Este trabajo fue apoyado por la Fundación Uehara Memorial de Japón (a ES), así como la Fundación de Whitehall, el Instituto Nacional de Trastornos Neurológicos y Accidentes Cerebrovasculares y el Premio Stein-Oppenheimer Fundación (a BSK).

PROCEDIMIENTO EXPERIMENTAL El procedimiento experimental consta de dos partes principales que se describen de una manera paso a paso a continuación. Parte 1: preparación de los cultivos de astrocitos del hipocampo En pocas palabras, mezcla de neuronas del hipocampo astrocitos culturas fueron preparados utilizando un protocolo bien establecido 1,2,3. Hemos optimizado el procedimiento para obtener saludable astrocitos cultivados. Todos los procedimientos que se enumeran a continuación deben ser llevadas a cabo en un ambiente estéril, como una campana de flujo laminar. Cubreobjetos de la preparación <ul><li> Cubreobjetos de 22 mm (VWR, 48380-068) </li><li> Poli-D-lisina (PDL, Sigma P0899), alícuotas (1 mg / ml) </li><li> Laminina alícuotas (20 ug / ml): agregar 49 ml de agua estéril a 1 mg / ml de solución de laminina (Sigma L2020), en versión 1.2 ml alícuotas y guardar a -20 ° C </li></ul><ol><li> Disolver PDL en agua estéril (50 mg / ml) </li><li> Autoclave de los cubreobjetos, enjuague con agua estéril y colocar en PDL (20 ml por 100 cubreobjetos). Dejar a temperatura ambiente durante la noche. </li><li> Vuelva a colocar PDL con agua estéril (3 lavados rápidos). Luego secar los cubreobjetos y se almacenan a 4 ° C. </li><li> El día de las placas, los astrocitos, cubreobjetos lugar individual en un pozo estéril en una placa de 6 pocillos (Multiwell placas de fondo plano con tapa, estériles de BD Bioscience). Ponga 400 l de laminina en cada cubreobjetos y se incuba durante la noche, en la incubadora para evitar la evaporación. </li></ol> Disección Medios de disección: <ul><li> 500 ml de Earle solución salina balanceada (EBSS) (1X), líquido (Invitrogen, 14.155-063) </li><li> 5 ml solución HEPES (Sigma, H0887) </li></ul> Los medios de comunicación del hipocampo: <ul><li> 412,5 ml de medio mínimo esencial (MEM) (1X), el líquido contiene sales de Earle, pero no rojo L-glutamina o fenol (Invitrogen, 51200-038) </li><li> 10 ml de glucosa (Sigma, D-(+)-glucosa anhidra SIGMA ULTRA G7528) 1 M en el MEM </li><li> 5 ml penicilina-estreptomicina (Invitrogen, 15140-122) </li><li> 5 ml de sodio piruvato solución (Sigma, S8636) </li><li> 12,5 ml de HEPES solución 1 M (Sigma, H0887) </li><li> 5 ml N-2 Suplemento (100X), líquido (Invitrogen, 17502-048) </li><li> 50 ml de suero de caballo, inactivado por calor (Invitrogen, 26050-088) </li></ul> La papaína solución: <ul><li> Añadir 5 ml de los medios de disección en Worthington papaína-022 vial (LK003178, la concentración final, 20 U / ml) y se incuba a 37 ° C durante 60 min. </li></ul><ol><li> Decapitar a las crías de rata entre P1 (normalmente 2 cachorros) siguiendo las directrices revisadas y aprobadas por los reglamentos nacionales y locales de su cuidado animal institucional y el empleo. </li><li> Quite la piel y el cráneo y el cerebro en lugar de una placa de Petri llena de medios de disección en frío. </li><li> Eliminado meninges, diseccionar los dos hemisferios y diseccionar hipocampo. </li><li> Picar el hipocampo en ~ 1X1 piezas mm y digerir en solución de papaína a 37 ° C durante 11-13 min. </li><li> Una vez que las piezas de tejido se han instalado, retire con cuidado la papaína y añadir 5 ml de medio de hipocampo para eliminar todo rastro de la enzima. Repita este paso y se resuspenden en medio del hipocampo (2 ml). </li><li> Triturar las células unas 5 veces hasta que no queden grumos izquierda, con tres pulido llama pipetas de agujeros cada vez más pequeños (1000 m, ~ 500 ~ 300 micras y micras). </li><li> Una vez triturado, pasar a las células a través de un colador de células (tamaño de poro, 70 m, BD Bioscience). Contar las células en 10 l de suspensión. Ajustar el volumen del hipocampo de medio a fin de tener 400.000 células / ml. </li><li> Aspirar la laminina de la cubreobjetos, y antes de que las tapas se pueden secar, la placa de 200 l de la suspensión de células por cubreobjetos. </li><li> Agregar a conectar durante 60 minutos en la incubadora, y luego agregar 2 ml de medio por pozo del hipocampo. </li><li> Al día siguiente, aspirado medio del hipocampo de edad para eliminar las células muertas y los desechos y añadir 2 ml de pre-calentado medio del hipocampo fresco. </li></ol> Mantenimiento Los medios de comunicación Neurobasal: <ul><li> 500 ml neurobasal (Invitrogen, 12348-017) </li><li> 10 ml de suero libre B27 suplemento (Invitrogen, 17504-044) </li><li> 5 ml penicilina-estreptomicina (Invitrogen, 15140-122) </li><li> 1,25 ml de L-glutamina (Invitrogen, 25030-149) </li></ul> Alimentar a los cultivos de neuronas astrocitos dos veces por semana con el medio neurobasal, comenzando cuatro días después de la siembra. Preincubación de los medios de comunicación alrededor de 30 minutos en la incubadora en un frasco de ventilación para equilibrar la temperatura y el CO 2. Parte 2: imágenes de calcio Búfer de grabación del hipocampo: 110 mM NaCl, 5,4 mMKCl, 1,8 mM CaCl 2, 0,8 mM MgCl2, 10 mM D-glucosa, 10 mM HEPES (Todos los productos químicos de Sigma) pH 7,4 (ajustado con NaOH). Carga de tintes de calcio indicador en los astrocitos <ol><li> Lugar de la cultura en un pozo en una placa de 6 pocillos lleno con 2 ml de tampón de la grabación del hipocampo que contiene 2,5 M Fluo-4, AM (Invitrogen, F-14217) y el 0,05% Pluronic ® solución F-127 del 20% en DMSO (Invitrogen, P-3000MP) e incubar a temperatura ambiente durante 10-30 min. </li><li> Quitar Fluo-4 solución y lave cubreobjetos 3 veces con tampón de grabación del hipocampo e incubar a temperatura ambiente durante 30 min. </li><li> Coloque el cubreobjetos sobre la cámara, y limpiar el otro lado del cubreobjetos con líquido de limpieza de lentes. </li><li> Ponga una pequeña gota de aceite de inmersión (aceite de inmersión DF TIPO de Cargille) sobre el objetivo y poner la cámara (cámara abierta de 25 mm cubreobjetos ronda de Warner Instruments) en la platina del microscopio (Olympus IX71). </li><li> Observar las células con transmisión de la luz para ver cómo se ven las células, y ponerlos en el foco. Luego iluminar las células con 488 nm de un monocromador (V policromada de TILL Vision) y determinar si la señal de fluorescencia de Fluo-4 es uniforme y detectable en los astrocitos. </li><li> El uso del PAI y la iluminación TIRF para medir el calcio en los astrocitos. </li></ol> Microscopía TIRF En pocas palabras, se utiliza un microscopio Olympus IX71 equipado con una cámara de Andor IXON DV887DCS EMCCD. El control de la excitación y la adquisición de imágenes se realiza mediante software TILLVision. Las vigas de 454/488/515 nm argón (100 mW) y 442 nm de estado sólido (45 mW) láser se combinan y se controla con un láser HASTA Polilínea combinador, condensador TIRF de doble puerto y un filtro sintonizable acoustoptical y el controlador (AOTF, todos de TILL Photonics) y se introduce en una fibra de banda ancha Kineflex para la entrada en el condensador TIRF. Utilizamos un lente Olympus 60X 1,45 NA para lograr TIRF. La ganancia de la cámara se ajusta para cada astrocito para ofrecer la mejor relación señal-ruido de las imágenes. El fondo y los principios de la microscopía TIRF se han revisado recientemente 4, 5. La mayoría de los componentes ópticos que utilizamos fueron adquiridos de TILL Photonics, que ahora es parte de Agilent Technologies (http://www.till-photonics.com/). La profundidad de penetración TIR se puede calcular a partir de las siguientes ecuaciones. <img style="float:left;" src="/files/ftp_upload/1142/equation.png" alt="Ecuación" /> d = profundidad de penetración n1 = índice de refracción del vidrio n2 = índice de refracción de las células a = ángulo de incidencia NAi = apertura numérica de la incidencia A fin de asegurar que el láser se alinea de manera óptima para TIRF creemos que es útil para observar 100 nm fluorescente talón (Invitrogen, F8803). Se presentan imágenes fijas y videos de cuentas con EPI y microscopía TIRF. Cuando en el TIRF, se observa un aumento dramático en relación señal-ruido y las cuentas de visualización difusión browniana. Creemos que es útil para observar el comportamiento de 100 nm cuentas con microscopía TIRF sobre una base regular (una vez por semana) para asegurarse de que TIRF óptimo se produce, en lugar de la iluminación oblicua en peligro que se produciría si el ángulo crítico no eran iguales a α (ver Fig. 1). La aplicación de agonistas de la proteína G-receptores acoplados Los astrocitos expresan una variedad de receptores acoplados a Gq 6, 7, incluyendo los receptores metabotrópicos de glutamato y los receptores P2Y (agonista, ATP, ADP). La activación de estos receptores conduce a un aumento significativo en los niveles de calcio intracelular en los astrocitos. Por ejemplo, uno puede fácilmente observar elevaciones de calcio intracelular durante la aplicación de la ATP (30 mM) a 8.10 astrocitos. Utilizamos una solución rápida conmutación de Warner Instrumentos llamado VC-77SP rápido paso del sistema de perfusión (http://www.warneronline.com/index.cfm). Con esta solución se puede aplicar el método en menos de ~ 10 ms. <img src="/files/ftp_upload/1142/Fig 1a.jpg" alt="Figura 1" /> <img src="/files/ftp_upload/1142/fig1c.jpg" alt="" /> Dibujos Figura 1. Y fotografías de la imagen creada. A. Muestra una fotografía de un microscopio montado en un airtable, mientras que (B) muestra una fotografía de la asamblea de láser, los controladores y las cajas de la viga. C. Muestra una fotografía de la platina del microscopio con la cámara montada para la imagen. A la izquierda del dispositivo de perfusión rápida se puede ver (junto con el motor paso a paso y el tubo de teta). A la derecha de la headstage de un amplificador Axopatch 200A se ve. La caricatura esquematiza la trayectoria de la luz en la puesta en marcha y la forma en TIRF se logra. El láser se centra en el plano focal posterior de la lente del objetivo 60X 1,45 NA y su posición se ajusta fuera del centro por lo que surge en el aceite de inmersión en el ángulo crítico α. En este punto, el rayo sufre de reflexión interna total y se desintegra con una distancia λ (véase la ecuación en el texto principal) en el medio de índice de refracción menor. En este caso, este es el búfer de grabación alrededor de los astrocitos y los propios astrocitos. El resultado es una excitación óptica (y por lo tanto de imagen) de las moléculas dentro de ~ 100 nm de la membrana plasmática. En el dibujo de la célula de esto se muestra en verde los receptores &quot;emocionado&quot; de la membrana, mientras que dentro de la celda o en la superficie superior de la celda, no muy contentos. Un informe completo de la microscopía TIRF ha sido proporcionada por Steyer y Almers 4. <img src="/files/ftp_upload/1142/fig2.png" alt="Figura 2" /> Figura 2. Imágenes de 100 nm cuentas fluorescentes adquiridos con EPI y microscopía TIRF. A. Muestra imágenes del PAI de un campo de visión con perlas de varias docenas de 100 nm fluorescentes. Las flechas señalan a los granos rojos que se han asentado en el portaobjetos de vidrio, mientras que las flechas azules señalan a los granos que se están difundiendo en el agua. B. Muestra una imagen TIRF del mismo campo de visión, como se muestra en A. En este punto de vista sólo las cuentas de adherentes mostrado por las flechas rojas son visibles. Esto es porque estos se habían asentado en el coverlsip de vidrio y fueron por lo tanto dentro del campo de ~ 100 nm evanescente. Las cuentas se muestra en una de las flechas azules no están dentro de esta región y por tanto son invisibles en las imágenes TIRF. Las parcelas inferiores muestran la representación 3D de las imágenes. Es evidente que un gran aumento en relación señal-ruido se produce porque las cuentas en el campo evanescente cuando se observan al microscopio TIRF. De hecho, para estas imágenes la relación señal-ruido de EPI fue de 7,1 ± 0,6, mientras que para TIRF fue de 20 ± 0,7. <img src="/files/ftp_upload/1142/fig3.png" alt="Figura 3" /> Figura 3. Imágenes de los astrocitos cargados con Fluo-4 tinte indicador de calcio adquiridos con EPI y microscopía TIRF. A. EPI imágenes de un campo de visión con cinco astrocitos. B. Un TIRF imagen del mismo campo de visión se muestra en B. Nótese que las imágenes de A y B son significativamente diferentes. Esto es porque con TIRF única iluminación de las regiones membrana plasmática en estrecha aposición a la cubreobjetos se va a examinar. Los paneles inferiores muestran ATP-evocó los transitorios de calcio intracelular con imágenes de EPI y microscopía TIRF.

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Es un hecho bien establecido que los astrocitos mostrar elevaciones de calcio intracelular. Esto ocurre de forma espontánea, puede ser desencadenada por la actividad neuronal o por la aplicación de agonistas para activar los receptores en la superficie de los astrocitos 11. Una cuestión importante y polémica es si los astrocitos elevaciones de calcio intracelular puede desencadenar la liberación de moléculas de señalización que activan los receptores en las neuronas 11, 12. Esto es controvertido porque no ha ha...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>VWR&#174; Micro Cover Slips, Round, No. 1</td>
<td>Tool</td>
<td>VWR</td>
<td>48380-068</td>
<td>&nbsp;</td>
<tr>
<td>Poly-D-lysine hydrobromide</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P0899</td>
<td>&nbsp;</td>
<tr>
<td>Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td>
<td>Reagent</td>
<td>Sigma</td>
<td>L2020</td>
<td>&nbsp;</td>
<tr>
<td>Earle&#x2019;s Balanced Salt Solution (EBSS) (1X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>14155-063</td>
<td>&nbsp;</td>
<tr>
<td>Minimum Essential Medium (MEM) (1X), liquid Contains Earle's salts, but no L-glutamine or phenol red</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>51200-038</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Sodium pyruvate solution</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S8636</td>
<td>&nbsp;</td>
<tr>
<td>HEPES solution 1 M</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H0887</td>
<td>&nbsp;</td>
<tr>
<td>N-2 Supplement (100X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>17502-048</td>
<td>&nbsp;</td>
<tr>
<td>Horse Serum, Heat-Inactivated</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>26050-088</td>
<td>&nbsp;</td>
<tr>
<td>PAPAIN-022</td>
<td>Reagent</td>
<td>Worthington</td>
<td>LK003178</td>
<td>&nbsp;</td>
<tr>
<td>Neurobasal&#x2122; Medium (1X) Liquid without Phenol Red</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>12348-017</td>
<td>&nbsp;</td>
<tr>
<td>B-27 Serum-Free Supplement (50X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine-200 mM (100X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>25030-149</td>
<td>&nbsp;</td>
<tr>
<td>Cell Strainers</td>
<td>Tool</td>
<td>BD Biosciences</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>BD Falcon Multiwell Flat-Bottom Plates with Lids, Sterile</td>
<td>Tool</td>
<td>BD Biosciences</td>
<td>353046</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S7653</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P3911</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2 hexahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>21108</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2 hexahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M2670</td>
<td>&nbsp;</td>
<tr>
<td>HEPES free acid</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H3375</td>
<td>&nbsp;</td>
<tr>
<td>D-(+)-glucose</td>
<td>Reagent</td>
<td>Sigma</td>
<td>G7528</td>
<td>&nbsp;</td>
<tr>
<td>Fluo-4, AM 1 mM solution in DMSO</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>F-14217</td>
<td>&nbsp;</td>
<tr>
<td>Pluronic&#174; F-127 20% solution in DMSO</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>P-3000MP</td>
<td>&nbsp;</td>
<tr>
<td>Immersion Oil TYPE DF</td>
<td>Microscope</td>
<td>Cargille</td>
<td>16242</td>
<td>&nbsp;</td>
<tr>
<td>Open chamber for 25 mm round coverslips, 100 &mu;l volume</td>
<td>Tool</td>
<td>WARNER Instruments</td>
<td>64-0362 (RC-21BDW)</td>
<td>&nbsp;</td>
<tr>
<td>P-2 platform for Series 20 chambers, non-heater</td>
<td>Tool</td>
<td>WARNER Instruments</td>
<td>64-0278 (P-2)</td>
<td>&nbsp;</td>
<tr>
<td>FluoSpheres carboxylate-modified microspheres, 0.1 &mu;m, yellow-green fluorescent (505/515) 2% solids</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>F8803</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

measuring near plasma membrane and global intracellular calcium dynamics in astrocytes

El cerebro contiene células gliales. Los astrocitos, un tipo de célula glial, han sabido de largo para proporcionar una función de apoyo pasivo a las neuronas. Sin embargo, la evidencia creciente sugiere que los astrocitos pueden también participar activamente en la función cerebral a través de interacciones funcionales con las neuronas. Sin embargo, muchos aspectos fundamentales de la biología de los astrocitos siguen siendo controvertidas, poco claras y / o explorar experimentalmente. Una cuestión importante es la dinámica de los transitorios de calcio intracelular en los astrocitos. Esto es importante porque el calcio se ha consolidado como un importante segundo mensajero y como se ha propuesto que las elevaciones de los astrocitos de calcio puede desencadenar la liberación de los transmisores de los astrocitos. Sin embargo, no ha habido ninguna descripción detallada o la satisfacción de calcio cerca de la membrana plasmática de señalización en los astrocitos. Total de fluorescencia de reflexión interna (TIRF) microscopía es una herramienta poderosa para analizar los eventos de señalización fisiológicamente relevantes dentro de unos 100 nm de la membrana plasmática de las células vivas. En este sentido, el uso de microscopía TIRF y describir la manera de supervisar cerca de la membrana plasmática y la dinámica global de calcio intracelular casi al mismo tiempo. El perfeccionamiento y la aplicación sistemática de este enfoque tiene el potencial de dar a conocer los detalles precisos de la señalización del calcio astrocitos. Una comprensión detallada de la dinámica del calcio astrocitos puede proveer una base para entender si, cómo, cuándo y por qué someterse a los astrocitos y neuronas dependientes de calcio interacciones funcionales.

Watch this Scientific Journal Video about Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes at JoVE.com

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

Se describe la forma de medir cerca de la membrana y la dinámica global de calcio intracelular en los astrocitos cultivados usando la reflexión total interna y microscopía de epifluorescencia.

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-near-plasma-membrane-global-intracellular-calcium-dynamics

Research

JoVE Journal

Biology

13.0K vistas.  David Geffen School of Medicine, University of California, Los Angeles. Se describe la forma de medir cerca de la membrana y la dinámica global de calcio intracelular en los astrocitos cultivados usando la reflexión total interna y microscopía de epifluorescencia.

Video: Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

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 ...

Investigación

Biología

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

La medición de flujos rápidos de calcio en los cardiomiocitos

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)1. Single-molecule videomicroscopy, offering millisecond and nanometric resolution1-11, allows a detailed representation of membrane organization12-14 by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.
We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis15,16. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.
The nanometric accuracy17 obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods17 such as STORM18, PALM19,20, RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.

Cartografía de la difusión molecular en la membrana plasmática de objetivos múltiples de Búsquedas (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim 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, ...

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 ...

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

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

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 ...

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein using the profiles of fluorescence intensity across the plasma membrane. Measured fluorescence profiles are fitted by a model for membrane and cytoplasm fluorescence distribution along a line applied perpendicularly to the cell periphery. This model is constructed from the fluorescence intensity values in reference cells expressing a fluorescently-tagged marker for cytoplasm and with FM 4-64-labeled plasma membrane. The method can be applied to various cell types and organisms; however, only plasma membranes of non-neighboring cells can be evaluated. This fast microscopy-based method is suitable for experiments, where subtle and dynamic changes of plasma membrane-associated markers are expected and need to be quantified, e.g., in the analysis of mutant versions of proteins, inhibitor treatments, and signal transduction observations. The method is implemented in a multi-platform R package that is coupled with an ImageJ macro that serves as a user-friendly interface.

Here, we present a protocol to perform a quantitative analysis of the level of plasma-membrane association for fluorescently-tagged peripherally-associated protein. The method is based on the computational decomposition of membrane and cytoplasmic component of signal observed in cells labeled with plasma membrane fluorescent marker.

Determinación de la membrana plasmática particionado en proteínas asociadas periféricamente

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and activates the mixed lineage kinase-like domain pseudokinase, MLKL, to rupture or permeabilize the plasma membrane. Necroptosis is an inflammatory pathway associated with multiple pathologies including autoimmunity, infectious and cardiovascular diseases, stroke, neurodegeneration, and cancer. Here, we describe protocols that can be used to characterize MLKL as the executioner of plasma membrane rupture in necroptosis. We visualize the process of necroptosis in cells using live-cell imaging with conventional and confocal fluorescence&#160;microscopy, and in fixed cells using electron microscopy, which together revealed the redistribution of MLKL from the cytosol to the plasma membrane prior to induction of large holes in the plasma membrane. We present in vitro nuclear magnetic resonance (NMR) analysis using lipids to identify putative modulators of MLKL-mediated necroptosis. Based on this method, we identified quantitative lipid-binding preferences and phosphatidyl-inositol phosphates (PIPs) as critical binders of MLKL that are required for plasma membrane targeting and permeabilization in necroptosis.

We report methods for characterization of MLKL-mediated plasma membrane rupture in necroptosis including conventional and confocal live-cell microscopy imaging, scanning electron microscopy, and NMR-based lipid binding.

Caracterización de la ruptura de la membrana plasmática mediado MLKL en Necroptosis

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Medidas simultáneas de calcio intracelular y el potencial de membrana en el endotelio Cerebral de ratón recién aislado e intacto

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Imagen de la dinámica del calcio en subpoblaciones de células de islotes pancreáticos de ratón

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...