Este trabajo fue apoyado por la Agencia de Investigación de Eslovenia con el proyecto de Z2-9229 y el programa P2-0249. Este video representa el material complementario para la &quot;base de electroporación, tecnologías y tratamientos&quot; taller científico y postgrado, organizado cada dos años por la Facultad de Ingeniería Eléctrica de la Universidad de Ljubljana, en Eslovenia.

Parte I: Pasos preliminares <ol><li> En este experimento ovario de hámster chino línea celular (CHO-K1) se utiliza. Las células se colocan en Lab-Tek II cámaras (dos pozos, 4 cm 2 cada uno) (Nalge Nunc, Alemania) a ~ 0.7x10 5 células / ml en la HAM-F12 medio de cultivo suplementado con 8% de suero fetal bovino, 0,15 mg / ml L-glutamina, 16 mg / ml de gentamicina (todos de Sigma-Aldrich, Steinheim, Alemania), y 200 unidades / ml Crystacillin (Pliva, Zagreb, Croacia), y se incubaron en el 5% de CO 2 a 37 ° C. Alternativamente, las células también pueden ser chapados en # 1 se desliza cubierta de vidrio (0,13 a 0,16 mm de espesor), recubierta con un adhesivo de celulares, tales como polilisina. </li><li> Se incuban las células en su medio de cultivo. La incubación dura de 2 a 4 Rendimientos horas las células que aún están más o menos esférico, pero firmemente adherida a la superficie con una pequeña parte de su membrana. Por otra parte, después de 16 a 20 horas de incubación, las células están totalmente adheridos a la superficie y tienen formas más complejas, pero la mayoría de ellos todavía no dividir. </li><li> Prepare una solución stock 10 mM de di-8-ANEPPS (Invitrogen, Eugene, Oregon, EE.UU.) mediante la adición de 843 l de DMSO (Sigma-Aldrich, Steinheim, Alemania) a 5 mg del colorante en el vial de Invitrogen original. La solución madre se pueden almacenar en un refrigerador a 4 ° C durante varios meses. Antes de comenzar los experimentos, calentar la solución hasta que los cristales se disuelven de DMSO. </li><li> Algunas líneas celulares pueden requerir el uso de Pluronic a facilitar la incorporación de contraste en la membrana celular. Pluronic se pueden comprar en solución de reserva del 20% en DMSO (F-127, Invitrogen, Eugene, Oregon, EE.UU.), o una solución de reserva de la misma concentración se puede preparar disolviendo plurónico en DMSO. Solución de archivo de Pluronic se pueden almacenar a temperatura ambiente. </li></ol> Parte II: Carga de las células con di-8-ANEPPS <ol><li> Mezcle 3 l de 10 mM di-8-ANEPPS y 2,5 l de 20% plurónico en 1 ml de la modificación Spinner (calcio agotado versión) de la SMEM medio mínimo esencial (M8167 o M4767 medio, Sigma-Aldrich, Steinheim, Alemania ) en un tubo Eppendorf de 1,5 ml. Esto produce una &quot;solución de carga&quot;, que contiene aproximadamente 30 M-8-di ANEPPS y 0,05% de Pluronic. Para otros tipos de células, sus medios de cultivo nativo se puede utilizar en lugar de SMEM. </li><li> Vuelva a colocar el medio de cultivo en la cámara Lab-Tek con la solución de carga. La transferencia de la cámara a la nevera durante 10 min a 4 ° C. A esta temperatura, la internalización del colorante a través de la membrana plasmática es muy reprimidas. </li><li> Después de la tinción, lave el exceso de colorante dos o tres veces con SMEM puro. </li><li> Agregar 1,5 ml de SMEM en la cámara. </li></ol> Parte III: Experimento y la adquisición de imágenes <ol><li> Las células se observan con un microscopio de fluorescencia (en nuestro caso Zeiss Axiovert 200, Zeiss, Alemania) equipado con un objetivo de inmersión en aceite (x63, NA 1.4), un VisiCam monocromador (policroma IV, Visitron, Alemania) y una cámara CCD enfriado ( 1280, Visitron, Alemania). Las imágenes se adquieren con MetaFluor 7.1.1 y procesada en MetaMorph 7.1.1 (tanto Molecular Devices, Downingtown, PA, EE.UU.), pero otro software de adquisición similar también se puede utilizar. La longitud de onda de excitación en el software de adquisición de 490 nm y elegir un filtro de emisión adecuados para ANEPPS, por ejemplo, un filtro pasa-banda centrado en 605 nm (605/55m, dicroico 565 DCXR, Chroma, Rockingham, Vermont, EE.UU.). Si un monocromador no está disponible, un filtro de excitación centrado en 490 nm se puede utilizar en su lugar. </li><li> Coloque la cámara con las células en el microscopio, la posición de los electrodos en la parte inferior de la cámara y su conexión con el generador de impulsos. El medio debe cubrir los electrodos. </li><li> Para mantener la viabilidad celular y reducir el calentamiento, el pulso debe ser de amplitud suficientemente baja y de corta duración. En este experimento, un pulso cuadrado de 35 V de amplitud y 50 ms de duración (lo suficientemente baja como para evitar la electroporación) se genera mediante una fuente de tensión continua y una medida controlado por microprocesador, el dispositivo de conmutación. Por otra parte, un generador de impulsos comerciales, tales como 33210A (Agilent, Santa Clara, CA, EE.UU.), conectado a un amplificador o un estimulador comercial, como el S88 (Grass, West Warwick, Rhode Island, EE.UU.), se puede utilizar. El pulso se entrega a dos electrodos paralelos de alambre de Pt / Ir con un diámetro de 0,8 mm y 4 mm de distancia entre ellos, creando un campo eléctrico de aproximadamente 88 V / cm entre los electrodos y ΔΦ inducción. La distribución exacta de campo entre los electrodos utilizados en este experimento es descrito en [1]. </li><li> Encuentra las células de interés. Aplicar un pulso eléctrico o una secuencia de pulsos. Para cada pulso, la adquisición de dos imágenes de fluorescencia: una inmediatamente antes del pulso (el control de la imagen), y uno durante el pulso (la imagen de pulso). Debido a la baja respuesta de los di-8-ANEPPS, los cambios en la fluorescencia son difíciles de percibir a simple vista y llegar a ser aparente only después del procesamiento. La entrega de pulso debe estar sincronizado con la adquisición de la imagen, que es una característica de la adquisición de software. Para evitar posibles photobleaching y calefacción, la iluminación puede ser limitada a la duración del pulso. </li></ol> Parte IV: El procesamiento de imágenes y análisis <ol><li> Abra las imágenes en el software MetaMorph. Para cada pulso, restar el fondo, tanto en el control y la imagen de pulso. </li><li> Elegir un conjunto de células y la región de interés por lo que corresponde a la membrana. Medir la intensidad de fluorescencia a lo largo de esta región en el control de la imagen y el pulso y la transferencia de los valores de una hoja de cálculo. </li><li> Para cada pulso, restar los datos de control de los datos de pulso, y dividir el resultado por los datos de control para obtener los cambios de fluorescencia relativa. Si una secuencia de pulsos se aplica, los valores de los cambios relativos de fluorescencia determinada por cada pulso se promedian para obtener una medición más confiable. </li><li> Transformar los cambios de fluorescencia relativa a ΔΦ utilizando una curva de calibración. Una estimación aproximada de la curva se puede obtener de la literatura, pero para mayor precisión, tiene que ser medida para cada instalación en particular. Para las células y la configuración utilizada en este experimento una disminución del 6% de la fluorescencia corresponde a un incremento de 100 mV en ΔΦ [2]. </li><li> Finalmente, grafique el voltaje en función de la longitud del arco relativa en un paquete de software de gráficos, tales como Excel (Microsoft Corp., Redmond, WA, EE.UU.), Sigma Plot (Systat Software Inc., San Jose, CA, EE.UU.), o origen (OriginLab Corp., Northampton, MA, EE.UU.). La curva también pueden ser suavizadas con un filtro adecuado, como el filtro de media móvil. </li></ol>

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Eng. 34, 642-652 (2006).</li><li>Huang, C. J., Harootunian, A., Maher, M. P., Quan, C., Raj, C. D., McCormack, K., Numann, R., Negulescu, P. A., Gonzalez, 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=Characterization+of+voltage-gated+sodium-channel+blockers+by+electrical+stimulation+and+fluorescence+detection+of+membrane+potential.">Characterization of voltage-gated sodium-channel blockers by electrical stimulation and fluorescence detection of membrane potential.</a> Nat. Biotechnol. 24, 439-446 (2006).</li><li>Bedlack, R. S., Wei, M., Fox, S. H., Gross, E., Loew, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+electric+potentials+in+soma+and+neurite+membranes.">Distinct electric potentials in soma and neurite membranes.</a> Neuron. 13, 1187-1193 (1994).</li><li>Cheng, D. K. L., Tung, L., Sobie, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonuniform+responses+of+transmembrane+potential+during+electric+field+stimulation+of+single+cardiac+cells.">Nonuniform responses of transmembrane potential during electric field stimulation of single cardiac cells.</a> Am. J. Physiol. 277, H351-H362 (1999).</li><li>Teissie, J., Eynard, N., Gabriel, B., Rols, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electropermeabilization+of+cell+membranes.">Electropermeabilization of cell membranes.</a> Adv. Drug. Del. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+properties+of+tissue+and+cell+suspensions.">Electrical properties of tissue and cell suspensions.</a> Adv. Biol. Med. Phys. 5, 147-209 (1957).</li><li>Gowrishankar, T. R., Weaver, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+approach+to+electrical+modeling+of+single+and+multiple+cells.">An approach to electrical modeling of single and multiple cells.</a> Proc. Natl. Acad. Sci. USA. 100, 3203-3208 (2003).</li><li>Joshi, R. P., Hu, Q., Schoenbach, K. 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Las mediciones de la membrana inducido (transmembrana) de tensión, ΔΦ, puede ser importante en diversos parámetros experimentales, tales como estudios de los canales de membrana voltaje-dependientes, propagación de potenciales de acción, la estimulación de células cardíacas, o la electroporación de la membrana celular [3, 4, 5, 6 , 7]. Con formas simples de células, ΔΦ se puede calcular analíticamente. Por ejemplo, para una célula esférica, ΔΦ está dada por la ecuación de Schwan s, que establece que la tensión es proporcional a la intensidad de campo y el tamaño celular y sigue a la función del coseno a lo largo de la membrana [8, 9]. Para más formas de células complicado, ΔΦ pueden desviarse considerablemente de la del coseno y debe ser determinado numéricamente, usando una computadora [2, 10, 11], o de forma experimental, utilizando un medio de potenciométrica [12, 13, 14, 15]. Uno de los colorantes potenciométricos utilizados para este propósito es di-8-ANEPPS (di-8-butil-amino-naftil-etilen-piridinio-propil-sulfonato), un tinte rápido con los espectros de excitación y de emisión depende de la tensión de la membrana, que permite que las observaciones no invasivo de las variaciones de la ΔΦ en la membrana celular y para medir su valor. En este vídeo, se muestra un enfoque experimental para la determinación de ΔΦ mediante el uso de di-8-ANEPPS. El colorante fue desarrollado por el profesor Leslie Loew y sus colegas [13, 14] en la Universidad de Connecticut, y pertenece a la clase de respuesta rápida colorantes. di-8-ANEPPS es no fluorescente en el agua y se convierte en fuertemente fluorescente cuando se incorpora en la bicapa lipídica de la membrana celular. Un cambio en los resultados de ΔΦ en un cambio de la distribución de carga intramolecular y los correspondientes cambios en el perfil espectral y la intensidad de la fluorescencia del colorante. La intensidad de fluorescencia de di-8-ANEPPS varía proporcionalmente al cambio de ΔΦ, la respuesta de la tintura es lineal para las tensiones que van desde -280 mV a 250 mV [4, 16]. Cambios relativamente pequeños en la fluorescencia de las manchas del tinte, la membrana irregular, y la internalización de tinte que di-8-ANEPPS menos adecuados para la medición absoluta de la tensión de la membrana, por ejemplo, su componente de descanso, a pesar de estos esfuerzos también se informó [17]. Es, sin embargo, adecuada para medir los cambios más grandes en el voltaje de la membrana, tales como la aparición de la tensión de la membrana en las células no excitables inducida expuestos a campos eléctricos externos [12, 13], o potenciales de acción en las células excitables [4, 5]. Aunque no se aplica aquí, di-8-ANEPPS también permite la determinación de las mediciones ΔΦ radiométrica de excitación de fluorescencia [18] o de las emisiones [19], lo que aumenta la sensibilidad de la respuesta y reduce los efectos antes mencionados. Como di-8-ANEPPS manchas de la membrana, sino que también puede ser utilizado simplemente como un marcador de membrana [2]. Uno de los inconvenientes de la tintura es que es propenso a photobleaching, por lo que la exposición prolongada a la luz fuerte se debe evitar. La calibración de la tintura se realiza ya sea con valinomicina ionóforo (i) de potasio y un conjunto de diferentes concentraciones de potasio en el medio externo [2,18], o (ii) de patch-clamp en el modo de fijación de voltaje [17]. Finalmente, con las medidas de ΔΦ en células esféricas y las células de formas más complejas, el video muestra la influencia de la forma de la célula en la distribución espacial de la amplitud y ΔΦ. Así, por células esféricas ΔΦ está cerca de un coseno, de acuerdo con la ecuación de Schwan, mientras que para las formas más complicadas de células de la distribución espacial de ΔΦ es más complejo [20] ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>di-8-ANEPPS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>D-3167</td>
<td>potentiometric fluorescent dye</td>
</tr>
<tr>
<td>pluronic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P3000MP</td>
<td>potassium ionophore</td>
</tr>
<tr>
<td>DMSO</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D2650</td>
<td>&nbsp;</td>
<tr>
<td>SMEM</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M8167 or M4767</td>
<td>Spinner modification of the Minimum Essential Medium</td>
</tr>
<tr>
<td>Ham-F12</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>N4888</td>
<td>culture medium</td>
</tr>
<tr>
<td>fetal calf serum</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>F4135</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G7513</td>
<td>&nbsp;</td>
<tr>
<td>crystacillin</td>
<td>&nbsp;</td>
<td>Pliva</td>
<td>625110</td>
<td>antibiotic</td>
</tr>
<tr>
<td>gentamicin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G1397</td>
<td>antibiotic</td>
</tr>
<tr>
<td>Lab-Tek II</td>
<td>&nbsp;</td>
<td>Nalge Nunc</td>
<td>155379</td>
<td>chamber</td>
</tr>
<tr>
<td>DC voltage supply</td>
<td>&nbsp;</td>
<td>Elektro-Automatik</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>microprocessor-controlled switcher</td>
<td>&nbsp;</td>
<td>Custom made</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>electrodes</td>
<td>&nbsp;</td>
<td>Custom made</td>
<td>&nbsp;</td>
<td>Pt/Ir</td>
</tr>		</tbody>
		</table>
		</div>

measuring the induced membrane voltage with di-8-anepps

La colocación de una celda en un campo eléctrico externo provoca una redistribución de carga en el interior local y exterior de la célula en los alrededores de la membrana celular, lo que resulta en un voltaje a través de la membrana. Este voltaje, llamado el voltaje de la membrana inducida (tensión inducida también transmembrana, o la diferencia de potencial transmembrana inducida) y se denota por ΔΦ, existe sólo mientras el campo externo está presente. Si la tensión de reposo está presente en la membrana, la tensión inducida superpone (agrega) en la misma. Mediante el uso de uno de los tintes fluorescentes potenciométrica, como di-8-ANEPPS, es posible observar las variaciones de ΔΦ en la membrana celular y para medir su valor forma no invasiva. di-8-ANEPPS se convierte en fuertemente fluorescente cuando se une a la bicapa lipídica de la membrana celular, con el cambio de la intensidad de fluorescencia proporcional al cambio de ΔΦ. Este video muestra el protocolo para la medición de ΔΦ con di-8-ANEPPS y también demuestra la influencia de la forma celular de la amplitud y la distribución espacial de ΔΦ.

Watch this Scientific Journal Video about Measuring the Induced Membrane Voltage with Di-8-ANEPPS at JoVE.com

Measuring the Induced Membrane Voltage with Di-8-ANEPPS

Campo eléctrico externo provoca una tensión en la membrana de una célula, denominada tensión de la membrana inducida (ΔΦ). Mediante el uso de la tintura potenciométrica di-8-ANEPPS, es posible medir el ΔΦ no invasiva. Este video muestra el protocolo para la medición de ΔΦ con di-8-ANEPPS.

La medición de la tensión de la membrana inducida con Di-8-ANEPPS

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell ...

measuring-the-induced-membrane-voltage-with-di-8-anepps

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JoVE Journal

Biology

17.3K vistas.  University of Ljubljana. Campo eléctrico externo provoca una tensión en la membrana de una célula, denominada tensión de la membrana inducida (ΔΦ). Mediante el uso de la tintura potenciométrica di-8-ANEPPS, es posible medir el ΔΦ no invasiva. Este video muestra el protocolo para la medición de ΔΦ con di-8-ANEPPS.

Video: Measuring the Induced Membrane Voltage with Di-8-ANEPPS

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

El estudio de biogénesis de la membrana con un ensayo de luciferasa basado en genes

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Investigación

Biología

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, ...

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

Medición de proteína de la membrana de plasma tarifas endocítica por biotinilación reversible

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

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 ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

La cristalización de proteínas de membrana en mesofases lipídico

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

Examinar la dinámica conformacional de las proteínas de membrana In situ Sitio-dirigida con fluorescencia de etiquetado

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

La<em&gt; Xenopus</em&gt; Ovocitos-Cut abierta vaselina Gap Técnica Voltage-clamp Con Fluorometría

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

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 ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

Medición inducida glutatión-Alimentación Respuesta en Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Microperfusión Técnica de investigar Reglamento de Microvessel Permeabilidad en Rata Mesenterio

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...