The authors are deeply indebted to all the members of the scientific community working on pH and ion transport, who have originated and improved the measurements described here. They particularly thank Dr. Jacques Pouyss&#233;gur who originated the H+-killing selection technique used here. They acknowledge the University of Nice-Sophia Antipolis, the CNRS, the ANR (JCJC SVSE1 NHEint) and the ICST Labex for support.

1. H + Selección Matar <ol><li> Líneas celulares <ol><li> Transfectar células establemente NHE-deficientes (por ejemplo, la línea celular PS120 CCL39 derivado 16) utilizando cualquier combinación de vector de expresión de mamífero y el método de transfección que produzca rendimientos de transfección eficientes y selección en una línea celular de fibroblastos. NOTA: Durante muchos años, Fosfato de Calcio precipitación 19 se utilizó con buenos rendimientos de transfección. Esto ha sido reemplazado más recientemente por reactivos comerciales tales como Lipofectamine2000, las transfecciones se llevaron a cabo siguiendo las instrucciones del fabricante. </li></ol></li><li> Soluciones <ol><li> Prepare tres soluciones estériles se describen a continuación. Esterilizar estas soluciones por filtración (0,22 micras). <ol><li> Preparar una solución de NH 4 + Loading (pH 7,4) compuesto por 50 mM NH 4 Cl, 70 mM de cloruro de colina, 5 mM KCl, 1 mM de MgCl 2, 2 mM CaCl 2, 5 mM glucose y 15 mM MOPS a pH 7,4. </li><li> Preparar una solución de enjuague (pH 7,0) compuesta de 120 mM de cloruro de colina, 5 mM KCl, 1 mM de MgCl 2, 2 mM CaCl 2, glucosa 5 mM y 15 mM de HEPES a pH 7,0. </li><li> Preparar una solución de recuperación (pH 7,4) compuesto por NaCl 120 mM, 5 mM KCl, 1 mM de MgCl 2, 2 mM CaCl 2, glucosa 5 mM y 15 mM de HEPES a pH 7,4. </li></ol></li></ol></li><li> Antes de comenzar la selección, obtener una incubadora de cultivo celular que puede ser utilizado a 37 ° C sin CO adicional 2 del tanque externo (denominado CO 2 - incubadora &quot;libre&quot;), tal como 5% de CO 2 resultará en búfer que pueden poner en peligro el acidificación. Amplificar la línea celular que expresa el NHE de interés hasta aproximadamente 2 x 10 8 células (típicamente alrededor de 20 confluentes 100 placas mm). </li><li> H + procedimiento matar: <ol><li> Se incuban las células que expresan el NHE intracelular de interés durante 1 hora en la carga de soluciones, en 37 ° C en el incubador de CO2 exento. </li><li> Aspirar la solución de carga y luego enjuague dos veces en la solución descrita aclarado anteriormente. Realice este paso de manera eficiente para eliminar el residual extracelular NH 4 Cl que puedan afectar a la creación de un gradiente de NH 4 + empinada que va a generar la acidificación. </li><li> Eliminar el medio de enjuague por aspiración y se incuban las células durante una hora en una solución de recuperación de nuevo a 37 ° C en la incubadora de CO 2 libre. </li><li> Reemplazar el medio de recuperación con medio de cultivo regular (típicamente DMEM con 7,5% de FCS) y crecer las células en condiciones de cultivo estándar (37 ° C en una atmósfera humidificada de 5% de CO2 y 95% de aire). </li></ol></li><li> Repita este ciclo de selección dos veces por semana hasta que los clones estables surgen. NOTA: Tenga especial cuidado en relación con el cultivo celular y las condiciones de selección. Meses de trabajo puede ser arruinado por la contaminación que es más propenso a oCCur después de un largo periodo de la cultura y las manipulaciones celulares repetidas. <ol><li> Aquí, elija si desea amplificar los clones y caracterizar de forma individual se describe con más detalle en las secciones 3.2 o agruparse entre sí para generar una población celular antes de la caracterización. </li></ol></li><li> Aplicar selección ocasional (aproximadamente una vez cada dos semanas) para retener las células resistentes a la acidificación por el contador-seleccionar aquellos que puedan haber revertido al fenotipo sensible a los ácidos de los padres (Figura 1B). </li></ol> 2. Las mediciones de pH intracelular 2.1) Imaging pH Fluorescencia <ol><li> Utilice la radiométrica sensible al pH colorante fluorescente BCECF / AM, que es sensible al pH cuando se excita a 490 nm y posee un punto isosbéstico nm 445, siguiendo los protocolos del fabricante. <ol><li> También puede utilizar otras sondas sensibles al pH, siguiendo los protocolos del fabricante. </li></ol></li><li> Utilice una estafa de imágenes setconsistente de un microscopio invertido acoplado a una cámara de vídeo de alta sensibilidad. Equipar este conjunto con 450 nm y 490 filtros de interferencia de banda estrecha nm emparejados con los filtros de densidad neutra cuarzo apropiada para la excitación. <ol><li> Si es posible, utilice el conjunto adecuado de los filtros y las condiciones de iluminación para configurar los valores de fluorescencia comparables para λ1 y λ2 manera que la relación se aproxima a 1. Evite también los valores de fluorescencia basal que se establecen ya sea saturar o demasiado baja, tanto para λ1 y / o λ2. Esto puede producir artefactos importantes como entonces la relación no puede sido detectable aunque pH intracelular está cambiando. NOTA: Este sistema debe permitir la perfusión rápida, la excitación de lapso de tiempo en las dos longitudes de onda antes mencionados y la adquisición en la longitud de onda de emisión adecuada (535 nm para BCECF). Dotar al sistema de software que permite la adquisición automática de datos y almacenamiento. </li></ol></li></ol> 2.2) Medición de NHE Forward Actividad (Extrusión deLos protones desde el citosol) <ol><li> Se acidifica células mediante una incubación de 1 hora en la solución de NH 4 + Carga (véase el paso 1.4) en ausencia de CO 2. </li><li> Añadir BCECF-AM (concentración final 5 mM) a la solución para los últimos cinco minutos y luego enjuague con NH 4 + solución de carga para eliminar la sonda extracelular. </li><li> Montar las células en el microscopio y tomar varias imágenes para grabar una línea de base estable. Perfundir la solución de lavado (véase más arriba). Esto se traduce en una caída de la fluorescencia correspondiente a la acidificación. </li><li> Después de la estabilización del pH, perfundir cualquiera de las soluciones dadas a continuación para medir las tasas de recuperación de pH mediadas por Li + / H +, Na + / H + o K + / H + de cambio. Cada solución contiene un catión de potencial de acoplamiento a ensayar para H + de cambio. Si uno de ellos es transportado, esto se traducirá en un aumento de fluorescencia a 495 nm que correspond a un aumento en el pH intracelular: 120 mM LiCl, 5 mM KCl, 1 mM de MgCl 2, 2 mM CaCl 2, glucosa 5 mM y 15 mM de HEPES a pH 7,4 NaCl 120 mM, 5 mM KCl, 1 mM de MgCl 2, 2 mM CaCl 2, glucosa 5 mM y 15 mM de HEPES a pH 7,4 125 mM KCl, 1 mM de MgCl 2, 2 mM CaCl 2, glucosa 5 mM y 15 mM de HEPES a pH 7,4 </li></ol> 2.3) Medición de NHE Actividad Inversa NOTA: El principio aquí es invertir los gradientes iónicos transmembrana. <ol><li> Antes de la medición, cargar las células con el catión intracelular de interés (véase más adelante). </li><li> Incubar durante 5 min con BCECF / AM (véase el paso 2.2.2); enjuagarlos, montarlos en el microscopio de vídeo. </li><li> Perfundir la solución de carga y tome varias imágenes a grabar una línea de base estable. </li><li> Después de la estabilización de la línea de base, la imagen de las variaciones de pH cuando las células se perfunden con un medio ácido extracelular que contiene 120 mM de cloruro de colina, 5 mM KCl, 1 mM de MgCl 2, 2 mM CaCl 2, 5 mM de glucosa y MES 15 mM a pH 6,5. </li><li> Para LiCl carga, incubar las células durante 2 horas en una solución compuesta de 120 mM LiCl, 5 mM KCl, 1 mM de MgCl 2, 2 mM CaCl 2, glucosa 5 mM y 15 mM de HEPES a pH 7,4. De litio intracelular medido por espectroscopia de absorción atómica produce un valor de alrededor de 60 mM. </li><li> Para Na + carga, incubar las células durante dos horas en una solución compuesta por NaCl 120 mM, 5 mM KCl, 1 mM de MgCl 2, 2 mM CaCl 2, glucosa 5 mM y 15 mM de HEPES a pH 7,4 en presencia de ouabaína 1 mM para bloquear la Na / K ATPasa. En estas condiciones, la concentración intracelular de Na + es en el intervalo de 40 mM. NOTA: K + carga no es necesario ya que K + citosólico concentración está en el rango de 140 mm y un gradiente de K + hacia el entorno es por lo tanto fácil de lograr. </li></ol> 2.4) Tratamiento y calibración de datos: NOTA: Este paso se aplica tanto a las mediciones de avance y retroceso. <ol><li> Al final de cada experimento, las células perfundir con una solución de 140 mM KCl, 20 mM HEPES y 5 mM nigericin ajustado a valores de pH entre 6,5 y 7,4. </li><li> Recopilar datos como medidas de fluorescencia de las imágenes (niveles de gris que representan los niveles de intensidad) y la exportación bajo el formato de texto y tratar como se explica a continuación utilizando un programa de hoja de cálculo. </li><li> Calcular los valores de pH intracelulares para cada celda o región de interés utilizando la siguiente ecuación individual: pH = pKa + (Log (R-Rmin) / (R max -R)) x F min (λ2) / F max (λ2) </li><li> Utilice el F min (λ2) / F ma x (λ2) Relación de si hay blanqueo sonda o fugas, lo que resulta en una disminución en la fluorescencia de 450 nm a lo largo de los experimentos. Esto sin embargo es muy raramente observada en las condiciones utilizadas en el desexperimentos crito. </li><li> Como los datos de calibración están presentes al final de cada medición de pH, comprobar si los correspondientes valores calculados difieren de los valores de pH utilizados para la calibración. Si es el caso, entonces ajustar todos los valores de pH determinados experimentalmente a la calibración mediante el siguiente procedimiento: <ol><li> Calcula el siguiente cociente (Q, la diferencia entre el medido valores / Diferencia entre los valores de calibración). Multiplique el conjunto de datos por Q. Haga el ajuste final de la adición o sustracción de manera que los valores calculados serán iguales a los valores de calibración correspondientes. </li></ol></li></ol> 3. Las mediciones de los precios iniciales de NHE7 por Fast Li + Captación <ol><li> Sembrar las células en placas de pocillos múltiples (de 6 a 24 pocillos) y se acidifica usando ya sea la técnica de carga + NH 4 descrito anteriormente, o alternativamente el / albúmina de suero bovino (BSA) descrito en la acidificación nigericin 20. </li><li&gt; placas Enjuague dos veces rápidamente utilizando la solución de lavado antes descrito y luego se incuban las células en soluciones de absorción que contienen litio 1-10 mM (ligeramente por debajo de los valores de Km) y las concentraciones deseadas de los cationes y / o inhibidores de interés para la cantidad deseada de tiempo. </li><li> Mantener duraciones cortas de captación y consistentes (típicamente un minuto, o más adelante) para asegurar que el transporte opera en condiciones iniciales tasas. También asegúrese de que todas las soluciones utilizadas para la captación son isotónicas. </li><li> Al final del tiempo de absorción, eliminar cuidadosamente el medio absorción y lavar las células cuatro veces con solución salina de fosfato enfriado con hielo tamponada (PBS). Realizar estos lavados tan rápido como sea posible (menos de 10 seg para los cuatro de ellos es ideal) para evitar que el eflujo de litio. </li><li> Lisar las células en ácido nítrico 25%. Aplicar 250 l por pocillo de ácido nítrico al 25% y dejar que repose durante al menos 1 hora, después desechar cada pocillo con el final de la punta de la pipeta y transferir toda la suspensión bien en un nuevo mitubo crocentrifuge. </li><li> Transferir el lisado en 1,5 ml tubos de microcentrífuga, centrifugar durante 5 minutos a 15.000 xg (temperatura ambiente) para eliminar los restos celulares. </li><li> Mida el contenido de litio de los sobrenadantes mediante espectroscopía de absorción atómica 21. NOTA: Diferentes empresas ofrecen los espectrómetros de absorción atómica, que tienen que estar equipados con una lámpara de cátodo hueco de litio. Siga las instrucciones del fabricante ya que estas máquinas son muy precisos pero también bastante delicada. </li></ol>

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The authors have nothing to disclose.

Este protocolo describe cómo seleccionar las células que expresan intracelular de Na + / H + intercambiadores en la membrana plasmática sin alterar su secuencia primaria por mutagénesis dirigida al sitio. Estos intercambiadores de ahora se pueden caracterizar. Este método se basa en la toxicidad celular de protones intracelulares para seleccionar líneas celulares que expresarán vesicular Na + / H + intercambiadores en la membrana plasmática...

La mayoría de los compartimentos intracelulares muestran un pH luminal ácida, que es un parámetro clave para la maduración, el tráfico, el reciclaje de las proteínas u hormonas y neurotransmisores de carga. Se ha demostrado que el gradiente de pH entre el citosol y vesicular contenido es generado por vacuolar H + ATPasas 1, acoplado a los transportadores vesiculares de cloruro ClC 2. Tanto en ratones knock-out (KO) y los pacientes humanos, la importancia de estos transportadores ha sido destacada por los fenotipos pesados ​​causadas por mutaciones en sus genes 3-6. Los miembros de la familia SLC9A intercambiadores de sodio-hidrógeno, también denominado NHEs de Na + / H + intercambiadores, han mostrado ser efectores clave en el pH intracelular y la regulación del volumen celular, así como en el transporte vectorial de equivalentes de ácido-base través de los epitelios . Además de los NHEs de la membrana plasmática, tres altamente conservadas Na + / H + intercambiadores, NHE 6, 7y 9 se expresan en la red trans-Golgi y en los endosomas tempranos 7. Las mutaciones en sus genes se han relacionado con Angelman similar o Christianson Síndromes 8-9, basado en la familia autismo 10 y Déficit de Atención e Hiperactividad 11-12. Estos intercambiadores también han estado involucrados en problemas neurodegenerativos como el Alzheimer y la susceptibilidad a la enfermedad 13 genes contiguos retraso mental ligado al cromosoma X síndromes 14. En conjunto, estos estudios ponen de relieve la importancia de estos NHEs intracelulares en el desarrollo y / o la función cerebral. La localización intracelular de estos intercambiadores impide mediciones precisas de su selectividad de iones, dirección de transporte, parámetros cinéticos, y la regulación. Como es el caso para todos los transportadores expresados ​​en compartimentos intracelulares, es extremadamente difícil de evaluar sus actividades bioquímicas y por lo tanto para comprender plenamente sus funciones fisiológicas y la mechanismos subyacente sus implicaciones patológicas. En base a la alta concentración citosólica K +, la hipótesis más comúnmente aceptada era que estaban trabajando como K +, junto transportadores de salida de protones. La existencia de una fuga de protones tales había planteado la hipótesis, ya que puede contrarrestar de bombeo de protones por las V-ATPasas con el fin de mantener un pH constante vesicular estado. El objetivo de este artículo es visual (i) para demostrar un método que permite la selección genética de líneas celulares que expresan tales transportadores vesiculares en su membrana plasmática, y (ii) para mostrar dos enfoques independientes para medir las funciones de estos transportadores. Hace tres décadas, Pouysségur y Franchi han sido pioneros en un enfoque genético que permitió la clonación molecular y caracterización de los miembros de la familia NHE 15. Esto se basó en la toxicidad de protones intracelulares como un método de cribado. El primer paso fue obtener líneas celulares deficientesen cualquier / H + intercambio Na + expresado en la membrana plasmática, utilizando la reversibilidad de este transportador. Los fibroblastos (línea de células CCL39) se precargados con Na + o Li + y luego se colocan en un medio extracelular ácido (pH 6,5) durante 2 horas. Esto condujo a la muerte de las células que expresan un funcional Na + / H + de cambio y para la selección de células-antiporter deficiente (línea celular PS120) 16. Cuando se cultiva en un medio libre de bicarbonato, estas células son muy sensibles a la acidificación intracelular aguda. En consecuencia, se seleccionará positivamente la expresión de cualquier mecanismo de eflujo de protones funcional en la membrana plasmática (véase 17), si dichas células se someten a acidificación intracelular agudas. Tales técnicas de acidificación pueden ser utilizados para aislar líneas celulares con el tráfico de defectos que permiten la expresión forzada de NHEs intracelulares WT en la membrana plasmática. Como eucariota Na + / H+ Intercambiadores son electroneutral, que no son mensurables por los métodos electrofisiológicos que se han utilizado con gran éxito para medir canales. Por tanto, este manuscrito demuestra cómo medir la actividad de este intercambiador por mediciones de pH intracelular y cinética rápida de la absorción de litio. Como los conceptos subyacentes son los mismos, es interesante notar que muchos de los procesos desarrollados para la sección de selección también se utilizan directamente para las mediciones funcionales. Curiosamente, hemos observado que el defecto tráfico presente en las líneas celulares seleccionadas usando el enfoque descrito en este manuscrito conduce a una mayor expresión de otras proteínas vesiculares en la membrana plasmática, tales como el canal de potasio vesicular TWIK1 18. Esto señala hacia la selección de un mecanismo general defecto de retención para proteínas transmembrana vesiculares. Por lo tanto este procedimiento de selección y las células que genera mayoconstituyen una herramienta prometedora para la comunidad científica que trabaja en las proteínas de la membrana de los compartimentos intracelulares. Además de las técnicas de medición presentados aquí podrían ser aplicables para el estudio de otros transportistas no electrógenos.

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functional characterization of na+/h+ exchangers of intracellular compartments using proton-killing selection to express them at the plasma membrane

La acidificación endosomal es crítica para una amplia gama de procesos, como el reciclaje de proteínas y la degradación, la desensibilización del receptor y neurotransmisor de carga en las vesículas sinápticas. Esta acidificación se describe estar mediado por ATPasas de protones, junto a los transportadores de cloruro ClC. Altamente conservadas protones electroneutral transportadores, la Na + / H + (NHE) intercambiadores de 6, 7 y 9 también se expresan en estos compartimentos. Las mutaciones en sus genes se han relacionado con enfermedades cognitivas y neurodegenerativas humanas. Paradójicamente, su papel sigue siendo difícil, ya que su localización intracelular ha impedido caracterización funcional detallada. Este manuscrito muestra un método para resolver este problema. Esta consiste en la selección de líneas celulares mutantes, capaces de sobrevivir a la acidificación citosólica aguda mediante la retención de NHEs intracelulares a la membrana plasmática. A continuación, representa dos protocolos complementarios para medir la selectividad y actividad de los ionesde estos intercambiadores de: (i) uno en base a mediciones de pH intracelulares mediante microscopía de fluorescencia de vídeo, y (ii) uno basado en la cinética rápida de la absorción de litio. Estos protocolos pueden ser extrapolados a medir otros transportistas no electrógenos. Además, el procedimiento de selección que aquí se presenta genera células con un fenotipo defectuoso retención intracelular. Por lo tanto estas células también expresar otras proteínas de membrana vesicular en la membrana plasmática. Por consiguiente, la estrategia experimental representado aquí puede constituir una herramienta potencialmente potente para estudiar otras proteínas intracelulares que se expresan a continuación en la membrana plasmática junto con el vesicular Na + / H + intercambiadores utilizados para la selección.

Selección: La selección + matando H se basa en la difusión de la base débil de amonio, tal como se representa en la Figura 1A. El efecto de las bases débiles y ácidos difusión sobre el pH intracelular ha sido promovida por Walter Boro y colaboradores 22. El elegante idea de utilizar este fenómeno para producir una acidificación letal para la selección genética positiva luego fue desarrollada por Jacques Pouysségur 17. En virtud de...

Watch this Scientific Journal Video about Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane at JoVE.c

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

Caracterización funcional de Na<sup&gt; +</sup&gt; / H<sup&gt; +</sup&gt; Intercambiadores de compartimentos intracelular usando selección Protón-matanza para expresarlos a la membrana plasmática

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

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JoVE Journal

Biology

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

9.2K vistas.  Université Nice-Sophia Antipolis, Laboratoire de Physiomédecine Moléculaire, CNRS UMR7370, and Laboratories of Excellence Ion Channel Science and Therapeutics. The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers. 

Video: Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

Cerca de la medición de la membrana plasmática y Global Dynamics calcio intracelular en los astrocitos

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Investigación

Biología

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

Cristalización de las proteínas de membrana para la Determinación de la estructura con mesofases lipídico

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

La medición cuantitativa de la translocación de GLUT4 a la membrana plasmática por citometría de flujo

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

La identificación de los efectos de las mutaciones BRCA1 en recombinación homóloga utilizando células que expresan endógeno de tipo salvaje BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

El uso de un robot para la cristalización de alto rendimiento de proteínas de membrana en mesofases lipídicas

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

Recolección y enfriamiento Cryo-cristales de proteínas de membrana cultivadas en mesofases lipídicas para la Determinación de la estructura por cristalografía macromolecular

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Microscopia intravital para formar imágenes de estructuras subcelulares en ratones vivos que expresan proteínas fluorescentes

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

Imaging membrana plasmática Deformaciones Con pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Investigar los efectos perjudiciales de baja presión Plasma esterilización sobre la supervivencia de vivir el Bacillus subtilis esporas usando el celular microscopia

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...