Este trabalho é apoiado pelo NIH R01 GM47607, CA163296 e NSF 1334493 a CET. Agradecemos a membros do laboratório Turner para a leitura crítica do manuscrito. Todos os dados apresentados neste relatório foram reproduzidas com permissão a partir de 2014 11 Wormer et al..

1. Preparação de Durotaxis Chambers <ol><li> Para preparar uma placa de 6 poços de cultura de tecidos, tarar a balança com um tubo cónico de 50 ml. Pesar aproximadamente 10 g de solução de base de PDMS no tubo de 50 ml (a solução é bastante viscosa). </li><li> Para uma 90: 1 substrato (Conformidade de ~ 1 kPa), dividir o peso medido da solução de base de PDMS no tubo por 90 para determinar a quantidade correcta de agente reticulante solução necessário. Adicionar a quantidade calculada da solução de agente reticulante PDMS ao mesmo tubo. Exemplo: 10 g / 90 = 0,11 g. Adicionar 0,11 g da solução de agente de reticulação à solução PDMS base de PDMS. NOTA: A base e do agente reticulante soluções são ambos fornecidos no kit de PDMS. </li><li> Misturar vigorosamente a mistura de base / agente de reticulação de PDMS durante 5 min à temperatura ambiente usando uma pequena espátula. Nesta fase, a mistura conterá um grande número de bolhas de ar. </li><li> Centrifuga-se o substrato de PDMS numa centrífuga de bancada durante 5 minutos a 50 xg à temperatura ambiente para remover a bubbles. Se ainda houver bolhas após a 5 min, centrifugar novamente. </li><li> Pipete 1 mL de 90: 1 PDMS substrato em cada poço de cultura de tecidos de 6 cavidades tratadas placa. As bolhas de ar restantes presentes nos PDMS pode ser eliminado nesta fase por popping-los usando uma agulha 21 G. Permitir que o PDMS para espalhar durante 30 min no poço. </li><li> Ferva 12 milímetros de vidro # 1 lamelas em água destilada por 5 min. Repita duas vezes e armazenar as lamelas em água destilada. </li><li> Coloque um, secou-se a lamela em cada poço da placa de cultura de tecidos tocando suavemente um lado da lamela na solução, em seguida, deixar cair o PDMS lamela para o PDMS. À medida que a lamela se instala, o PDMS começará a invadir ao longo dos bordos da lamela, mas não se cobri-lo completamente. Isto vai gerar uma interface entre o PDMS e de vidro após a cura (ver Figura 1A, B). </li><li> Incubar a placa a 70 ° C num forno durante 16 horas para curar (Harden) do PDMS. Coloque o plate em uma capa de cultura de células e UV esterilizar durante 10 min. NOTA: É melhor fazer as placas dentro de alguns dias de uso. No entanto, as placas podem ser envoltos em parafilme sem qualquer tampão e armazenado a 4 ° C durante até 2 semanas, sem qualquer queda significativa na qualidade. </li></ol> 2. celular Galvanização NOTA: Se se estudar o efeito do knockdown mediada por siRNA, executar o knockdown utilizando as instruções do fabricante ou o protocolo optimizado para o tipo de célula de escolha. <ol><li> Revestimento durotaxis cada câmara com 1 ml de 10 ug / ml de fibronectina em PBS sem cálcio e magnésio durante 1 hora a 37 ° C. Alternativamente, a fibronectina pode ser aplicado no dia antes da análise por incubação das PDMS com 10 ug / ml de fibronectina em PBS durante 16 horas a 4 ° C. Certifique-se de toda a superfície é submersa na solução de fibronectina em PBS. </li><li> Prepare desnaturado termicamente 1% BSA. Pesar 0,5 g de BSA e dissolvê-lo em 50 ml de PBS.Filtro de esterilizar a solução através de um filtro de 0,22 um e de calor a 80 ° C durante 12 min. Nota: Preparar esta solução no dia antes da célula de galvanização e armazenar a 4 ° C. </li><li> Aspirar a solução de fibronectina e lavar 3 vezes com PBS. Adicionar 1 ml de BSA desnaturado por calor em PBS a cada poço e incubar durante 30 min à temperatura ambiente. </li><li> Trypsinize e contar as células de escolha, enquanto as câmaras durotaxis estão bloqueando com o calor desnaturado BSA. </li><li> Placa 1 x 10 5 células em um volume de 2 ml em cada poço da câmara durotaxis, usando os meios necessários para o tipo particular de célula de escolha. Permitir que as células aderir e espalhada sobre o substrato durante 4 horas numa incubadora humidificada a 37 ° C com 5% de CO 2. NOTA: As células U2OS são rotineiramente mantidas em DMEM com 10% de FBS, suplementado com 2 mM de L-glutamina, piruvato de sódio 1 mM, e 10 UI / ml de penicilina, 10 ug / ml de estreptomicina. NOTA: O número de células utilizado neste exemplo é optimizado paraU2OS células. Optimização para outros tipos de células pode não ser necessária. Esta densidade dá as células espaço suficiente para migrar sem interações significativas com outras células. </li></ol> Imagem 3. Em células vivas <ol><li> Execute imagens de células vivas em um microscópio invertido, usando contraste de fase com uma objetiva de 10X. O microscópio deve ser equipado com uma câmara humidificada ambiente fechado, permitindo o controlo de temperatura a 37 ° C e 5% de CO 2 durante o exame a longo prazo. </li><li> Depois que as células se espalharam por cerca de 3,5 horas, montar a placa na câmara de microscópio. Deixar a amostra (s) para equilibrar na câmara durante 30 min. </li><li> Configure automatizado, multi-ponto de visitação no microscópio, se disponível. Concentre-se na interface entre as 90: 1 PDMS ea lamela de vidro e escolher aponta para imagem em todo o interface com um média de 40 pontos por durotaxis câmara. Imagem das células a cada 10 minutos até 16 horas. NOTA: A região de interesse aparece como duas linhas, com a linha exterior, correspondente ao bordo da lamela e a linha interna correspondente à interface real entre o PDMS e a lamela. Ver Figura 1B. </li></ol> Análise 4. Dados <ol><li> Gerar uma planilha como na Tabela 1. </li><li> Contar o número de eventos de passagem a partir dos PDMS à superfície do vidro e vice-versa de cada filme gerado. Grave o número de eventos de passagem na coluna apropriada na planilha excel. NOTA: Um evento de cruzamento é definido como o núcleo da célula, passando através da fronteira entre o PDMS e de vidro em um ou outro sentido. </li><li> Para quantificar múltiplos cruzamentos, contar o número de vezes que a célula atravessada a interface. Este número deve ser gravado na coluna Excel correspondente ao substrato sobre o qual a célula foi localizado no final do filme. Repita a análise para cada célula que atravessa a interface em tfilme que ele. Excluir células que migram para fora do campo de visão durante o exame. NOTA: É também importante para verificar, por contagem de células no início da experiência, as células que são capazes de aderir igualmente aos PDMS revestido com fibronectina e superfícies de lamela de vidro. </li><li> Calcula-se a percentagem de células que migraram a partir de PDMS para a superfície do vidro (isto é, submetidos a durotaxis). Adicione o número de cruzar eventos de suave a difícil e os múltiplos eventos de passagem, que terminou no disco e dividir pelo número total de eventos de passagem. </li><li> Calcula-se a percentagem de passagens múltiplas através da divisão do número de passagens múltiplas pelo número total de cruzamentos. </li></ol>

<ol><li>Engler, A. J., Sen, S., Sweeney, H. L., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Matrix+elasticity+directs+stem+cell+lineage+specification%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Matrix elasticity directs stem cell lineage specification.</a> Cell. 126 (4), 677-689 (2006).</li>
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<li>Levental, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Matrix+crosslinking+forces+tumor+progression+by+enhancing+integrin+signaling%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Matrix crosslinking forces tumor progression by enhancing integrin signaling.</a> Cell. 139 (5), 891-906 (2009).</li>
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<li>Choquet, D., Felsenfeld, D. P., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+matrix+rigidity+causes+strengthening+of+integrin-cytoskeleton+linkages%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Extracellular matrix rigidity causes strengthening of integrin-cytoskeleton linkages.</a> Cell. 88 (1), 39-48 (1997).</li>
<li>Balaban, N. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Force+and+focal+adhesion+assembly%3A+a+close+relationship+studied+using+elastic+micropatterned+substrates%5BTitle%5D)+AND+%22Nature+Cell+Biology%22%5BJournal%5D)">Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates.</a> Nature Cell Biology. 3 (5), 466-472 (2001).</li>
<li>Provenzano, P. P., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanical+signaling+through+the+cytoskeleton+regulates+cell+proliferation+by+coordinated+focal+adhesion+and+Rho+GTPase+signaling%5BTitle%5D)+AND+%22Journal+of+Cell+Science%22%5BJournal%5D)">Mechanical signaling through the cytoskeleton regulates cell proliferation by coordinated focal adhesion and Rho GTPase signaling.</a> Journal of Cell Science. 124 (8), 1195-1205 (2011).</li>
<li>Wang, Y. L., Pelham, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preparation+of+a+flexible%2C+porous+polyacrylamide+substrate+for+mechanical+studies+of+cultured+cells%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">Preparation of a flexible, porous polyacrylamide substrate for mechanical studies of cultured cells.</a> Methods in Enzymology. 298, 489-496 (1998).</li>
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<li>Wormer, D. B., Davis, K. A., Henderson, J. H., Turner, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+focal+adhesion-localized+CdGAP+regulates+matrix+rigidity+sensing+and+durotaxis%5BTitle%5D)+AND+%22PloS+ONE%22%5BJournal%5D)">The focal adhesion-localized CdGAP regulates matrix rigidity sensing and durotaxis.</a> PloS ONE. 9 (3), e91815(2014).</li>
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The authors have no conflicts to disclose.

Aqui nós descrevemos um ensaio simples para estudar durotaxis em células migrando. Uma grande força deste ensaio é a facilidade de preparação das câmaras durotaxis utilizando PDMS. A rigidez dos substratos pode ser facilmente manipulado por alteração da proporção de solução de base de PDMS de agente de reticulação para permitir o estudo de vários elementos de rigidez no ensaio. No entanto, uma limitação potencial do sistema é que apenas as células são expostas a uma única mudança na rigidez do sub...

A matriz extracelular (ECM) é constituído por uma matriz complexa de proteínas estruturais e de reticulação, incluindo colagénio, fibronectina e laminina. Embora seja bem estabelecido que a ECM fornece um suporte estrutural importante para os tecidos celulares, há evidências crescentes de que indicam que as células respondem activamente a mudanças físicas no seu ambiente de ECM para regular processos celulares, incluindo diversas sobrevivência celular, diferenciação e migração da célula. Por exemplo, diferenças na rigidez do ECM podem conduzir as células estaminais mesenquimais para linhagens diferentes, com substratos macios (~ 1 kPa) a promoção de linhagens neurogénicos enquanto substratos rígidos (~ 25 kPa) promover a diferenciação osteogénica 1. Da mesma forma, foi demonstrado um aumento na rigidez da matriz estromal para promover a tumorigénese célula epitelial mamaria e invasão do tecido circundante 2,3. Um aspecto particularmente interessante deste mechanosignaling resultados da actividade em um processo conhecido como durotaxis, em que as células migram preferencialmente para um substrato mais rígido 4,5. Células sentir constantemente as características físicas do ambiente extracelular através da ligação do receptor de integrina ao ECM. Este, por sua vez, promove a acumulação de numerosas proteínas estruturais e de sinalização para os seus domínios citoplasmáticos para conduzir a formação de estruturas adesivas conhecidas como adesões focais ou contactos focais 6,7. Dado que as integrinas não têm actividade enzimática inerente, os sinais são transmitidos a partir do ECM através destas proteínas acessórias para coordenar a resposta da célula ao seu ambiente em mudança 8. Consequentemente, a identificação e caracterização das proteínas-chave envolvidas na regulação mechanosignaling durotaxis e é uma área importante de investigação. Vários sistemas de modelos têm sido desenvolvidos para estudar durotaxis in vitro, mas a maioria têmutilizados substratos de poliacrilamida revestidos com colagénio 4. No entanto, a preparação dos substratos de poliacrilamida pode ser tecnicamente exigente e o colagénio utilizado nestes ensaios devem ser quimicamente reticulado ao substrato 9. Polidimetilsiloxano (PDMS) substratos têm mostrado exibir propriedades mecânicas comparáveis ​​aos substratos de poliacrilamida a 10. No entanto, os substratos PDMS são preparadas simplesmente misturando uma proporção de base para o agente de reticulação e esses substratos podem ser revestidos com proteínas de ECM sem a necessidade de reticulação química, tornando assim mais fácil PDMS uma ferramenta para estudar os efeitos de rigidez no comportamento de células. Aqui, nós descrevemos como preparar uma câmara durotaxis simples em que um substrato macio PDMS é integrado com uma lamela de vidro rígida. O ensaio, conforme descrito abaixo, proporciona um método rápido e simples para estudar durotaxis. Para este estudo foram utilizadas células de osteossarcoma U2OS humanos, combinado com siRNA mediadaknockdown de cdGAP para estudar o papel desta proteína de adesão focal em durotaxis 11. É importante ressaltar que este protocolo pode ser facilmente adaptado às necessidades individuais. Outros tipos celulares podem ser substituídos para as células U2OS e qualquer proteína pode ser batido ou sobre-expresso para determinar os efeitos sobre o comportamento das células durante durotaxis. Além disso, este protocolo pode ser adaptado para incorporar proteínas fluorescente etiquetado para analisar sua dinâmica e comportamento usando FRAP ou FRET abordagens.

<table><tbody><tr><td>Polydimethylsiloxane (PDMS)</td><td>Dow Corning</td><td>3097358-1004</td><td>Sylgard 184 Silicone Elastomer Kit</td></tr><tr><td>#1 Cover glass 12 mm</td><td>Fisher Scientific</td><td>12-545-82</td><td></td></tr><tr><td>6-well plate</td><td>Celltreat</td><td>229106</td><td></td></tr><tr><td>DMEM</td><td>Cellgro</td><td>15-017-CM</td><td></td></tr><tr><td>L-Glutamine</td><td>Cellgro</td><td>25-005-CI</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Fisher Scientific</td><td>BP356-100</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Cellgro</td><td>30-002-CI</td><td></td></tr><tr><td>Fibronectin</td><td>BD Biosciences</td><td>610077</td><td></td></tr><tr><td>PBS</td><td>Invitrogen</td><td>21600-044</td><td></td></tr><tr><td>Falcon tubes</td><td>Celltreat</td><td>229456</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Atlanta Biologicals</td><td>S11150</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>U2OS cells</td><td>ATCC</td><td>HTB-96</td><td></td></tr></tbody></table>

a simplified system for evaluating cell mechanosensing and durotaxis in vitro

As propriedades mecânicas da composição e da matriz extracelular são altamente variáveis ​​entre tipos de tecidos. Este tecido conjuntivo diversidade estroma grandemente impactos comportamento celular para regular os processos normais e patológicos, incluindo a proliferação celular, diferenciação, migração e adesão de sinalização direccional. A este respeito, a capacidade inata de determinados tipos de células para migrar em direcção um substrato mais rígido, ou menos complacente matriz é referida como durotaxis. Este fenómeno desempenha um papel importante durante o desenvolvimento embrionário, cicatrização de feridas e a invasão de células do cancro. Aqui, nós descrevemos um ensaio simples para estudar durotaxis, in vitro, utilizando PDMS (polidimetilsiloxano) substratos. Preparação do descrito durotaxis câmaras cria uma interface entre a rigidez do gel PDMS relativamente macio e uma lamela de vidro rígida. No exemplo fornecido, temos usado estes durotaxis câmaras para demonstrar um papel para o cdc42 / Rac1 GTPase ativando proteem, cdGAP, em mechanosensing e durotaxis regulamentação em células U2OS osteossarcoma humano. Este ensaio é facilmente adaptável a outros tipos e / ou knockdown de outras proteínas de interesse celulares para explorar seus respectivos papéis na mechanosignaling e durotaxis.

Um esquema da câmara durotaxis é mostrado na Figura 1A. Substrato PDMS macio (uma 90: 1 mistura de base de PDMS de reticulador soluções) é espalhada em uma 6 bem prato e uma lamela de vidro é colocado no topo do PDMS, que, em seguida, cobre parcialmente a superfície superior da lamela, criando, assim, uma interface entre os dois substratos de conformidade diferente. A rigidez do substrato macio PDMS é ~ 1 kPa, o que é comparável ao cumprimento típica do tecido do cérebro, en...

Watch this Scientific Journal Video about A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro at JoVE.com

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

Um sistema simplificado para a Avaliação celular Mechanosensing e Durotaxis<em&gt; In Vitro</em

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...

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Research

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Biology

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

8.2K visualizações.  SUNY Upstate Medical University. Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

Vídeo: A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to understand morphogenetic processes is to directly measure cellular forces and mechanical properties in vivo during embryogenesis. Here, we present a setup of optical tweezers coupled to a light sheet microscope, which allows to directly apply forces on cell-cell contacts of the early Drosophila embryo, while imaging at a speed of several frames per second. This technique has the advantage that it does not require the injection of beads into the embryo, usually used as intermediate probes on which optical forces are exerted. We detail step by step the implementation of the setup, and propose tools to extract mechanical information from the experiments. By monitoring the displacements of cell-cell contacts in real time, one can perform tension measurements and investigate cell contacts&#39; rheology.

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

We present a setup of optical tweezers coupled to a light sheet microscope, and its implementation to probe cell mechanics without beads in the Drosophila embryo.

Sondando a célula mecânica com pinça óptica de grânulo-livre no embrião da drosófila

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to ...

Biologia do Desenvolvimento

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila larvae, customized metal nickel-titanium alloy (nitinol) filaments. These mechanical probes are similar to the von Frey filaments used in vertebrates to measure mechanical nociception. Here, we demonstrate how to make and calibrate these mechanical probes and how to generate a full behavioral dose-response from subthreshold (innocuous or non-noxious range) to suprathreshold (low to high noxious range) stimuli. To demonstrate the utility of the probes, we investigated tissue damage-induced hypersensitivity in Drosophila larvae. Mechanical allodynia (hypersensitivity to a normally innocuous mechanical stimulus) and hyperalgesia (exaggerated responsiveness to a noxious mechanical stimulus) have not yet been established in Drosophila larvae. Using mechanical probes that are normally innocuous or probes that typically elicit an aversive behavior, we found that Drosophila larvae develop mechanical hypersensitization (both allodynia and hyperalgesia) after tissue damage. Thus, the mechanical probes and assay that we illustrate here will likely be important tools to dissect the fundamental molecular/genetic mechanisms of mechanical hypersensitivity.

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

The goal of this protocol is to show how to perform an improved assay for mechanical nociception in Drosophila larvae. We use the assay here to demonstrate that mechanical hypersensitivity (allodynia and hyperalgesia) exists in Drosophila larvae.

Um ensaio e ferramentas aprimoradas para medir o nocicepção mecânico em larvas de Drosophila

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila ...

Comportamento

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

Resposta celular ao vivo para Estimulação mecânicas estudadas por Microscopia de Força Atômica e Óptica Integrada

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Biologia

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes.

Here, we present a protocol to investigate the intracellular mechanical properties of isolated embryonic zebrafish cells in three-dimensional confinement with direct force measurement by an optical trap.

Medições de força direta de mecânica subcelular em confinamento usando pinças ópticas

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Clones em um ensaio comportamental para Mechanosensation de MarcM baseada em<em&gt; Drosophila melanogaster</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Neurociência

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

Medição de Quimiotaxia celulares com ECIS / Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

Mechano-Node-Pore Sensing: A Rapid, Label-Free Platform for Multi-Parameter Single-Cell Viscoelastic Measurements

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer metastasis. Conventional methods for measuring these properties, such as atomic force microscopy (AFM) and micropipette aspiration (MA), capture rich information, reflecting a cell's full viscoelastic response; however, these methods are limited by very low throughput. High-throughput approaches, such as real-time deformability cytometry (RT-DC), can only measure limited mechanical information, as they are often restricted to single-parameter readouts that only reflect a cell's elastic properties. In contrast to these methods, mechano-node-pore sensing (mechano-NPS) is a flexible, label-free microfluidic platform that bridges the gap in achieving multi-parameter viscoelastic measurements of a cell with moderate throughput. A direct current (DC) measurement is used to monitor cells as they transit a microfluidic channel, tracking their size and velocity before, during, and after they are forced through a narrow constriction. This information (i.e., size and velocity) is used to quantify each cell's transverse deformation, resistance to deformation, and recovery from deformation. In general, this electronics-based microfluidic platform provides multiple viscoelastic cell properties, and thus a more complete picture of a cell's mechanical state. Because it requires minimal sample preparation, utilizes a straightforward electronic measurement (in contrast to a high-speed camera), and takes advantage of standard soft lithography fabrication, the implementation of this platform is simple, accessible, and adaptable to downstream analysis. This platform's flexibility, utility, and sensitivity have provided unique mechanical information on a diverse range of cells, with the potential for many more applications in basic science and clinical diagnostics.

Presented here is a method to mechanically phenotype single cells using an electronics-based microfluidic platform called mechano-node-pore sensing (mechano-NPS). This platform maintains moderate throughput of 1-10 cells/s while measuring both the elastic and viscous biophysical properties of cells.

Mechano-Node-Pore Sensing: Uma plataforma rápida e sem rótulos para medições viscoelásticas de célula única multiparâmetro

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer ...

Bioengenharia

Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such as aspiration or coverslip compression have been used to mechanically stimulate whole embryos. These approaches limit experimental design as they are imprecise, require manual handling, and can process only a couple of embryos simultaneously. Microfluidic systems have great potential for automating such experimental tasks while increasing throughput and precision. This article describes a microfluidic system developed to precisely compress whole Drosophila melanogaster (fruit fly) embryos. This system features microchannels with pneumatically actuated deformable sidewalls and enables embryo alignment, immobilization, compression, and post-stimulation collection. By parallelizing these microchannels into seven lanes, steady or dynamic compression patterns can be applied to hundreds of Drosophila embryos simultaneously. Fabricating this system on a glass coverslip facilitates the simultaneous mechanical stimulation and imaging of samples with high-resolution microscopes. Moreover, the utilization of biocompatible materials, like PDMS, and the ability to flow fluid through the system make this device capable of long-term experiments with media-dependent samples. This approach also eliminates the requirement for manual mounting which mechanically stresses samples. Furthermore, the ability to quickly collect samples from the microchannels enables post-stimulation analyses, including -omics assays which require large sample numbers unattainable using traditional mechanical stimulation approaches. The geometry of this system is readily scalable to different biological systems, enabling numerous fields to benefit from the functional features described herein including high sample throughput, mechanical stimulation or immobilization, and automated alignment.

The present protocol describes the design, fabrication, and characterization of a microfluidic system capable of aligning, immobilizing, and precisely compressing hundreds of Drosophila melanogaster embryos with minimal user intervention. This system enables high-resolution imaging and recovery of samples for post-stimulation analysis and can be scaled to accommodate other multicellular biological systems.

Mecanoestimulação de organismos multicelulares através de um sistema de compressão microfluídica de alto rendimento

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such ...

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

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

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 ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

Avaliação de Dictyostelium discoideum resposta à estimulação mecânica aguda

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

Um sistema simplificado para a Avaliação celular Mechanosensing e Durotaxis

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