Este trabalho foi financiado pelo Prémio Carreira Burroughs Wellcome na Interface Científica (ARD), o Instituto Nacional de Saúde através do Programa Novo Diretor do NIH Innovator Award 1-DP2-OD007078 (ARD), o William Jr. Bowes Stanford Graduate Fellowship (ASA ), e do Stanford Cardiovascular Institute Fellowship Younger predoctoral (JC). Os autores agradecem James Spudich para emprestar equipamentos de microscopia.

1. Preparação célula de fluxo <ol><li> Lamelas (# 1.5, 22x22 mm e 22x40, VWR) são limpos usando sonicação. <ol><li> Adicionar as lamelas de um pequeno recipiente de vidro capaz de manter lamelas e encaixando no sonicador (ver passo 2). </li><li> Encher o recipiente com isopropanol e sonicar num sonicador de banho durante 20 minutos. </li><li> Descartar o isopropanol e enxaguar as lamelas com quantidades copiosas de água desionizada produzido por um aparelho de MilliQ Barnsted ou dispositivo semelhante. Encher o recipiente com água e sonicar durante 20 minutos. </li><li> Após sonicação, secar as lamelas em uma corrente de filtrado, o ar livre de poeira. Use apenas lamelas que aparecem limpos (sem manchas). </li><li> Gentilmente chama as lamelas por alguns segundos para limpá-los de qualquer poeira restante, e umidade: passar as lamelas através de uma chama de gás produzido com um bico de Bunsen, tomando cuidado para não deformar a lamela devido ao excesso de calor. Este passo remove superfície residualcontaminantes. </li></ol></li><li> Usando dupla face fita adesiva, prenda as duas lamelas juntos. Cortar a fita ~ 3 cm de comprimento e 0,3 cm de largura. Coloque a fita sobre lamínula 22x40 milímetros deixando um canal de 1,6 centímetros no meio. Usar uma ponta de pipeta suavemente para pressionar para baixo sobre a lamela para assegurar que a fita tem adequadamente aderido. </li></ol> 2. Configuração Pinças magnética e Calibração <ol><li> Pinças magnéticas foram configurados usando permanentes de terras raras ímãs, como descrito anteriormente 10,12. Um alumínio suporte em L foi construído com um furo de diâmetro 1 mm e montado sobre uma vertical z-tradutor (Thorlabs) para modular a altura da pinça. Dois permanentes de terras raras ímans foram ligados ao suporte em ambos os lados do orifício para criar a armadilha magnética (Figura 1). </li><li> Preparação de ADN: DNA de fago lambda rotulado a sua 5 &#39;e 3&#39; termina com biotina e digoxigenina (IDT DNA, San Diego, CA) foi utilizado para calibrar o magpinças magnéticos. <ol><li> Misturar 50 uL de 4 nM λ-DNA com 2 ul de 40 nM de biotina conjugado oligonucleótido e aquecer a 60 ° C durante 10 minutos. Em seguida, arrefecer lentamente à temperatura ambiente durante 1 hora. </li><li> Adicionar ligase de DNA de acordo com as especificações do fabricante e ligar à temperatura ambiente durante 3 horas. </li><li> Adicionar 2 uL de 400 nM oligonucleótido digoxigenina conjugado e incube à temperatura ambiente durante 45 minutos. </li><li> Adicionar 1,5 uL de 10 mM ATP e continuar a ligadura do oligonucleótido digoxigenina conjugado com o λ-DNA durante 3 horas. </li><li> Remover os oligonucleótidos em excesso eo usando DNA-ligase de 100 kDa filtros de spin (Microcon Ultracell YM-100). Um total de 1000 vezes troca de tampão em tampão TE é adequado para eliminar os oligos em excesso. Nota: É importante não utilizar velocidades de rotação&gt; 500xG porque irá conduzir à ruptura do ADN. </li><li> Examinar uma amostra do ADN através de electroforese num gel de agarose a 0,7% para assegurar que o DNA inão é desnaturado ou cortado. </li><li> Medir a concentração de DNA. A utilização de um espectrómetro de UV-Vis Nanodrop (Thermo Scientific) é recomendado porque o volume de amostra disponível pode ser muito pequena. </li></ol></li><li> Prepare-se células de fluxo, tal como descrito na seção 1. </li><li> Para a fixação do DNA para a célula de fluxo, adicionar o anticorpo anti-digoxigenina (ovinos IgG; 1 ug mL -1; Roche Bioscience, Manheim, Alemanha) e permitir que ele adere à superfície do vidro durante 15 minutos. </li><li> Passivar a superfície da célula de fluxo para evitar colagem não-específica do DNA por adição de 2 mg mL -1 de BSA, 0,1% de Tween-20 em 1x PBS. Incubar a temperatura ambiente durante 45 minutos. </li><li> Repita o passo 2.5. </li><li> Adicionar 50 pM de fuctionalized λ-DNA e permitir que a associar à lamela durante 15 minutos. </li><li> Lava-DNA em excesso com 1x PBS. </li><li> Adicionar grânulos revestidos estreptavidina-superparamagnéticas (1 grânulos iM = 2 ug mL -1, 2,8 grânulos iM = 20 ug mL -1 </sup&gt;) e permitir que eles atribuem ao DNA imobilizado por 15 minutos. </li><li> Lavar o excesso de grânulos com 1x PBS. </li><li> Calibração Pinça Magnética <ol><li> Mova os ímãs permanentes para uma posição que está longe da superfície da amostra (&gt; 15 mm). </li><li> Coloque a célula de fluxo preparado no microscópio. </li><li> Mova os ímãs permanentes para a posição acima da célula de fluxo. </li><li> Escolha o plano focal da λ-DNA contas funcionalizados, concentrando-se nas contas de distância de ambas as superfícies de vidro. O plano focal correcta é aquele em que os grânulos têm um centro desejados brilhante. </li><li> Faça um vídeo de movimento talão de 80 Hz ou superior a 500 frames. </li><li> Mova o íman mais perto da superfície da amostra. Para distâncias superiores a 5 mm, se mover em incrementos de 1 mm. Para distâncias entre 1 mm e 5, mover-se em incrementos de 0,5 mm. Para distâncias inferiores a 1 mm, mover-se em incrementos de 0,25 mm. </li><li> Repita os passos 2.11.5 e 2.11.6 em cada posição novo ímã. </li><li> Para cada dadodefinido, controlar o centro dos grânulos utilizando um ajuste gaussiano 2D. </li><li> Calcule a força através de flutuações browniano por meio de: </li></ol><img alt="Equação 1" src="/files/ftp_upload/3520/3520eq1.jpg" /> onde k B T é a energia térmica Boltzmann, L é o comprimento de λ-DNA, e &lt;x 2&gt; é a variância na posição centróide. <ol start="10"><li> Confirmar a exatidão das forças verificando o cálculo da força através de um ajuste de energia do espectro Lorentzian a fim de encontrar a freqüência rolloff. Força aplicada é relacionada com a frequência rolloff pela relação: </li></ol><img alt="Equação 2" src="/files/ftp_upload/3520/3520eq2.jpg" /> Onde 0 f é a frequência rolloff, a é o raio do grânulo, e μ é a viscosidade do fluido 12. </li></ol> 3. Collagen Peptide anexo para as células de fluxo A fim de proporcionar um exemplo concreto para a aplicação da nossa metodologia, descrevemos trabalho recente no nosso laboratório, que caracteriza a proteólise de um péptido trimérico colagénio modelo. Prevemos que esta abordagem geral pode ser amplamente aplicado a outras proteínas e polinucleótidos. <ol><li> O péptido modelo de colagénio consiste de um N-terminal His-6x para a purificação, seguido por uma etiqueta de 5x myc, (GPP) 10 para aplicar a formação de hélice tripla, o colagénio a1 resíduos 772-786 (GPQGIAGQRGVVGL), que formam a MMP- 1 local de reconhecimento, a sequência de foldon (GSGYIPEAPRDGQAYVRKDGEWVLLSTFL), e uma KKCK C-terminal para facilitar a marcação com biotina-maleimida. Foldon deriva do fibritin proteína T4-fago, e estabiliza trímeros modelo de colagénio quando fundido em uma ou outra a N - ou C-terminal 13,14. </li><li> O péptido de colagénio e MMP-1 proteínas foram expressas e purificada como descrito anteriormente 4,15. &lt;/ Li&gt; <li> Adicionar anti-myc (15 ug ml -1) para a célula de fluxo e incube à temperatura ambiente durante 20 minutos para permitir que o anti-myc para anexar à superfície da célula de fluxo. </li><li> Passivar a célula de fluxo para evitar ligação não específica de proteínas utilizando 5 mg mL -1 BSA da mesma forma como descrito no passo 2.5. </li><li> Adicionar 150 pM péptido de colagénio e permitir que ele para anexar o anticorpo através da marca de myc durante 45 minutos. </li><li> Lave o colágeno em excesso com tampão PBS. </li><li> Adicionar contas superparamagnéticas revestidas com estreptavidina (1 contas mM = 2 mg mL -1 e 2,8 mM contas = 20 mg mL -1) e permitir que eles atribuem às moléculas de colágeno por 45 minutos. As pérolas de 1 uM deve ser diluído até à concentração desejada em PBS. As pérolas de 3 uM deve ser separado da solução usando ímans fortes, em seguida, resolubilized em PBS. Este processo deve ser repetido 3 vezes. Percebemos que este passo é essencial para a get as pérolas de 3 uM de se ligar a superfície imobilizada péptido de colagénio. </li></ol> 4. Ensaio Proteólise Força <ol><li> Uma vez que a célula de fluxo é montado com o péptido de colagénio e as esferas magnéticas, a imagem da célula de fluxo na armadilha magnético sob baixa, ~ força pN 1. Na nossa experiência, uma subpopulação das pérolas é por vezes fracamente aderida à superfície de uma forma não específica. A aplicação de força modesto assegura que estas pérolas são libertados. </li><li> Adicionar enzima activada para a célula de fluxo. Neste caso a MMP-1 foi pré-activado por adição de 3,5 mM APMA (4-aminophenylmercuric acetato) e incubados a 37 ° C durante 3 horas. A ativação foi verificada utilizando SDS-PAGE. </li><li> Logo que a célula de fluxo é re-introduzido no aparelho pinças magnético, gravar um vídeo spanning alguns campos de visão, tipicamente rendendo várias centenas de pérolas anexas. Este será t = 0. </li><li> Repita o mesmo processo de gravação de vários campos de vista (quando regressava to o mesmo campo de visão) em pontos regulares de tempo, até que todas as esferas de separar ou não proteólise ainda é perceptível. </li><li> A análise dos dados cinéticos <ol><li> Conte com as contas em vários campos de vista em diferentes momentos e média-los. Normalizar o número de esferas, dividindo o número médio de grânulos em cada ponto de tempo pelo número de pérolas em t = 0. Isso garante que podemos comparar as taxas de proteólise de experiências diferentes, porque o número absoluto de contas será diferente de experimento para experimento. </li><li> Traça-se a razão como uma função do tempo. Neste caso, equipado-lo para um decaimento exponencial mais uma constante. </li></ol><img alt="Equação 3" src="/files/ftp_upload/3520/3520eq3.jpg" /> onde f (t) é a razão entre as esferas associadas (ou moléculas de colagénio unproteolyzed), t é o tempo, uma é a fracção de grânulos ligados por um tirante de colagénio, b é a taxa de decaimento constante, e cé a fracção de grânulos que estão ligados não especificamente. <ol start="3"><li> A mesma experiência é repetida em diferentes força e MMP-1 concentrações para elucidar o efeito da força sobre a proteólise de colagénio por MMP-1. </li></ol></li></ol> 5. Os resultados representativos O protocolo acima descreve uma nova utilização de uma pinça magnética (Figura 1) para estudar o efeito da força sobre a proteólise enzimática. Nós calibrado as pinças para 1 mícron e 3 uM grânulos utilizando tanto a magnitude das oscilações observadas brownianos e cálculo da frequência roll-off em posições diferentes de íman (Figura 2). Nas experiências de proteólise de força, a configuração é semelhante, excepto que o ADN é substituído com colagénio (Figuras 3, 4). O número normalizado de pérolas restantes podem ser traçados em função do tempo para encontrar as taxas de proteólise (Figura 5), e este processo pode serrepetido para várias concentrações de enzimas e de forças. <img alt="A Figura 1" src="/files/ftp_upload/3520/3520fig1.jpg" /> Figura 1. Esquemática do processo magnético armadilha de calibração (não à escala). Dois permanentes imans de terras raras criar um campo magnético que puxa a superparamagnético grânulo. Traduzir o íman cima e para baixo ajusta a força aplicada. As contas são fotografada utilizando um microscópio de campo brilhante convencional com a luz que passa através de um orifício entre os dois imans Inset:. Imagem tirada com um objectivo de ar de 40x. Os afiadas, pontos redondos correspondem aos grânulos que aderem à superfície lamela. Os objetos fora de foco são esferas separadas. <img alt="A Figura 2" src="/files/ftp_upload/3520/3520fig2.jpg" /> Figura 2. A representação gráfica da força calibrado como uma função da distância a partir do magneto superfície da amostra durante 1 mM (esquerda) e 2,8 mM (direita) do grânulos. Os dados estavam aptos para a função empírica <img alt="Equation4" src="/files/ftp_upload/3520/3520eq4.jpg" /> , Onde x é a distância entre o íman. A = 31,8, b = 5,61, e c = 4,39 para os grânulos de 1 pm e uma = 140, b = 3,10 e c = 1,86 para os 3 grânulos uM. Estes valores são específicos para o nosso instrumento específico e geometria experimental, e cada instrumento deve ser calibrado individualmente. As barras de erro em cada ponto representa uma variabilidade ~ 10% em força aplicada sobre um grânulo de base do grânulo, devido à variabilidade no tamanho do grânulo. A força aplicada é proporcional ao volume dos grânulos, e que o volume do grânulo varia ~ 9% sobre a média (de acordo com as especificações do fabricante). <img alt="A Figura 3" src="/files/ftp_upload/3520/3520fig3.jpg" /> Figura 3. Força configuração ensaio proteólise (não à escala). O trímero modelo de colagénio está ligado à superfície do lamela via myc / anti-myc conjugação. As pérolas revestidas de estreptavidina-superparamagnéticas estão ligados a os trímeros de colagénio através de uma ligação biotina-estreptavidina. Activada MMP-1 cortes do colagénio ao longo do tempo, causando as pérolas para separar a partir da superfície e afastar-se do plano focal. <img alt="A Figura 4" src="/files/ftp_upload/3520/3520fig4.jpg" /> Figura 4. Dos desenhos esquemático de proteólise como uma função do tempo. O desenho mostra um campo de vista da amostra ao longo do tempo como proteólise ocorre. Ao longo do tempo, a MMP-1 cortes o colagénio e as contas de vidro separar e afastar-se do plano focal sob a influência do campo magnético. <img alt="A Figura 5" src="/files/ftp_upload/3520/3520fig5.jpg" /> As taxas de proteólise Figura 5. Dependem força aplicada 16. São mostrados os dados coligidos a 1,0 pN (3 uM MMP-1; preto), 6,2 pN (3 uM MMP-1; vermelho) e 13 pN (0,2 uM MMP-1; magonta). As taxas de proteólise (parâmetros de ajuste) são: 0,22 ± 0,02 min -1 (1 pN), 0,46 ± 0,09 min -1 (6,2 pN), e 2,08 ± 0,18 min -1 (13 pN). A fracção de grânulos unproteolyzed em pontos de tempo longo (&gt; 15 minutos) permanece aproximadamente constante em toda a ~ 0,25 experiências diferentes. As barras de erro correspondem ao estatísticas Poisson reflectindo o número de observações em cada ponto de tempo. O erro em cada ponto de tempo para as esferas n é n 1/2. O erro na fracção de grânulos ligados foi calculada por erro propagation.1 grânulos mM foram usados ​​para 1,0 pN e experiências 6.2 pN e 2,8 uM grânulos foram utilizados para experiências de 13 pN.

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Este protocolo descreve uma nova utilização para uma técnica clássica molécula única. Pinças magnéticas permitir médio e alto rendimento ensaios única molécula de uma forma custo-eficiente. No entanto, como todas as técnicas experimentais que existem desafios e armadilhas potenciais. Limitações de pinças magnéticas Em comparação com uma armadilha óptica a resolução espacial e temporal de um aparelho de MT é baixa. Além disso, as forças gerada...

<table border="1"><tbody><tr><td> Nome do Reagente </td><td> Companhia </td><td> Número de Catálogo </td></tr><tr><td> Micro tampa de vidro # 1.5 (22x22) </td><td> VWR </td><td> 48366-067 </td></tr><tr><td> Micro tampa de vidro # 1.5 (22x40) </td><td> VWR </td><td> 48393-048 </td></tr><tr><td> Lambda DNA </td><td> Invitrogen </td><td> 25250-010 </td></tr><tr><td> DNA-ligase de T4 </td><td> Invitrogen </td><td> 15224-041 </td></tr><tr><td> Microcon Ultracel YM-100 </td><td> Millipore </td><td> 42413 </td></tr><tr><td> Anti-digoxigenina </td><td> Roche Diagnostics </td><td> 11-333-089-001 </td></tr><tr><td> Tween 20 </td><td> Sigma </td><td> P9416-100ML </td></tr&gt; <tr><td> Anti-myc Anticorpo </td><td> Invitrogen </td><td> 46-0603 </td></tr><tr><td> Albumina de Soro Bovino </td><td> Sigma </td><td> B4287-5G </td></tr><tr><td> Dynabeads M-280 Estreptavidina </td><td> Invitrogen </td><td> 658.01D </td></tr><tr><td> Dynabeads MyOne T1 Estreptavidina </td><td> Invitrogen </td><td> 658.01D </td></tr><tr><td> p-Aminophenylmercuric Acetato </td><td> Calbiochem </td><td> 164610 </td></tr><tr><td> Biotina-maleimida </td><td> Sigma Aldrich </td><td> B1267 </td></tr><tr><td> Marcado com biotina oligo </td><td> IDT DNA </td><td> Síntese feita sob encomenda </td></tr><tr><td> Digoxigenina marcada oligo </td><td> IDT DNA </td><td> Síntese feita sob encomenda </td></tr><tr><td> Colágenogene peptídeo </td><td> DNA 2.0 </td><td> Síntese feita sob encomenda </td></tr><tr><td> MMP-1 ADNc </td><td> Harvard Banco de Dados Plasmid </td><td></td></tr><tr><td> z-tradutor </td><td> Thorlabs </td><td> MTS50 </td></tr><tr><td> Controlador de Servo para tradutor </td><td> Thorlabs </td><td> TDC001 </td></tr></tbody></table>

multiplexed single-molecule force proteolysis measurements using magnetic tweezers

A geração e detecção de forças mecânicas é um aspecto onipresente de fisiologia celular, com relevância direta para câncer de metástase 1, 2 e aterogênese cicatrização 3. Em cada um destes exemplos, as células tanto exercer força sobre os seus arredores e, simultaneamente, remodelação enzimaticamente a matriz extracelular (ECM). O efeito das forças sobre ECM tornou-se assim uma área de grande interesse devido à sua provável importância biológica e médica 4-7. Técnicas única molécula, como a captura óptica 8, microscopia de força atômica 9, e pinças magnéticas 10,11 permitir aos pesquisadores sondar a função de enzimas em um nível molecular, exercendo forças em proteínas individuais. Destas técnicas, pinças magnéticas (MT) são notáveis ​​pelo seu baixo custo e alto rendimento. MT exercem forças na gama de 1-100 ~ pN e pode fornecer milissegundo resolução temporal,qualidades que são bem adaptado para o estudo do mecanismo de enzima no nível molécula única 12. Relatamos aqui um ensaio de MT altamente paralelizável para estudar o efeito da força sobre a proteólise das moléculas de proteína única. Apresentamos o exemplo específico da proteólise de um péptido de colagénio trimérico por metaloproteinase de matriz 1 (MMP-1), no entanto, este ensaio pode ser facilmente adaptado para estudar outros substratos e proteases.

Watch this Scientific Journal Video about Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers at JoVE.com

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

Neste artigo, descrevem a utilização de uma pinça magnéticos para estudar o efeito da força sobre a proteólise enzimática no nível única molécula de uma maneira altamente paralelizável.

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 ...

multiplexed-single-molecule-force-proteolysis-measurements-using

Research

JoVE Journal

Bioengineering

11.5K visualizações.  Stanford University. Neste artigo, descrevem a utilização de uma pinça magnéticos para estudar o efeito da força sobre a proteólise enzimática no nível única molécula de uma maneira altamente paralelizável.

Vídeo: Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers

Single-molecule techniques for stretching DNA of contour lengths less than a kilobase are fraught with experimental difficulties. However, many interesting biological events such as histone binding and protein-mediated looping of DNA1,2, occur on this length scale. In recent years, the mechanical properties of DNA have been shown to play a significant role in fundamental cellular processes like the packaging of DNA into compact nucleosomes and chromatin fibers3,4. Clearly, it is then important to understand the mechanical properties of short stretches of DNA. In this paper, we provide a practical guide to a single-molecule optical tweezing technique that we have developed to study the mechanical behavior of DNA with contour lengths as short as a few hundred basepairs.
The major hurdle in stretching short segments of DNA is that conventional optical tweezers are generally designed to apply force in a direction lateral to the stage5,6, (see Fig. 1). In this geometry, the angle between the bead and the coverslip, to which the DNA is tethered, becomes very steep for submicron length DNA. The axial position must now be accounted for, which can be a challenge, and, since the extension drags the microsphere closer to the coverslip, steric effects are enhanced. Furthermore, as a result of the asymmetry of the microspheres, lateral extensions will generate varying levels of torque due to rotation of the microsphere within the optical trap since the direction of the reactive force changes during the extension.
Alternate methods for stretching submicron DNA run up against their own unique hurdles. For instance, a dual-beam optical trap is limited to stretching DNA of around a wavelength, at which point interference effects between the two traps and from light scattering between the microspheres begin to pose a significant problem. Replacing one of the traps with a micropipette would most likely suffer from similar challenges. While one could directly use the axial potential to stretch the DNA, an active feedback scheme would be needed to apply a constant force and the bandwidth of this will be quite limited, especially at low forces.
We circumvent these fundamental problems by directly pulling the DNA away from the coverslip by using a constant force axial optical tweezers7,8. This is achieved by trapping the bead in a linear region of the optical potential, where the optical force is constant-the strength of which can be tuned by adjusting the laser power. Trapping within the linear region also serves as an all optical force-clamp on the DNA that extends for nearly 350 nm in the axial direction. We simultaneously compensate for thermal and mechanical drift by finely adjusting the position of the stage so that a reference microsphere stuck to the coverslip remains at the same position and focus, allowing for a virtually limitless observation period.

We illustrate the use of a constant force axial optical tweezers to explore the mechanical properties of short DNA molecules. By stretching DNA axially, we minimize steric hindrances and artifacts arising in conventional lateral manipulation, allowing us to study DNA molecules as short as ~100 nm.

Alongamento curtas seqüências de DNA com Pinça força constante óptico axial

Single-molecule techniques for stretching DNA of contour lengths  less than a kilobase are fraught with experimental difficulties. However, many ...

Bioengenharia

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires microtubules that are stiff and straight enough to span a significant fraction of the cell diameter. As a result, the microtubule persistence length, a measure of stiffness, has been actively studied for the past two decades1. Nonetheless, open questions remain: short microtubules are 10-50 times less stiff than long microtubules2-4, and even long microtubules have measured persistence lengths which vary by an order of magnitude5-9.
	Here, we present a method to measure microtubule persistence length. The method is based on a kinesin-driven microtubule gliding assay10. By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores attached to the microtubule, the gliding trajectories of single microtubules are tracked with nanometer-level precision. The persistence length of the trajectories is the same as the persistence length of the microtubule under the conditions used11. An automated tracking routine is used to create microtubule trajectories from fluorophores attached to individual microtubules, and the persistence length of this trajectory is calculated using routines written in IDL.
	This technique is rapidly implementable, and capable of measuring the persistence length of 100 microtubules in one day of experimentation. The method can be extended to measure persistence length under a variety of conditions, including persistence length as a function of length along microtubules. Moreover, the analysis routines used can be extended to myosin-based acting gliding assays, to measure the persistence length of actin filaments as well.

A method to measure the persistence length or flexural rigidity of biopolymers is described. The method uses a kinesin-driven microtubule gliding assay to experimentally determine the persistence length of individual microtubules and is adaptable to actin-based gliding assays.

Flexão Medidas rigidez de Biopolímeros usando ensaios de deslizamento

Microtubules are cytoskeletal polymers which  play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Investigando Sistemas receptor-ligante do Celulossoma com base-AFM única molécula Força Espectroscopia

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

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 ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

De alta resolução espacial e temporal Análise de Receptor Dynamics por uma única molécula de microscopia de fluorescência

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

Bicamadas única molécula de microscopia de fluorescência em Planar Apoiado

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

Único Detecção de Moléculas livre-Label Usando Microtoroid ópticos Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

Rápida digitalização ressonância paramagnética eletrônica abre novos caminhos para criação de imagens fisiologicamente parâmetros importantes<em&gt; In Vivo</em

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Fabricação de uma matriz de microambiente celular Artificial multiplexado

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as atomic force microscopy have limited temporal resolution, while F&#246;rster resonance energy transfer (FRET) only allow conformations to be inferred. Fluorescence microscopy, on the other hand, allows real-time in situ visualization of single molecules in various flow conditions. Our protocol describes the steps to capture conformational changes of single biomolecules under different shear flow environments using fluorescence microscopy. The shear flow is created inside microfluidic channels and controlled by a syringe pump. As demonstrations of the method, von Willebrand factor (VWF) and lambda DNA are labeled with biotin and fluorophore and then immobilized on the channel surface. Their conformations are continuously monitored under variable shear flow using total internal reflection (TIRF) and confocal fluorescence microscopy. The reversible unraveling dynamics of VWF are useful for understanding how its function is regulated in human blood, while the conformation of lambda DNA offers insights into the biophysics of macromolecules. The protocol can also be widely applied to study the behavior of polymers, especially biopolymers, in varying flow conditions and to investigate the rheology of complex fluids.

We present a protocol for immobilizing single macromolecules in microfluidic devices and quantifying changes in their conformations under shear flow. This protocol is useful for characterizing the biomechanical and functional properties of biomolecules such as proteins and DNA in a flow environment.

Caracterizando mudanças conformais de molécula única fluxo de cisalhamento com microscopia de fluorescência

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as ...