Estamos muy en deuda con Jonathon Howard, cuyo grupo desarrolló el protocolo básico para un ensayo de la motilidad de deslizamiento, que fue adaptado posteriormente por nosotros. El apoyo financiero de NSF concesión DMR0645023 se agradece.

1.) Buffers y Reactivos Estas soluciones deben prepararse con antelación y se almacena en alícuotas de tamaño conveniente. Una alícuota deberá contener solución suficiente para un experimento típico y una alícuota se debe utilizar para cada ensayo de motilidad. Las condiciones de almacenamiento y los tamaños típicos alícuota también se mencionan en los siguientes protocolos. 1. BRB80 buffer, (80 TUBOS mM, MgCl2 1 mM, EGTA 1 mM en agua desionizada destilada (dd) de agua, pH ajustado a 6,9 por KOH) <ul><li> Constituyen una solución de 100 ml de caldo de 0,5 M EGTA en agua dd. Ajustar el pH a 7.0 con 2 M NaOH. </li><li> Constituyen una solución de 100 ml de caldo de 1 M de MgCl 2 en el agua dd. Autoclave la solución. </li><li> Añadir 24,2 g de tuberías y 3,1 g de pellets de KOH en aproximadamente 800 ml de agua dd y revuelva hasta disolver. Ajustar el pH a 6.9 con 1 M solución de KOH. Añadir 2 ml de solución 0,5 M de valores EGTA y 1 ml de una solución de reserva M MgCl 2. Llevar el volumen a 1000 ml con agua dd. </li><li> Alícuota de 50 ml en tubos falcon y se congelan a -20 ° C para su uso futuro. El tubo BRB80 actualmente se utilizan se pueden almacenar a 4 ° C oa temperatura ambiente. </li></ul> 2. Cloruro de magnesio, MgCl2 (100 mM en agua dd) <ul><li> Diluido en agua dd para conseguir una concentración final de 100 mM. </li><li> Alícuota (10 l de volumen) en 0,5 ml de tubos de microcentrífuga y se almacena a -20 ° C para su uso futuro </li></ul> 3. Guanosina-5&#39;-trifosfato, sal disódica, GTP (25 mM dd agua, se ajustó el pH a 7 con NaOH) <ul><li> Se pesa y se disuelve en agua dd y ajustar el pH a 7 por 2 M de NaOH. </li><li> Verificar la concentración midiendo la absorbancia UV a 260 nm. (Use un coeficiente de extinción de 11,7 x 103 M -1 cm -1). </li><li> Alícuota (10 l de volumen) en 0,5 ml de tubos de microcentrífuga y se almacena a -20 ° C para su uso futuro. </li></ul> 4. Dimetilsulfóxido, DMSO <ul><li> Alícuota (10 l de volumen) en 0,5 ml de tubos de microcentrífuga y se almacena a -20 ° C para su uso futuro. </li></ul> 5. Taxol (1 mM en DMSO) <ul><li> Se pesa y se disuelven en DMSO bajo campana de extracción para lograr una concentración final de 1 mM. </li><li> Alícuota (20 l de volumen) en 0,5 ml de tubos de microcentrífuga y se almacena a -20 ° C para su uso futuro. </li></ul> 6. D-(+)-glucosa, (2 M en agua dd) <ul><li> Se pesa y se disuelve en agua dd para conseguir una concentración final de 2 M. </li><li> Alícuota (20 l de volumen) en 0,5 ml de tubos de microcentrífuga y se almacena a -20 ° C para su uso futuro. </li></ul> 7. Glucosa oxidasa, (2 mg / ml en BRB80) <ul><li> Disolver en BRB80 para lograr una concentración final de 2 mg / ml. </li><li> Alícuota (20 l de volumen) en 0,5 ml de tubos de microcentrífuga y se almacena a -20 ° C para su uso futuro. </li></ul> 8. Ditiotreitol, TDT (1 M en agua dd) <ul><li> Disolver en agua dd bajo campana de extracción para lograr una concentración final de 1 M. </li><li> Alícuota (20 l de volumen) en 0,5 ml de tubos de microcentrífuga y se almacena a -20 ° C para su uso futuro. </li></ul> 9. Catalasa, (0,8 mg / ml en BRB80) <ul><li> Disolver en BRB80 en al menos dos etapas para lograr una concentración final de 0,8 mg / ml. Determinar la concentración en cada etapa mediante la medición de absorbancia UV a 276 nm y 406 nm (Use un coeficiente de extinción de 3,1 x 10 5 M -1 cm -1 a 276 nm y 2.2 x 10 5 M -1 cm -1 a 406 nm y la ecuación A = cL). </li><li> Alícuota (20 l de volumen) en 0,5 ml de tubos de microcentrífuga y se almacena a -20 ° C para su uso futuro. </li></ul> 10. Adenosina-5&#39;-trifosfato, ATP (100 mM en 100 mM MgCl 2) <ul><li> Prepare una solución stock de 100 mM de MgCl 2 en el agua dd. Pesar de polvo seco y se disuelven en esta solución y para lograr una concentración final de 100 mm. </li><li> Verificar la concentración midiendo la absorbancia UV a 260 nm. (Use un coeficiente de extinción de 15,4 x 10 3 M -1 cm -1). </li><li> Alícuota (20 l de volumen) en 0,5 ml de tubos de microcentrífuga y se almacena a -20 ° C para su uso futuro. </li></ul> 11. Caseína solución (20 mg / ml de caseína en BRB80) <ul><li> Añadir aproximadamente 3 g de caseína a 30 ml de agua dd en un tubo Falcon de 50 ml. Vortex durante 1 hora aproximadamente hasta que la solución se desarrolla consistencia espesa. </li><li> Se centrifuga a aproximadamente 15.000 g durante 30 minutos. Filtrar el sobrenadante a través de 0.5 micras y 0.2 micras filtros de jeringa. </li><li> Determinar la concentración del sobrenadante midiendo la absorbancia UV a 280 nm (Use un coeficiente de extinción de 0,67 cm mg mL -1 -1). Diluir a 20 mg / ml en BRB80. </li><li> Alícuota (20 l de volumen) enpara tubos de 0,5 ml de microcentrífuga y se almacena a -20 ° C para su uso futuro. </li></ul> 2.) Soluciones estándar Estos se preparan en el día de la prueba y debe ser desechado después de que el experimento ha terminado. Prepare 1 ml de cada uno. 1. BRB80CS0.5 <ul><li> Solución diluida de caseína en BRB80 a una concentración final de 0,5 mg / ml y almacenar más de hielo. Esta solución se introduce en la celda de flujo antes de la kinesina y ayuda a mantener la actividad después de kinesin superficie de adsorción. </li></ul> 2. BRB80CA <ul><li> Prepare 0,2 mg / ml de caseína y un ATP mM en BRB80 y almacenar más de hielo. Kinesin se diluye aún más con esta solución antes de introducirlo en la celda de flujo. </li></ul> 3. BRB80T <ul><li> Solución diluida de taxol en BRB80 a una concentración final de 10 mM y se almacena a temperatura ambiente. Esta solución se utiliza para estabilizar los microtúbulos. </li></ul> 4. BRB80CT <ul><li> Preparar 10 taxol M y 0,2 mg / ml de caseína en BRB80 y almacenar a temperatura ambiente. Esto se utiliza para preparar a la Antifade y soluciones de los microtúbulos. </li></ul> 5. BRB80AF <ul><li> Prepare 20 mM D-glucosa, 20 mg / ml de glucosa oxidasa, 8 mg / ml de catalasa, 10 mM DTT, y 20 mM de ATP en BRB80CT y almacenar a temperatura ambiente. Esta solución se utiliza para diluir la estreptavidina y las nanoesferas y &quot;eliminar&quot; el exceso de estreptavidina en la celda de flujo. La velocidad de kinesina se puede controlar mediante el ajuste de la concentración de ATP en esta solución. </li></ul> 3.) Kinesin Preparación <ul><li> Expresar una kinesin construcción que consiste en el tipo salvaje, de larga duración en la cadena de Drosophila melanogaster kinesin pesada y un C-terminal-Su etiqueta en Escherichia coli y purificar utilizando una columna de Ni-NTA como se describe en 6. </li><li> Hacer alícuotas (10 l cada uno) en tubos de 0,5 mL microcentrífuga y almacenar a -80 ° C para su uso futuro. La concentración de activos en estos kinesin alícuotas es de aproximadamente 200 nM 7. </li><li> Para un experimento típico, se diluye la solución kinesin 20 veces en BRB80CA. La etiqueta del KIN20 solución y almacenar más de hielo. </li></ul> 4.) Microtúbulos Preparación <ul><li> En un tubo de microcentrífuga de 0,5 mL, preparar 25 L de solución de crecimiento: 4 mM MgCl 2, 1 mM GTP y un 5% DMSO (v / v) en BRB80 buffer. </li><li> Añadir 6,25 l de esta solución a una alícuota de 20 mg de biotina tubulina liofilizado. </li><li> Vortex, luego colóquelo en un baño de calor a 37 ° C durante 30 minutos para polimerizar. Diluir 100 veces en BRB80T y agitar suavemente. La etiqueta del MT100 solución y almacenar a temperatura ambiente. </li><li> Hacer una dilución de 10 veces en BRB80AF MT100. La etiqueta del MT1000 solución. </li></ul> 5.) Estreptavidina y Nanosphere Solución Prepare AlexaFluor568 estreptavidina marcada con una concentración de 100 nM en BRB80AF. Etiqueta que STV100 y almacenar más de hielo. Del mismo modo, diluir nanoesferas 5000 veces en la solución de BRB80AF. Etiqueta que NS5000 y almacenar más de hielo. 6.) Flujo de construcción de la célula Construir una celda de flujo con dos cubreobjetos de vidrio separados por cinta de doble cara. Esta celda de flujo es de aproximadamente 2 cm de largo, 1 cm de ancho y 100 micras, y tiene un volumen de aproximadamente 20 mL. Las soluciones se introdujeron en la celda de flujo de un lado con una pipeta y con los malos de los otros con papel de filtro. 7.) Asamblea de ensayo invertido Superficie del vidrio es la primera cubierta con la caseína que permite kinesina para mantener su funcionalidad en la adsorción. Después de kinesina se absorbe, los microtúbulos se introducen, que están en manos de quinesina. Los microtúbulos son luego recubiertos con estreptavidina fluorescentes. Después de lavar el exceso de estreptavidina, biotina fluoresceína nanoesferas de poliestireno (40 nm de diámetro) se introducen. Superficie adsorbido nanoesferas estacionaria chocan con los microtúbulos en movimiento y se cargan sobre ellos (Figura 1). El orden de flujo de las soluciones y el tiempo permitido antes de la introducción de la solución a continuación se enumeran a continuación. <ul><li> BRB80CS0.5, a 5 minutos </li><li> KIN20, a 5 minutos </li><li> MT1000, a 5 minutos </li><li> STV100, a 5 minutos </li><li> BRB80AF, 3x </li><li> NS5000 </li></ul> 8.) Microscopía Montar la celda de flujo en la platina del microscopio inmediatamente después de la introducción nanoesfera. En este experimento, un Eclipse TE2000-U microscopio de fluorescencia (Nikon, Melville, NY) equipado con un objetivo de aceite de 100X (AN 1.45), un X-Cite 120 de la lámpara (EXFO, Ontario, Canadá) y un ixon EMCCD cámara (ANDOR, South Windsor, CT) se utilizó. Un filtro FITC cubo (# 48001) y un cubo de filtro TRITC (# 48002, Tecnologías de Chroma, Rockingham, VT) se utilizaron para la imagen y nanoesferas respecti microtúbulosvamente en la superficie inferior de las celdas de flujo. El tiempo de exposición fue de 0,2 s, mientras que el tiempo entre la exposición fue de 2 s. <img src="/files/ftp_upload/2006/2006fig1.jpg" alt="Figura 1" /> Figura 1. Esquema del servicio de transporte molecular.

<ol>
	<li>Agarwal, A., Hess, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomolecular+motors+at+the+intersection+of+nanotechnology+and+polymer+science.">Biomolecular motors at the intersection of nanotechnology and polymer science.</a> Progress in Polymer Science. 35 (1-2), 252-252 (2010).</li><li>Howard, J., Hunt, A. J., Baek, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+microtubule+movement+driven+by+single+kinesin+molecules.">Assay of microtubule movement driven by single kinesin molecules.</a> Methods Cell Biol. 39, 137-137 (1993).</li><li>Agarwal, A., Katira, P., Hess, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond+curing+time+of+a+molecular+adhesive+causes+velocity-dependent+cargo-loading+of+molecular+shuttles.">Millisecond curing time of a molecular adhesive causes velocity-dependent cargo-loading of molecular shuttles.</a> Nano Lett. 9 (3), 1170-1170 (2009).</li><li>Diez, S., Reuther, C., Dinu, C., Seidel, R., Mertig, M., Pompe, W., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretching+and+Transporting+DNA+Molecules+Using+Motor+Proteins.">Stretching and Transporting DNA Molecules Using Motor Proteins.</a> Nano Lett. 3 (9), 1251-1251 (2003).</li><li>Bachand, G. D., Rivera, S. B., Boal, A. K., Gaudioso, J., Liu, J., Bunker, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+transport+of+nanocrystal+CdSe+quantum+dot+nanocomposites+using+microtubules+and+kinesin+motor+proteins.">Assembly and transport of nanocrystal CdSe quantum dot nanocomposites using microtubules and kinesin motor proteins.</a> Nano Lett. 4 (5), 817-817 (2004).</li><li>Coy, D. L., Wagenbach, M., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesin+takes+one+8-nm+step+for+each+ATP+that+it+hydrolyzes.">Kinesin takes one 8-nm step for each ATP that it hydrolyzes.</a> J. Biol. Chem. 274 (6), 3667-3667 (1999).</li><li>Katira, P., Agarwal, A., Fischer, T., Chen, H. -. Y., Jiang, X., Lahann, J., Hess, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+performance+of+protein-resisting+surfaces+at+ultra-low+protein+coverages+using+kinesin+motor+proteins+as+probes.">Quantifying the performance of protein-resisting surfaces at ultra-low protein coverages using kinesin motor proteins as probes.</a> Advanced Materials. 19, 3171-3171 (2007).</li><li>Vigers, G. P. A., Coue, M., McIntosh, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Microtubules+Break+Up+Under+Illumination.">Fluorescent Microtubules Break Up Under Illumination.</a> J. Cell Biol. 107, 1011-1011 (1988).</li><li>Brunner, C., Hess, H., Ernst, K. -. H., Vogel, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifetime+of+biomolecules+in+hybrid+nanodevices.">Lifetime of biomolecules in hybrid nanodevices.</a> Nanotechnology. 15 (10), S540-S540 (2004).</li></ol>

Con pequeñas modificaciones, este protocolo ha sido utilizado con éxito por una variedad de grupos para montar kinesina-microtúbulo ensayos de movilidad basado en. 10 mM de TDT en la solución final de la motilidad puede ser reemplazado con un 0,5% β-mercaptoetanol. Soluciones estándar (BRB80AF, KIN20 y MT1000) de más de 2 horas de vida no se debe utilizar. Cualquier solución que contiene microtúbulos taxol y sobre todo nunca deben ser colocados en el hielo. La exposición excesiva de la celda de flujo a los ray...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Adenosine-5&#x2019;-triphosphate (ATP)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A1049</td>
<td>&nbsp;</td>
<tr>
<td>Biotin tubulin</td>
<td>&nbsp;</td>
<td>Cytoskeleton Inc.</td>
<td>T333</td>
<td>&nbsp;</td>
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<td>Casein</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>C-0376</td>
<td>&nbsp;</td>
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<td>Catalase</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>C-9322</td>
<td>&nbsp;</td>
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<td>D-(+)-Glucose</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G-7528</td>
<td>&nbsp;</td>
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<td>Dimethylsulfoxide (DMSO)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D-8779</td>
<td>&nbsp;</td>
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<td>Bio-Rad</td>
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<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#x2032;,N&#x2032;-tetraacetic acid (EGTA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>E-4378</td>
<td>&nbsp;</td>
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<td>FluoSpheres Biotinylated microspheres, 40 nm, yellow-green fluorescent (505/515)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>F-8766</td>
<td>&nbsp;</td>
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<td>Glucose oxidase</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G-7016</td>
<td>&nbsp;</td>
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<td>Guanosine-5&#x2019;-triphosphate (GTP)</td>
<td>&nbsp;</td>
<td>Roche Diagnostic</td>
<td>106399</td>
<td>&nbsp;</td>
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<td>Magnesium Chloride (MgCl2)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>63069</td>
<td>&nbsp;</td>
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<td>Paclitaxel (Taxol)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T1912</td>
<td>&nbsp;</td>
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<td>1,4-Piperazinediethanesulfonic acid, Piperazine-1,4-bis(2-ethanesulfonic acid), Piperazine-N,N&#x2032;-bis(2-ethanesulfonic acid) (PIPES)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P-6757</td>
<td>&nbsp;</td>
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<td>Potassium hydroxide (KOH)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P-6310</td>
<td>&nbsp;</td>
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<td>Sodium hydroxide (NaOH)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>480878</td>
<td>&nbsp;</td>
<tr>
<td>Streptavidin Alexa Fluor 568 conjugate</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>S11226</td>
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cargo loading onto kinesin powered molecular shuttles

Las células han desarrollado mecanismos moleculares sofisticadas, como las proteínas motoras kinesina y filamentos de microtúbulos, para apoyar el transporte activo intracelular de la carga. Mientras que la cola de dominio kinesins se une a una variedad de cargas, los dominios kinesins cabeza utilizar la energía química almacenada en moléculas de ATP al paso a lo largo de la red de microtúbulos. Los microtúbulos largas y rígidas sirven como pistas para el transporte de larga distancia intracelular. Estos motores y los filamentos se pueden emplear también en microfabricated entornos sintéticos como componentes del servicio de transporte molecular 1. En un diseño de uso frecuente, los motores de quinesina se anclan a la superficie de la pista a través de sus colas, y los microtúbulos funcionalizados servir como elementos de transporte de carga, que son impulsados ​​por estos motores. Estas naves se pueden cargar con la carga mediante la utilización de la unión fuerte y selectiva entre biotina y estreptavidina. Los componentes clave (tubulina biotina, estreptavidina, biotina y de carga) están disponibles comercialmente. Basándose en el ensayo de la motilidad clásico invertido 2, la construcción de lanzaderas moleculares se detalla aquí. Kinesin proteínas motoras son adsorbidos en una superficie recubiertas con caseína, los microtúbulos de tubulina se polimerizan con biotina, se adhirió a la quinesina y posteriormente recubiertos con estreptavidina marcada con rodamina. La concentración de ATP se mantiene a una concentración subsaturating para lograr una velocidad de deslizamiento de microtúbulos óptima para operaciones de carga 3. Por último, biotina marcado con fluoresceína nanoesferas se agregan como carga. Nanoesferas se adhieren a los microtúbulos, como resultado de colisiones entre los microtúbulos deslizándose y nanoesferas adherirse a la superficie. El protocolo puede ser fácilmente modificado para cargar una gran variedad de cargas, como el ADN biotinilado 4, 5 o puntos cuánticos una amplia variedad de antígenos mediante anticuerpos con biotina 4-6.

Watch this Scientific Journal Video about Cargo Loading onto Kinesin Powered Molecular Shuttles at JoVE.com

Cargo Loading onto Kinesin Powered Molecular Shuttles

Servicio de transporte molecular que consiste en deslizarse sobre los microtúbulos funcionalizados superficie adherido kinesin proteínas motoras pueden servir como un sistema de transporte a nanoescala. En este caso, el montaje de un sistema de transporte típico se describe.

Las operaciones de carga en Kinesin molecular Traslados Desarrollado

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport ...

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10.4K vistas.  University of Florida. Servicio de transporte molecular que consiste en deslizarse sobre los microtúbulos funcionalizados superficie adherido kinesin proteínas motoras pueden servir como un sistema de transporte a nanoescala. En este caso, el montaje de un sistema de transporte típico se describe.

Video: Cargo Loading onto Kinesin Powered Molecular Shuttles

Ex vivo Mechanical Loading of Tendon

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37&deg;C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.

A new in vitro system for simultaneously loading four tendons in culture is described.

Ex vivo Carga mecánica del tendón

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace.  Most are associated with ...

Investigación

Biología

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

Cargando Drosophila terminales nerviosas con indicadores de calcio

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

Molecular Evolution of the Tre Recombinase

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. 
We started with Cre, a 38-kDa recombinase, that recognizes a 34-bp double-stranded DNA sequence known as loxP. Because Cre can effectively eliminate genomic sequences, we set out to tailor a recombinase that could remove the sequence between the 5'-LTR and 3'-LTR of an integrated HIV-1 provirus. As a first step we identified sequences within the LTR sites that were similar to loxP and tested for recombination activity. Initially Cre and mutagenized Cre libraries failed to recombine the chosen loxLTR sites of the HIV-1 provirus. As the start of any directed molecular evolution process requires at least residual activity, the original asymmetric loxLTR sequences were split into subsets and tested again for recombination activity. Acting as intermediates, recombination activity was shown with the subsets. Next, recombinase libraries were enriched through reiterative evolution cycles. Subsequently, enriched libraries were shuffled and recombined. The combination of different mutations proved synergistic and recombinases were created that were able to recombine loxLTR1 and loxLTR2. This was evidence that an evolutionary strategy through intermediates can be successful. After a total of 126 evolution cycles individual recombinases were functionally and structurally analyzed. The most active recombinase -- Tre -- had 19 amino acid changes as compared to Cre. Tre recombinase was able to excise the HIV-1 provirus from the genome HIV-1 infected HeLa cells (see "HIV-1 Proviral DNA Excision Using an Evolved Recombinase", Hauber J., Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany). While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of "molecular surgery" and molecular medicine.

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of molecular surgery and molecular medicine.

Evolución molecular de la recombinasa Tre

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within ...

From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF plates to feeder cell-free Matrigel plates.


This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 2 of 3.

Desde MEFs a Matrigel 2: La división de hESCs MEFs en Matrigel

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF ...

From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated.

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 3 of 3.

Desde MEFs a Matrigel 3: hESCs pases de Matrigel en Matrigel

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

Concentración, la carga, y la alineación de una célula de ultracentrifugación analítica de muestras

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Aislamiento y purificación de Kinesin desde Drosophila Los embriones

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

Direct Imaging de calcio ER con Targeted-esterasa inducida colorante de carga (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

Generating a &#34;Humanized&#34; Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper distribution of the genetic material requires that sister kinetochores on every chromosome become bioriented by attaching to microtubules from opposite spindle poles before progressing into anaphase. However, erroneous, non-bioriented attachment states are common and cellular pathways exist to both detect and correct such attachments during cell division. The process by which improper kinetochore-microtubule interactions are destabilized is referred to as error correction. To study error correction in living cells, incorrect attachments are purposely generated via chemical inhibition of kinesin-5 motor, which leads to monopolar spindle assembly, and the transition from mal-orientation to biorientation is observed following drug washout. The large number of chromosomes in many model tissue culture cell types poses a challenge in observing individual error correction events. Drosophila S2 cells are better subjects for such studies as they possess as few as 4 pairs of chromosomes. However, small molecule kinesin-5 inhibitors are ineffective against Drosophila kinesin-5 (Klp61F). Here we describe how to build a Drosophila cell line that effectively replaces Klp61F with human kinesin-5, which renders the cells sensitive to pharmacological inhibition of the motor and suitable for use in the cell-based error correction assay.

Generating a "Humanized" Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

This protocol describes how to generate a Drosophila S2 cell line that is sensitive to small molecule inhibitors of kinesin-5. The use of these cells in a cell-based error correction assay is also outlined.

Generación de un &quot;humanizado&quot;<em&gt; Drosophila</em&gt; S2 Línea Celular Sensible a farmacológico Inhibición de la Kinesin-5

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper ...

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear transcription factor c-MYC for proteosomal degradation in the cytoplasm. The method described here involves the study of a motorless KIF5B mutant for fluorescence microscopy. The wild-type and motorless KIF5B proteins are tagged with the fluorescent protein tdTomato. The wild-type tdTomato-KIF5B appears homogenously in the cytoplasm, while the motorless tdTomato-KIF5B mutant forms aggregates in the cytoplasm. Aggregation of the motorless KIF5B mutant induces aggregation of its cargo c-MYC in the cytoplasm. Hence, this method provides a visual means to identify the cargos of Kinesin-1. A similar strategy can be utilized to identify cargos of other motor proteins.

Here, a protocol is presented to identify Kinesin-1 cargos. A motorless mutant of the Kinesin-1 heavy chain (KIF5B) aggregates in the cytoplasm and induces aggregation of its cargos. Both aggregates are detected under fluorescence microscopy. A similar strategy can be employed to identify cargos of other motor proteins.

Identificación de Kinesin-1 Cargos El uso de microscopía de fluorescencia

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear ...