Name: a cell-free assay using xenopus laevis embryo extracts to study mechanisms of nuclear size regulation
Uploaded: 2016-08-08T14:00:00.000+00:00
Duration: 14 min 27 s
Description: Watch this Scientific Journal Video about A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation at JoVE.com

Members of the Levy and Gatlin labs as well as colleagues in the Department of Molecular Biology offered helpful advice and discussions. Rebecca Heald provided support in the early stages of developing this protocol. This work was supported by the NIH/NIGMS (R15GM106318) and the American Cancer Society (RSG-15-035-01-DDC).

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The authors have nothing to disclose.

Here is presented a novel method to study mechanisms of nuclear size regulation during X. laevis development. Developmental progression is associated with dramatic changes in cell physiology, metabolism, division rates, and migration, as well as alterations in the sizes of cells and intracellular structures. These varied processes are complex and essential, so it is difficult to study just one of these aspects of development in an in vivo setting. The X. laevis embryo extract and nuclear shrink...

Los tamaños de los orgánulos celulares típicamente escala con el tamaño de la célula, y esto ha sido quizás mejor documentado para el escalado de tamaño nuclear con tamaño de celda 1-10. Esto es particularmente cierto durante la embriogénesis y la diferenciación celular, cuando reducciones dramáticas en tanto celular y tamaño nuclear se observan a menudo 11,12. Además, alterado tamaño nuclear es un parámetro clave en el diagnóstico de cáncer y el pronóstico 13-17. Mecanismos que contribuyen a la regulación del tamaño nuclear son en gran parte desconocidos, en parte debido a la complejidad y la naturaleza esencial de la estructura y la función nuclear. El método aquí descrito se desarrolló como un ensayo in vitro para la escala del tamaño nuclear que es susceptible a la manipulación bioquímica y la elucidación de los mecanismos de regulación del tamaño nuclear. Xenopus laevis extracto de huevo es un sistema bien establecido para recapitular y estudiar procesos celulares complejos en un in vitro </em&gt; Contexto. Estos extractos han revelado nueva información fundamental acerca de varios procesos celulares, incluyendo el montaje y la función del huso mitótico, retículo endoplasmático, y el núcleo 18-22. Una ventaja clave del sistema es que el extracto de X. extractos de huevo laevis representan un citoplasma casi sin diluir cuya composición puede ser alterado fácilmente, por ejemplo mediante la adición de proteínas o immunodepletion recombinantes. Además, uno es capaz de manipular los procesos esenciales mediante el empleo de tratamientos que de otro modo podrían ser letales en un contexto in vivo. Las modificaciones del procedimiento de extracto de huevo permiten para el aislamiento de extractos de X. laevis embriones en lugar de huevos, y estos extractos de embriones son igualmente susceptibles de manipulación bioquímica 23. Durante X. laevis desarrollo, el embrión fertilizado de una sola célula (~ 1 mm de diámetro) se somete a una serie de doce divisiones celulares rápidas (etapas 1-8) para generar varios miles de 50 μm de diámetro y células más pequeñas, llegando a una etapa de desarrollo denominan la transición midblastula (MBT) o 24-26 de la etapa 8. El MBT se caracteriza por el inicio de la transcripción cigóticos, la migración celular, divisiones celulares asíncronos, la adquisición de fases Gap, y el establecimiento de tamaños de estado estable nucleares en lugar de la expansión nuclear continua como en el embrión de pre-MBT. Desde la etapa 4 a la gastrulación (etapas 10,5-12), el volumen de los núcleos individuales se reduce en más de 10 veces 11. Aquí, el objetivo es identificar los mecanismos responsables de estas reducciones en el tamaño nuclear durante la progresión del desarrollo. El enfoque es montar primero núcleos en X. laevis huevo y el extracto de aislar esos núcleos desde el citoplasma del huevo / extracto. Estos núcleos se resuspendieron a continuación en el citoplasma de embriones en estadio de gástrula tardía. Después de un período de incubación, los núcleos de extracto de huevo se hacen más pequeños a finales de extracto de la etapa embrionaria. Pensamos que este would ser un ensayo útil para la identificación de los componentes citoplasmáticos presentes en los embriones en etapas avanzadas que contribuyen al desarrollo de escala del tamaño nuclear. Utilizando este ensayo, junto con la validación in vivo, hemos demostrado que la proteína quinasa C (PKC) contribuye a la reducción del desarrollo en el tamaño nuclear en X. laevis 23.

<table><tbody><tr><td>Alexa Fluor 568 Donkey anti-mouse IgG</td><td>Molecular Probes</td><td>A10037</td><td></td></tr><tr><td>ATP disodium salt</td><td>Sigma Aldrich</td><td>A2383</td><td></td></tr><tr><td>Benzocaine</td><td>Sigma Aldrich</td><td>E1501</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma Aldrich</td><td>A3059</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>C3306</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman</td><td>J2-21M</td><td></td></tr><tr><td>Centrifuge rotor</td><td>Beckman</td><td>JS 13.1</td><td></td></tr><tr><td>chymostatin</td><td>Sigma Aldrich</td><td>C7268</td><td></td></tr><tr><td>creatine phosphate disodium</td><td>Calbiochem</td><td>2380</td><td></td></tr><tr><td>cycloheximide</td><td>Sigma Aldrich</td><td>C6255</td><td></td></tr><tr><td>cytochalasin D</td><td>Sigma Aldrich</td><td>C8273</td><td></td></tr><tr><td>disposable wipes (kimwipes)</td><td>Sigma Aldrich</td><td>Z188956</td><td></td></tr><tr><td>L-cysteine</td><td>Sigma Aldrich</td><td>W326306</td><td></td></tr><tr><td>EGTA</td><td>Sigma Aldrich</td><td>E4378</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma Aldrich</td><td>F8775</td><td></td></tr><tr><td>Glass crystallizing dish (150 x 75 mm)</td><td>VWR</td><td>89090-662</td><td></td></tr><tr><td>Glycerol</td><td>Macron</td><td>5094-16</td><td></td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H4034</td><td></td></tr><tr><td>Hoechst - bisBenzimide H 33342 trihydrochloride</td><td>Sigma Aldrich</td><td>B2261</td><td></td></tr><tr><td>HCG - Human Chorionic Gonadotropin&amp;nbsp;</td><td>Prospec</td><td>hor-250-c</td><td></td></tr><tr><td>L15 Media</td><td>Sigma Aldrich</td><td>L4386</td><td></td></tr><tr><td>leupeptin</td><td>Sigma Aldrich</td><td>L2884</td><td></td></tr><tr><td>Lysolecithin</td><td>Sigma Aldrich</td><td>L1381</td><td></td></tr><tr><td>mAb414</td><td>Abcam</td><td>ab24609</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>EMD</td><td>MX0045-2</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>M9397</td><td></td></tr><tr><td>Maltose</td><td>Sigma Aldrich</td><td>M5885</td><td></td></tr><tr><td>NP40</td><td>BDH</td><td>56009</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Penicillin + Streptomycin</td><td>Sigma Aldrich</td><td>Pp0781</td><td></td></tr><tr><td>pepstatin</td><td>Sigma Aldrich</td><td>P5318</td><td></td></tr><tr><td>PIPES</td><td>Sigma Aldrich</td><td>P6757</td><td></td></tr><tr><td>Plastic paraffin film (parafilm)</td><td>Sigma Aldrich</td><td>P7793</td><td></td></tr><tr><td>KCl</td><td>Sigma Aldrich</td><td>P9541</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Mallinckrodt</td><td>70100</td><td></td></tr><tr><td>KOH</td><td>Baker</td><td>5 3140</td><td></td></tr><tr><td>PMSG - Pregnant Mare Serum Gonadotropin</td><td>Prospec</td><td>hor-272-a</td><td></td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S3014</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher</td><td>BP328</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>EMD</td><td>SX0720-1</td><td></td></tr><tr><td>NaOH</td><td>EMD</td><td>SX0590</td><td></td></tr><tr><td>Pestle</td><td>Thomas Scientific</td><td>3411D56</td><td></td></tr><tr><td>Round bottom glass tubes, 15 ml</td><td>Corex</td><td>8441</td><td></td></tr><tr><td>Secondary antibody (Alexa Fluor 568 donkey anti-mouse IgG)</td><td>ThermoFisher</td><td>A10037</td><td></td></tr><tr><td>sucrose</td><td>Calbiochem</td><td>8550</td><td></td></tr><tr><td>thermal cycler</td><td>Bio-Rad</td><td>T100</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman</td><td>L8-80M</td><td></td></tr><tr><td>Ultracentrifuge rotor</td><td>Beckman</td><td>SW 50.1</td><td></td></tr><tr><td>Vectashield (anti-fade mounting medium)</td><td>Vector</td><td>H-1000</td><td></td></tr></tbody></table>

a cell-free assay using xenopus laevis embryo extracts to study mechanisms of nuclear size regulation

Una pregunta fundamental en biología celular es cómo se regulan los tamaños de células y orgánulos. Durante mucho tiempo se ha reconocido que el tamaño del núcleo escalas generalmente con el tamaño de la celda, en particular durante la embriogénesis cuando se producen reducciones dramáticas en tanto celular y tamaños nucleares. Mecanismos de regulación del tamaño nuclear son en gran parte desconocida y puede ser relevante para el cáncer, donde el tamaño nuclear alterada es un parámetro clave de diagnóstico y pronóstico. En vivo para la identificación de los reguladores nucleares de tamaño se complica por la naturaleza esencial y compleja de la función nuclear. El enfoque in vitro descrito aquí para estudiar el control del tamaño nuclear se aprovecha de las reducciones normales en tamaño nuclear que se producen durante el desarrollo de Xenopus laevis. En primer lugar, los núcleos se ensamblan en X. laevis extracto de huevo. A continuación, estos núcleos se aíslan y se resuspendieron en el citoplasma de embriones de etapa tardía. Después de un 30 - 90 min período de incubación, la superficie nucleardisminuye en 20 a 60%, proporcionando un ensayo útil para identificar los componentes citoplasmáticos presentes en embriones de etapa tardía que contribuyen al desarrollo de escala tamaño nuclear. Una ventaja importante de este enfoque es la facilidad relativa con la que los extractos de huevo y embrión puede ser bioquímicamente manipulados, lo que permite la identificación de nuevas proteínas y actividades que regulan el tamaño nuclear. Como con cualquier enfoque en vitro, la validación de los resultados en un sistema in vivo es importante, y la microinyección de X. laevis embriones es particularmente apropiado para estos estudios.

Asamblea de los núcleos en extracto de huevo Los primeros pasos de este protocolo son para preparar X. extracto de huevo laevis (Protocolo 1) y núcleos de los espermatozoides demembranated (Protocolo 2). Estos reactivos se utilizan para ensamblar núcleos de novo (Protocolo 3). La figura 1 muestra algunos datos representativos. La adición de calcio conduce el extr...

Watch this Scientific Journal Video about A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation at JoVE.com

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

Mechanisms of cellular and intra-cellular scaling remain elusive. The use of Xenopus embryo extracts has become increasingly common to elucidate mechanisms of organelle size regulation. This method describes embryo extract preparation and a novel nuclear scaling assay through which mechanisms of nuclear size regulation can be identified.

Un ensayo libre de células Utilizando<em&gt; Xenopus laevis</em&gt; Embrión Extrae para estudiar los mecanismos de regulación del tamaño nuclear

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

8.3K vistas.  University of Wyoming. Mechanisms of cellular and intra-cellular scaling remain elusive. The use of Xenopus embryo extracts has become increasingly common to elucidate mechanisms of organelle size regulation. This method describes embryo extract preparation and a novel nuclear scaling assay through which mechanisms of nuclear size regulation can be identified.

Video: A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

La manipulación y<em&gt; In Vitro</em&gt; La maduración de<em&gt; Xenopus laevis</em&gt; Los ovocitos, seguido de inyección intracitoplasmática de espermatozoides, para estudiar el desarrollo embrionario

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Investigación

Biología del Desarrollo

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

Uso del análisis confocal de<em&gt; Xenopus laevis</em&gt; Encargado de investigar moduladores de Wnt y Shh morfógeno Degradados

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts prepared from Xenopus laevis eggs have been powerful in dissecting biochemical networks underlying the cell-cycle progression, their ensemble average measurement typically leads to a damped oscillation, despite each individual oscillator being sustained. This is due to the difficulty of perfect synchronization among individual oscillators in noisy biological systems. To retrieve the single-cell dynamics of the oscillator, we developed a droplet-based artificial cell system that can reconstitute mitotic cycles in cell-like compartments encapsulating cycling cytoplasmic extracts of Xenopus laevis eggs. These simple cytoplasmic-only cells exhibit sustained oscillations for over 30 cycles. To build more complicated cells with nuclei, we added demembranated sperm chromatin to trigger nuclei self-assembly in the system. We observed a periodic progression of chromosome condensation/decondensation and nuclei envelop breakdown/reformation, like in real cells. This indicates that the mitotic oscillator functions faithfully to drive multiple downstream mitotic events. We simultaneously tracked the dynamics of the mitotic oscillator and downstream processes in individual droplets using multi-channel time-lapse fluorescence microscopy. The artificial cell-cycle system provides a high-throughput framework for quantitative manipulation and analysis of mitotic oscillations with single-cell resolution, which likely provides important insights into the regulatory machinery and functions of the clock.

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

We present a method for the generation of in vitro self-sustained mitotic oscillations at the single-cell level by encapsulating egg extracts of Xenopus laevis in water-in-oil microemulsions.

Reconstitución de las oscilaciones del ciclo celular en microemulsiones de extractos de huevo libre de Xenopus

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts ...

Bioquímica

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Preparación y fraccionamiento de los extractos de huevo Xenopus laevis

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Biología

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

En la Asamblea Vitro nucleares utilizando fraccionado extractos de huevo Xenopus

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Células vivas, imágenes y análisis cuantitativo de las células epiteliales embrionarias en Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

La producción de transgénicos Xenopus laevis Integración mediada por la enzima de restricción y transferencia nuclear

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

Producción de<em&gt; Xenopus tropicalis</em&gt; Huevos Extrae para identificar los ARN asociada a los microtúbulos

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

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 &#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 ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

Una célula de ensayo libre para el Estudio de la cromatina descondensación en la final de la mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...

Un ensayo libre de células Utilizando

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Producción de

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