50MG</td><td>Bovine Apo-Transferrin</td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7906-500G</td><td></td></tr><tr><td>DMEM high glucose, GlutaMAX supplement, pyruvate&amp;nbsp;</td><td>Life Technologies</td><td>31966-021</td><td>DMEM+GlutaMAX-I 500 ml&amp;nbsp;</td></tr><tr><td>60*15 mm culture dish</td><td>Falcon</td><td>353004</td><td></td></tr></tbody></table>

analyzing cell surface adhesion remodeling in response to mechanical tension using magnetic beads

complexos de adesão da superfície celular Mechanosensitive permitir que as células para detectar as propriedades mecânicas dos seus arredores. Estudos recentes identificaram duas moléculas de detecção de força em locais de adesão e fatores de transcrição dependente da força que regulam a expressão de genes específicos de linhagem e impulsionam resultados fenotípicos. No entanto, as redes de sinalização de conversão de tensão mecânica em vias bioquímicas têm permanecido elusivos. Para explorar as vias de sinalização envolvidos em cima aplicada a tensão mecânica receptor da superfície celular, micropérolas superparamagnéticas pode ser usado. Aqui é apresentado um protocolo para a utilização de contas magnéticas para aplicar forças de proteínas de adesão da superfície celular. Utilizando esta abordagem, é possível investigar não apenas as vias de sinalização citoplasmática dependente da força por várias abordagens bioquímicas, mas também pode ser notada aderência por isolamento magnético de complexos de adesão ligados às esferas revestidas com ligando. Este protocolo inclui a preparação do ligando-coesferas superparamagnéticas ciados, e a aplicação de definir forças de tração, seguido por análises bioquímicas. Além disso, nós fornecemos uma amostra representativa de dados que demonstram que a tensão aplicada a aderência à base de integrina desencadeia adesão remodelação e altera a fosforilação da tirosina de proteína.

O esquema da técnica é ilustrada na figura 1a. Após a conjugação ligando, esferas magnéticas são incubados com as células durante 20 minutos, e, em seguida, um íman permanente é usada para aplicar forças de tracção de cerca de 30-40 NP para várias quantidades de tempo. A Figura 1b mostra 2,8 ^ M FN-esferas magnéticas revestidas com destino a receptores de adesão de células MRC5. Os passos de lavagem de grânulos superparamagnéticas após li...

Watch this Scientific Journal Video about Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads at JoVE.com

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

aderências na superfície celular são centrais na mecanotransdução, como eles transmitem tensão mecânica e iniciar as vias de sinalização envolvidos na homeostase e desenvolvimento do tecido. Aqui, apresentamos um protocolo para dissecar as vias bioquímicas que são ativadas em resposta à tensão, utilizando microesferas magnéticas revestidas com ligando e aplicação de força para receptores de adesão.

Analisando celular superfície de adesão remodelação em resposta à tensão mecânica usando pérolas magnéticas

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...

analyzing-cell-surface-adhesion-remodeling-response-to-mechanical

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

8.4K visualizações.  Centre de recherche UGA - INSERM U1209 - CNRS UMR.  aderências na superfície celular são centrais na mecanotransdução, como eles transmitem tensão mecânica e iniciar as vias de sinalização envolvidos na homeostase e desenvolvimento do tecido. Aqui, apresentamos um protocolo para dissecar as vias bioquímicas que são ativadas em resposta à tensão, utilizando microesferas magnéticas revestidas com ligando e aplicação de força para receptores de adesão. 

Vídeo: Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

Multiplexados molécula única Força Medidas proteólise, usando uma pinça magnética

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

Bioengenharia

Magnetic Tweezers for the Measurement of Twist and Torque

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a &#8220;conventional&#8221; magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the &#8220;conventional&#8221; magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.

Magnetic tweezers, a powerful single-molecule manipulation technique, can be adapted for the direct measurements of the twist (using a configuration called freely-orbiting magnetic tweezers) and torque (using a configuration termed magnetic torque tweezers) in biological macromolecules. Guidelines for performing such measurements are given, including applications to the study of DNA and associated nucleo-protein filaments.

Pinças magnéticas para a Medição do Twist and Torque

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques ...

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

Conta Agregação ensaios para a caracterização dos putativos Moléculas de Adesão Celular

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

Um novo método para localizar Repórter fluorescentes Beads perto da superfície de cultivo celular para Tração Microscopia de Força

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Magnetic Tweezers in a Microplate Format

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these approaches are limited single-molecule methods, which restricts their availability. To address this need, a microplate assay was developed that enables mechanical manipulation while performing standard biochemical assays. This is achieved using magnets incorporated into a microplate lid to create multiple magnetic tweezers. In this format, force is exerted across biomolecules connected to paramagnetic beads, equivalent to a typical magnetic tweezer. The study demonstrates the application of this tool with FRET-based assays to monitor protein conformations. However, this approach is widely applicable to different biological systems ranging from measuring enzymatic activity through to the activation of signaling pathways in live cells.

Here, we describe the use of a novel microplate assay to enable mechanical manipulation of biomolecules while performing ensemble biochemical assays. This is achieved using a microplate lid modified with magnets to create multiple static magnetic tweezers across the microplate.

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these ...

Bioquímica

High-Speed Magnetic Tweezers for Nanomechanical Measurements on Force-Sensitive Elements

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are therefore poised to be useful in the field of mechanobiology. Since the method commonly relies on image-based tracking of magnetic beads, the speed limit in recording and analyzing images, as well as the thermal fluctuations of the beads, has long hampered its application in observing small and fast structural changes in target molecules. This article describes detailed methods for the construction and operation of a high-resolution MT setup that can resolve nanoscale, millisecond dynamics of biomolecules and their complexes. As application examples, experiments with DNA hairpins and SNARE complexes (membrane-fusion machinery) are demonstrated, focusing on how their transient states and transitions can be detected in the presence of piconewton-scale forces. We expect that high-speed MTs will continue to enable high-precision nanomechanical measurements on molecules that sense, transmit, and generate forces in cells, and thereby deepen our molecular-level understanding of mechanobiology.

Here, we describe a high-speed magnetic tweezer setup that performs nanomechanical measurements on force-sensitive biomolecules at the maximum rate of 1.2 kHz. We introduce its application to DNA hairpins and SNARE complexes as model systems, but it will be also applicable to other molecules involved in mechanobiological events.

Pinças magnéticas de alta velocidade para medições nanomecânicas em elementos sensíveis à força

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are ...

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

Controle da adesão celular usando técnicas de padronização de hidrogel para aplicações em microscopia de força de tração

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...

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 ...

Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate underlying a cell must be manipulated. While hydrogels with graded stiffness and long-term migration assays have proven useful in durotaxis studies, immediate, acute responses to local changes in substrate tension allow focused study of individual cell movements and subcellular signaling events. To repeatably test the ability of cells to sense and respond to the underlying substrate stiffness, a modified method for application of acute gradients of increased tension to individual cells cultured on deformable hydrogels is used which allows for real time manipulation of the strength and direction of stiffness gradients imparted upon cells in question. Additionally, by fine tuning the details and parameters of the assay, such as the shape and dimensions of the micropipette or the relative position, placement, and direction of the applied gradient, the assay can be optimized for the study of any mechanically sensitive cell type and system. These parameters can be altered to reliably change the applied stimulus and expand the functionality and versatility of the assay. This method allows examination of both long term durotactic movement as well as more immediate changes in cellular signaling and morphological dynamics in response to changing stiffness.

Mechanical forces are important for controlling cell migration. This protocol demonstrates the use of elastic hydrogels that can be deformed using a glass micropipette and a micromanipulator to stimulate cells with a local stiffness gradient to elicit changes in cell structure and migration.

Ensaio de Durotaxis de célula única para avaliar o controle mecânico do movimento celular e eventos de sinalização relacionados

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate ...

Biologia

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.