D. El Sames gracias Harold G. &amp; Y. Mathers Leila Charitable Foundation y la Universidad de Columbia iniciativas en ciencia e ingeniería. D. Sames y Sulzer D. agradecer a la Fundación McKnight de las innovaciones tecnológicas en McKnight Premio de Neurociencia D. Sulzer gracias NIDA, NIMH, y el Picower y Fundaciones Enfermedad de Parkinson. H. Zhang NARSAD gracias. HR Edwards gracias al Michael J. Fox Foundation, la Fundación Nacional de Parkinson, el NIDA y el NIMH. Damos las gracias a Robert Burke por 6-OHDA inyecciones y asesoramiento, Sonders Marcar útil para el debate, Merek Siu para la programación de análisis de imágenes, y Schmoranzer Jan de apoyo técnico con la configuración de la microscopía TIRF.

Este método se utilizó en la investigación publicada en <a href="http://www.sciencemag.org/cgi/content/full/324/5933/1441">Gubernator et al., Science 324 (5933). 1441-1444 (2009)</a> . 1. Preparación aguda rebanadas estriatales <ol><li> Antes de preparar las rebanadas estriatal aguda, se debe oxigenar el líquido cefalorraquídeo artificial (ASCF) y enfriar con hielo durante al menos 15 minutos antes de la extracción del cerebro. Mantener el frío de hielo y ACSF oxigenada durante la disección. ACSF (en mM): NaCl 125, KCl 2,5, NaHCO3 26, CaCl2 2,4, MgSO4 1,3, KH 2 PO 4 0,3, glucosa 10, HEPES 5, pH 7,3-7,4, 290-295 mOsm. </li><li> Decapitar ratón macho sin anestesia. </li><li> Extraer todo el cerebro del ratón, y montarlo en la bandeja de vibratome colocado en el vibratome utilizando pegamento Krazy ®. Extracción del cerebro y el montaje se debe hacer en 2 minutos. Durante este tiempo, asegúrese de mantener el frío de hielo ACSF y oxigenado para mantener el tejido sano. </li><li> Corte coronal rodajas de cerebro del cuerpo estriado en espesor de 250 micras entre bregma desde 1.54 hasta 0.62 1. Tres rebanadas se obtiene que puede ser cortado en 6 rodajas de media con una aguja. </li><li> Se incuban las rodajas de cerebro en ACSF oxigenada a temperatura ambiente durante al menos 1 hora antes de la carga de la sonda. Rebanadas de buena exhibición FFN511 de carga y permanecer activos para obtener imágenes de hasta 4 horas después de la preparación rebanada. </li></ol> 2. Cargando FFN511 <ol><li> Prepare FFN511 solución de carga (10μM FFN511 en ACSF); también preparan 100μM ADVASEP-7 en ACSF. Todas las soluciones deben ser recién preparadas, oxigenada por lo menos 15 minutos antes de cargarlos en los cortes. </li><li> Individualmente se incuban con una solución de corte de carga FFN511 durante 30 minutos a temperatura ambiente. </li><li> A continuación, retire el tinte unido al tejido extracelular mediante la incubación de la corte en carga oxigenada 100μM ADVASEP-7 ACSF durante 30 minutos a temperatura ambiente 2. Rodajas ya están listos para la imagen. </li></ol> 3. Imágenes FFN511 en las rebanadas de cerebro Imágenes de FFN511 en los cortes se realiza mediante microscopio de barrido láser multifotónica. Estamos utilizando una pradera Ultima multifotónica microscopio de barrido láser (Estábamos utilizando un Zeiss LSM 510 NLO multifotónica microscopio de barrido láser anteriormente y algunos resultados se obtuvieron con el microscopio Zeiss). <ol><li> Coloque la rebanada FFN511 cargada en la cámara de grabación (RC-27L, Warner) y superfuse con ACSF oxigenada con un caudal de 2.1 ml por minuto. A continuación, coloque un arpa de platino con cuerdas de nylon en la parte superior de la rebanada para minimizar el movimiento durante el experimento. Para minimizar aún más el movimiento, permiten el corte se estabilice por lo menos 10 minutos en la cámara antes de la imagen. </li><li> Visualizar y localizar el cuerpo estriado dorsal, con la luminiscencia de campo claro en un objetivo de inmersión en agua 10 veces. </li><li> Lugar y la posición de los electrodos de tungsteno bipolar trenzado en esta región si se realiza un experimento de estimulación. El área de imagen ideal se encuentra a menos de 300 m entre las dos puntas del electrodo bipolar estimulante. Situar a la región en el centro del campo visual. </li><li> Imagen FFN511 marcado con terminales estriatales en un 63x (0,9 NA) objetivo de inmersión de agua ultravioleta. FFN511 se excita a 760 nm con un láser Mai Tai de la Física y de fluorescencia espectral óptimo de los terminales del cuerpo estriado se ve a través de un filtro pasa banda (480-520 nm). Las imágenes se capturan en formato de 12-bit con 75 x 75 m de las regiones de interés de 512 píxeles de resolución x 512. Para estimular los experimentos de decoloración, para compensar el eje Z de turno, un z-series de imágenes 5.7, separados por un m en el plano z, se obtiene para cada período de tiempo. </li></ol> 4. FFN511 decolorante La etiqueta puede ser FFN511 desteñido, ya sea por alta concentración de KCl (utilizamos 70 mM KCl), (+)-anfetamina sulfato (AMPH, 20 M), o la estimulación eléctrica. En el experimento de decoloración KCl, cuando una solución de potasio ACSF alta se aplica a la porción del cerebro, el FFN511 destains etiqueta en 2 minutos. Registro xyz-t imágenes para realizar un seguimiento FFN511 decoloración con el tiempo. A continuación se describe la estimulación eléctrica dependiente de decoloración de terminales de dopamina: <ol><li> Para realizar el seguimiento FFN511 decoloración con el tiempo, xyz-t se graban las imágenes. A fin de garantizar un mínimo movimiento de la porción (menos de 4 m en el eje Z) y no photobleaching por el láser, imágenes de la serie de control de tiempo de la z-stack se debe obtener por lo menos 5 minutos antes de la estimulación. En caso contrario, ajustar la potencia del láser. </li><li> A raíz de estas imágenes de control, continuar con la imagen xyz-t, mientras que iniciar la estimulación a frecuencias de 1, 4 o 20 Hz. Estímulos a los 1, 4 o 20 Hz (300 ms x 1 mA) se aplican en el cuerpo estriado a nivel local por un aislador de estímulo Iso-Flex provocado por un maestro-8 generador de impulsos (AMPI, Jerusalén, Israel), utilizando electrodos bipolares. Para reducir al mínimola variación en la despolarización de los sitios de liberación, hemos utilizado un protocolo de estimulación de aplicar el 150% de la intensidad de la estimulación máxima determinada por voltametría cíclica. </li><li> Utilice el software adecuado para el análisis de imágenes. En nuestro laboratorio, el uso de la imagen J (Wayne Rosband, Institutos Nacionales de Salud, Rockville, MD) y software a medida, escrito en IDL (Research Systems, Boulder, CO) para cuantificar la fluorescencia de puntos lagrimales y los cambios con tiempo3. </li></ol> Resultados represenative La figura 1 muestra FFN511 carga en parte del cuerpo estriado se ha completado. Imágenes representativas con el objetivo de 10x y 60x se muestran en la Figura 1. El etiquetado selectiva de los terminales de dopamina puede ser desteñido por las anfetaminas M 20. La figura 2 muestra decoloración de FFN511 etiquetado por KCl alta. La Figura 3 muestra decoloración de FFN511 etiquetado mediante la estimulación eléctrica local. <img src="/files/ftp_upload/1562/1562fig1.jpg" alt="Figura 1" /> Figura 1 FFN511 etiquetas de terminales de dopamina en vivo corticotalamoestriatales rodajas aguda (A) Etiquetado de FFN511 aguda en vivo corticotalamoestriatales corte:.. Etiquetado abundante en el cuerpo estriado (STR), etiquetado más escaso en la corteza (CTX), y no en la etiqueta cuerpo calloso (CC). Barra de escala:. 100 micras (B) Decoloración de FFN511 del cuerpo estriado por las anfetaminas. Paneles de la izquierda: antes de las anfetaminas, los paneles de la derecha: después de 20 minutos de la anfetamina M 20. Barra de escala: 10 micras. <img src="/files/ftp_upload/1562/1562fig2.jpg" alt="Figura 2" /> Figura 2. Decoloración de FFN511 etiquetado por KCl alta. Etiquetado FFN511 es destained dentro de la aplicación de 2 minutos de KCl 70 en ACSF. Barra de escala: 10μm. <img src="/files/ftp_upload/1562/1562fig3.jpg" alt="Figura 3" /> Figura 3. Dependiente de la frecuencia de decoloración de FFN511 etiquetado en el cuerpo estriado. (A) la estimulación local de 4 Hz como resultado de decoloración de los terminales. La estimulación se inició en t = 0. Barra de escala: 5 micras (B) Decoloración de FFN511 a 4 Hz es Ca 2 + - y dependientes de la frecuencia.. Controles no recibieron la estimulación (153 puntos lagrimales de 3 rebanadas). Decoloración con cloruro de cadmio (200 M) era idéntico a los controles sin estimular (475 puntos lagrimales de 5 rebanadas). Las curvas de decoloración para cada frecuencia de estimulación se forma por una función de decaimiento exponencial de una sola y la vida media (t 1 / 2) los valores calculados como τ x 0.693 (1 Hz: 765 puntos lagrimales de 9 rebanadas, 4 Hz: 410 puntos lagrimales de 7 segmentos, 20 Hz: 416 puntos lagrimales de 6 porciones). Por favor refiérase a la micrografía de vídeo de 2-fotones microscopía de FFN511 decoloración durante la exocitosis en la preparación del cerebro rebanada del cuerpo estriado.

<ol>
	<li>Franklin, K. B. J., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The mouse brain in stereotaxi coordinators. , (1997).</li><li>Kay, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+synaptic+activity+in+intact+brain+and+slices+with+FM1-43+in+C.+elegans,+lamprey,+and+rat.">Imaging synaptic activity in intact brain and slices with FM1-43 in C. elegans, lamprey, and rat.</a> Neuron. 24, 8098-8117 (1999).</li><li>Bamford, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterosynaptic+dopamine+neurotransmission+selects+sets+of+corticostriatal+terminals.">Heterosynaptic dopamine neurotransmission selects sets of corticostriatal terminals.</a> Neuron. 42, 653-653 (2004).</li></ol>

The authors have nothing to disclose.

En este vídeo, se demuestra un método para visualizar la neurotransmisión ópticamente con análogos fluorescentes de la dopamina. FFN511 es la primera generación de FFNs que hemos desarrollado. A pesar de que fue diseñado por objetivo el transportador vesicular de monoaminas neuronales (VMAT2) que lleva la monoamino neurotransmisores desde el citoplasma en vesículas sinápticas y, específicamente, las etiquetas de terminales de dopamina en el estriado y catecolaminas presunta y / o terminales de la serotonina en la corteza (como se muestra en la ciencia papel), un período de carga adecuada es fundamental para la especificidad desde FFN511 es relativamente hidrofóbica. Se encontró que la incubación de más de 40 minutos se traducirá en manchas extensas inespecíficos en rebanadas estriatales. La especificidad puede ser directamente determinado por decoloración utilizando una alta concentración de KCl, que hace que la liberación de FFN dentro de las vesículas sinápticas funcionales. La exposición de la división para FFN511 de menos de 15 minutos se traducirá en una señal débil fluorescencia debido a la carga insuficiente de la tintura en las terminales. En este protocolo, el uso de 100μM ADVASEP-7 para quitar el tinte obligado a tejido extracelular. Este paso no es necesario, pero si se omite, el tiempo de lavado en ACSF debe ser prolongado. En resumen, dependiendo de la preparación que se elija, tendrá que modificar el período de concentración y de carga para determinar el etiquetado óptima.

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>FFN511 (8-(2-Amino-ethyl)-2,3,5,6-tetrahydro-1H,4H-11-oxa-3a-aza-benzo[de]anthracen-10-one)</td>
<td>&nbsp;</td>
<td>Dalibor Sames&#x2019;s laboratory at Columbia University</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>ADVASEP-7 </td>
<td>&nbsp;</td>
<td>CyDex, Overland Park, KS</td>
<td>AR-OA7-005</td>
<td>&nbsp;</td>
<tr>
<td>RC-27L Recording chamber</td>
<td>&nbsp;</td>
<td>Warner Instrument</td>
<td>64-0375</td>
<td>&nbsp;</td>
<tr>
<td>PELCO PrepEze 6-Well Holder</td>
<td>&nbsp;</td>
<td>Ted Pella, Inc.</td>
<td>36157-1</td>
<td>For slice incubation</td>
</tr>
<tr>
<td>Master-8 pulse stimulator and Iso- Flex stimulus isolator</td>
<td>&nbsp;</td>
<td>AMPI, Jerusalem, Israel</td>
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dopamine release at individual presynaptic terminals visualized with ffns

El sistema nervioso transmite señales entre las neuronas a través de la liberación de neurotransmisores durante la fusión de vesículas sinápticas. Para observar la captación de neurotransmisores y liberación de terminales presinápticas individuales directamente, hemos diseñado fluorescentes neurotransmisores falsos como sustrato para el transportador de monoamina vesicular sináptica. El uso de estas sondas para liberar la imagen de la dopamina en el estriado, que hizo varias observaciones pertinentes a la plasticidad sináptica. Hemos encontrado que la fracción de la liberación de las vesículas sinápticas del neurotransmisor por estímulo depende de la frecuencia del estímulo. A cinéticamente distintas &quot;reserva&quot; de la población de vesículas sinápticas no se observó en estas condiciones experimentales. Una heterogeneidad depende de la frecuencia de las terminales presinápticas se reveló que se depende en parte de los receptores D2 de la dopamina, lo que indica un mecanismo para dependientes de la frecuencia de codificación de la selección pre-sináptica. Hui Zhang y Gubernator Niko G. contribuyeron igualmente a este trabajo.

Watch this Scientific Journal Video about Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs at JoVE.com

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

Una nueva forma de medir la neurotransmisión ópticamente con análogos fluorescentes de la dopamina.

La liberación de dopamina en los terminales presinápticos individuales visualizados con FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and ...

dopamine-release-at-individual-presynaptic-terminals-visualized-with

Research

JoVE Journal

Biology

24.7K vistas.  Columbia University. Una nueva forma de medir la neurotransmisión ópticamente con análogos fluorescentes de la dopamina.

Video: Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

Cargando Drosophila terminales nerviosas con indicadores de calcio

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

Investigación

Biología

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

Etiquetado y hESCs hMSCs con nanopartículas de óxido de hierro para no invasivo en el seguimiento in vivo mediante RM

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Etiquetado de las células madre con tintes fluorescentes para la detección no invasiva con imágenes ópticas

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Primaria disociada Mesencéfalo culturas dopamina celulares de roedores recién nacidos

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.
Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice2,3, where it has been recently characterized4.
In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 &mu;L of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents5. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.

Murine Model for Parkinson&#039;s Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

Modelo murino de enfermedad de Parkinson: de la lesión 6-OH dopamina a prueba de comportamiento

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

Electrofusión de células visualiza con microscopía de fluorescencia

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

iClip - transcriptoma en todo el mapeo de la proteína-ARN interacciones con la Resolución de nucleótidos individuales

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

Preparación Individual Drosophila Huevos Cámaras de Imágenes en directo

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Interacciones proteína-proteína se visualizó por fluorescencia Bimolecular Complementación en protoplastos de tabaco y hojas

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

Imágenes de fluorescencia con un nanómetros Precisión (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...