Name: purification of the membrane compartment for endoplasmic reticulum-associated degradation of exogenous antigens in cross-presentation
Uploaded: 2017-08-21T14:00:00.000+00:00
Duration: 12 min 48 s
Description: Watch this Scientific Journal Video about Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation at JoVE.com

Este trabajo es apoyado por la Universidad de Takasaki de la salud y el bienestar.

1. cultivo de células y la adición de antígenos exógenos bOVA <ol><li>preparar utilizando un etiquetado de biotina-proteína kit siguiendo el fabricante &#39; Protocolo s. 
	Nota: Normalmente, bOVA contiene biotina de 2 M por 1 M huevos en promedio.</li>
	<li>DC2.4 crecer las células en RPMI-1640 suplementado con 2 mM L-glutamina, piruvato de sodio 1 mM, 0.1 mM aminoácidos no esenciales, 100 U/mL de penicilina-estreptomicina, 2-Mercaptoetanol de 55 mM, 10 mM HEPES (pH 7,5) y suero de ternera fetal 10% (en lo sucesivo RPMI) a 37 ° C en 5 % CO 2 en una incubadora humidificada (en lo sucesivo sin mención, las células se incuban en esta condición). También es posible utilizar DMEM suplementado con 10% de suero fetal de becerro, 3,7 g/L de NaHCO 3 (en adelante DMEM) en lugar del RPMI.</li>
	<li>Un día antes de la purificación, dividir las células en RPMI a 1 x 10 5 células/mL en una placa de cultivo de tejidos. Evitar mantener las células en un estado confluente. Las células DC2.4 pueden altamente incorporar antígenos exógenos hasta un estado semi-confluente, pero pierde rápidamente la capacidad después de alcanzar un estado de excesiva confluentes.</li>
	<li>Antes la DC2.4 las células son parcialmente confluentes, Añadir 250 μg/mL de bOVA para 1 x 10 6 células/mL e incube durante 2-4 h. 
	Nota: Durante los experimentos posteriores, todos los buffers y reactivos se mantienen a 4 ° C a menos que se indique lo contrario.</li>
</ol> 2. Preparación de los microsomas <ol><li>las células DC2.4 de la cosecha mediante pipeteo suave de la placa de tejido en un tubo cónico de 50 mL nuevo.</li>
	<li>Centrifugar a 1.000 x g durante 5 min, 4 ° C y retirar cuidadosamente el medio con bOVA por la aspiración de.</li>
	<li>Lavar las DC2.4 las células dos veces con 40 mL de tampón PBS (1,37 mM NaCl, 8,1 mM Na 2 HPO 4, 2,68 mM KCl, 1,47 mM KH 2 PO 4) por centrifugación a 1.000 x g durante 5 min, 4 ° C y descarte el sobrenadante cada vez por la aspiración de.</li>
	Células <li>Resuspender el precipitado en el paso 2.3 en 1-2 mL (1/20-1/10 volumen de medio de cultivo) de homogeneización medio (0.25 M sacarosa, 1 mM EDTA, 10 mM HEPES-NaOH [pH 7.4]) con el volumen de 1/1.000 de cócteles inhibidores de proteasa y transferencia DC2.4 resuspendidos en un helada homogeneizadores Dounce.</li>
	<li>Romper las células DC2.4 resuspendidas suavemente por 10-20 movimientos con la helada homogeneizadores Dounce. 
	Nota: Durante la etapa de interrupción, los homogeneizadores Dounce es enfriado en hielo.</li>
	<li>La suspensión celular interrumpió la transferencia a un nuevo tubo cónico de 15 mL.</li>
	<li>Medio de homogeneización de 8-9 mL total 10 mL y centrifugar el tubo cónico 10 min a 2.000 x g y 4 ° C.</li>
	<li>Transferir el sobrenadante a un nuevo tubo cónico de 15 mL para eliminar las células intactas y núcleos y centrifugar el tubo cónico nuevamente durante 10 minutos a 2.000 x g y 4 ° C.</li>
	<li>Transferir el sobrenadante a un tubo nuevo de ultracentrifugación y centrifugar durante 45 min a 100.000 x g y 4 ° C.</li>
	<li>Aspirar el sobrenadante y resuspender el precipitado cuidadosamente en 1-2 mL de medio de homogeneización con volumen de 1/1.000 de cócteles inhibidores de proteasa por pipetear la solución arriba y abajo varias veces para hacer una fracción del microsoma para experimentos posteriores.</li>
	<li>Transferencia de la fracción del microsoma en un nuevo tubo de fondo redondo 5 mL. 
	Nota: Después de este paso, es posible enriquecer los microsomas objetivo de iodixanol centrifugación gradiente de densidad según el fabricante &#39; protocolo de s, para quitar los microsomas no específicos. Las fracciones de pico para los antígenos exógenos son recogidas y luego sometidas al siguiente paso de purificación (paso 3).</li>
</ol> 3. Purificación de los microsomas con bOVA que ERAD <ol><li>Agregar de 1/100 volumen de frescos granos SA-magnéticos para la fracción del microsoma de paso 2.11 en 5 mL redondo tubo inferior. 
	Nota: Antes de este paso, es posible borrar previamente la fracción del microsoma por perlas de control magnético para reducir la contaminación de los microsomas no específico.</li>
	<li>Mezclar bien suavemente y gire el tubo de fondo redondo despacio durante 30 minutos a 4 ° C.</li>
	<li>Añadir 2-3 mL de medio de homogeneización total 4 ml en el tubo de fondo redondo y suavemente mezclar bien.</li>
	<li>Coloque el tubo de fondo redondo sobre un soporte magnético e incubar 10 min a 4 ° C. Puesto que los granos a las vesículas son atraídos por el imán, micro-beads marrón poco a poco se acumularán a la pared del tubo más cercana al imán.</li>
	<li>Con el tubo restante en el soporte magnético, descartar cuidadosamente el sobrenadante por aspiración para eliminar las vesículas.</li>
	<li>Lavado los granos magnéticos enlazados a las vesículas dos veces con 5 mL de medio de homogeneización por el magnético reposar 10 min a 4 ° C y descarte el sobrenadante cada vez por aspiración cuidadosamente. 
	Nota: Sin el soporte magnético, purificación por centrifugados a 2.000 x g durante 10 min, 4 ° C está también disponible.</li>
	<li>Resuspender las bolas magnéticas vinculadas a las vesículas con cuidado en 100 μl de medio de homogeneización por pipetear la solución arriba y abajo varias veces.</li>
	<li>Transferir las vesículas resuspendidas en un microtubo nuevo como del microsoma purificado para experimentos posteriores. 
	Nota: Típicamente, 50 fracción del microsoma de μl que contiene 1 x 10 7 células de 5-20 μg proteínas puede ser aislado.</li>
</ol> 4. Análisis de los microsomas purificada perlas <ol><li>Resuspender el magnético a las vesículas de paso 3.8 por 100-200 μL de tampón de TNE (pH de 20 mM Tris-HCl 7.4, 150 mM NaCl, 0.5 M de EDTA, de 1% Nonidet P-40) con volumen de 1/1.000 de cócteles inhibidores de proteasa en lugar del medio de homogeneización.</li>
	<li>Transferir el lisado de paso 4.1 a un nuevo microtubo de 1.5ml.</li>
	<li>Determinar la concentración de proteína de paso 4.2 usando un kit de ensayo BCA por el fabricante &#39; Protocolo s. 
	Nota: Normalmente, 5-10 μl lisado de paso 4.2 es suficiente para determinar la concentración de proteína.</li>
	<li>Transferir el lisado de paso 4.2 (2 μg de proteína para la tinción de plata y 10 μg de proteína para el borrar occidental) en microtubos nuevo.</li>
	<li>Colocar los microtubos en un bloque de calor las proteínas 95 ° C y hervir en 1 x de tampón de carga de gel SDS (100 mM Tris-HCl de pH 6.8, azul de bromofenol 200 mM Ditiotreitol, 4% SDS, de 0,2%, 20% de glicerol) por 5 min</li>
	<li>Centrifugar microtubos 10 min a 21.500 x g y 4 º C. recoger el sobrenadante en nuevos microtubos para quitar las fracciones insolubles.</li>
	<li>Analizar las proteínas resueltas en el paso 4.6 (2 μg) por página de SD standard.</li>
	<li>Visualiza las bandas de proteínas por la tinción utilizando la tinción de plata de plata kit siguiendo el fabricante &#39; Protocolo s.</li>
	<li>Analizar las proteínas resueltas en el paso 4.6 (10 μg) por estándar SD-PAGE y Western-blotting. Utilizar el reactivo (SA-HRP) por el fabricante &#39; s protocolo y visualizar por fluorografía.</li>
</ol> 5. In Vitro Reconstitución de ubiquitinación ERAD de bOVA usando microsomas de purificada <ol><li>transferencia la purificada microsomas (5-10 μg de proteína) de paso 3.8 con un volumen de 50% de reticulocitos lisado (RL), en 1 x buffer de reacción (50 mM Tris, pH 7.4, 3 mM ATP, 0,5 mM MgCl 2) y 12:02 bandera etiqueta ubiquitina en un microtubo nuevo. El volumen final de este experimento es 20-40 μl.</li>
	<li>Incubar los microtubos por 2 h a 37 ° C. 
	Nota: Si la cantidad de ubiquitinated bOVA en paso 5.12 es demasiado pequeña para detección por Western blotting, agregue 10 μm de MG132 o 2 μm de lactacystin para inhibir el procesamiento de la bOVA ubiquitinated por proteasomas.</li>
	<li>Detener la reacción mediante la colocación de los microtubos en hielo.</li>
	<li>Resolver los microsomas por agregar 500 μl de tampón de TNE con volumen de 1/1.000 de cócteles inhibidores de proteasa.</li>
	<li>Centrifugar microtubos 10 min a 21,500 x g y 4 º C. recoger el sobrenadante a un nuevo microtubo para quitar las fracciones insolubles, que contienen ubiquitinated bOVA en DC2.4 antes del ensayo de ubiquitinación en vitro.</li>
	<li>Transferir el sobrenadante a un microtubo nuevo y añadir volumen 1/100 de nuevos granos SA-magnético (generalmente 5 μL para 500 μl del sobrenadante).</li>
	<li>Mezclar bien suavemente y gire el microtubo lentamente durante 30 minutos a 4 ° C.</li>
	<li>Centrifugar microtubos 10 min a 21.500 x g y 4 ° C. desechar el sobrenadante por aspiración a recuperar la bOVA y bOVA ubiquitinated atado con las bolas magnéticas SA.</li>
	<li>Lavar dos veces los granos SA-magnético recogidos con 1 mL de tampón de la TNE por centrifugación 10 min a 21.500 x g y 4 ° C. descarte el sobrenadante cada vez por la aspiración de.</li>
	<li>Hervir los granos SA-magnético en 1 x gel SDS cargando buffer por 5 min a 95 ° C en un bloque de calor para resolver las proteínas purificadas por las bolas magnéticas SA.</li>
	<li>Centrifugar microtubos 10 min a 21.500 x g y 4 º C. recoger el sobrenadante a un nuevo microtubo para quitar las fracciones insolubles.</li>
	<li>Analizar los granos SA-magnético enlazados a las proteínas por estándar SD-PAGE y Western-blotting. Es el uso de anticuerpos (Anti-Flag, anti-multi-Ub y anti-mouse IgG-HRP) y el reactivo (SA-HRP) por el fabricante &#39; Protocolo s y visualizados por fluorografía.</li>
</ol>

<ol>
	<li>van Endert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Providing+ligands+for+MHC+class+I+molecules.">Providing ligands for MHC class I molecules.</a> Cell Mol Life Sci. 68 (9), 1467-1469 (2011).</li><li>Janeway, C., Travers, P., Walport, M., Shlomchik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunobiology: The Immune System in Health and Disease. , (2001).</li><li>McDevitt, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovering+the+role+of+the+major+histocompatibility+complex+in+the+immune+response.">Discovering the role of the major histocompatibility complex in the immune response.</a> Annu Rev Immunol. 18 (1), 1-17 (2000).</li><li>Bedoui, 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=Cross-presentation+of+viral+and+self+antigens+by+skin-derived+CD103++dendritic+cells.">Cross-presentation of viral and self antigens by skin-derived CD103+ dendritic cells.</a> Nat Immunol. 10 (5), 488-495 (2009).</li><li>Segura, E., Villadangos, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+presentation+by+dendritic+cells+in+vivo.">Antigen presentation by dendritic cells in vivo.</a> Curr Opin Immunol. 21 (1), 105-110 (2009).</li><li>Cheong, C., 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=Microbial+stimulation+fully+differentiates+monocytes+to+DC-SIGN/CD209(+)+dendritic+cells+for+immune+T+cell+areas.">Microbial stimulation fully differentiates monocytes to DC-SIGN/CD209(+) dendritic cells for immune T cell areas.</a> Cell. 143 (3), 416-429 (2010).</li><li>Henri, 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=CD207++CD103++dermal+dendritic+cells+cross-present+keratinocyte-derived+antigens+irrespective+of+the+presence+of+Langerhans+cells.">CD207+ CD103+ dermal dendritic cells cross-present keratinocyte-derived antigens irrespective of the presence of Langerhans cells.</a> J Exp Med. 207 (2), 189-206 (2010).</li><li>Shortman, K., Heath, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CD8++dendritic+cell+subset.">The CD8+ dendritic cell subset.</a> Immunol. Rev. 234 (1), 18-31 (2010).</li><li>Carbone, F. R., Kurts, C., Bennett, S. R., Miller, J. F., Heath, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-presentation:+a+general+mechanism+for+CTL+immunity+and+tolerance.">Cross-presentation: a general mechanism for CTL immunity and tolerance.</a> Immunol Today. 19 (8), 368-373 (1998).</li><li>Nair-Gupta, P., Blander, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+updated+view+of+the+intracellular+mechanisms+regulating+cross-presentation.">An updated view of the intracellular mechanisms regulating cross-presentation.</a> Front Immunol. , (2013).</li><li>Joffre, O. P., Segura, E., Savina, A., Amigorena, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-presentation+by+dendritic+cells.">Cross-presentation by dendritic cells.</a> Nat Rev Immunol. 12 (8), 557-569 (2012).</li><li>Kurts, C., 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=Constitutive+class+I-restricted+exogenous+presentation+of+self+antigens+in+vivo.">Constitutive class I-restricted exogenous presentation of self antigens in vivo.</a> J Exp Med. 184 (3), 923-930 (1996).</li><li>Kurts, C., Kosaka, H., Carbone, F. R., Miller, J. F., Heath, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Class+I-restricted+cross-presentation+of+exogenous+self-antigens+leads+to+deletion+of+autoreactive+CD8(+)+T+cells.">Class I-restricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive CD8(+) T cells.</a> J Exp Med. 186 (2), 239-245 (1997).</li><li>Bonifaz, L., 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=Efficient+targeting+of+protein+antigen+to+the+dendritic+cell+receptor+DEC-205+in+the+steady+state+leads+to+antigen+presentation+on+major+histocompatibility+complex+class+I+products+and+peripheral+CD8++T+cell+tolerance.">Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance.</a> J Exp Med. 196 (12), 1627-1638 (2002).</li><li>Heath, W. R., Carbone, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-presentation+in+viral+immunity+and+self-tolerance.">Cross-presentation in viral immunity and self-tolerance.</a> Nat Rev Immunol. 1 (2), 126-134 (2001).</li><li>Imai, J., Hasegawa, H., Maruya, M., Koyasu, S., Yahara, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exogenous+antigens+are+processed+through+the+endoplasmic+reticulum-associated+degradation+(ERAD)+in+cross-presentation+by+dendritic+cells.">Exogenous antigens are processed through the endoplasmic reticulum-associated degradation (ERAD) in cross-presentation by dendritic cells.</a> Int Immunol. 17 (1), 45-53 (2005).</li><li>Houde, M., 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=Phagosomes+are+competent+organelles+for+antigen+cross-presentation.">Phagosomes are competent organelles for antigen cross-presentation.</a> Nature. 425 (6956), 402-406 (2003).</li><li>Guermonprez, P., 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=ER-phagosome+fusion+defines+an+MHC+class+I+cross-presentation+compartment+in+dendritic+cells.">ER-phagosome fusion defines an MHC class I cross-presentation compartment in dendritic cells.</a> Nature. 425 (6956), 397-402 (2003).</li><li>Burgdorf, S., Scholz, C., Kautz, A., Tampe, R., Kurts, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+mechanistic+separation+of+cross-presentation+and+endogenous+antigen+presentation.">Spatial and mechanistic separation of cross-presentation and endogenous antigen presentation.</a> Nat Immunol. 9 (5), 558-566 (2008).</li><li>Liz&#233;e, G., 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=Control+of+dendritic+cell+cross-presentation+by+the+major+histocompatibility+complex+class+I+cytoplasmic+domain.">Control of dendritic cell cross-presentation by the major histocompatibility complex class I cytoplasmic domain.</a> Nat Immunol. 4 (11), 1065-1073 (2003).</li><li>Basha, G., 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=A+CD74-dependent+MHC+class+I+endolysosomal+cross-presentation+pathway.">A CD74-dependent MHC class I endolysosomal cross-presentation pathway.</a> Nat Immunol. 13 (3), 237-245 (2012).</li><li>Saveanu, L., Carroll, O., Hassainya, Y., van Endert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complexity,+contradictions,+and+conundrums:+studying+post-proteasomal+proteolysis+in+HLA+class+I+antigen+presentation.">Complexity, contradictions, and conundrums: studying post-proteasomal proteolysis in HLA class I antigen presentation.</a> Immunol Rev. 207 (1), 42-59 (2005).</li><li>Wiertz, E. 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=Sec61-mediated+transfer+of+a+membrane+protein+from+the+endoplasmic+reticulum+to+the+proteasome+for+destruction.">Sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction.</a> Nature. 384 (6608), 432-438 (1996).</li><li>Hampton, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ER-associated+degradation+in+protein+quality+control+and+cellular+regulation.">ER-associated degradation in protein quality control and cellular regulation.</a> Curr Opin Cell Biol. 14 (4), 476-482 (2002).</li><li>Tsai, B., Ye, Y., Rapoport, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-translocation+of+proteins+from+the+endoplasmic+reticulum+into+the+cytosol.">Retro-translocation of proteins from the endoplasmic reticulum into the cytosol.</a> Nat Rev Mol Cell Biol. 3 (4), 246-255 (2002).</li><li>Nyathi, Y., Wilkinson, B. M., Pool, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-translational+targeting+and+translocation+of+proteins+to+the+endoplasmic+reticulum.">Co-translational targeting and translocation of proteins to the endoplasmic reticulum.</a> Biochim Biophys Acta. 1833 (11), 2392-2402 (2013).</li><li>Varabyova, A., Stojanovski, D., Chacinska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+protein+homeostasis.">Mitochondrial protein homeostasis.</a> IUBMB Life. 65 (3), 191-201 (2013).</li><li>Shen, Z., Reznikoff, G., Dranoff, G., Rock, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloned+dendritic+cells+can+present+exogenous+antigens+on+both+MHC+class+I+and+class+II+molecules.">Cloned dendritic cells can present exogenous antigens on both MHC class I and class II molecules.</a> J Immunol. 158 (6), 2723-2730 (1997).</li><li>Imai, J., Otani, M., Sakai, T., Hatta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+the+subcellular+compartment+in+which+exogenous+antigens+undergo+endoplasmic+reticulum-associated+degradation+from+dendritic+cells.">Purification of the subcellular compartment in which exogenous antigens undergo endoplasmic reticulum-associated degradation from dendritic cells.</a> Heliyon. , (2016).</li><li>Kovacsovics-Bankowski, M., Rock, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phagosome-to-cytosol+pathway+for+exogenous+antigens+presented+on+MHC+class+I+molecules.">A phagosome-to-cytosol pathway for exogenous antigens presented on MHC class I molecules.</a> Science. 267 (5195), 243-246 (1995).</li><li>Ackerman, A. L., Giodini, A., Cresswell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+the+endoplasmic+reticulum+protein+retro+translocation+machinery+during+cross+presentation+by+dendritic+cells.">A role for the endoplasmic reticulum protein retro translocation machinery during cross presentation by dendritic cells.</a> Immunity. 25 (4), 607-617 (2006).</li><li>Ackerman, A. L., Kyritsis, C., Tamp&#233;, R., Cresswell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+phagosomes+in+dendritic+cells+form+a+cellular+compartment+sufficient+for+cross+presentation+of+exogenous+antigens.">Early phagosomes in dendritic cells form a cellular compartment sufficient for cross presentation of exogenous antigens.</a> Proc Natl Acad Sci U S A. 100 (22), 12889-12894 (2003).</li><li>Saveanu, L., 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=IRAP+identifies+an+endosomal+compartment+required+for+MHC+class+I+cross-presentation.">IRAP identifies an endosomal compartment required for MHC class I cross-presentation.</a> Science. 325 (5937), 213-217 (2009).</li><li>Zehner, M., 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=The+translocon+protein+Sec61+mediates+antigen+transport+from+endosomes+in+the+cytosol+for+cross-presentation+to+CD8(+)+T+cells.">The translocon protein Sec61 mediates antigen transport from endosomes in the cytosol for cross-presentation to CD8(+) T cells.</a> Immunity. 42 (5), 850-863 (2015).</li></ol>

Los autores no tienen nada que revelar.

En estudios previos de CP, los antígenos exógenos incorporados acumulan en el área restricta del último endosome o ER por microscopía inmunofluorescente16,30,31,32. Se estima que ERAD-como transporte y procesamiento de antígenos exógenos se llevan a cabo en estas áreas especializadas de la ER o último endosome, el compartimento celular fue identificado por sacarosa o iodixanol centrifu...

El MHC las moléculas se expresan en la superficie de todas las células nucleadas, con cortos péptidos antigénicos derivados de antígenos endógenos, que son procesados por el sistema ubiquitina-proteasoma en el citosol1. Después de procesar, se transportan péptidos antigénicos en el lumen del retículo endoplásmico (ER) por el transportador de péptidos TAP. En el lumen del re, una serie de chaperonas específicas ayudan a la carga del péptido y el plegamiento correcto del MHC I complejo. Esta serie de moléculas se llama el complejo de carga (PLC), que indica que el ER es un compartimiento central para péptido cargando al MHC y2. Después de la carga, de péptidos del MHC las moléculas son transportadas a la superficie de la célula y juega un papel clave en el sistema inmune adaptativo auto marcadores, como permite el CD8+ los linfocitos T citotóxicos (CTLs) para detectar células cancerosas o agentes infecciosos por antigénica péptidos de las proteínas del mismo3.
En el antígeno que presenta las células (APC), péptidos antigénicos de los antígenos exógenos son también presentados en MHC4,5,6,7,8 a través de CP, que es fundamentalmente por DCs9,10,11. CP es esencial tanto para la activación de ingenuo CD8+ T memoria CD8 y células+ T células antiinfecciosas y anti-tumoral CTLs12,13y en el mantenimiento de la tolerancia inmune mediante la inactivación de autoactuante ingenuo T células14,15. El CP juega un papel importante muchos en el sistema inmune adaptativo, sin embargo, los mecanismos moleculares de CP tienen todavía ser descrito en detalle. Estudios previos de CP revelaron que antígenos exógenos fueron localizados en el retículo endoplasmático y el endosome y fueron procesados por ERAD, lo que sugiere que los antígenos exógenos son transportados desde el endosome a ER para ERAD-como proceso y péptido carga16 . Sin embargo, evidencia acumulada indica que la carga de péptido de CP se realiza no en la sala de emergencia sino en compartimentos endocíticos no clásica, que también tienen características distintivas de la ER (figura 1)17,18 ,19,20,21. Para evitar la degradación de los precursores del péptido antigénico por la alta actividad de aminopeptidasa22 en el citosol, el procesamiento y el péptido carga en CP se produce en la zona proximal de estos compartimentos endocíticos no clásica (figura 1). Aunque las caracterizaciones de estos compartimentos endocíticos son polémicas, hay no hay moléculas específicas existentes distintos antígenos exógenos en este compartimiento.
ERAD es una vía celular, que elimina específicamente proteínas mal plegadas de la ER. En el camino ERAD, proteínas mal plegadas retrogradely son transportadas a través de la membrana del ER al citoplasma y procesadas por el sistema de ubiquitina-proteasoma23,24,25. Cuando las moléculas grandes, como las proteínas, son transportadas a través de la bicapa lipídica, estas moléculas pasan a través de un aparato molecular llamado un translocon, tales como el complejo Sec61 y Derlin complejo en el ER26y el complejo de Tom y Tim complejo en la las mitocondrias27. Cuando exógeno agregado antígenos son transportados a través de la membrana del ER, que deben penetrar la bicapa lipídica en complejo con translocons, como el complejo Sec61. El método descrito aquí purificada la vesícula específica mediante la utilización de estas moléculas de penetración de la membrana como marcadores para los compartimentos endocíticos.
El método descrito aquí es un nuevo protocolo de purificación de vesícula usando el celular de DC como línea DC2.428 y biotinilado ovoalbúmina (bOVA) como un antígeno exógeno. Los compartimentos endocíticos se purificaron por estreptavidina (SA)-granos magnéticos mediante la penetración de la membrana bOVA como fabricante. En esta purificado microsoma, algunos añaden exógenamente bOVA se conservan en fracciones de membrana pero se transporta al exterior del microsoma y entonces ubiquitinated, procesaron en vitro29. Este microsome purificada contiene proteínas específicas del compartimiento endocíticas, sino también proteínas ER residente para ERAD y el péptido carga compleja; lo que sugiere que el compartimiento celular el compartimiento endocítico prospectivo para CP29. Este protocolo no es dependiente en el tipo de antígenos exógenos y también es aplicable para otros subconjuntos de DC y otros tipos de células, como macrófagos, células B y las células endoteliales, para aclarar el mecanismo molecular exacto de DCs para perito CP.

<table><tbody><tr><td>RPMI 1640</td><td>gibco by life technologies</td><td>11875-093</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Equitech bio</td><td>SFB30</td><td></td></tr><tr><td>Sodium pyruvate</td><td>gibco by life technologies</td><td>11360-070</td><td></td></tr><tr><td>MEM non-essential amino acids</td><td>gibco by life technologies</td><td>11140-050</td><td></td></tr><tr><td>HEPES</td><td>gibco by life technologies</td><td>15630-080</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>gibco by life technologies</td><td>21985-023</td><td></td></tr><tr><td>L-glutamine</td><td>gibco by life technologies</td><td>25030-164</td><td></td></tr><tr><td>Penisicillin-Sreptomycin</td><td>gibco by life technologies</td><td>15140-122</td><td></td></tr><tr><td>DMEM</td><td>gibco by life technologies</td><td>12100-46</td><td></td></tr><tr><td>OVA</td><td>SIGMA</td><td>A5503</td><td></td></tr><tr><td>Biotin-protein labelling kit</td><td>Thermo Fisher Scientific</td><td>F6347</td><td></td></tr><tr><td>MG-132</td><td>Santa Cruz Biotechnology</td><td>201270</td><td></td></tr><tr><td>lactacystin</td><td>SIGMA</td><td>L6785</td><td></td></tr><tr><td>Dounce homogenizer</td><td>IUCHI</td><td>131703</td><td></td></tr><tr><td>protease inhibitor cocktails</td><td>SIGMA</td><td>P8340</td><td></td></tr><tr><td>iodixanol</td><td>Cosmo bio</td><td>1114542</td><td></td></tr><tr><td>SA-magnetic beads</td><td>New England Biolabs</td><td>201270</td><td></td></tr><tr><td>control magnetic beads</td><td>Chemagen</td><td>M-PVA012</td><td></td></tr><tr><td>magnetic stand</td><td>BD Biosciences</td><td>552311</td><td></td></tr><tr><td>BCA protein assay kit</td><td>Thermo Fisher Scientific</td><td>23225</td><td></td></tr><tr><td>silver staining kits</td><td>Cosmo bio</td><td>423413</td><td></td></tr><tr><td>Reticulocyte Lysate</td><td>Promega</td><td>1730714</td><td></td></tr><tr><td>Flag-tagged ubiquitin</td><td>SIGMA</td><td>U5382</td><td></td></tr><tr><td>anti-ovalbumin (OVA,mouse)</td><td>Antibody Shop</td><td>HYB 094-06</td><td></td></tr><tr><td>ant-multi-ubiquitin (mouse)</td><td>MBL</td><td>D058&amp;minus;3</td><td></td></tr><tr><td>anti-Flag (mouse)</td><td>SIGMA</td><td>F3165</td><td></td></tr><tr><td>trypsin</td><td>SIGMA</td><td>85450C</td><td></td></tr></tbody></table>

purification of the membrane compartment for endoplasmic reticulum-associated degradation of exogenous antigens in cross-presentation

Las células dendríticas (DCs) son altamente capaces de procesar y presentar antígenos exógenos internalizados a clase principal de histocompatibilidad (MHC) las moléculas también conocido como Cruz-presentación (CP). CP desempeña un papel importante no sólo en la estimulación de la ingenua CD8+ T memoria CD8 y células+ T las células infecciosas e inmunidad del tumor sino también en la inactivación de automática ingenuo T células por anergia de células T o células T de supresión. Aunque el mecanismo molecular crítico de CP queda por ser aclarada, acumulando indicios que antígenos exógenos se procesan a través del retículo endoplasmático asociado degradación (ERAD) después de su exportación de no clásicas endocíticas compartimientos. Hasta hace poco, las caracterizaciones de estos compartimentos endocíticos fueron limitadas porque no había marcadores moleculares específicos distintos antígenos exógenos. El método descrito aquí es un nuevo protocolo de aislamiento de la vesícula, que permite la purificación de estos compartimentos endocíticos. Usando esta purificado microsoma, hemos reconstituido la ERAD-como transporte, ubiquitinación y procesamiento de los antígenos exógenos en vitro, sugiriendo que el sistema ubiquitina-proteasoma procesado el antígeno exógeno después de la exportación de este compartimiento celular. Este protocolo se puede aplicar más a otros tipos de células para aclarar el mecanismo molecular de CP.

Para dilucidar el mecanismo molecular de la CP, es necesario identificar los compartimentos celulares donde someterse a antígenos exógenos ERAD-como transporte y procesamiento. Mientras que observaciones por microscopía de inmunofluorescencia o microscopia electrónica identificaron el compartimento celular donde antígenos exógenos acumularon16,17,18,1<sup ...

Watch this Scientific Journal Video about Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation at JoVE.com

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

El método descrito aquí es un nuevo protocolo de aislamiento de la vesícula, que permite la purificación de los compartimentos celulares donde se procesan los antígenos exógenos por degradación retículo endoplasmático asociado en presentación cruzada.

Purificación del compartimento de la membrana de retículo endoplasmático asociado degradación de antígenos exógenos en presentación cruzada

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

purification-membrane-compartment-for-endoplasmic-reticulum

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

8.2K vistas.  Takasaki University of Health and Welfare. El método descrito aquí es un nuevo protocolo de aislamiento de la vesícula, que permite la purificación de los compartimentos celulares donde se procesan los antígenos exógenos por degradación retículo endoplasmático asociado en presentación cruzada.

Video: Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

La purificación y la agregación de la proteína precursora amiloide del dominio intracelular

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

Investigación

Medicina

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic cues. Their membranes are often juxtaposed at defined contact sites, which become hubs for the exchange of signaling molecules and membrane components1,2,3,4. The inter-organellar membrane microdomains that are formed between the endoplasmic reticulum (ER) and the mitochondria at the opening of the IP3-sensitive Ca2+ channel are known as the mitochondria associated-ER membranes or MAMs4,5,6. The protein/lipid composition and biochemical properties of these membrane contact sites have been extensively studied particularly in relation to their role in regulating intracellular Ca2+ 4,5,6. The ER serves as the primary store of intracellular Ca2+, and in this capacity regulates a myriad of cellular processes downstream of Ca2+ signaling, including post-translational protein folding and protein maturation7. Mitochondria, on the other hand, maintain Ca2+ homeostasis, by buffering cytosolic Ca2+ concentration thereby preventing the initiation of apoptotic pathways downstream of Ca2+ unbalance4,8. The dynamic nature of the MAMs makes them ideal sites to dissect basic cellular mechanisms, including Ca2+ signaling and regulation of mitochondrial Ca2+ concentration, lipid biosynthesis and transport, energy metabolism and cell survival 4,9,10,11,12. Several protocols have been described for the purification of these microdomains from liver tissue and cultured cells13,14. 
	Taking previously published methods into account, we have adapted a protocol for the isolation of mitochondria and MAMs from the adult mouse brain. To this procedure we have added an extra purification step, namely a Triton X100 extraction, which enables the isolation of the glycosphingolipid enriched microdomain (GEM) fraction of the MAMs. These GEM preparations share several protein components with caveolae and lipid rafts, derived from the plasma membrane or other intracellular membranes, and are proposed to function as gathering points for the clustering of receptor proteins and for protein&#x2013;protein interactions4,15.

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial preparations.

Las mitocondrias asociadas ER membranas (MAM) y glicoesfingolípidos microdominios enriquecidos (GEMS): Aislamiento de cerebro de ratón

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic ...

Neurociencias

Reconstitution of Msp1 Extraction Activity with Fully Purified Components

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function depends on a robust quality control system to maintain protein homeostasis (proteostasis). Declines in mitochondrial proteostasis have been linked to cancer, aging, neurodegeneration, and many other diseases. Msp1 is a AAA+ ATPase anchored in the outer mitochondrial membrane that maintains proteostasis by removing mislocalized tail-anchored proteins. Using purified components reconstituted into proteoliposomes, we have shown that Msp1 is necessary and sufficient to extract a model tail-anchored protein from a lipid bilayer. Our simplified reconstituted system overcomes several of the technical barriers that have hindered detailed study of membrane protein extraction. Here, we provide detailed methods for the generation of liposomes, membrane protein reconstitution, and the Msp1 extraction assay.

Here, we present a detailed protocol for reconstitution of Msp1 extraction activity with fully purified components in defined proteoliposomes.

Reconstitución de la actividad de extracción de Msp1 con componentes totalmente purificados

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function ...

Bioquímica

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca2+ in the ambient buffer. Subsequent depletion of Ca2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation.

Solid-supported, protein-free, double phospholipid bilayer membranes (DLBM) can be transformed into complex and dynamic lipid nanotube networks and can serve as 2D bottom-up models of the endoplasmic reticulum.

Formación espontánea y cambio de redes de nanotubos lipídica Artificial como un modelo Bottom-Up de retículo endoplasmático

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense ...

Bioingeniería

Purification of the Sarco-Endoplasmic Reticulum Ca2+-ATPase from Rabbit Muscle

Some P-type ATPases, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), are inherently labile membrane proteins that require specific physicochemical conditions during purification to obtain them with high purity and structural quality and in a catalytically active form. The disaccharide trehalose is a compatible solute that is synthesized and accumulated in high concentrations in the yeast cytoplasm to stabilize the membranes and proteins. The use of trehalose as an additive in the protocol for the purification of plasma membrane H+-ATPase results in a high-quality preparation, the hexameric structure of which is shown by biochemical analytical methods. Trehalose can, therefore, be used as a stabilizing additive for the purification of membrane proteins (P-ATPases). This protocol describes the modification of the classical protocol for SERCA purification by subjecting SERCA to centrifugation on a trehalose concentration gradient. The inclusion of this carbohydrate led to the purification of SERCA in a catalytically active form with high purity and, importantly, in a stable form. Partial biochemical characterization of the purified SERCA (SDS-PAGE, enzyme kinetics, FITC labeling, circular dichroism spectroscopy) showed that the enzyme is suitable for functional and structural studies. The use of trehalose in the purification protocol of P-type ATPases and other labile membrane (and cytosolic) proteins is suggested.

This protocol describes an improved SERCA purification method, which includes the disaccharide trehalose in the final centrifugation step. This carbohydrate stabilizes proteins under harsh conditions. The purified SERCA was catalytically active and displayed high purity, making it suitable for structural and functional studies.

Purificación del retículo sarcoendoplásmico Ca2+-ATPasa a partir de músculo de conejo

Some P-type ATPases, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), are inherently labile membrane proteins that require specific ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

La visualización de los ARNm retículo endoplásmico localizados en células de mamíferos

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Biología

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

Visualización del retículo endoplasmático subdominios en células cultivadas

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Protein abundance reflects the rates of both protein synthesis and protein degradation. This article describes the use of cycloheximide chase followed by western blotting to analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast).

Análisis cicloheximida persecución de degradación de proteínas en<em&gt; Saccharomyces cerevisiae</em

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein ...

The Endoplasmic Reticulum

The endoplasmic reticulum or ER makes up for more than half of the membranes in a cell and accounts for 10% of total cell volume. It is also the primary protein and lipid synthesis factory for most cell organelles, such as the Golgi apparatus, lysosomes, secretory vesicles, and the plasma membrane. Despite being the most extensive and functionally complex subcellular organelle, ER was the last to be discovered. After years of deliberation, Keith Porter and George Palade in the year 1954, produced the first high-resolution electron microscope images to affirm the presence of ER in the eukaryotic cell.
The ER membranes were identified as sites for protein synthesis and crucial for intracellular transport using radio-labeled and fluorescent-labeled amino acids. The isolation of ER is a tricky task as it forms an intricate mesh with other subcellular organelles. However, on cell homogenization, the disrupted ER membranes reseal into small closed vesicles called microsomes. These vesicles form a microsystem capable of sustaining all ER-related functions like protein and lipid synthesis, calcium signaling, and glycosylation. Subcellular fractionation is the best and often-used technique for the purification of these membranes. When separating using a sucrose gradient, the rough ER microsomes sediments at a higher density than smooth ER microsomes.
The ER network in a cell is dynamic. It is constantly shape-shifting along with the cytoskeleton to bolster mechanical support for the cell structure. Despite the distinct cisternae and tubule morphologies of the ER membrane, interconversion between the two is possible and is governed by the expression of membrane proteins. The ER network rearranges by tubule growth, retraction, and fusion of adjacent ER-ER membranes.
As mentioned above, the ER network is required for optimal overall cellular health. Disruption of ER morphology is linked to pathological conditions, including neurological disorders like Alzheimer&#39;s disease, hereditary spastic paraplegia, and viral infections like hepatitis C virus, and dengue virus.

El retículo endoplasmático

The endoplasmic reticulum or ER makes up for more than half of the membranes in a cell and accounts for 10% of total cell volume. It is also the primary ...

Educación

JoVE Core

Biología Celular

Unit 1

Transporte transmembrana en el retículo endoplasmático y peroxisomas

TÉRMINOS Y CONCEPTOS CLAVES

ER Retrieval Pathway

In the secretory pathway, vesicles transport proteins from one cellular compartment to another in forward transport to deliver the protein to its correct location. Occasionally, misfolded proteins and incorrect proteins escape their original compartments, and a retrieval pathway is used to return the escaped proteins to their original compartment.
The ER uses many checkpoints to prevent the entry of incorrectly folded or a resident protein as cargo onto a transport vesicle. These mechanisms include cargo selectivity of the receptors, aggregation of functionally similar proteins, and retention sequences on resident proteins.
Biochemical studies characterizing the cargo-sorting receptors have shown that they are ligand-specific and bind only at specific pH (pH 5.0 in yeast and mammals). Functionally similar ER-resident can proteins aggregate to form large complexes that cannot be loaded onto transport vesicles, thus avoiding the accidental escape of these proteins from the organelle. Additionally, the membrane-spanning domain of several ER and Golgi proteins contain a retention sequence that marks these proteins for staying back in the ER, so receptors do not bind them.
If an ER-resident protein escapes these checkpoints and gets loaded onto a Golgi-bound vesicle, a specific amino acid sequence called a retrieval signal helps the protein get recognized and transported back to the ER. The receptors involved in retrieval mechanisms recognize the retrieval signals at the carboxyl terminus of soluble proteins of the ER and in the cytoplasmic domain of some ER and Golgi membrane proteins. The receptors selectively capture such proteins and package them in vesicles that transport them back to the ER.
Two well-characterized retrieval signals are the carboxy-terminal tetrapeptide KDEL and KKXX found in many ER lumen resident proteins. BiP, a molecular chaperone, plays a vital role in preventing the aggregation of misfolded proteins. Interaction between BiP and unfolded proteins is mediated by a substrate-binding domain and a nucleotide-binding domain for ATPase activity. KDEL receptor in the post-ER compartments such as ERGIC and Golgi binds with the carboxyl-terminal retrieval signal of BiP and returns BiP to the ER via COPI vesicles.

Ruta de recuperación del RE

In the secretory pathway, vesicles transport proteins from one cellular compartment to another in forward transport to deliver the protein to its correct ...

Purificación del compartimento de la membrana de retículo endoplasmático asociado degradación de antígenos exógenos en presentación cruzada

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