Name: ensemble force spectroscopy by shear forces
Uploaded: 2022-07-26T13:00:00
Description: Watch this Scientific Journal Video about Ensemble Force Spectroscopy by Shear Forces at JoVE.com

Este trabajo de investigación fue apoyado por la National Science Foundation [CBET-1904921] y los Institutos Nacionales de Salud [NIH R01CA236350] a H. M.

NOTA: Todos los tampones y los reactivos químicos utilizados en este protocolo se enumeran en la tabla Materiales.
1. Preparación del microscopio de fuerza de cizallamiento
NOTA: El microscopio de fuerza de cizallamiento contiene dos partes, una unidad de reacción (homogeneizador) y una unidad de detección (microscopio de fluorescencia). El aumento del ocular es 10x, y el aumento de la lente del objetivo (aire) es 4x.
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<ol>
	<li>Neuman, K. C., Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+force+spectroscopy:+Optical+tweezers,+magnetic+tweezers+and+atomic+force+microscopy.">Single-molecule force spectroscopy: Optical tweezers, magnetic tweezers and atomic force microscopy.</a> Nature Methods. 5 (6), 491-505 (2008).</li><li>Woodside, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomechanical+measurements+of+the+sequence-dependent+folding+landscapes+of+single+nucleic+acid+hairpins.">Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (16), 6190-6195 (2006).</li><li>Grandbois, M., Beyer, M., Rief, M., Clausen-Schaumann, H., Gaub, H. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+single-molecule+force+spectroscopy+using+a+centrifuge.">Multiplexed single-molecule force spectroscopy using a centrifuge.</a> Nature Communications. 7, 11026 (2016).</li><li>Su, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-responsive+polymer+particles+as+force+clamps+for+the+mechanical+unfolding+of+target+molecules.">Light-responsive polymer particles as force clamps for the mechanical unfolding of target molecules.</a> Nano Letters. 18 (4), 2630-2636 (2018).</li><li>Kirkness, M. W. H., Forde, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+assay+for+proteolytic+susceptibility:+Force-induced+collagen+destabilization.">Single-molecule assay for proteolytic susceptibility: Force-induced collagen destabilization.</a> Biophysical Journal. 114 (3), 570-576 (2018).</li><li>Astumian, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamics+and+kinetics+of+molecular+motors.">Thermodynamics and kinetics of molecular motors.</a> Biophysical Journal. 98 (11), 2401-2409 (2010).</li><li>Bekard, I. B., Asimakis, P., Bertolini, J., Dunstan, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+shear+flow+on+protein+structure+and+function.">The effects of shear flow on protein structure and function.</a> Biopolymers. 95 (11), 733-745 (2011).</li><li>Chistiakov, D. A., Orekhov, A. N., Bobryshev, Y. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+shear+stress+on+endothelial+cells:+go+with+the+flow.">Effects of shear stress on endothelial cells: go with the flow.</a> Acta Physiologica. 219 (2), 382-408 (2017).</li><li>Hu, C., Jonchhe, S., Pokhrel, P., Karna, D., Mao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+unfolding+of+ensemble+biomolecular+structures+by+shear+force.">Mechanical unfolding of ensemble biomolecular structures by shear force.</a> Chemical Science. 12 (30), 10159-10164 (2021).</li><li>Sedghi Masoud, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+interactions+between+telomeric+i-motif+DNA+and+a+cyclic+tetraoxazole+compound.">Analysis of interactions between telomeric i-motif DNA and a cyclic tetraoxazole compound.</a> ChemBioChem. 19 (21), 2268-2272 (2018).</li><li>Abraham Punnoose, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+and+specific+recognition+of+telomeric+G-quadruplexes+via+polyvalency+induced+unstacking+of+binding+units.">Adaptive and specific recognition of telomeric G-quadruplexes via polyvalency induced unstacking of binding units.</a> Journal of the American Chemical Society. 139 (22), 7476-7484 (2017).</li><li>Dhakal, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coexistence+of+an+ILPR+i-motif+and+a+partially+folded+structure+with+comparable+mechanical+stability+revealed+at+the+single-molecule+level.">Coexistence of an ILPR i-motif and a partially folded structure with comparable mechanical stability revealed at the single-molecule level.</a> Journal of the American Chemical Society. 132 (26), 8991-8997 (2010).</li><li>Hu, C., Tahir, R., Mao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+mechanochemical+sensing.">Single-molecule mechanochemical sensing.</a> Accounts of Chemical Research. 55 (9), 1214-1225 (2022).</li></ol>

El protocolo descrito en este manuscrito permite la investigación en tiempo real del despliegue de un conjunto de estructuras biomoleculares por fuerza de corte. Los resultados presentados aquí subrayan que las estructuras del motivo i del ADN pueden desplegarse mediante fuerza de corte. El despliegue del i-motivo ligado al ligando y las reacciones de clic accionadas por fuerza de cizallamiento fueron aplicaciones de prueba de concepto para este método de espectroscopia de fuerza de conjunto.
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En la espectroscopia de fuerza de molécula única1 (SMFS), las propiedades mecánicas de estructuras moleculares individuales han sido estudiadas por instrumentos sofisticados como el microscopio de fuerza atómica, pinzas ópticas y pinzas magnéticas 2,3,4. Restringido por el mismo requisito de direccionalidad de las moléculas en las configuraciones generadoras / de detección de fuerza o el pequeño campo de visión en pinzas magnéticas y el microscopio de fuerza centrífuga en miniatura (MCF)5,6,7,8, solo se puede investigar simultáneamente un número limitado de moléculas utilizando SMFS. El bajo rendimiento de SMFS impide su amplia aplicación en el campo del reconocimiento molecular, que requiere la participación de un gran conjunto de moléculas.
El flujo de cizallamiento proporciona una solución potencial para aplicar fuerzas a un conjunto masivo de moléculas9. En un flujo de líquido dentro de un canal, cuanto más cerca de la superficie del canal, más lento será el caudal10. Tal gradiente de velocidad de flujo causa un esfuerzo cortante que es paralelo a la superficie límite. Cuando una molécula se coloca en este flujo de corte, la molécula se reorienta para que su eje largo se alinee con la dirección del flujo, ya que la fuerza de cizallamiento se aplica al eje largo11. Como resultado de esta reorientación, se espera que todas las moléculas del mismo tipo (tamaño y longitud de los mangos) se alineen en la misma dirección mientras experimentan la misma fuerza de corte.
Este trabajo describe un protocolo para usar dicho flujo de cizallamiento para ejercer fuerza de cizallamiento sobre un conjunto masivo de estructuras moleculares, como lo ejemplifica el motivo i del ADN. En este protocolo, se genera un flujo de corte entre el rotor y el estator en una punta homogeneizadora. El presente estudio encontró que la estructura plegada del i-motivo del ADN podría desplegarse mediante velocidades de cizallamiento de 9724-97245 s−1. Además, se encontró una constante de disociación de 36 μM entre el ligando L2H2-4OTD y el motivo i. Este valor es consistente con el de 31 μM medido por el ensayo de desplazamiento de gel12. Además, la técnica actual se utiliza para desplegar el i-motivo, que puede exponer el cobre quelado (I) para catalizar una reacción de clic. Por lo tanto, este protocolo permite desplegar un gran conjunto de estructuras i-motif con instrumentos de bajo costo en un tiempo razonable (menos de 30 min). Dado que la técnica de fuerza de cizallamiento aumenta drásticamente el rendimiento de la espectroscopia de fuerza, llamamos a esta técnica espectroscopía de fuerza de conjunto (EFS). Este protocolo tiene como objetivo proporcionar directrices experimentales para facilitar la aplicación de este EFS basado en la fuerza de corte.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3K MWCO Amicon</td>
			<td>Millipore Sigma</td>
			<td>ufc900324</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>VWR</td>
			<td>VWRC0143-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Calfluor 488 azide</td>
			<td>Click Chemistry Tools</td>
			<td>1369-1</td>
			<td></td>
		</tr>
		<tr>
			<td>CuCl</td>
			<td>Thermo&#160;</td>
			<td>ACRO270525000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispersion tip</td>
			<td>Switzerland</td>
			<td>PT-DA07/2EC-B101</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA oligos</td>
			<td>IDT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dye</td>
			<td>IDT</td>
			<td>/5Cy5/</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Janpan</td>
			<td>Nikon TE2000-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>Switzerland</td>
			<td>PT 3100D</td>
			<td></td>
		</tr>
		<tr>
			<td>HPG</td>
			<td>Santa Cruz Biotechnology</td>
			<td>cs-295271</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>VWR</td>
			<td>VWRC26760.295</td>
			<td></td>
		</tr>
		<tr>
			<td>MES</td>
			<td>VWR</td>
			<td>VWRCE169-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Quencher</td>
			<td>IDT</td>
			<td>/3IAbRQSp/</td>
			<td></td>
		</tr>
		<tr>
			<td>TBTA</td>
			<td>Tokyo Chemical Industry</td>
			<td>T2993</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>VWR</td>
			<td>VWRCE133-100G</td>
			<td></td>
		</tr>
	</tbody>
</table>

ensemble force spectroscopy by shear forces

Las técnicas de molécula única basadas en principios de fluorescencia y mecanoquímicos proporcionan una sensibilidad superior en la detección biológica. Sin embargo, debido a la falta de capacidades de alto rendimiento, la aplicación de estas técnicas es limitada en biofísica. La espectroscopia de fuerza de conjunto (EFS) ha demostrado un alto rendimiento en la investigación de un conjunto masivo de estructuras moleculares mediante la conversión de estudios mecanoquímicos de moléculas individuales en los de conjuntos moleculares. En este protocolo, las estructuras secundarias de ADN (i-motivos) se desplegaron en el flujo de corte entre el rotor y el estator de una punta homogeneizadora a velocidades de cizallamiento de hasta 77796 / s. Se demostraron los efectos de las tasas de flujo y los tamaños moleculares sobre las fuerzas de cizallamiento experimentadas por el i-motivo. La técnica EFS también reveló la afinidad de unión entre los i-motivos de ADN y los ligandos. Además, hemos demostrado una reacción química de clic que puede ser accionada por fuerza de cizallamiento (es decir, química de clic mecánico). Estos resultados establecen la efectividad del uso de la fuerza de cizallamiento para controlar la conformación de estructuras moleculares.

La Figura 1 describe el despliegue mecánico y la detección en tiempo real de moléculas de conjunto en EFS. En la Figura 1B, se observó que la intensidad de fluorescencia del ADN i-motif aumentaba con una velocidad de cizallamiento que oscilaba entre 9.724 s−1 y 97.245 s−1 en un tampón MES de pH 5,5. Como control, la intensidad de fluorescencia no aumentó cuando el mismo ADN i-motif se cortó a una velocidad de 63...

Watch this Scientific Journal Video about Ensemble Force Spectroscopy by Shear Forces at JoVE.com

Ensemble Force Spectroscopy by Shear Forces

La espectroscopia de fuerza de conjunto (EFS) es una técnica robusta para el despliegue mecánico y la detección en tiempo real de un conjunto de estructuras biomoleculares en campos biofísicos y biosensibles.

Espectroscopía de fuerza de conjunto por fuerzas de cizallamiento

Single-molecule techniques based on fluorescence and mechanochemical principles provide superior sensitivity in biological sensing. However, due to the ...

Este protocolo proporciona una técnica de alto rendimiento para estudiar la unión intermolecular entre el ADN y los ligandos. Esta técnica ha demostrado un alto rendimiento en la investigación de un conjunto masivo de estructuras biomoleculares. Lo hace convirtiendo las propiedades químicas mecánicas de moléculas individuales en las de conjuntos moleculares.Creo que es un experimento muy avanzado, pero hay pocos pasos críticos. Por ejemplo, la alineación del microscopio, el homogeneizador y el tubo de reacción. Estas tres cosas deben estar perfectamente alineadas para rastrear con precisión el cambio en la fluorescencia.Comience ensamblando el homogeneizador y el microscopio en una mesa de montaje. Después de usar gafas, encienda el microscopio de fluorescencia y luego ajuste el homogeneizador para asegurarse de que el haz de luz de excitación pase a través del centro de la punta dispersa del homogeneizador. Luego prepare una cámara de reacción de fondo plano que tenga cinco centímetros de altura y 1.5 por 1.5 centímetros cuadrados de sección transversal, asegurándose de que el material de la cámara seleccionado no emita fluorescencia para reducir el fondo.Después, sujete la cámara de reacción en la etapa de muestra del microscopio de fluorescencia y luego ajuste la cámara para sostener la punta dispersante del homogeneizador, asegurándose de que la punta esté ligeramente por encima de la superficie inferior de la cámara de reacción. A continuación, ajuste la posición vertical del homogeneizador y la cámara juntos para asegurarse de que el foco del microscopio esté en la superficie de la punta de dispersión. A continuación, ajuste la posición horizontal de la punta de dispersión para asegurarse de que el área de detección se establece entre el rotor y el estador.Encienda los canales fluorescentes, de acuerdo con el tinte fluorescente utilizado en el experimento. Antes del experimento de cizallamiento de alta velocidad, se utilizó agua desionizada para probar el cizallamiento con una velocidad de cizallamiento baja. Primero, prepare un ADN telomérico humano con un e-motivo marcado con un tinte y un quencher en sus dos extremos, respectivamente, en agua desionizada como se describe en el manuscrito.A continuación, diluya el ADN en cinco micromolares en tampón MES de 30 milimolares a pH 5.5 o pH 7.4. A la solución de ADN, agregue el ligando L2H24OTD" en un rango de concentración de cero a 60 micromolares y mezcle la solución suavemente durante 10 minutos para doblar las estructuras del motivo i sin luz. Verifique y minimice la intensidad de fluorescencia de fondo de la cámara de reacción que se llena con agua desionizada utilizando el microscopio fluorescente sin cizallamiento.Luego configure los parámetros de la cámara CCD utilizando el software con un tiempo de exposición de 0,5 segundos, sensibilidad CCD de 1, 600 y un tiempo de grabación igual a 20 minutos. Con una pipeta larga, agregue la solución de ADN a la cámara de reacción vacía y limpia y cubra la cámara de reacción con una caja negra. Luego inicie el homogeneizador para realizar el cizallamiento a una velocidad de cizallamiento seleccionada que oscila entre 9, 724 por segundo y 97, 245 por segundo durante 20 minutos, con la cámara CCD encendida para grabar los datos.Después del experimento, retire la cámara y lávela con agua desionizada. Preparar ADN i-motif y agua desionizada. Luego, incubar 10 micromolares de ADN i-motif y 300 microlitros de 30 milimolar tris buffer suplementado con 150 cloruro cúprico micromolar y 300 ácido ascórbico micromolar durante 10 minutos para doblar las estructuras i-motivo.A continuación, ultra filtre la solución con un dispositivo de ultrafiltración a una fuerza centrífuga de 14, 300 veces G.Reponga la solución a aproximadamente 500 microlitros con 30 tampones tris milimolares suplementados con 300 ácido ascórbico micromolar después de cada filtración. Después de recoger la solución residual, hacer un volumen final de 300 microlitros añadiendo 30 tris milimolares suplementados con 300 micromolares de ácido ascórbico junto con 20 micromolares CalFluor 488 Azide, 20 micromolares HPG y 10 micromolares TBTA. Una vez que se agreguen los reactivos, mueva la solución al cuarto oscuro.Luego verifique y minimice la intensidad de fluorescencia de fondo de la cámara de reacción llena de agua desionizada usando el microscopio antes de los experimentos de cizallamiento. A continuación, agregue la solución de ADN en la cámara de reacción vacía con una pipeta larga y luego inicie el cizallamiento del homogeneizador a una velocidad de cizallamiento de 63, 209 por segundo durante 20 minutos con la cámara CCD encendida. Después del experimento, retire la cámara y lávela con agua desionizada.Se observó que la intensidad de fluorescencia del ADN i-motif aumentaba con una velocidad pura que oscilaba entre 9, 724 por segundo y 97, 245 por segundo en un tampón MES de pH 5.5. Sin embargo, la intensidad de fluorescencia no aumentó cuando el mismo ADN del motivo i se cortó a una velocidad de 63, 209 por segundo, ya que no se pliega a pH 7.4 en el tampón MES. Se evaluó la unión del ligando a las moléculas de ADN i-motivo sometidas al flujo puro, lo que mostró que el incremento de la intensidad de fluorescencia se hizo menos obvio con el aumento de la concentración del ligando.La constante de disociación se determinó para la interacción entre el ligando y el i-motivo y se encontró que eran 34 micromoles. El motivo i-quelatado de cobre se desplegó a una velocidad de cizallamiento de 63, 209 por segundo, y el de cobre liberado desencadenó la reacción de clic fluorgénico, lo que resultó en una mayor intensidad de fluorescencia con el tiempo. En primer lugar, asegúrese de que el área de detección esté establecida entre el rotor y el estador.En segundo lugar, verifique y minimice el fondo de fluorescencia. Y, por último, asegúrese de filtrar la reacción antes de realizar la reacción de clic. Este método abre una puerta para explorar la resistencia mecánica, plegando y desplegando todo tipo de polímeros.Podrían ser otras estructuras de ADN, proteínas u otros polímeros, y su interacción con otros tipos de moléculas. Como se dijo, varias otras macromoléculas podrían estudiarse utilizando este enfoque.

ensemble-force-spectroscopy-by-shear-forces

Research

JoVE Journal

Bioengineering

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, &Delta;G&deg;H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.

This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.

Termodinámica del plegado de proteínas de membrana medido por espectroscopia de fluorescencia

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

Investigación

Bioingeniería

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

Inmovilización de bacterias para tratamiento de imágenes de microscopía de fuerza atómica

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

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.

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

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important applications in the quest for the development of novel therapeutics. Assays using static conditions fail to capture the dependence of adhesion on shear, limiting their correlation with in vivo environment. Parallel plate flow chambers that quantify adhesion under physiological fluid flow need multiple experiments for the generation of a shear adhesion map. In addition, they do not represent the in vivo scale and morphology and require large volumes (~ml) of reagents for experiments. In this study, we demonstrate the generation of shear adhesion map from a single experiment using a microvascular network based microfluidic device, SynVivo-SMN. This device recreates the complex in vivo vasculature including geometric scale, morphological elements, flow features and cellular interactions in an in vitro format, thereby providing a biologically realistic environment for basic and applied research in cellular behavior, drug delivery, and drug discovery. The assay was demonstrated by studying the interaction of the 2 &#181;m biotin-coated particles with avidin-coated surfaces of the microchip. The entire range of shear observed in the microvasculature is obtained in a single assay enabling adhesion vs. shear map for the particles under physiological conditions.

Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables.

Generación de Shear Adhesión Mapa Utilizando SynVivo redes microvasculares sintético

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important ...

Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using sonication, mechanical agitation, microfluidic devices, or homogenization. Recently, lipid-based oxygen microbubbles (LOMs) have been designed to deliver oxygen intravenously during medical emergencies, reversing life-threatening hypoxemia, and preventing subsequent organ injury, cardiac arrest, and death. We present methods for scaled-up production of highly oxygenated microbubbles using a closed-loop high-shear homogenizer. The process can produce 2 L of concentrated LOMs (90% by volume) in 90 min. Resulting bubbles have a mean diameter of ~2 &#956;m, and a rheologic profile consistent with that of blood when diluted to 60 volume %. This technique produces LOMs in high capacity and with high oxygen purity, suggesting that this technique may be useful for translational research labs.

We describe methods for the manufacture of large volumes of lipid-based oxygen microbubbles (LOMs) designed for intravenous oxygen delivery using high-shear homogenization and serial concentration.

Fabricación de concentrado, de oxígeno a base de lípidos microburbujas Emulsiones por High Shear Homogeneización y Concentración de serie

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using ...

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

Medición de la fuerza isométrica máxima generada por las fibras musculares esqueléticas permeabilizadas

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

Fuerza Atómica Microscopía Imaging y Fuerza Espectroscopia de admitidos Bilayers lípidos

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

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

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.

Un protocolo para el uso de Transferencia de energía de resonancia de Förster (FRET) y forzar Biosensores para medir fuerzas mecánicas en todo el complejo nuclear de LINC

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

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Detección de endotoxinas en las formulaciones Nano mediante ensayos de Limulus Amoebocyte Lysate (LAL)

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell cultures. Microcarrier culture technology has become one of the main techniques in cytological research and is commonly used in the field of large-scale cell expansion. Microcarriers have also been shown to play an increasingly important role in in vitro tissue engineering construction and clinical drug screening. Current methods for preparing microcarriers include microfluidic chips and inkjet printing, which often rely on complex flow channel design, an incompatible two-phase interface, and a fixed nozzle shape. These methods face the challenges of complex nozzle processing, inconvenient nozzle changes, and excessive extrusion forces when applied to multiple bioink. In this study, a 3D printing technique, called alternating viscous-inertial force jetting, was applied to enable the construction of hydrogel microcarriers with a diameter of 100-300 &#181;m. Cells were subsequently seeded on microcarriers to form tissue engineering modules. Compared to existing methods, this method offers a free nozzle tip diameter, flexible nozzle switching, free control of printing parameters, and mild printing conditions for a wide range of bioactive materials.

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Presented here is a mild 3D printing technique driven by alternating viscous-inertial forces to enable the construction of hydrogel microcarriers. Homemade nozzles offer flexibility, allowing easy replacement for different materials and diameters. Cell binding microcarriers with a diameter of 50-500 &#181;m can be obtained and collected for further culturing.

Impresión 3D de microportadores de hidrogel in vitro mediante chorro de fuerza viscoso-inercial alternado

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell ...