Este trabajo fue apoyado por el NIH R01 GM50322 a PM Kane. Los autores agradecen al Dr. Rajini Rao, de la Universidad Johns Hopkins para la prestación de los plásmidos pHluorin levadura y asesoramiento sobre medidas de pH radiométricos y Dra. Gloria A. Martínez Muñoz para la elaboración de estos protocolos para nuestro laboratorio.

1. Medida del pH vacuolar in vivo usando BCECF-AM <ol><li> Crecer un cultivo líquido de 50 ml de la cepa de levadura a medir en el medio deseado durante la noche. El objetivo es que las células en fase semilogarítmica (OD600 (densidad óptica a 600 nm) de medición de aproximadamente 0,8 para la suspensión). </li><li> Sedimentar las células de levadura mediante centrifugación. Resuspender el precipitado en 0,6 ml de medio de crecimiento y la transferencia a un tubo de microcentrífuga que ha sido previamente pesado. Pellet de nuevo las células en una microcentrífuga a 2000 xg durante 60 seg. Eliminar el sobrenadante tan completamente como sea posible y a continuación, pesar el sedimento celular. Resuspender el pellet a una densidad final de 0,5 g / ml (w / v); una cultura de este volumen dará un sedimento celular de aproximadamente 200 mg, y 200 l de tampón se añadiría para dar un volumen final de 400 l. </li><li> Añadir BCECF-AM a la suspensión de células a una concentración final de 50 mM de una reserva de 12 mM en DMSO. Mezclar bien y luego incUbaté las células a 30 ° C durante 30 min. en una plataforma oscilante o vibratorios. </li><li> Mientras que las células están incubando, preparar buffers de calibración. El tampón de calibración contiene 50 mM de MES (2 - (N-morfolino) etanosulfónico), 50 mM HEPES (4 - (2-hidroxietil)-1-piperazinetanosulfónico ácido), 50 mM KCl, 50 mM de NaCl, acetato de amonio 0,2 M, azida de sodio 10 mM, y 10 mM de 2-desoxiglucosa, y se ajusta el pH a valores apropiados para su rango de calibración con NaOH o HCl. (Atención: La azida sódica es altamente tóxica y debe manipularse con cuidado.) Para la medición de pH en las células de tipo salvaje, podríamos preparar varias mezclas de calibración a pH 5,5 a 6,5, pero para los mutantes que se espera que tenga un pH vacuolar más alcalino ( vma mutantes), nos prepare, tampones de pH superiores adicionales. </li><li> Alícuota de 2 ml de tampón de calibración para cada pH en tubos cónicos de 15 ml, y añadir la monensina (15 mM de existencias) y nigericina (2 mM stock) para dar concentraciones finales de 110 mM monensina y 15 micras, nigericina, respectively. Mezclar bien con un vórtex. (Atención: Tanto monensina y nigericina son tóxicos y deben manejarse con cuidado.) </li><li> Cuando el tiempo de incubación BCECF-AM se ha completado, la suspensión de células de pellets por centrifugación durante 30 segundos a 2.000 x g en una microcentrífuga. Resuspender las células es 1 ml de medio de crecimiento que carece de glucosa y se centrifuga como anteriormente. Repita este paso de lavado una vez, y luego volver a suspender el precipitado en 200 l de medio de cultivo sin glucosa. Coloque sobre hielo. </li><li> Añadir 20 l de la suspensión celular a cada tubo de calibración de pH preparado anteriormente. Incubar a 30 ° C durante 30-60 min. en un tambor de rodillos. </li><li> Ajuste el fluorímetro para medir alternativamente en longitudes de onda de excitación de 450 nm y 490 nm, ambos con una longitud de onda de emisión de 535 nm. Ajuste la temperatura de la cámara de la muestra a 30 ° C. Llevar a cabo todas las mediciones con agitación continua de la mezcla en la cubeta. </li><li> Añadir 1,96 ml de 1 mM de MES (ajustado a pH 5 o 7 dependiendo del pH del medio de crecimiento de las células de unnd el diseño experimental). Añadir 20 l de suspensión de células en la cubeta. Iniciar las mediciones de fluorescencia. Recopilamos tanto cinética continua y datos de tiempo de punto en diferentes experimentos. Para los datos cinéticos continuos, tomamos mediciones cada 6 segundos durante 5 minutos, a continuación, añadir glucosa a una concentración final de glucosa de 50 mM, y continuar la medición durante 5-10 min. Para las mediciones de tiempo de un solo punto, por lo general, tomamos medidas a 1 y 5 min. después de la adición de las células a la cubeta, añadir la glucosa como en las mediciones cinéticas, y luego tomar otra medición 5 min. después de la adición de glucosa. Estas adiciones pueden ser variados, y también se pueden añadir componentes adicionales (inhibidores, etc). </li><li> Después de las mediciones experimentales, retire los tubos de calibración de la incubación de 30 ° C y la transferencia de la totalidad del volumen de 2 ml para cada uno a la cubeta fluorímetro. Medir la fluorescencia en la misma configuración (ver 1.8) cada 5 segundos durante un total de 30 segundos para cada muestra. </li><li>Exportar datos de fluorescencia para Microsoft Excel. (Para nuestro fluorímetro, esto requiere exportación de datos como texto en forma delimitado por tabuladores y la importación en Excel.) Obtener una curva de calibración mediante el cálculo de la proporción de fluorescencia a 490 nm a 450 nm para cada mezcla de calibración. El ratio de fluorescencia se representa gráficamente a continuación, vs pH para obtener una curva de calibración. </li><li> Convertir los datos experimentales a la proporción de fluorescencia y calcular pH usando la curva estándar. PH vacuolar puede entonces representado frente al tiempo (cinética de los cambios de pH) o en comparación bajo diversas condiciones (Figura 1) o en diferentes cepas mutantes. </li></ol> 2. La medición de pH citosólico En Vivo Con pHluorin levadura <ol><li> Transformación de la cepa de levadura deseada con el plásmido por protocolos estándar de levadura pHluorin, y seleccionar los transformantes en medio mínimo suplementado carece de uracilo (SC-uracilo). </li><li> Crecer un cultivo líquido de 50 ml de las células transformadas en SC-uracilo a la fase logarítmica media (OD <sub&gt; 600 = 0,8 o inferior). </li><li> Preparar los estándares de calibración como se describe para el pH vacuolar, pero tampón a un intervalo de pH apropiado para mediciones de pH citosólicas, generalmente de pH 6-8. Alícuota de 2 ml de tampón de calibración ajusta al pH deseado en varios tubos cónicos de 15 ml. Añadir monensina y nigericina como se describe anteriormente (1.4) y mezclar bien. </li><li> Recoger las células por centrifugación como se describió anteriormente, y resuspender el sedimento en 600 l de medio de crecimiento. Transferir a un tubo de microcentrífuga de pesado, y precipitar las células por centrifugación a 5000 rpm durante 30 seg. Resuspender el precipitado en 1 ml de medio de crecimiento sin glucosa (SC-uracilo,-glucosa), sedimentar las células de nuevo y repetir. Después de la centrifugación final, eliminar el sobrenadante tan a fondo como sea posible y pesar el sedimento celular. Resuspender las células a una densidad final de 0,5 g / ml en SC-uracilo sin glucosa. </li><li> Añadir 20 l de suspensión celular a cada tubo de tampón de calibración y mezcle bien en un mezclador de vórtice. Incubar a 30 ° C en un rotor de tambor giratorio durante 60 minutos. </li><li> Configurar el fluorímetro para la excitación a longitudes de onda 405 y 485 nm y una longitud de onda de emisión de 508 nm. Proceder con el punto sola vez o mediciones cinéticas y la adición de glucosa como se describió anteriormente para la medición de BCECF. </li><li> Medir la fluorescencia de las muestras de calibración, y la construcción de una curva de calibración como para BCECF (ver 1.10 a 1.11). Una gráfica de la proporción de fluorescencia vs pH es lineal en el rango de la mayoría de las mediciones de pH citosólico (pH 6,0-8,0) 9, medidas de relación de fluorescencia así experimentales se convierten fácilmente a pH. </li></ol>

<ol>
	<li>Casey, J. R., Grinstein, S., Orlowski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensors+and+regulators+of+intracellular+pH.">Sensors and regulators of intracellular pH.</a> Nat. Rev. Mol. Cell Biol. 11, 50-61 (2010).</li><li>Orij, R., Brul, S., Smits, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+pH+is+a+tightly+controlled+signal+in+yeast.">Intracellular pH is a tightly controlled signal in yeast.</a> Biochim. Biophys. Acta. 1810, 933-944 (2011).</li><li>Diakov, T. T., Kane, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+vacuolar+proton-translocating+ATPase+activity+and+assembly+by+extracellular+pH.">Regulation of vacuolar proton-translocating ATPase activity and assembly by extracellular pH.</a> J. Biol. Chem. 285, 23771-23778 (2010).</li><li>Martinez-Munoz, G. A., Kane, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vacuolar+and+plasma+membrane+proton+pumps+collaborate+to+achieve+cytosolic+pH+homeostasis+in+yeast.">Vacuolar and plasma membrane proton pumps collaborate to achieve cytosolic pH homeostasis in yeast.</a> J. Biol. Chem. 283, 20309-20319 (2008).</li><li>Tarsio, M., Zheng, H., Smardon, A. M., Martinez-Munoz, G. A., Kane, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+loss+of+Vph1+protein-containing+vacuolar+ATPases+(V-ATPases)+for+overall+cellular+pH+homeostasis.">Consequences of loss of Vph1 protein-containing vacuolar ATPases (V-ATPases) for overall cellular pH homeostasis.</a> J. Biol. Chem. 286, 28089-28096 (2011).</li><li>Plant, P. J., Manolson, M. F., Grinstein, S., Demaurex, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+mechanisms+of+vacuolar+acidification+in+H(+)-ATPase-deficient+yeast.">Alternative mechanisms of vacuolar acidification in H(+)-ATPase-deficient yeast.</a> J. Biol. Chem. 274, 37270-37279 (1999).</li><li>Ali, R., Brett, C. L., Mukherjee, S., Rao, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+sodium/proton+exchange+by+a+Rab-GTPase-activating+protein+regulates+endosomal+traffic+in+yeast.">Inhibition of sodium/proton exchange by a Rab-GTPase-activating protein regulates endosomal traffic in yeast.</a> J. Biol. Chem. 279, 4498-4506 (2004).</li><li>Miesenbock, G., De Angelis, D. A., Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+secretion+and+synaptic+transmission+with+pH-sensitive+green+fluorescent+proteins.">Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.</a> Nature. 394, 192-195 (1998).</li><li>Brett, C. L., Tukaye, D. N., Mukherjee, S., Rao, 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+yeast+endosomal+Na+K+/H++exchanger+Nhx1+regulates+cellular+pH+to+control+vesicle+trafficking.">The yeast endosomal Na+K+/H+ exchanger Nhx1 regulates cellular pH to control vesicle trafficking.</a> Mol. Biol. Cell. 16, 1396-1405 (2005).</li><li>Owen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+spectrum-shifting+intracellular+pH+probes+5'(and+6')-carboxy-10-dimethylamino-3-hydroxyspiro[7H-benzo[c]xanthene-7,+1'(3'H)-isobenzofuran]-3'-one+and+2',7'-biscarboxyethyl-5(and+6)-carboxyfluorescein.">Comparison of spectrum-shifting intracellular pH probes 5'(and 6')-carboxy-10-dimethylamino-3-hydroxyspiro[7H-benzo[c]xanthene-7, 1'(3'H)-isobenzofuran]-3'-one and 2',7'-biscarboxyethyl-5(and 6)-carboxyfluorescein.</a> Anal. Biochem. 204, 65-71 (1992).</li><li>Dechant, R., 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=Cytosolic+pH+is+a+second+messenger+for+glucose+and+regulates+the+PKA+pathway+through+V-ATPase.">Cytosolic pH is a second messenger for glucose and regulates the PKA pathway through V-ATPase.</a> Embo J. 29, 2515-2526 (2010).</li><li>Gustavsson, M., Barmark, G., Larsson, J., Muren, E., Ronne, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomics+of+monensin+sensitivity+in+yeast:+implications+for+post-Golgi+traffic+and+vacuolar+H+-ATPase+function.">Functional genomics of monensin sensitivity in yeast: implications for post-Golgi traffic and vacuolar H+-ATPase function.</a> Mol. Genet. Genomics. 280, 233-248 (2008).</li><li>Kovac, L., Bohmerova, E., Butko, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionophores+and+intact+cells.+I.+Valinomycin+and+nigericin+act+preferentially+on+mitochondria+and+not+on+the+plasma+membrane+of+Saccharomyces+cerevisiae.">Ionophores and intact cells. I. Valinomycin and nigericin act preferentially on mitochondria and not on the plasma membrane of Saccharomyces cerevisiae.</a> Biochim. Biophys. Acta. 721, 341-348 (1982).</li><li>Braun, N. A., Morgan, B., Dick, T. P., Schwappach, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yeast+CLC+protein+counteracts+vesicular+acidification+during+iron+starvation.">The yeast CLC protein counteracts vesicular acidification during iron starvation.</a> J. Cell Sci. 123, 2342-2350 (2010).</li><li>Orij, R., Postmus, J., Beek, T. e. r., Brul, A., S, G. J., Smits, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+cytosolic+and+mitochondrial+pH+using+a+pH-sensitive+GFP+derivative+in+Saccharomyces+cerevisiae+reveals+a+relation+between+intracellular+pH+and+growth.">In vivo measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in Saccharomyces cerevisiae reveals a relation between intracellular pH and growth.</a> Microbiology. 155, 268-278 (2009).</li><li>Zhang, Y. Q., 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=Requirement+for+ergosterol+in+V-ATPase+function+underlies+antifungal+activity+of+azole+drugs.">Requirement for ergosterol in V-ATPase function underlies antifungal activity of azole drugs.</a> PLoS Pathog. 6, e1000939 (2010).</li><li>Brett, C. 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=Genome-wide+analysis+reveals+the+vacuolar+pH-stat+of+Saccharomyces+cerevisiae.">Genome-wide analysis reveals the vacuolar pH-stat of Saccharomyces cerevisiae.</a> PLoS One. 6, e17619 (2011).</li><li>Roberts, C. J., Raymond, C. K., Yamashiro, C. T., Stevens, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+studying+the+yeast+vacuole.">Methods for studying the yeast vacuole.</a> Methods Enzymol. 194, 644-661 (1991).</li><li>Chan, C. Y., 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=Inhibitors+of+V-ATPase+proton+transport+reveal+uncoupling+functions+of+tether+linking+cytosolic+and+membrane+domains+of+V0+subunit+a+(Vph1p).">Inhibitors of V-ATPase proton transport reveal uncoupling functions of tether linking cytosolic and membrane domains of V0 subunit a (Vph1p).</a> J. Biol. Chem. 287, 10236-10250 (2012).</li><li>Johnson, R. 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=Identification+of+inhibitors+of+vacuolar+proton-translocating+ATPase+pumps+in+yeast+by+high-throughput+screening+flow+cytometry.">Identification of inhibitors of vacuolar proton-translocating ATPase pumps in yeast by high-throughput screening flow cytometry.</a> Anal. Biochem. 398, 203-211 (2010).</li></ol>

Los autores no tienen conflictos de intereses a revelar.

Hemos utilizado estos protocolos para hacer frente a una serie de aspectos de la homeostasis del pH. Por ejemplo, hemos comparado las respuestas citosólicas y el pH de las células mutantes de tipo salvaje y V-ATPasa deficiente en 4,5. También hemos examinado los efectos de las condiciones de crecimiento alterado, pH extracelular, particularmente en respuesta a la glucosa pH vacuolar 3. Es importante destacar que las respuestas que se observan son a la vez coherente con otros métodos de medició...

la homeostasis del pH es un proceso dinámico y altamente regulado en todos los organismos 1,2. Los procesos bioquímicos están fuertemente regulados por el pH, y los ambientes intracelulares están sintonizados con rangos estrechos de pH para permitir la óptima actividad de las enzimas residentes. Sin embargo, la homeostasis del pH intracelular puede ser impugnada por los rápidos cambios en el pH del medio ambiente, los cambios metabólicos y algunas vías de señalización. Además, el pH intracelular puede servir por sí misma como una señal importante. Por último, muchos orgánulos mantener los valores pH de la luz que son distintos desde el citosol circundante y esenciales para las funciones específicas de orgánulo. Los Saccharomyces cerevisiae de levadura acciones un número de mecanismos de homeostasis de pH con eucariotas superiores 2. En los orgánulos ácidos de la vía endocítica / lisosomal, el pH se controla principalmente por el muy conservadas de protones ATPasa vacuolar translocación (V-ATPasa), en actuación conjunta con muchos intercambiadoresgers dependientes del gradiente de pH. Todas las células eucariotas también tienen mecanismos de exportación de protones. En los hongos y las plantas, un segundo, de la bomba de protones distinto en la membrana plasmática, PMA1, las exportaciones protones metabólicas y se cree que es el principal determinante de pH citosólico y el potencial de la membrana plasmática. La flexibilidad genética de S. cerevisiae y su importancia comercial, se han convertido en un modelo muy interesante e importante para el estudio de la homeostasis del pH 2. Además de ser los principales impulsores de la acidificación de orgánulos, V-ATPasas están altamente regulados enzimas y nuestro laboratorio está interesado en comprender los mecanismos de regulación V-ATPasa. Para lograr este objetivo, hemos utilizado en las mediciones de pH in vivo de pH vacuolar y citosólica: 1) para monitorear las respuestas a las cambiantes condiciones extracelulares, tales como la privación de glucosa y readdition, 2) para examinar los efectos de las mutaciones que comprometen la actividad V-ATPasa, y 3) para explorar la coorsobre coordinación de los orgánulos y plasma bombas de protones de membrana 3-5. Estos experimentos sólo se hizo posible mediante el desarrollo de indicadores de pH ratiometric sólidas susceptibles de utilizar en células de levadura. . Planta et al primero mostró que BCECF (2&#39;7&#39;-bis-(2-carboxietil) -5 - (y 6)-carboxifluoresceína), que se ha utilizado ampliamente para medir el pH citosólico en células de mamífero, se acumula en la vacuola de levadura en lugar de la citosol 6. Esta diferencia en la localización de BCECF se ha atribuido a las muchas enzimas hidrolíticas en la vacuola, que probablemente responsable de la escisión del éster de metilo acetoxi de BCECF-AM (éster de acetoximetilo de BCECF) y vacuolar de retención 6. Ali et al. 7 desarrollado la medición del pH vacuolar con BCECF y adaptado estas mediciones en un formato de lector de placas de fluorescencia. Brett et al. Introducido pHluorin levadura como un medio de medir el pH citosólico en la levadura mediante la expresión de un plásmido r transmitidasatiometric GFP sensible al pH 8 bajo el control de un promotor de levadura-específica 9. Los espectros de excitación de ambos BCECF y la levadura pHluorin son sensibles al pH, por lo que se utilizan como indicadores de pH radiométricos en los que la proporción de fluorescencia a dos longitudes de onda de excitación, medidos a una única longitud de onda de emisión, proporciona una medida de pH 8,10. Estas levaduras sensores de pH vacuolar y citosólica se han utilizado tanto para mediciones de una sola célula y poblacional. Mediciones de celda única 6,11 se realizan por microscopía de fluorescencia y análisis de imágenes. Fluorescencia vacuolar o citosólica en las dos longitudes de onda se mide para cada célula. Las mediciones basadas en la población se llevan a cabo ya sea en un lector de microplacas con capacidades de fluorescencia apropiados o en un fluorímetro. Hemos hecho generalmente nuestras mediciones en un fluorímetro, ya que proporciona un fácil acceso para la adición de componentes, tales como la glucosa durante el conmediciones cinéticas continuas. Nuestros protocolos de laboratorio actuales para la medición de pH vacuolar y citosólica se enumeran a continuación, ambos también se adaptan fácilmente a los ensayos de microplaca.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Spectrofluorometer</td>
 <td>Horiba Jobin Yvon</td>
 <td>Model Fluoromax-4</td>
 <td>Temperature control and stirring capability are desirable.</td>
 </tr>
 <tr>
 <td>BCECF-AM</td>
 <td>Invitrogen/Molecular Probes</td>
 <td>B1150</td>
 <td>Prepare a 12 mM stock in dry DMSO, store as aliquots at -20 &deg;C </td>
 </tr>
 <tr>
 <td>monensin</td>
 <td>Sigma</td>
 <td>M5273</td>
 <td>Toxic. </td>
 </tr>
 <tr>
 <td>nigericin</td>
 <td>Sigma</td>
 <td>N7143</td>
 <td>Toxic. </td>
 </tr>
 <tr>
 <td>MES</td>
 <td>Sigma</td>
 <td>M8250</td>
 <td></td>
 </tr>
 </tbody>
</table>

measurement of vacuolar and cytosolic ph in vivo in yeast cell suspensions

Vacuolar y el pH citosólico están altamente regulados en células de levadura y ocupan un papel central en la homeostasis global de pH. Se describen protocolos para la medición radiométrica de pH in vivo utilizando fluoróforos sensibles al pH localizadas en la vacuola o citosol. PH vacuolar se mide usando BCECF, que se localiza en la vacuola en la levadura cuando se introduce en las células en su forma de éster de acetoximetilo. PH citosólico se mide con una GFP sensible al pH expresado bajo el control de un promotor de levadura, levadura pHluorin. Se describen métodos para la medición de fluorescencia ratios en suspensiones de células de levadura en un fluorímetro. A través de estos protocolos, mediciones puntuales de tiempo individuales de pH bajo condiciones diferentes o en diferentes mutantes de levadura se han comparado y los cambios de pH a través del tiempo han sido controlados. Estos métodos también se han adaptado a un formato de lector de placas de fluorescencia para experimentos de alto rendimiento. Ventajas de las mediciones de pH ratiometric más de otros APTambién se describen los enfoques actualmente en uso, posibles problemas y soluciones experimentales, y las perspectivas para el futuro uso de estas técnicas.

La Figura 1 presenta los datos de pH vacuolar obtenidos en células de levadura de tipo salvaje cultivadas en medio rico (extracto de levadura-peptona-dextrano; YEPD) tamponada a pH 5 con 50 mM de MES. A menudo crecer las células en medio tamponado debido a que el pH del medio puede cambiar de manera espectacular durante el crecimiento durante la noche, en particular para un medio mínimo, y hemos encontrado que el pH del medio de crecimiento puede afectar a las respuestas de pH vacuolar 3. ...

Watch this Scientific Journal Video about Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions at JoVE.com

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

PH vacuolar y citosólica se puede medir en la levadura en vivo (<em&gt; S. cerevisiae</em&gt;) Células usando colorantes fluorescentes ratiometric localizados en compartimentos celulares específicos. Se describen procedimientos para la medición de pH vacuolar con BCECF-AM, que se localiza en la vacuola en levaduras, y el pH citosólico con una GFP sensible al pH radiométrica citosólica (levadura pHluorin).

La medición de pH vacuolar y citosólica<em&gt; En Vivo</em&gt; En suspensiones de células de levadura

Vacuolar and  cytosolic pH are highly regulated in yeast cells and occupy a central role in  overall pH homeostasis. We describe  protocols for ...

measurement-vacuolar-cytosolic-ph-vivo-yeast-cell

Research

JoVE Journal

Biology

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

19.8K vistas.  SUNY Upstate Medical University. PH vacuolar y citosólica se puede medir en la levadura en vivo ( S. cerevisiae) Células usando colorantes fluorescentes ratiometric localizados en compartimentos celulares específicos. Se describen procedimientos para la medición de pH vacuolar con BCECF-AM, que se localiza en la vacuola en levaduras, y el pH citosólico con una GFP sensible al pH radiométrica citosólica (levadura pHluorin).

Video: Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

En vivo la micro-circulación de medición en el músculo esquelético de Intra-vital Microscopía

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Investigación

Biología

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

La medición cuantitativa de invadopodios mediada por proteolisis de la matriz extracelular en contextos simples y multicelulares

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.

Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.

Medición pH simultánea en Endocítica y citosólicas compartimentos en las células vivas usando microscopía confocal

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Medición de niveles hemo síntesis en células de mamífero

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Las mediciones del calcio citosólico en las células epiteliales renales mediante citometría de flujo

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Análisis de suspensiones celulares aisladas de tejidos sólidos por Spectral Citometría de Flujo

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

Imágenes de alta resolución y análisis de la dinámica de microtúbulos astrales individuales de levadura en ciernes

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

Microscopía 4D de levadura

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

Efecto de proteínas fluorescentes de fusión socios en ensayos de toxicidad de poliglutaminas de levadura

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

Medición de las tasas de consumo de oxígeno en intacto Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...