Investigación en el laboratorio Sokol es patrocinado por el institutos agregados Nacional de Salud.

1. Fertilización In Vitro de Xenopus huevos <ol><li> Obtener huevos de las ranas hembra que fueron inyectados con gonadotrofina coriónica humana (400 unidades / rana) 12 horas antes del experimento. </li><li> Coloque los huevos en una pequeña cantidad (0,5-1 ml) de 1 x Marc solución de Ringer modificadas (MMR) 10 a los huevos y fertilizar in vitro con un pequeño trozo de testículo disecado. Después de 2-3 minutos, añadir 0,1 x MMR para cubrir toda la superficie de los huevos. En 20 minutos, el huevo capa gelatinosa se elimina en un 3% cisteína - HCl (pH ajustado a 8 con hidróxido de sodio). Los huevos son lavados con 0,1 x MMR tres veces y se fue en una fría incubadora (13 ° C) para inyección. </li><li> Los huevos fertilizados se les permite desarrollarse a la etapa de células 2-4. Para las inyecciones, los embriones se transfieren a la solución que contiene 3% de Ficoll, 0,5 x MMR. </li></ol> 2. ARN microinyección <ol><li> ARN se sintetiza a partir de plantillas de ADN lineal con mMessage mMachine kit (Ambion) y se diluye con agua libre de RNasa en la concentración de acciones de 0,1-1 mg / l. Las dosis óptimas de ARN para la inyección son determinados en experimentos piloto. ARN para Diversin-RFP, el marcador de membrana GFP-CAAX Frizzled y 8 se utilizan en 0.1-1 ng por inyección. </li><li> Agujas de inyección se preparan con un extractor de la aguja de un capilar y luego con un molinillo de aguja. Antes de la inyección, cada aguja se calibra con agua para expulsar 10 nl de inyección de líquido por habitante. </li><li> Para las inyecciones, los embriones se colocan en un plato de plástico en una gran gota de 3% Ficoll, 0,5 x MMR. Un microlitro de solución de ARN se aspira en una aguja de inyección con Narishige microinyector. 10 nl de ARN se inyecta en animales de 8 blastómeros de embriones de células 2-3 veces. Los embriones inyectados se transfieren a un pozo de la placa de 12 pocillos. </li></ol> 3. Preparación de explantes ectodérmica <ol><li> Cuando los embriones inyectados llegar a las primeras etapas de gástrula, se transfieren a 0,6 x solución de MMR en un plato de 3 cm de plástico recubiertas con agarosa al 1%. Membrana vitelina se elimina manualmente con un par de pinzas. Explantes ectodérmica son extirpados de los embriones con una aguja de tungsteno y un hairloop. </li><li> Explantes ectodérmica se transfieren a un frasco de vidrio y se fija con 3,7% de formaldehído en tampón fosfato salino (PBS) durante 30 minutos. Explantes fija se lavan tres veces con PBS (10 minutos cada uno). DAPI se incluye en el tercer lavado para teñir los núcleos. </li><li> Los explantes se montan en un portaobjetos de vidrio. Dos tiras de cinta adhesiva se unen a la diapositiva y los explantes se colocan entre las dos bandas. Puesto que la superficie exterior de los explantes se pigmenta, la parte interna de los explantes debe mirar hacia el objetivo del microscopio. Añadir dos-tres 20 gotas l de la solución de montaje (70% de glicerol en PBS como 25 mg / ml DABCO, anti-fading reactivo) 11. Ponga un cubreobjetos en la parte superior. </li></ol> 4. Imágenes de explantes bajo un microscopio fluorescente <ol><li> Las muestras son examinadas bajo microscopio Zeiss Axioplan fluorescente con filtros adecuados. </li><li> Las imágenes son tomadas con el archivo adjunto apótoma de visualizar un plano específico de varios explantes independiente. </li></ol> 5. Muestras criostáticas Muestras criostáticas es una forma alternativa de visualizar la distribución de las proteínas fluorescentes en la célula y más aplicable para inmunotinción. En la 10 ª etapa, los embriones son fijos durante 1-2 horas con el fijador de Dent (20% DMSO, 80% de metanol), se lava con PBS, y embebidos en una solución de sacarosa al 15% de pescado gelatin/15 11%. Los embriones son incorporados rápidamente congelado en hielo seco y cryosections se generan en el criostato Leica. Las secciones transversales se incluyen las células ectodérmicas que heredan ARN inyectada y sus productos proteicos traducido. Las secciones de mantener la fluorescencia y se immunostained con anticuerpos específicos y con anticuerpos secundarios conjugados con fluorescencia. Los núcleos se tiñeron con DAPI. Los medios de montaje son las mismas que las descritas anteriormente. Imágenes se pueden realizar como se describe anteriormente. 6. Los resultados representativos: <img src="/files/ftp_upload/2700/2700fig1.jpg" alt="Figura 1" /> Figura 1. Receptor rizado reclutas Diversin a la membrana celular. Ectodermo células que expresan Frizzled 8 (Xfz8) y ARN Div-RFP revelan Div-RFP en la membrana celular, en lugar del centrosoma (según lo revelado por g-tubulina co-tinción). El esquema del experimento es en la parte superior, una sección transversal típica se muestra.

<ol>
	<li>Gurdon, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+induction+---+molecular+aspects.">Embryonic induction --- molecular aspects.</a> Development. 99, 285-306 (1987).</li><li>Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Developmental Genetics. , (2007).</li><li>Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/beta-catenin+signaling+in+development+and+disease.">Wnt/beta-catenin signaling in development and disease.</a> Cell. 127, 469-480 (2006).</li><li>Klaus, A., Birchmeier, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signalling+and+its+impact+on+development+and+cancer.">Wnt signalling and its impact on development and cancer.</a> Nat Rev Cancer. 8, 387-398 (2008).</li><li>Gordon, M. D., Nusse, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signaling:+multiple+pathways,+multiple+receptors,+and+multiple+transcription+factors.">Wnt signaling: multiple pathways, multiple receptors, and multiple transcription factors.</a> J Biol Chem. 281, 22429-22433 (2006).</li><li>Schwarz-Romond, T., Asbrand, C., Bakkers, J., Kuhl, M., Schaeffer, H. J., Huelsken, J., Behrens, J., Hammerschmidt, M., Birchmeier, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ankyrin+repeat+protein+Diversin+recruits+Casein+kinase+Iepsilon+to+the+beta-catenin+degradation+complex+and+acts+in+both+canonical+Wnt+and+Wnt/JNK+signaling.">The ankyrin repeat protein Diversin recruits Casein kinase Iepsilon to the beta-catenin degradation complex and acts in both canonical Wnt and Wnt/JNK signaling.</a> Genes Dev. 16, 2073-2084 (2002).</li><li>Moeller, H., Jenny, A., Schaeffer, H. J., Schwarz-Romond, T., Mlodzik, M., Hammerschmidt, M., Birchmeier, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversin+regulates+heart+formation+and+gastrulation+movements+in+development.">Diversin regulates heart formation and gastrulation movements in development.</a> Proc Natl Acad Sci U S A. 103, 15900-15905 (2006).</li><li>Itoh, K., Jenny, A., Mlodzik, M., Sokol, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosomal+localization+of+Diversin+and+its+relevance+to+Wnt+signaling.">Centrosomal localization of Diversin and its relevance to Wnt signaling.</a> J. Cell Sci. 122, 3791-3798 (2009).</li><li>Itoh, K., Jacob, J., Sokol, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+Xenopus+Frizzled+8+in+dorsal+development.">A role for Xenopus Frizzled 8 in dorsal development.</a> Mech Dev. 74, 145-157 (1998).</li><li>Newport, J., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+major+developmental+transition+in+early+Xenopus+embryos:+I.+characterization+and+timing+of+cellular+changes+at+the+midblastula+stage.">A major developmental transition in early Xenopus embryos: I. characterization and timing of cellular changes at the midblastula stage.</a> Cell. 30, 675-686 (1982).</li><li>Itoh, K., Brott, B. K., Bae, G. U., Ratcliffe, M. J., Sokol, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+localization+is+required+for+Dishevelled+function+in+Wnt/beta-catenin+signaling.">Nuclear localization is required for Dishevelled function in Wnt/beta-catenin signaling.</a> J Biol. 4, 3-3 (2005).</li></ol>

Hemos utilizado el protocolo anterior para caracterizar la localización subcelular de Diversin. En los explantes de polo animal, Diversin-RFP se detectó al lado del núcleo y colocalized con g-tubulina, un marcador centrosomal, en cryosections (Figura 1). Una vez que la localización subcelular de una proteína se identifica, supresión construcciones se pueden generar para establecer dominios de la proteína que son necesarias y suficientes para la localización subcelular. Con este enfoque, los dominios de la locali...

polarized translocation of fluorescent proteins in xenopus ectoderm in response to wnt signaling

Polaridad celular es una propiedad fundamental de las células eucariotas que es dinámica regulada por factores tanto intrínsecos como extrínsecos durante el desarrollo embrionario de 1, 2. Una de las vías de señalización implicadas en el presente Reglamento es la vía Wnt, que se utiliza muchas veces durante la embriogénesis y la crítica de la enfermedad humana 3, 4, 5. Múltiples componentes moleculares de esta vía de señalización de forma coordinada regular de manera espacialmente restringida, pero los mecanismos subyacentes no se comprenden totalmente. Xenopus células epiteliales embrionarias es un excelente sistema para estudiar la localización subcelular de las diferentes proteínas de señalización. Las proteínas fluorescentes de fusión se expresan en los embriones de Xenopus por microinyección de ARN, los explantes ectodérmica se preparan y localización de la proteína es evaluada por epifluorescencia. En este protocolo experimental se describe cómo la localización subcelular de Diversin, una proteína citoplasmática que se ha implicado en la señalización celular y la determinación de la polaridad de 6, 7 se visualiza en Xenopus células ectodérmicas para estudiar la transducción de señales Wnt 8. Co-expresión de un ligando Wnt o un receptor Frizzled altera la distribución de Diversin fusionada con la proteína fluorescente de color rojo, RFP, y recluta a la membrana celular de una manera polarizada 8, 9. Este protocolo ex vivo debe ser una adición útil a los estudios in vitro de células de mamífero cultivadas, en el que el control espacial de la señalización difiere de la del tejido intacto y es mucho más difícil de analizar.

Watch this Scientific Journal Video about Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling at JoVE.com

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Ectodermo embrionario de Xenopus se ha convertido en un atractivo modelo para el estudio de la polaridad celular. Un ensayo se describe, en el que la distribución subcelular de las proteínas fluorescentes se evalúa en las células de ectodermo. Este protocolo ayudará a contestar preguntas relacionadas con el control espacial de la señalización.

Translocación polarizado de proteínas fluorescentes en Xenopus Ectodermo en respuesta a la señalización de Wnt

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic ...

polarized-translocation-fluorescent-proteins-xenopus-ectoderm

Research

JoVE Journal

Biology

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

10.7K vistas.  Mount Sinai School of Medicine. Ectodermo embrionario de Xenopus se ha convertido en un atractivo modelo para el estudio de la polaridad celular. Un ensayo se describe, en el que la distribución subcelular de las proteínas fluorescentes se evalúa en las células de ectodermo. Este protocolo ayudará a contestar preguntas relacionadas con el control espacial de la señalización.

Video: Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

La cuantificación de γH2AX focos en respuesta a la radiación ionizante

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Investigación

Biología

Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of fluorescence can be changed by exposure to light of a specific wavelength. Optical highlighting allows noninvasive marking of a subpopulation of fluorescent molecules, and is therefore ideal for tracking single cells or organelles.
Critical parameters for efficient photoconversion are the intensity and the exposure time of the photoconversion light. If the intensity is too low, photoconversion will be slow or not occur at all. On the other hand, too much intensity or too long exposure can photobleach the protein and thereby reduce the efficiency of photoconversion.
This protocol describes a general approach how to set up a confocal laser scanning microscope for pc-FP photoconversion applications. First, we describe a procedure for preparing purified protein droplet samples. This sample format is very convenient for studying the photophysical behavior of fluorescent proteins under the microscope. Second, we will use the protein droplet sample to show how to configure the microscope for photoconversion. And finally, we will show how to perform optical highlighting in live cells, including dual-probe optical highlighting with mOrange2 and Dronpa.

This protocol describes a general approach to perform photoconversion of fluorescent proteins on a confocal laser scanning microscope. We describe procedures for the photoconversion of puried protein samples, as well as for dual-probe optical highlighting in live cells with mOrange2 and Dronpa.

Fotoconversión purificada de proteínas fluorescentes y de doble sonda óptica destacar en células vivas

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Patch clamp y técnicas de perfusión para el estudio de los canales iónicos expresados ​​en Xenopus Ovocitos

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

El análisis genético rápido de epitelio-mesenquimal de señalización durante la regeneración del cabello

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

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, ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

Los trasplantes faciales en<em&gt; Xenopus laevis</em&gt; Los embriones

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

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

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

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

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

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

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

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

Reconstitution Of &amp;#946;-catenin Degradation In Xenopus Egg Extract

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

La reconstitución de la degradación de β-catenina en<em&gt; Xenopus</em&gt; Extracto de Huevo

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

Quantification of Orofacial Phenotypes in Xenopus

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial development will allow for more rigorous analysis of orofacial phenotypes upon abrogation with substances that can genetically or molecularly manipulate gene expression or protein function. Using two dimensional images of the embryonic heads, traditional size dimensions-such as orofacial width, height and area- are measured. In addition, a roundness measure of the embryonic mouth opening is used to describe the shape of the mouth. Geometric morphometrics of these two dimensional images is also performed to provide a more sophisticated view of changes in the shape of the orofacial region. Landmarks are assigned to specific points in the orofacial region and coordinates are created. A principle component analysis is used to reduce landmark coordinates to principle components that then discriminate the treatment groups. These results are displayed as a scatter plot in which individuals with similar orofacial shapes cluster together. It is also useful to perform a discriminant function analysis, which statistically compares the positions of the landmarks between two treatment groups. This analysis is displayed on a transformation grid where changes in landmark position are viewed as vectors. A grid is superimposed on these vectors so that a warping pattern is displayed to show where significant landmark positions have changed. Shape changes in the discriminant function analysis are based on a statistical measure, and therefore can be evaluated by a p-value. This analysis is simple and accessible, requiring only a stereoscope and freeware software, and thus will be a valuable research and teaching resource.

Quantification of Orofacial Phenotypes in Xenopus

A method to quantify the orofacial size and shape of Xenopus laevis embryos has been developed. In this protocol, traditional size measurements are combined with geometric morphometrics to allow for more sophisticated analyses of orofacial development and defects.

La cuantificación de fenotipos en Orofacial<em&gt; Xenopus</em

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial ...

Measuring Glutathione-induced Feeding Response in Hydra

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

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

Medición inducida glutatión-Alimentación Respuesta en Hydra

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