Name: xenopus laevis egg extract preparation and live imaging methods for visualizing dynamic cytoplasmic organization
Uploaded: 2021-06-06T13:00:00
Description: Watch this Scientific Journal Video about Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization at JoVE.com

We thank J. Kamenz, Y. Chen, and W. Y. C. Huang for comments on the manuscript. This work was supported by grants from the National Institutes of Health (R01 GM110564, P50 GM107615, and R35 GM131792) awarded to James E. Ferrell, Jr.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Stanford University.
1. Preparation of slides and coverslips
<ol>
	<li>Apply a layer of fluorinated ethylene propylene (FEP) adhesive tape to a glass slide with a roller applicator. Cut off excessive tape over the edges with a clean razor blade. Prepare FEP tape-coated coverslips in the same way (Figure 1A).</li>
	<li>Apply a double-sided sticky imaging sp...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+visualization+of+cell+cycle-dependent+changes+in+microtubule+dynamics+in+cytoplasmic+extracts.">Real-time visualization of cell cycle-dependent changes in microtubule dynamics in cytoplasmic extracts.</a> Cell. 62 (3), 579-589 (1990).</li><li>Krauss, S. W., Lee, G., Chasis, J. A., Mohandas, N., Heald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+protein+4.1+domains+essential+for+mitotic+spindle+and+aster+microtubule+dynamics+and+organization+in+vitro.">Two protein 4.1 domains essential for mitotic spindle and aster microtubule dynamics and organization in vitro.</a> Journal of Biological Chemistry. 279 (26), 27591-27598 (2004).</li><li>Wang, S., Romano, F. B., Field, C. M., Mitchison, T. J., Rapoport, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+mechanisms+determine+ER+network+morphology+during+the+cell+cycle+in+Xenopus+egg+extracts.">Multiple mechanisms determine ER network morphology during the cell cycle in Xenopus egg extracts.</a> Journal of Cell Biology. 203 (5), 801-814 (2013).</li><li>Field, C. M., Nguyen, P. A., Ishihara, K., Groen, A. C., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+egg+cytoplasm+with+intact+actin.">Xenopus egg cytoplasm with intact actin.</a> Methods in Enzymology. 540, 399-415 (2014).</li><li>Good, M. 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Xenopus laevis egg extracts have emerged as a powerful model system for imaging-based&#160;studies of various subcellular structures10,14,15,16,17,18,21,31,32,33,<sup ...

The cytoplasm constitutes the main volume of a cell and has a distinct organization. The ingredients of the eukaryotic cytoplasm can self-assemble into a wide range of spatial structures, such as microtubule asters and the Golgi apparatus, which in turn are dynamically arranged and turned over depending on the cell&#39;s identity and physiological state. Understanding the spatial organization of the cytoplasm and its link to cellular functions is thus important for understanding how the cell works. Xenopus laevis egg extracts have traditionally been used for bulk biochemical assays1,2,3,4,5,6,7,8, but recent work establishes them as a powerful live imaging system for mechanistic studies of cytoplasmic structures and their cellular functions9,10,11,12,13,14,15,16,17,18. These undiluted extracts preserve many structures and functions of the cytoplasm, while allowing direct manipulations of cytoplasmic contents not achievable in conventional cell-based models19,20. This makes them ideal for characterizing cytoplasmic phenomena and dissecting their mechanistic underpinnings.
Existing methods for imaging extracts require chemical surface modifications, or fabrication of microfluidic devices. One coverslip-based method requires polyethylene glycol (PEG) passivation of glass coverslips21. A microemulsion-based method requires vapor deposition of trichloro(1H,1H,2H,2H-perfluorooctyl)silane on glass surfaces22,23. Microfluidic-based systems allow precise control of the volume, geometry and composition of extract droplets, but require specialized microfabrication facilities11,12,24.
Here we introduce an alternative method for imaging egg extracts that is easy to implement and utilizes readily available, low-cost materials. This includes preparation of an imaging chamber with a slide and a coverslip coated with fluorinated ethylene propylene (FEP) tape. The chamber can be used for imaging extracts with a variety of microscopy systems, including stereoscopes and upright and inverted microscopes. This method requires no chemical treatment of surfaces while achieving similar optical clarity obtained with existing glass-based methods discussed above. It is designed to image a layer of extracts with a uniform thickness across a 2D field, and can be easily extended to image a 3D volume of extracts. It is well suited for time-lapse imaging of collective cytoplasmic behavior over a large field of view.
We have used interphase-arrested egg extracts to demonstrate our imaging method. The extract preparation follows the protocol of Deming and Kornbluth19. Briefly, eggs naturally arrested in metaphase of meiosis II are crushed by a low speed spin. This spin releases the cytoplasm from meiotic arrest and allows the extract to proceed into interphase. Normally, cytochalasin B is added prior to the crushing spin to inhibit F-actin formation. However, it can be omitted if F-actin is desired. Cycloheximide is also added prior to the crushing spin to prevent the interphase extract from entering the next mitosis. The extracts are subsequently placed in the aforementioned imaging chambers and placed on a microscope. Finally, images are recorded over time at defined intervals by a camera connected to the microscope, producing time-lapse image series that capture the dynamical behavior of the extract in a 2D field.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>17 ml centrifuge tube</td>
			<td>Beckman Coulter</td>
			<td>337986</td>
			<td></td>
		</tr>
		<tr>
			<td>22x22 mm square #1 cover glass</td>
			<td>Corning</td>
			<td>284522</td>
			<td></td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>MilliporeSigma</td>
			<td>10236624001</td>
			<td>Protease inhibitor</td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>MilliporeSigma</td>
			<td>01810</td>
			<td>Protein synthesis inhibitor</td>
		</tr>
		<tr>
			<td>Cytochalasin B</td>
			<td>MilliporeSigma</td>
			<td>C6762</td>
			<td>Actin polymerization inhibitor</td>
		</tr>
		<tr>
			<td>Female Xenopus laevis frogs</td>
			<td>Nasco</td>
			<td>LM00535MX</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent HiLyte 488 labeled tubulin protein</td>
			<td>Cytoskeleton, Inc.</td>
			<td>TL488M-A</td>
			<td>For visualizing the microtubule cytoskeleton</td>
		</tr>
		<tr>
			<td>Fluorescent HiLyte 647 labeled tubulin protein</td>
			<td>Cytoskeleton, Inc.</td>
			<td>TL670M-A</td>
			<td>For visualizing the microtubule cytoskeleton</td>
		</tr>
		<tr>
			<td>Fluorinated ethylene propylene (FEP) optically clear tape</td>
			<td>CS Hyde company</td>
			<td>23-FEP-2-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pasteur pipette</td>
			<td>Fisher Scientific</td>
			<td>13-678-20C</td>
			<td></td>
		</tr>
		<tr>
			<td>Human chorionic gonadotropin (hCG)</td>
			<td>MilliporeSigma</td>
			<td>CG10</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging spacer</td>
			<td>Electron Microscopy Sciences</td>
			<td>70327-8S</td>
			<td></td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>MilliporeSigma</td>
			<td>11017101001</td>
			<td>Protease inhibitor</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-518-100B</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>MilliporeSigma</td>
			<td>330760</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker Red CMXRos</td>
			<td>Thermo Fisher Scientific</td>
			<td>M7512</td>
			<td>For visualizing mitochondria</td>
		</tr>
		<tr>
			<td>Pregnant mare serum gonadotropin (PMSG)</td>
			<td>BioVendor</td>
			<td>RP1782725000</td>
			<td></td>
		</tr>
		<tr>
			<td>Roller applicator</td>
			<td>Amazon</td>
			<td>B07HMBJSP8</td>
			<td>For applying the FEP tape to the glass slides and coverslips</td>
		</tr>
		<tr>
			<td>Single-edged razor blades</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td>For removing excessive FEP tape</td>
		</tr>
		<tr>
			<td>Transfer pipette</td>
			<td>Fisher Scientific</td>
			<td>13-711-7M</td>
			<td></td>
		</tr>
	</tbody>
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xenopus laevis egg extract preparation and live imaging methods for visualizing dynamic cytoplasmic organization

Traditionally used for bulk biochemical assays, Xenopus laevis egg extracts have emerged as a powerful imaging-based tool for studying cytoplasmic phenomena, such as cytokinesis, mitotic spindle formation and assembly of the nucleus. Building upon early methods that imaged fixed extracts sampled at sparse time points, recent approaches image live extracts using time-lapse microscopy, revealing more dynamical features with enhanced temporal resolution. These methods usually require sophisticated surface treatments of the imaging vessel. Here we introduce an alternative method for live imaging of egg extracts that require no chemical surface treatment. It is simple to implement and utilizes mass-produced laboratory consumables for imaging. We describe a system that can be used for both wide-field and confocal microscopy. It is designed for imaging extracts in a 2-dimensional (2D) field, but can be easily extended to imaging in 3D. It is well-suited for studying spatial pattern formation within the cytoplasm. With representative data, we demonstrate the typical dynamic organization of microtubules, nuclei and mitochondria in interphase extracts prepared using this method. These image data can provide quantitative information on cytoplasmic dynamics and spatial organization.

Xenopus laevis egg extracts can be used to study the self-organization of the cytoplasm during interphase. Figure 2A shows results from a successful experiment. We supplemented interphase-arrested extracts with demembranated Xenopus laevis sperm nuclei19 at a concentration of 27 nuclei/&#181;L and 0.38 &#181;M purified GST-GFP-NLS27,28,<sup class="x...

Watch this Scientific Journal Video about Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization at JoVE.com

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

We describe a method for the preparation and live imaging of undiluted cytoplasmic extracts from Xenopus laevis eggs.

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

Traditionally used for bulk biochemical assays, Xenopus laevis egg extracts have emerged as a powerful imaging-based tool for studying cytoplasmic ...

This method is suitable for live imaging of cytoplasmic dynamics using frog egg extracts, enabling the study of spatial pattern information in the cytoplasm. Samples can be shaped into a layer with a uniform and adjustable thickness and imaged across a 2D field or 3D volume. This method is cost effective and easy to implement.Begin by applying a layer of FEP adhesive tape to a glass slide with a roller applicator. Cut off excessive tape over the edges with a clean razor blade. Next, prepare FEP tape coated cover slips in the same way.Then apply a double-sided sticky imaging spacer to the FEP tape coated side of the slide leaving the protective liner unpeeled. Transfer eggs to a 400 milliliter glass beaker and remove as much egg-laying buffer as possible by decanting. Then incubate the eggs in 100 milliliters of freshly prepared dejellying solution and gently swirl them.After about three minutes, pour off the solution and add 100 milliliters of fresh dejellying solution. Continue the incubation until the eggs are tightly packed, but avoid leaving eggs in the dejellying solution for more than a total of five minutes. After the incubation, remove the dejellying solution as much as possible and wash the eggs in 0.25x MMR buffer.Swirl the eggs, then pour off the buffer. Repeat a few times until a total of one liter of the buffer is used for the washes. Next, wash the eggs a few times with a total of 400 milliliters of egg lysis buffer, removing abnormal eggs between the washes.Then use a transfer pipette with a wide cut tip to transfer the eggs to a 17 milliliter round bottom centrifuge tube containing one milliliter of egg lysis buffer. Spin the tube in a clinical centrifuge at 400 g for 15 seconds to pack the eggs. Then remove as much of the egg lysis buffer as possible using a Pasteur pipette.Determine the approximate volume of the packed eggs. then add five micrograms per milliliter each of aprotinin, leupeptin and cytochalasin B and 50 micrograms per milliliter of cyclohexamide directly on top of the packed eggs. Crush the eggs by centrifusing the tube for 15 minutes at 12, 000 g and four degrees Celsius in a swinging bucket rotor.Next, attach an 18 gauge needle to a syringe. With the needle tip bevel facing up puncture the tube from the side at the bottom of the cytoplasmic layer and recover the extract by drawing slowly. Transfer the recovered cytoplasmic extract to a new micro centrifuge tube and keep it on ice.When ready to image, supplement the extract with the desired reagents and fluorescence imaging probes. Remove the top protective liner from the imaging spacer on the prepared slide and deposit approximately seven microliters of extract at the center of the well. Immediately apply the FEP tape coated cover slip with the FEP side facing the extract to seal the well and quickly proceed to imaging.Set the slide on an inverted or upright microscope with a motorized stage and a digital camera. Image the extracts at the desired spatial positions in time intervals in both bright field and fluorescence channels. A time-lapse experiment using xenopus laevis egg extracts to study the self-organization of the cytoplasm during interphase is shown here.After about 20 minutes of incubation at room temperature areas depleted of light scattering cytoplasmic components were visible in both bright field and mitochondria channels. By about 60 minutes at room temperature, a spatial pattern consisting of cell-like compartments should be well established with microtubules forming a hollow wreath-like structure and mitochondria clearly partitioned into each compartment. A comparison of extract performance in imaging chambers with and without FEP tapes on glass is shown here.In the chamber made with FEP taped glass the extract self-organized into normal cell-like patterns. In the chamber where the glass surfaces were not covered by the FEP tape, the extract showed abnormal bright field and microtubule patterns that became increasingly disrupted over time. No significant differences were observed in the nuclear import of the GST-mCherry-NLS protein.The important things to remember are to passivate the glass surfaces with FEP tape and to remove all bad eggs during extract preparation. With this system, we can easily control the thickness of the extract sample, paving the way for imaging cytoplasmic patterns in 3D, using light sheet microscopy.

xenopus-laevis-egg-extract-preparation-live-imaging-methods-for

Research

JoVE Journal

Biology

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

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.

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

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

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.

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

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.

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

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.

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

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a &#34;double decker&#34; microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm.

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

The giant ciliate Stentor coeruleus is a classical system for studying regeneration and wound healing in single cells.&#160;By imaging Stentor cells simultaneously at low and high magnification it is possible to measure cytoplasmic flows before, during, and after wounding.

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

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.

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