Este trabajo fue financiado por el NIH subvención # DA15169 de HEM

Descripción general del procedimiento: Con este enfoque, proteínas de superficie celular son covalentemente marcado con biotina de disposición de residuos de lisina extracelular utilizando un impermeant membrana, disulfuro de acoplamiento reactivo biotinilación (sulfo-NHS-SS-biotina) en el tráfico de condiciones restrictivas (temperatura baja) (ver Fig. 1. por ejemplo). Un conjunto de células se desplaza a la trata de condiciones permisivas (37 ° C) e internalizar las proteínas con biotina. El otro conjunto de células se mantienen a baja temperatura, como los controles para: 1) la proteína de la superficie total en el momento = 0, y 2) el control de extracción. Después de un corto período de interiorización, las células se desplazan de nuevo a baja temperatura para detener la internalización, y cualquier biotina superficie residual se elimina mediante el tratamiento de células con un agente reductor, que rompe la biotina disulfuro de acoplamiento. Proteínas con biotina que surgió de la superficie celular y se internaliza están protegidos de la etapa de extracción, y será la única proteínas con biotina que quedan. Después de la lisis celular, proteínas con biotina se encuentran aisladas por estreptavidina cromatografía de afinidad y la proteína de interés se detectaron por inmunotransferencia cuantitativa. Para determinar la tasa de endocitosis, la cantidad de proteína es internalizada en comparación con el control total de la superficie de la etiqueta en el tiempo = 0. Hemos utilizado con éxito este método para medir la tasa de internalización de la norepinefrina y la dopamina neuronal 1 transportistas 1-4. Protocolo detallado: Día 1: <ol><li> Células de la placa en 6 y placas de tal manera que se confluente ~ 80% en el Día 2. Por otra parte, si las células transfectadas se están utilizando, transfectar a una densidad tal que van a ser confluentes ~ 80% en el momento se mide la tasa de internalización. Si las células no están fuertemente adheridas, artículos de cultivo de tejidos deben ser tratados con un sustrato de adhesión celular (por ejemplo, poli-D-lisina) para evitar la pérdida de células durante las etapas de lavado extenso. </li><li> Para cada proteína está probando, 2 pozos de la placa en una placa para ser utilizado como la proteína de la superficie total (t = 0) y los controles de extracción. En una segunda placa, la placa de un bien para cada condición de internalización se está probando (es decir, la tasa basal endocítica vs tratadas con el medicamento). </li><li> Prepare las siguientes soluciones y almacenar en las temperaturas indicadas para su uso en el Día 2: <ul><li> PBS 2 +: tampón fosfato salino (pH 7,4) suplementado con 1,5 mM de MgCl 2, 0,2 mM CaCl 2, (4 ° C) </li><li> Biotinilación Quench Solución: 2 + PBS suplementado con 100 mM glicina, (4 ° C) </li><li> NT búfer: 150 mM NaCl, 1,0 mM EDTA, 0,2% BSA, 20 mM Tris, pH 8.6, (4 ° C) </li><li> RIPA buffer: 10 mM Tris, pH 7,4, 150 mM NaCl, 1,0 mM EDTA, SDS 0,1%, 1,0% Triton X 100, 1,0% de desoxicolato de sodio, (4 ° C) </li><li> Sulfo-NHS-SS-biotina acciones: Se disuelven en dimetilsulfóxido (DMSO) a 200 mg / ml, (-20 ° C) </li><li> Tris (2-carboxietil) Clorhidrato de fosfina (TCEP) Solución: 500 mm de H 2 O, (-20 ° C, cubierto de papel de aluminio para bloquear la luz) </li></ul></li></ol> Día 2: <ol><li> Prepare 2 + PBS suplementado con glucosa 0.18g/ml, el 0,2% de IgG / proteasa libre de albúmina de suero bovino (PBS 2 + / g / BSA). Pre-caliente esta solución a 37 ° C en un baño de agua. </li><li> Descongelar la solución madre sulfo-NHS-SS-biotina en la parte superior del banco para fundir el DMSO. Inmediatamente antes de su uso, preparar nuevas sulfo-NHS-SS-biotina solución (2,5 mg / ml en PBS enfriado con hielo 2 +, suficiente para 0,75 ml / pocillo). Vortex vigorosamente la solución para solubilizar el DMSO. Tenga en cuenta que el reactivo NHS-biotina se hidroliza fácilmente en solución acuosa. Por lo tanto, todas las soluciones deben prepararse inmediatamente antes de su uso. </li><li> Biotinilación: Coloque las placas en un baño de hielo en la cámara frigorífica y enjuague 3 x 2 ml con PBS enfriado con hielo 2 +. Asegúrese de que las placas son ligeramente inclinada para permitir el drenaje completo y la eliminación de la solución tampón. Añadir 0,75 ml / pocillo de la nueva sulfo-NHS-SS-biotina a cada pocillo. Incubar x 15 &#39;, 4 ° C en el baño de hielo con agitación vigorosa. Después de la incubación, preparar otra nueva sulfo-NHS-SS-biotina solución. Reemplazar la antigua solución con la solución fresca e incubar x &#39;15, 4 ° C. </li><li> Enfriamiento: Es fundamental que todos los que no reaccionan NHS-biotina moléculas se apaga, por lo que no va a reaccionar con las proteínas intracelulares y biotinylate vez que las células se lisan. Lavar las células 3 x 2 ml con una solución de enfriamiento y se incuba dos veces en 2 ml de solución de enfriamiento x 15 &quot;, 4 ° C con agitación suave. </li><li> Internalización: Si los tratamientos de drogas están siendo probadas, añadir la concentración del fármaco apropiado para PBS 2 + / g / BSA. Mantenga el control de la placa a 4 ° C y llevar la placa de la internalización de la cámara frigorífica. Lavar 3 x 2 ml con pre-calentado PBS 2 + / g / BSA (+ / - drUGS) y dejar en las mismas soluciones (2 ml / pocillo). Rellene los pocillos vacíos con pre-calentado solución para asegurar una temperatura de toda la placa. Transferencia de las células a una incubadora a 37 ° C durante 10 &#39;. Inmediatamente antes de la final de 37 ° C de incubación, preparar nuevas soluciones 50 mM en tampón TCEP NT que se utilizará para el paso extracción y almacenamiento en hielo. Prepare la cantidad suficiente para 1,0 ml / pocillo. </li><li> Stripping: Inmediatamente placa de transferencia (s) de baño de hielo y volver a la cámara frigorífica. Lavarse rápidamente las células con helada NT buffer, 3 x 2 ml para detener la endocitosis. También lave las tiras de control de los pozos de 3 x 2 ml con tampón NT. Añadir 1,0 ml de solución fresca despojar a cada pocillo. Incubar en hielo, 15, 4 ° C con agitación suave. Vuelva a colocar los pozos con solución removedora fresco e incubar otros 15 &quot;, 4 ° C en hielo. </li><li> Lisis: Lave todos los pozos que fueron expuestos a la solución de extracción, 3 x 2 ml NT buffer. A continuación, lavar los pocillos (incluidos los controles en total) con 3 x 2 ml de PBS 2 +. Lisis en 300μl/well RIPA buffer (buffer de lisis o otros compatibles con la proteína de interés) que contienen inhibidores de la proteasa fresca (1,0 mM PMSF, 1,0 mg / ml leupeptina, aprotinina y pepstatina). Lyse agitando x 20 &quot;, 4 ° C. Transferencia a los tubos de microcentrífuga y los restos celulares por centrifugación claro 18.000 xg, 10, 4 ° C. </li><li> Determinación de concentración de proteínas: Utilice un ensayo de la proteína compatible con sus condiciones de lisis (por ejemplo, ensayo de proteínas DC, Bio-Rad) para determinar la concentración de proteínas de los lisados ​​en comparación con un estándar de la curva de BSA. </li><li> Estreptavidina cromatografía de afinidad: los tubitos de microcentrífuga con cantidades equivalentes de proteína para cada muestra. Añadir tampón de lisis para llevar el volumen final de cada muestra de 200μl. Vortex la estreptavidina perlas de agarosa con fuerza para llevar a una suspensión uniforme. Utilizando una pipeta 200μl con la punta cortada, cuentas de pipeta en cada tubo. Recomendado 20μl beads/50 lisado mg. Incubar toda la noche, 4 ° C en un rotor de tubo. </li></ol> Día 3: <ol><li> Lavado de esferas: las muestras de centrífuga, 18.000 xg, 2 &#39;para recoger perlas. Aspirar de lisado, teniendo cuidado de no acercarse a bolas de pellets con aspirador. Utilice una punta de pipeta de plástico en el extremo del aspirador para un mejor control. Añadir 0,75 ml de solución amortiguadora de lisis a cada tubo y agitar para lavar cuentas. Centrifugue las muestras, 18.000 xg, 2 &#39;para recoger los granos y repetir la aspiración y lavado dos veces (tres lavados en total). Después del último lavado, aspirado, como gran parte de la memoria intermedia como sea posible sin perturbar el precipitado de cuentas. De inflexión de la trompa hacia el aspirador ayuda con este paso. </li><li> Elución de la muestra: Agregar 20-25μl 2x tampón de muestra Laemmli (reducir) a cada tubo. El agente reductor se unirá el enlace disulfuro NHS-SS-biotina, la liberación de las proteínas aisladas en la solución. La mayoría de las proteínas de membrana son muy vulnerables a la agregación cuando se hierve, y no debe ser calentado antes de ser cargados en geles de SDS-PAGE. Determinar la sensibilidad al calor de la proteína de interés antes de realizar estos experimentos. Si la proteína no puede tolerar el calor de ebullición /, se incuban en un rotor, 30 &#39;, la temperatura ambiente antes de su análisis por SDS-PAGE. Esto es suficiente para romper los enlaces disulfuro y eluir las proteínas. </li><li> SDS-PAGE e inmunoblotting: proteínas separadas por SDS-PAGE. Para cada muestra, lo más fácil es ejecutar los ejemplos en el siguiente orden: la superficie total (tiempo = 0), la tira, la condición internalización # 1, Condición # 2, etc transferencia a la membrana de inmunotransferencia y borrar con el anticuerpo adecuado para su proteína de interés. La captura de bandas inmunorreactivas mediante un sistema de gel CCD de la cámara de documentación, con la certeza de que no hay píxeles saturados. Cuantificar la densidad de cada banda utilizando gel de análisis / densitometría software. </li></ol> Los resultados representativos: Un resultado inmunoblot representativo se muestra en la Figura 2A. La señal más fuerte es en el &quot;total&quot; de carril (T), que es la cantidad total de proteína de la superficie antes de la internalización. La &quot;tira&quot; de control (S), debe cerca de blanco, lo que demuestra que la banda era eficiente para el experimento. La eficiencia de banda se calcula mediante la comparación de la densidad de la &quot;tira&quot; de carril a la de la &quot;total&quot; de carril (por ejemplo, proteínas que se biotinilado en paralelo con la banda, pero no se calentó a 37 ° C, ni expuestos a la solución de extracción). La siguiente fórmula se utiliza: [1 - (tira / total)] * 100% Utilizando esta fórmula, la tira en la Figura 2 fue del 99,8% de eficiencia. Finalmente, podrás ver las bandas de menor intensidad que el total en el carril de la internalización (s) (I). En el ejemplo (Figura 2), las células tratadas fueron tratados con vehículo o con 1μM acetato de forbol miristato (PMA) en un 10 &#39;tasas de internalización de la internalización y el transportador de dopamina se midieron en un 10&#39; período de internalización inicial. La tasa se calcula como la internalizaciónsiguiente manera: internalizado / total * 100 Como se observa en la Figura 2B, el 10,4% de la superficie DAT interiorizado más de 10 &#39;en los vehículos tratados con condiciones. PMA tratamiento aumentó las tasas de DAT internalización al 23,2% de la superficie total de DAT. <img src="/files/ftp_upload/1669/1669fig1.jpg" alt="Figura 1" /> Figura 1. Las células ilustración protocolo. Biotina son a 4 ° C para marcar exclusivamente la población de la superficie, y se desplazan a 37 ° C para iniciar la internalización. Después de la internalización, las células se enfría rápidamente para detener los procesos de endocitosis y la biotina superficie residual es eliminado por el tratamiento de células con un agente reductor. Las proteínas sólo con biotina que permanecen son aquellos que surgieron desde la superficie en t = 0 y se internaliza, lo cual les protege del tratamiento de extracción. Proteínas con biotina se encuentran aisladas por cromatografía de afinidad por lotes con cuentas estreptavidina y la proteína de interés se detectaron por inmunotransferencia. <img src="/files/ftp_upload/1669/1669fig2.jpg" alt="Figura 2" /> Figura 2. Activación de la PKC aumenta la tasa de DAT endocítica. Internalización de ensayo. PC12 células que expresan establemente DAT se biotina, 4 ° C como se describe en &quot;protocolo detallado&quot;. Las células se calienta rápidamente a 37 ° C ± 1 M y se incubaron PMA &quot;10, 37 ° C. Biotina residual fue despojado por la reducción, las células fueron lisadas y las proteínas con biotina se aislaron por cromatografía de afinidad estreptavidina. (A) inmunoblot Representante mostrando DAT superficie total en t = 0 (T), tira de control (S), e internalizado DAT (I) en las condiciones indicadas. (B) Las bandas fueron capturadas con una cámara CCD y se cuantifica con la cantidad de datos de software (Bio-Rad). Los datos se expresan como% total min DAT internalized/10. <img src="/files/ftp_upload/1669/1669fig3.jpg" alt="Figura 3" /> Figura 3. Inmunoblot ejemplo que representa una mala banda de biotina. Internalización de ensayo. PC12 células que expresan establemente DAT se biotina, 4 ° C como se describe en &quot;protocolo detallado&quot;. Las células se calienta rápidamente a 37 ° C y se incubaron &#39;10, 37 ° C. Biotina residual fue despojado por la reducción, las células fueron lisadas y las proteínas con biotina se aislaron por cromatografía de afinidad estreptavidina. La inmunotransferencia muestra DAT superficie total en t = 0 (T), tira de control (S), e internalizado DAT (I). Nota de la banda visible en el carril de la tira de control, lo que indica la eficiencia de la tira pobres.

<ol>
	<li>Holton, K. L., Loder, M. K., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonclassical+distinct+endocytic+signals+dictate+constitutive+and+PKC-regulated+neurotransmitter+transporter+internalization.">Nonclassical distinct endocytic signals dictate constitutive and PKC-regulated neurotransmitter transporter internalization.</a> Nat Neurosci. 8, 881-888 (2005).</li><li>Boudanova, E., Navaroli, D. M., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine-induced+decreases+in+dopamine+transporter+surface+expression+are+protein+kinase+C-independent.">Amphetamine-induced decreases in dopamine transporter surface expression are protein kinase C-independent.</a> Neuropharmacology. 54, 605-612 (2008).</li><li>Boudanova, E., Navaroli, D. M., Stevens, Z., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+transporter+endocytic+determinants:+carboxy+terminal+residues+critical+for+basal+and+PKC-stimulated+internalization.">Dopamine transporter endocytic determinants: carboxy terminal residues critical for basal and PKC-stimulated internalization.</a> Mol Cell Neurosci. 39, 211-217 (2008).</li><li>Loder, M. K., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dopamine+transporter+constitutively+internalizes+and+recycles+in+a+protein+kinase+C-regulated+manner+in+stably+transfected+PC12+cell+lines.">The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines.</a> J Biol Chem. 278, 22168-22174 (2003).</li></ol>

Los problemas más comunes: El problema más común que surge en estos experimentos es la eficiencia tira pobres. La eficiencia de la tira es fundamental para poder interpretar los resultados. A menos que la tira fue muy eficiente, no es posible concluir que todas las proteínas con biotina en los carriles de interiorización, de hecho, interiorizado de la superficie. Tiras de la eficiencia ≥ 90% son óptimas, y descartamos cualquier resultado si la tira cae por debajo de este nivel. Un ejemplo de una banda de ...

measuring plasma membrane protein endocytic rates by reversible biotinylation

Las proteínas de la membrana plasmática son un grupo grande y diverso de proteínas formado por los receptores, canales iónicos, transportadores y bombas. Actividad de estas proteínas es responsable de una variedad de importantes eventos celulares, incluyendo la entrega de nutrientes, la excitabilidad celular, y las señales químicas. Muchas proteínas de la membrana plasmática se regula de forma dinámica por el tráfico endocítica, que modula la función de proteínas mediante la alteración de la expresión de proteínas de la superficie. Los mecanismos que facilitan la endocitosis de proteínas son complejos y no son totalmente entendido por muchas proteínas de la membrana. Con el fin de entender completamente los mecanismos que controlan el tráfico endocítica de una proteína, es fundamental que la tasa de endocitosis de la proteína s medirse con precisión. Para muchos receptores, mediciones directas de la tasa de endocitosis son con frecuencia logra utilizando la etiqueta ligandos del receptor. Sin embargo, para muchas clases de proteínas de la membrana, como los transportistas, las bombas y los canales de iones, no hay ligando conveniente que puede ser usado para medir la tasa de endocitosis. En el presente informe, se describe un método reversible biotinilación que empleamos para medir el transportador de dopamina (DAT) Tasa de endocítica. Este método proporciona un método directo para medir las tasas de internalización, y puede ser fácilmente empleados para los estudios de tráfico de proteínas de membrana de la mayoría.

Watch this Scientific Journal Video about Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation at JoVE.com

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Endocitosis regulado regula los niveles de expresión de la superficie celular de la mayoría de las proteínas de la membrana. Aquí se utilizan los reactivos reducible, la membrana impermeant biotinilación para medir la tasa de endocitosis del transportador de dopamina (DAT), una proteína de membrana politópica. El método facilita un método directo para medir la tasa de endocitosis de la mayoría de las proteínas de la membrana plasmática.

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 ...

measuring-plasma-membrane-protein-endocytic-rates-reversible

Research

JoVE Journal

Biology

16.9K vistas.  University of Massachusetts Medical School. Endocitosis regulado regula los niveles de expresión de la superficie celular de la mayoría de las proteínas de la membrana. Aquí se utilizan los reactivos reducible, la membrana impermeant biotinilación para medir la tasa de endocitosis del transportador de dopamina (DAT), una proteína de membrana politópica. El método facilita un método directo para medir la tasa de endocitosis de la mayoría de las proteínas de la membrana plasmática.

Video: Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

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

Measuring the Induced Membrane Voltage with 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 membrane, resulting in a voltage across the membrane. This voltage, termed the induced membrane voltage (also induced transmembrane voltage, or induced transmembrane potential difference) and denoted by &Delta;&Phi;, exists only as long as the external field is present. If the resting voltage is present on the membrane, the induced voltage superimposes (adds) onto it. By using one of the potentiometric fluorescent dyes, such as di-8-ANEPPS, it is possible to observe the variations of &Delta;&Phi; on the cell membrane and to measure its value noninvasively. di-8-ANEPPS becomes strongly fluorescent when bound to the lipid bilayer of the cell membrane, with the change of the fluorescence intensity proportional to the change of &Delta;&Phi;. This video shows the protocol for measuring &Delta;&Phi; using di-8-ANEPPS and also demonstrates the influence of cell shape on the amplitude and spatial distribution of &Delta;&Phi;.

External electric field induces a voltage on the membrane of a cell, termed the induced membrane voltage (&Delta;&Phi;). By using the potentiometric dye di-8-ANEPPS, it is possible to measure the &Delta;&Phi; noninvasively. This video shows the protocol for measuring &Delta;&Phi; using 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 ...

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 ...

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 ...

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 ...

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor's vitamin A uptake mechanism.

Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.

En tiempo real de análisis de transporte de retinol en el receptor de la membrana de plasma Proteína Fijadora del Retinol

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

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 ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Fluorescente verde a base de proteína Expresión Proyección de proteínas de membrana en<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable efforts have been made in this field, but techniques enabling automated high-throughput transport analysis in combination with solvent-free lipid bilayer techniques required for the analysis of membrane transporters are rare. This class of transporters however is crucial in cell homeostasis and therefore a key target in drug development and methodologies to gain new insights desperately needed.
The here presented manuscript describes the establishment and handling of a novel biochip for the analysis of membrane protein mediated transport processes at single transporter resolution. The biochip is composed of microcavities enclosed by nanopores that is highly parallel in its design and can be produced in industrial grade and quantity. Protein-harboring liposomes can directly be applied to the chip surface forming self-assembled pore-spanning lipid bilayers using SSM-techniques (solid supported lipid membranes). Pore-spanning parts of the membrane are freestanding, providing the interface for substrate translocation into or out of the cavity space, which can be followed by multi-spectral fluorescent readout in real-time. The establishment of standard operating procedures (SOPs) allows the straightforward establishment of protein-harboring lipid bilayers on the chip surface of virtually every membrane protein that can be reconstituted functionally. The sole prerequisite is the establishment of a fluorescent read-out system for non-electrogenic transport substrates.
High-content screening applications are accomplishable by the use of automated inverted fluorescent microscopes recording multiple chips in parallel. Large data sets can be analyzed using the freely available custom-designed analysis software. Three-color multi spectral fluorescent read-out furthermore allows for unbiased data discrimination into different event classes, eliminating false positive results.
The chip technology is currently based on SiO2 surfaces, but further functionalization using gold-coated chip surfaces is also possible.

The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

Membrana de transporte de procesos analizados por un sistema de nanoporos viruta altamente paralela a la Resolución de la proteína individual

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable ...

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 ...