Name: a direct force probe for measuring mechanical integration between the nucleus and the cytoskeleton
Uploaded: 2018-07-29T13:30:00.000+00:00
Duration: 5 min 47 s
Description: Watch this Scientific Journal Video about A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton at JoVE.com

Este trabalho foi financiado pelo NIH R01 EB014869.

1. preparar células para a imagem latente
Nota: A sonda de força direta (DFP) pode ser usada para qualquer tipo de células aderentes. Aqui, fibroblastos de rato do NIH 3T3 são usados como a linha celular do modelo para este protocolo.
<ol><li>Células de fibroblastos cultura NIH 3T3 em modificado águia de Dulbecco médio (DMEM) suplementado com 10% de soro de bovino doador e 1% penicilina-estreptomicina em um fundo de vidro 35mm prato até confluência desejada. Manter as células em 37 ° C e 5% de CO2.<ol>
<li>Não se esqueça de revestir todos os pratos de fundo de vidro 35mm com 5 µ g/mL de fibronectina (ou proteína de ECM semelhante), antes da semeadura das células 3T3 NIH para a imagem latente. Nota: As células devem ser totalmente espalhadas e aderente sobre o prato para o experimento. Não existem quaisquer restrições em termos de confluência para o método DFP trabalhar.</li>
	</ol>
</li>
	<li>Imediatamente antes do experimento, as células de duas lavagens com PBS, seguido por uma simples lavagem com meio de cultura completo.</li>
	<li>Adicione 3 mL de meio de cultura completo para o prato fundo de vidro.</li>
</ol>2. microscopia e aquisição de imagem
Nota: Um invertido fluorescência microscópio (ou equivalente) com micromanipulador instalado para o lado do braço, de acordo com as recomendações do fabricante. O microscópio também deve ser equipado com uma câmara ambiental para manter a temperatura a 37 ° C e o nível de CO2 em 5%. Também é necessário um micromanipulador e microinjector anexado ao microscópio. Um óleo de imersão x 40 / 1,3 at ou 60 x / 1.49 at (ou objectivos equivalentes) são recomendados para os experimentos. O microscópio deve ser montado sobre uma mesa de isolamento de vibração.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58038/58038fig1.jpg"> Figura 1 . Deformação nuclear e microscópio com foco R. máxima deformação nuclear e relaxamento da deformação nuclear. Antes de calcular a máxima deformação nuclear, as bordas traseiras das formas nucleares foram primeiro coincidiu para corrigir para a tradução do núcleo deformado. A forma do núcleo no momento do desprendimento de ponta micropipeta era revestida na forma nuclear inicial antes de puxar. A diferença de área entre as duas formas foi medida como Δum1. A máxima deformação nuclear foi definida como ΔA1 dividido pela área nuclear original. Da mesma forma, um segundo parâmetro, ΔA2, podem ser definidas por sobrepondo a forma nuclear final estado estacionário após descolamento micropipeta na forma nuclear original. B. a célula no avião A concentrar-se e mova o plano focal até avião B para encontrar a ponta da micropipeta. Durante a imagem latente, a micropipeta foi traduzida para a direita (sentido da seta laranja). Esta figura foi modificada de Eelam et al. 15. <a href="https://www.jove.com/files/ftp_upload/58038/58038fig1large.jpg" target="_blank">clique aqui para ver uma versão maior desta figura.</a>
<ol><li>Ligue o microinjector acordo com o protocolo do fabricante.</li>
	<li>Usando um gotas de óleo de imersão, aplica uma gota de óleo de imersão em cima da lente objetiva.</li>
	<li>Fixar o prato firmemente no suporte do prato e carregar o suporte para o palco. Nota: As células devem ser mantidas a 37 ° C e 5% CO2 durante todo o experimento.</li>
	<li>Ajuste a altura do objectivo de colocar as células em foco (A do avião, figura 1B).</li>
	<li>Mova o estágio de microscópio para encontrar uma célula de interesse.</li>
	<li>Gire o joystick no micromanipulador para mover o titular pipeta para a posição superior. Carrega a micropipeta de ponta de diâmetro de 0,5 µm no suporte pipeta.<ol>
<li>Para evitar aderência de célula para a micropipeta, pré-tratamento a ponta da micropipeta com 0,3 mg/mL solução de PLL-g-PEG por 1h à temperatura ambiente. Teste de aderência tocando a micropipeta para o núcleo sem qualquer pressão de sucção, e então traduzir a micropipeta longe do núcleo. A ausência de adesão pode ser discernida por falta completa de deformação nuclear e tradução. Nota: Por favor siga sugestões de fabrico para abrir o pacote.</li>
	</ol>
</li>
	<li>Elevar o plano focal objectivo acima A do avião e o topo da célula para o plano B, ajustando o controle fino (figura 1B, consulte a etapa 2.4).</li>
	<li>Conjunto o micromanipulador para controle de grosseira . Traga lentamente a micropipeta até avião B observando para a silhueta da micropipeta, até a micropipeta totalmente entra em foco.</li>
	<li>Uma vez que a ponta da micropipeta está em foco, defina o micromanipulador para controle fino .</li>
	<li>Baixe o objectivo para o plano equatorial da célula (avião A, figura 1B) e inferior a micropipeta para cerca de 15 µm acima plano A (figura 1B, micropipeta tracejada).</li>
	<li>Ajustar a pressão de compensação (Pc) sobre o microinjector a pressão desejada; Aguarde alguns segundos até a pressão estabilizar. Nota: O ponto de ajuste de pressão ideal depende de tipo de célula e os objetivos específicos do experimento. Para a maioria dos casos, 300 hPa seria um bom ponto de partida.</li>
	<li>Certifique-se de que a micropipeta não está entupida usando a configuração de limpeza no painel micromanipulador e verificar para certificar-se de bolhas de ar emergem da ponta da micropipeta.</li>
	<li>Introduza a ponta dentro da célula, diminuindo gradualmente a micropipeta, até que a ponta é levemente tocar a superfície nuclear. Nota: Ao descer a micropipeta, a silhueta da ponta da micropipeta se tornará clara como entra em foco. Antes a micropipeta toca o núcleo, aumentar o foco da objetivo e alinhe a ponta da micropipeta com núcleo (mesma coordenada x-y, z-plano superior). Retornar o foco para o plano equatorial do núcleo (A de avião, figura 1B) e gradualmente diminuir a ponta da micropipeta.</li>
	<li>Crie um selo entre a ponta da micropipeta e a membrana nuclear, desconectar o tubo de alimentação de pressão do sistema de microinjeção, abrindo assim a extremidade do tubo micropipeta para a atmosfera. Esta etapa cria uma pressão negativa igual a Pc na superfície nuclear.</li>
	<li>Adquira imagens com o software de coleta de imagem de microscópios. Configure um avi-aquisição (vídeo) ou nd-aquisição do software de coleta de imagem (imagens). Nota: Para qualquer imagem adquirir o software, criar imagens de vídeo em tempo real ou aquisições de imagem de lapso de tempo com um curto intervalo de tempo.</li>
	<li>Alternar para o fluorescente correspondente canal de imagem (ou seja, as boas práticas agrícolas, RFP, etc.) e começar a imagem latente.</li>
	<li>Definição da palavra a ponta da micropipeta longe do corpo da célula (para a direita, figura 1B) até que o núcleo se desprende da micropipeta. Nota: Puxe a ponta ao longo de x-direção positiva (à direita no campo de visão). A taxa de tração pode ser programada e controlada por computador ou o joystick pode ser movido manualmente. Não encontramos qualquer correlação entre a tirar taxa e deformação nuclear15 sugerindo uma resposta principalmente elástica para forçar.</li>
</ol>3. análise de dados
<ol><li>Realizar análise de imagem com qualquer software de processamento de imagem básico disponível. O grau de deformação nuclear pode ser quantificado pela estirpe de comprimento (Ɛ) ou a tensão da área (figura 1A). Quantificar a estirpe de comprimento usando a equação 1, onde L e L0 representam os comprimentos do núcleo na deformação máxima e a posição inicial, respectivamente. <img alt="Equation 1" src="/files/ftp_upload/58038/58038eq1.jpg">(Equação 1)</li>
</ol>

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Os autores não têm nada para divulgar.

Medindo a integração mecânica do núcleo com o citoesqueleto é um desafio para métodos mais atuais, como micropipeta aspiração16, pois eles exigem ou núcleos isolados (onde o núcleo é dissociado do citoesqueleto) ou núcleos em células suspensos (onde forças extracelulares, como forças de tração, estão ausentes). Força aplicou-se para o núcleo aplicando tensão biaxial de aderente de células a uma membrana17,,18; no enta...

Patologias como câncer envolvem alterações à forma nuclear e estrutura1,2, que geralmente são acompanhados de um amolecimento do núcleo3,4. Nuclear resistência à deformação mecânica tem sido geralmente caracterizada pela aplicação de uma força de núcleos isolados5.
O núcleo nas células molecularmente está ligado ao citoesqueleto pelo vinculador de Nucleoskeleton e citoesqueleto (LINC) complexo6,7,8,9. Como resultado, o núcleo mecanicamente é integrado com o citoesqueleto e, através de aderências célula-substrato, a matriz extracelular. Mecanicamente, sondar o núcleo no interior das células aderentes pode fornecer insights sobre essa integração mecânica. Métodos para manipular os núcleos em células vivas incluem micropipeta aspiração10,11e microscopia de força atômica a12,13,14. Recentemente descrevemos uma sonda de força direta (DFP) que se aplica forças mecânicas diretamente no núcleo em uma vida aderentes célula15.
Aqui, podemos descrever o procedimento para usar um sistema de microinjeção que está geralmente disponível em instalações de microscopia para aplicar uma força mecânica de nano-escala conhecida, diretamente para o núcleo em uma célula aderente. Um femtotip (0,5 µm ponta de micropipeta de diâmetro) é montado e ligado ao sistema de microinjeção por um tubo. A ponta, posicionada em um ângulo de 45° em relação à superfície do prato, cultura é abaixada até adjacente à superfície nuclear. O tubo é então desligado e aberto para a atmosfera, o que cria uma pressão negativa de sucção na superfície nuclear e lacra a ponta da micropipeta contra a superfície nuclear. Através da tradução da ponta da micropipeta, o núcleo é deformado e eventualmente (dependendo da magnitude da força aplicada), extraído da micropipeta. Este desprendimento ocorre quando as forças de restaura (oposição), exercidas pelo núcleo e célula, igual a força de sucção aplicada pela micropipeta. Análise pode ser realizada medindo-se o deslocamento do núcleo, a estirpe de comprimento (equação 1), ou a tensão da área (figura 1A).

<table><tbody><tr><td>FluoroDish</td><td>WPI</td><td>FD35</td><td></td></tr><tr><td>SYTO 59</td><td>ThermoFisher Scientific</td><td>S11341</td><td></td></tr><tr><td>Femtotips&amp;nbsp;</td><td>Eppendorf</td><td>930000043</td><td></td></tr><tr><td>InjectMan NI2</td><td>Eppendorf</td><td>NA</td><td>discontinued, current equivalent model: InjectMan 4</td></tr><tr><td>FemtoJet</td><td>Eppendorf</td><td>NA</td><td>Current model FemtoJet 4i</td></tr><tr><td>Plan Fluor oil immersion 40x</td><td>Nikon</td><td>NA</td><td></td></tr><tr><td>Apo TIRF oil immersion 60x</td><td>Nikon</td><td>NA</td><td></td></tr><tr><td>Donor Bovine Serum (DBS)</td><td>ThermoFisher Scientific</td><td>16030074</td><td>NIH 3T3 serum</td></tr><tr><td>Dulbecco&#039;s Modification of Eagle&#039;s (DMEM)</td><td>Mediatech cellgro</td><td>MT10013CVRF</td><td>NIH 3T3 medium</td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>Mediatech</td><td>MT30004CIRF</td><td>NIH 3T3 medium supplement</td></tr><tr><td>Immersion Oil Type LDF Non-Fluorescing</td><td>Nikon</td><td>77007</td><td>Immersion oil for objective lens&amp;nbsp;</td></tr></tbody></table>

a direct force probe for measuring mechanical integration between the nucleus and the cytoskeleton

As propriedades mecânicas do núcleo determinam sua resposta às forças mecânicas geradas nas células. Porque o núcleo é molecularmente contínuo com o citoesqueleto, métodos são necessários para sondar seu comportamento mecânico em células aderentes. Aqui, vamos discutir a sonda de força direta (DFP) como uma ferramenta para aplicar a força diretamente para o núcleo em uma célula aderente viva. Atribuímos uma micropipeta estreita à superfície nuclear com sucção. A micropipeta é traduzida longe do núcleo, o que faz com que o núcleo se deforma e traduzir. Quando a força restauradora é igual à força de sucção, o núcleo desconecta e elasticamente, relaxa. Porque o pressão de sucção é precisamente conhecido, é conhecida a força na superfície nuclear. Este método revelou-se que as forças de nano-escala são suficientes para deformar-se e traduzir o núcleo nas células aderentes e identificaram elementos do citoesqueleto que permitem que o núcleo resistir às forças. O DFP pode ser usado para dissecar as contribuições dos componentes celulares e nucleares para propriedades mecânicas nucleares em células vivas.

Figura 2A mostra a força de um núcleo de fibroblasto NIH 3T3 rato. Como a ponta da micropipeta é traduzida para a direita, o núcleo deforma e eventualmente desanexa da ponta da micropipeta. A estirpe do comprimento do núcleo é vista a aumentar com o aumento da força de sucção (Figura 2B). Na extremidade dianteira do núcleo (micropipeta puxando a borda) forma uma protusão nuclear e a borda direita é deslocada de sua po...

Watch this Scientific Journal Video about A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton at JoVE.com

A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton

Uma sonda de força direta para medir a integração mecânica entre o núcleo e o citoesqueleto

Neste protocolo, descrevemos um método de micropipeta para aplicar directamente uma força controlada para o núcleo de uma célula viva. Este ensaio permite interrogatório das propriedades mecânicas nucleares na célula viva, aderente.

The mechanical properties of the nucleus determine its response to mechanical forces generated in cells. Because the nucleus is molecularly continuous ...

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Research

JoVE Journal

Biochemistry

15.1K visualizações.  University of Florida. Neste protocolo, descrevemos um método de micropipeta para aplicar directamente uma força controlada para o núcleo de uma célula viva. Este ensaio permite interrogatório das propriedades mecânicas nucleares na célula viva, aderente.

Vídeo: A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

Um protocolo para usar a transferência de Förster Ressonância Energia (FRET) Biossensores -force medir forças mecânicas em todo o Complexo LINC Nuclear

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

Bioengenharia

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 ...

Biologia do Desenvolvimento

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

Medição da dinâmica de força-sensível da proteína em células vivas, usando uma combinação de técnicas fluorescentes

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

Ensaios biofísicos para investigar as propriedades mecânicas do núcleo da célula Interphase: Aplicação de substrato Strain e Manipulação de microagulhas

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Biologia

DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical for regulating many processes ranging from development to immunology. Therefore, developing the tools to study these forces at the molecular scale is critical. Our group developed a suite of molecular tension sensors to quantify and visualize the forces generated by cells and transmitted to specific ligands. The most sensitive class of molecular tension sensors are comprised of nucleic acid stem-loop hairpins. These sensors use fluorophore-quencher pairs to report on the mechanical extension and unfolding of DNA hairpins under force. One challenge with DNA hairpin tension sensors is that they are reversible with rapid hairpin refolding upon termination of the tension and thus transient forces are difficult to record. In this article, we describe the protocols for preparing DNA tension sensors that can be "locked" and prevented from refolding to enable "storing" of mechanical information. This allows for the recording of highly transient piconewton forces, which can be subsequently "erased" by the addition of complementary nucleic acids that remove the lock. This ability to toggle between real-time tension mapping and mechanical information storing reveals weak, short-lived, and less abundant forces, that are commonly employed by T cells as part of their immune functions.

This paper describes a detailed protocol for using DNA-based tension probes to image the receptor forces applied by immune cells. This approach can map receptor forces &gt;4.7pN in real-time and can integrate forces over time.

Sondas de Tensão de DNA para Mapear as Forças Transitórias do Receptor de Piconewton por Células Imunes

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical ...

Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction forces have been well-characterized, but there is a lack of techniques allowing the measurement of the protrusion forces exerted by cells orthogonally to their substrate. We designed an experimental setup to measure the protrusion forces exerted by adherent cells on their substrate. Cells plated on a compliant Formvar sheet deform this substrate and the resulting topography is mapped by atomic force microscopy (AFM) at the nanometer scale. Force values are then extracted from an analysis of the deformation profile based on the geometry of the protrusive cellular structures. Hence, the forces exerted by the individual protruding units of a living cell can be measured over time. This technique will enable the study of force generation and its regulation in the many cellular processes involving protrusion. Here, we describe its application to measure the protrusive forces generated by podosomes formed by human macrophages.

Here, we detail the experimental techniques used to evaluate the protrusion forces that podosomes apply on a compliant film, from the preparation of the film to the automated analysis of topographical images.

Microscopia de força de protrusão: Um método para quantificar as forças desenvolvidas pela célula saliências

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction ...

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

Isolando Myofibrils de Biópsias musculares esqueléticas e determinando função contractile com um transdutor de força de resolução nano-Newton

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of ...

Tension Gauge Tether Probes for Quantifying Growth Factor Mediated Integrin Mechanics and Adhesion

Multicellular organisms rely on interactions between membrane receptors and cognate ligands in the surrounding extracellular matrix (ECM) to orchestrate multiple functions, including adhesion, proliferation, migration, and differentiation. Mechanical forces can be transmitted from the cell via the adhesion receptor integrin to ligands in the ECM. The amount and spatial organization of these cell-generated forces can be modulated by growth factor receptors, including epidermal growth factor receptor (EGFR). The tools currently available to quantify crosstalk-mediated changes in cell mechanics and relate them to&#160;focal adhesions, cellular morphology, and signaling are limited. DNA-based molecular force sensors known as tension gauge tethers (TGTs) have been employed to quantify these changes. TGT probes are unique in their ability to both modulate the underlying force threshold and report piconewton scale receptor forces across the entire adherent cell surface at diffraction-limited spatial resolution. The TGT probes used here rely on the irreversible dissociation of a DNA duplex by receptor-ligand forces that generate a fluorescent signal. This allows quantification of the cumulative integrin tension (force history) of the cell. This article describes a protocol employing TGTs to study the impact of EGFR on integrin mechanics and adhesion formation. The assembly of&#160;the TGT mechanical sensing platform is systematically detailed and the procedure to image forces, focal adhesions, and cell spreading is outlined. Overall, the ability to modulate the underlying force threshold of the probe, the adhesion ligand, and the type and concentration of growth factor employed for stimulation make this a robust platform for studying the interplay of diverse membrane receptors in regulating integrin-mediated forces.

TGT surface is an innovative platform to study growth factor-integrin crosstalk. The flexible probe design, specificity of the adhesion ligand, and precise modulation of stimulation conditions allow robust quantitative assessments of EGFR-integrin interplay. The results highlight EGFR as a 'mechano-organizer' tuning integrin mechanics, influencing focal adhesion assembly and cell spreading.

Sondas de tether medidor de tensão para quantificar fator de crescimento mediado mecânica integrin e adesão

Multicellular organisms rely on interactions between membrane receptors and cognate ligands in the surrounding extracellular matrix (ECM) to orchestrate ...

Directly Measuring Forces Within Reconstituted Active Microtubule Bundles

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as specialized arrays during mitosis to regulate chromosome segregation. Proteins that interact with microtubules include motors such as kinesins and dynein, which can generate active forces and directional motion, as well as non-motor proteins that crosslink filaments into higher-order networks or regulate filament dynamics. To date, biophysical studies of microtubule-associated proteins have overwhelmingly focused on the role of single motor proteins needed for vesicle transport, and significant progress has been made in elucidating the force-generating properties and mechanochemical regulation of kinesins and dyneins. However, for processes in which microtubules act both as cargo and track, such as during filament sliding within the mitotic spindle, much less is understood about the biophysical regulation of ensembles of the crosslinking proteins involved. Here, we detail our methodology for directly probing force generation and response within crosslinked microtubule minimal networks reconstituted from purified microtubules and mitotic proteins. Microtubule pairs are crosslinked by proteins of interest, one microtubule is immobilized to a microscope coverslip, and the second microtubule is manipulated by an optical trap. Simultaneous total internal reflection fluorescence microscopy allows for multichannel visualization of all the components of this microtubule network as the filaments slide apart to generate force. We also demonstrate how these techniques can be used to probe pushing forces exerted by kinesin-5 ensembles and how viscous braking forces arise between sliding microtubule pairs crosslinked by the mitotic MAP PRC1. These assays provide insights into the mechanisms of spindle assembly and function and can be more broadly adapted to study dense microtubule network mechanics in diverse contexts, such as the axon and dendrites of neurons and polar epithelial cells.

Here, we present a protocol for reconstituting microtubule bundles in vitro and directly quantifying the forces exerted within them using simultaneous optical trapping and total internal reflection fluorescence microscopy. This assay allows for nanoscale-level measurement of the forces and displacements generated by protein ensembles within active microtubule networks.

Forças de Medição Direta Dentro de Feixes de Microtúbulos Ativos Reconstituídos

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as ...

Combining 3D Magnetic Force Actuator and Multi-Functional Fluorescence Imaging to Study Nucleus Mechanobiology

A fundamental question in mechanobiology is how living cells sense extracellular mechanical stimuli in the context of cell physiology and pathology. The cellular mechano-sensation of extracellular mechanical stimuli is believed to be through the membrane receptors, the associated protein complex, and the cytoskeleton. Recent advances in mechanobiology demonstrate that the cell nucleus in cytoplasm itself can independently sense mechanical stimuli simultaneously. However, a mechanistic understanding of how the cell nucleus senses, transduces, and responds to mechanical stimuli is lacking, mainly because of the technical challenges in accessing and quantifying the nucleus mechanics by conventional tools. This paper describes the design, fabrication, and implementation of a new magnetic force actuator that applies precise and non-invasive 3D mechanical stimuli to directly deform the cell nucleus. Using CRISPR/Cas9-engineered cells, this study demonstrates that this tool, combined with high-resolution confocal fluorescent imaging, enables the revelation of the real-time dynamics of a mechano-sensitive yes-associated protein (YAP) in single cells as a function of nucleus deformation. This simple method has the potential to bridge the current technology gap in the mechanobiology community and provide answers to the knowledge gap that exists in the relation between nucleus mechanotransduction and cell function.

This study presents a new protocol to directly apply mechanical force on the cell nucleus through magnetic microbeads delivered into the cytoplasm and to conduct simultaneous live-cell fluorescent imaging.

Combinando o Atuador de Força Magnética 3D e a Imagem de Fluorescência Multifuncional para Estudar a Mecanologia do Núcleo

A fundamental question in mechanobiology is how living cells sense extracellular mechanical stimuli in the context of cell physiology and pathology. The ...