1. Generation of X. tropicalis Eggs
All Xenopus tropicalis frogs are ordered from NASCO. Our frogs are housed in an Aquatic Habitats recirculating water system kept at 27 &deg;C. There are many options for water systems for care of X. tropicalis. Some good general information on this frog species can be found on the web sites of the Harland and Grainger labs (<a href="http://tropicalis.berkeley.edu/home/" target="_blank">http://tropicalis.berkeley.edu/home/</a>, <a href="http://www.faculty.virginia.edu/xtropicalis/" target="_blank">http://www.faculty.virginia.edu/xtropicalis/</a>). Our frogs are maintained in tankwater consisting of (0.4 g Ciclid Lake Salts, 0.6 g marine salt, 0.625 g NaHCO3 per liter of water, pH 7.0)20. This recipe results in a conductivity of ~1800 &mu;S, which is a high salinity for X. tropicalis. However, we have found that our frogs thrive in this environment and oocyte quality is improved. Alternative tankwater recipes can be found above at the resources listed for general X. tropicalis care.
<ol>
 <li>Frogs are injected with human Chorionic Gonadotropin (hCG) on three successive days to stimulate egg laying: First, prepare two concentrations of hCG solution. Resuspend 10,000 U of lyophilized hCG powder in 10 ml sterile, deionized H2O for a final concentration of 1,000 U/ml. Then, dilute 1 ml of 1,000 U/ml hCG solution in 9 ml H2O for a final concentration of 100 U/ml. Store both solutions at 4 &deg;C.</li>
 <li>On day 1, prepare 4-6 frogs for egg laying by injecting with hCG between 2:00-3:00 PM. Inject each frog in the dorsal lymph sac near the cloaca with 0.2 ml 100 U/ml hCG solution. Having the frogs fast during the subsequent two injections will minimize the amount of frog waste present during egg laying, but is optional.</li>
 <li>On day 2, inject the same frogs with 0.2 ml 100 U/ml hCG solution between 2:00-3:00 PM.</li>
 <li>On day 3, inject the same frogs with 0.2 ml 1,000 U/ml hCG solution, between 7:00-10:00 AM. Set up frogs to lay eggs: fill a 6-quart plastic bucket with fresh tankwater, add frogs and place in the dark at 25 &deg;C. After this injection, egg laying will begin after 4 hr and will be complete by 7 hr. Frogs should lay eggs in an environment that is maintained at a minimum of 25 &deg;C.</li>
 <li>Make extract solutions and have equipment ready immediately before collecting eggs.</li>
</ol>
20X MMR: 100 mM HEPES, pH 7.8; 2 mM EDTA pH 7.8; 2 M NaCl; 40 mM KCl; 20 mM MgCl2; 40 mM CaCl2. Autoclave and store at room temperature. Prepare 1 L of 1X MMR just prior to extract preparation.
10X XB: 100 mM HEPES, pH7.7; 10 mM MgCl2; 1 mM CaCl2; 1 M KCl; 500 mM sucrose. Autoclave and store at 4 &deg;C. Prepare 1 L of 1X XB just prior to extract preparation. Dejelly solution: Prepare 250 ml 3% cysteine solution in deionized H2O and pH to 7.8-8.0 with 10 N NaOH. Prepare just prior to extract preparation.
CSF-XB: take 200 ml of 1X XB and add 2 ml 0.5 M EGTA pH 7.7 and 200 &mu;l 1 M MgCl2. Prepare just prior to extract preparation.
CSF-XB+: take 50 ml of CSF-XB and add 50 &mu;l of LPC (10 mg/ml each stock of Leupeptin, Pepstatin, and Chymostatin in DMSO). Add 50 &mu;l Cytochalasin D (10 mg/ml in DMSO). Prepare just prior to extract preparation.
Prepare a 0.2% gelatin solution in deionized H2O, microwave to dissolve and filter sterilize. Store at room temperature.
Reserve 2 Beckman 2 x &frac12; inch Ultracentrifuge tubes.
Prepare two 15 ml glass round-bottomed centrifuge tubes with 0.5 ml of H2O in each to cushion the ultracentrifuge tube.
Make fire-polished glass Pasteur pipettes. Snap the end off of 5 &frac34; inch glass pipettes to expose a broad opening, and expose to flame to smooth the new exposed pipette tip.
<ol start="6">
 <li>Prepare a 500 ml glass beaker for storing eggs by swirling a 0.2% gelatin solution around to coat the walls of the beaker. Discard gelatin solution from beaker after use.</li>
 <li>Collect eggs from the plastic bucket used for laying 6-7 hr after the third injection on day 3. If desired, gently squeeze each frog once to get any remaining eggs. Wash eggs once with fresh tankwater and transfer to the 500 ml glass beaker coated with 0.2% gelatin solution.</li>
</ol>
2. Preparation of Extract from X. tropicalis Eggs
All steps of extract preparation can be performed at room temperature, approximately 25 &deg;C. Throughout the washes, it is important to keep the eggs submerged under liquid so that they remain wet. Exposure to air can cause the eggs to escape cell cycle arrest or lyse.
<ol>
 <li>Decant as much tankwater as possible while reserving enough liquid to keep the eggs wet. Tilt the beaker containing eggs to the side and add ~300 ml 1X MMR slowly to the wall of the beaker, so that physical agitation of the eggs is minimized. Let eggs settle, then decant off supernatant containing debris. X. tropicalis eggs are stringy at this step, so removal of activated eggs is done after dejellying. Repeat for a total of three 1X MMR washes.</li>
 <li>Dejelly the eggs. Decant off as much MMR as possible and add half of the dejelly solution. Swirl continuously for approximately 5 min. Dissolving jelly coats will be visible in the supernatant after a couple of minutes. Decant off and add the remaining dejelly solution. Continue to swirl continuously until eggs pack very tightly and all orient with their vegetal pole (the pole with white pigment) toward the bottom of the dish. Quickly decant off as much dejelly solution as possible. Once the eggs are dejellied they are very sensitive to mechanical manipulations.</li>
 <li>Carefully add XB to the eggs. In the first XB wash, remove eggs that have escaped CSF arrest by removing lysed, puffy, white, and pseudocleavage eggs. Activated X. tropicalis eggs tend to settle in the top center, so use a plastic transfer pipette to pull these out. Also remove pieces of skin and frog waste. Wash eggs a total of three times with ~ 300 ml 1X XB solution, gently swirling eggs between washes and allowing them to settle on the bottom of the beaker. As before, decant as much of each wash solution as possible while keeping eggs wet.</li>
 <li>Wash eggs twice with CSF-XB and decant.</li>
 <li>Add CSF-XB+ to eggs. Using a gelatin-treated fire-polished Pasteur pipette, transfer eggs to Ultra-centrifuge tubes with CSF-XB+, taking care not to expose the eggs to air. Place inside the 15 ml glass centrifuge tubes with the water cushion.</li>
 <li>Spin eggs in a clinical centrifuge at 200 x g for 1 min, increase speed to 800 x g and spin for 30 sec.</li>
 <li>Use an aspirator to remove as much buffer as possible from eggs. They should be almost dry on top. Quickly move eggs to a Sorvall RC-6 centrifuge equipped with a HB-6 rotor (or equivalent) and spin 17,000 x g for 15 min at 20 &deg;C.</li>
 <li>Remove the yellow cytoplasmic layer between the pigment and lipid layers using an 18 gauge needle attached to a 1 ml syringe. Puncture the side of the tube and pull the syringe barrel slowly to obtain the cytoplasmic extract layer. Avoid pigment granules as much as possible.</li>
 <li>Transfer cytoplasm to new ultracentrifugation tube. It is normal for the extract to appear slightly cloudy at this step. Place inside the 15 ml glass centrifuge tube with water cushion. Spin again 17,000 x g for 10 min at 20 &deg;C. Repeat extraction with 18 gauge needle.</li>
 <li>Transfer cytoplasm to a 1.5 ml microfuge tube. Estimate the extract volume and dilute Cytochalasin D and LPC 1:1,000 into the extract. Mix well with a 1 ml pipette tip, pipetting up and down many times without introduction of air bubbles. A typical yield from a healthy frog colony is approximately 300-500 &mu;l of extract/frog. To preserve maximum activity, it is necessary to store the extract and perform experimental manipulations at room temperature (20-25 &deg;C).</li>
</ol>
3. Purification Taxol-stabilized Microtubules from X. tropicalis Extract
<ol>
 <li>Add Taxol to a 100-200 &mu;l aliquot of extract at a final concentration of 10 &mu;M and incubate at room temperature for 30 min. For control reactions, treat an equivalent volume of extract with the microtubule-destablilzing drug Nocodazole (10 &mu;M). Reserve 100 &mu;l of untreated extract for analysis.</li>
 <li>Dilute the drug-treated extract with 10 volumes BRB-80 (80 mM PIPES pH 6.8, 1 mM MgCl2, 1 mM EGTA) + 30% glycerol. Assemble 14 ml round-bottom polypropylene tubes containing 10 ml of BRB-80 + 60% glycerol cushion. Using a wide bore pipette tip, layer the drug-treated extract reaction gently on top of the BRB-80 + 60% glycerol cushion. Centrifuge for 10 min at 17,000 x g at 20 &deg;C in a Sorvall RC-6 centrifuge equipped with a HB-6 rotor (or equivalent) and tube adapters.</li>
 <li>Aspirate the supernatant containing unsedimented extract material, and wash the interface twice with deionized H2O. Aspirate the remaining cushion volume slowly, taking care not to disturb the gel-like pellet containing microtubules, microtubule-associated proteins, and microtubule-associated RNAs in the Taxol-treated sample. The Nocodazole-treated sample does not contain visible material. Resuspend the pellet in 1 ml TRIzol and proceed with the manufacturer's instructions for isolating RNA. Untreated extract (up to 100 &mu;l) can be resuspended directly in 1 ml TRIzol.</li>
 <li>There are now commercially available kits for preparing transcriptome libraries suitable for RNA-seq. These may be purchased through <a href="http://www.illumina.com/" target="_blank">http://www.illumina.com/</a> and <a href="http://www.454.com/" target="_blank">http://www.454.com/</a>.</li>
</ol>

<ol>
	<li>Martin, K. C., Ephrussi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+localization:+gene+expression+in+the+spatial+dimension.">mRNA localization: gene expression in the spatial dimension.</a> Cell. 136, 719-730 (2009).</li><li>Besse, F., Ephrussi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+control+of+localized+mRNAs:+restricting+protein+synthesis+in+space+and+time.">Translational control of localized mRNAs: restricting protein synthesis in space and time.</a> Nat. Rev. Mol. Cell Biol. 9, 971-980 (2008).</li><li>Lawrence, J. B., Singer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+localization+of+messenger+RNAs+for+cytoskeletal+proteins.">Intracellular localization of messenger RNAs for cytoskeletal proteins.</a> Cell. 45, 407-415 (1986).</li><li>Mingle, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+all+seven+messenger+RNAs+for+the+actin-polymerization+nucleator+Arp2/3+complex+in+the+protrusions+of+fibroblasts.">Localization of all seven messenger RNAs for the actin-polymerization nucleator Arp2/3 complex in the protrusions of fibroblasts.</a> J. Cell Sci. 118, 2425-2433 (2005).</li><li>Blower, M. D., Feric, E., Weis, K., 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=Genome-wide+analysis+demonstrates+conserved+localization+of+messenger+RNAs+to+mitotic+microtubules.">Genome-wide analysis demonstrates conserved localization of messenger RNAs to mitotic microtubules.</a> J. Cell Biol. 179, 1365-1373 (2007).</li><li>Eliscovich, C., Peset, I., Vernos, I., Mendez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spindle-localized+CPE-mediated+translation+controls+meiotic+chromosome+segregation.">Spindle-localized CPE-mediated translation controls meiotic chromosome segregation.</a> Nat. Cell Biol. 10, 858-865 (2008).</li><li>Sharp, J. A., Plant, J. J., Ohsumi, T. K., Borowsky, M., Blower, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+the+microtubule-interacting+transcriptome.">Functional analysis of the microtubule-interacting transcriptome.</a> Mol. Biol Cell. 22, 4312-4323 (2011).</li><li>Lecuyer, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+mRNA+localization+reveals+a+prominent+role+in+organizing+cellular+architecture+and+function.">Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function.</a> Cell. 131, 174-187 (2007).</li><li>Mili, S., Moissoglu, K., Macara, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+screen+reveals+APC-associated+RNAs+enriched+in+cell+protrusions.">Genome-wide screen reveals APC-associated RNAs enriched in cell protrusions.</a> Nature. 453, 115-119 (2008).</li><li>Holt, C. E., Bullock, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+mRNA+localization+in+animal+cells+and+why+it+matters.">Subcellular mRNA localization in animal cells and why it matters.</a> Science. 326, 1212-1216 (2009).</li><li>Desai, A., Murray, A., Mitchison, T. J., Walczak, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+Xenopus+egg+extracts+to+study+mitotic+spindle+assembly+and+function+in+vitro.">The use of Xenopus egg extracts to study mitotic spindle assembly and function in vitro.</a> Methods Cell Biol. 61, 385-412 (1999).</li><li>Gache, V., Waridel, P., Luche, S., Shevchenko, A., Popov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+mass+spectrometry+identification+of+microtubule-binding+proteins+from+Xenopus+egg+extracts.">Purification and mass spectrometry identification of microtubule-binding proteins from Xenopus egg extracts.</a> Methods Mol. Med. 137, 29-43 (2007).</li><li>Gache, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+meiotic+microtubule-associated+interactome.">Xenopus meiotic microtubule-associated interactome.</a> PLoS One. 5, e9248 (2010).</li><li>Cross, M. K., Powers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obtaining+eggs+from+Xenopus+laevis+females.">Obtaining eggs from Xenopus laevis females.</a> J. Vis. Exp. (18), e890 (2008).</li><li>Cross, M. K., Powers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+fractionation+of+Xenopus+laevis+egg+extracts.">Preparation and fractionation of Xenopus laevis egg extracts.</a> J. Vis. Exp. (18), e891 (2008).</li><li>Cross, M., Powers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+nuclear+assembly+using+fractionated+Xenopus+egg+extracts.">In vitro nuclear assembly using fractionated Xenopus egg extracts.</a> J. Vis. Exp. (19), e908 (2008).</li><li>Harland, R. M., Grainger, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+research:+metamorphosed+by+genetics+and+genomics.">Xenopus research: metamorphosed by genetics and genomics.</a> Trends Genet. 27, 507-515 (2011).</li><li>Hellsten, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genome+of+the+Western+clawed+frog+Xenopus+tropicalis.">The genome of the Western clawed frog Xenopus tropicalis.</a> Science. 328, 633-636 (2010).</li><li>Brown, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+tropicalis+egg+extracts+provide+insight+into+scaling+of+the+mitotic+spindle.">Xenopus tropicalis egg extracts provide insight into scaling of the mitotic spindle.</a> J. Cell Biol. 176, 765-770 (2007).</li><li>Godfrey, E. W., Sanders, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+water+hardness+on+oocyte+quality+and+embryo+development+in+the+African+clawed+frog+(Xenopus+laevis).">Effect of water hardness on oocyte quality and embryo development in the African clawed frog (Xenopus laevis).</a> Comp. Med. 54, 170-175 (2004).</li><li>Groisman, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CPEB,+maskin,+and+cyclin+B1+mRNA+at+the+mitotic+apparatus:+implications+for+local+translational+control+of+cell+division.">CPEB, maskin, and cyclin B1 mRNA at the mitotic apparatus: implications for local translational control of cell division.</a> Cell. 103, 435-447 (2000).</li><li>Budde, P. P., Desai, A., 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=Analysis+of+microtubule+polymerization+in+vitro+and+during+the+cell+cycle+in+Xenopus+egg+extracts.">Analysis of microtubule polymerization in vitro and during the cell cycle in Xenopus egg extracts.</a> Methods. 38, 29-34 (2006).</li><li>Kalab, P., Pu, R. T., Dasso, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ran+GTPase+regulates+mitotic+spindle+assembly.">The ran GTPase regulates mitotic spindle assembly.</a> Curr. Biol. 9, 481-484 (1999).</li><li>Gaglio, T., Saredi, A., Compton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NuMA+is+required+for+the+organization+of+microtubules+into+aster-like+mitotic+arrays.">NuMA is required for the organization of microtubules into aster-like mitotic arrays.</a> J. Cell Biol. 131, 693-708 (1995).</li><li>Hughes, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microtubule+interactome:+complexes+with+roles+in+cell+cycle+and+mitosis.">A microtubule interactome: complexes with roles in cell cycle and mitosis.</a> PLoS Biol. 6, e98 (2008).</li><li>Suprenant, K. A., Tempero, L. B., Hammer, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+ribosomes+with+in+vitro+assembled+microtubules.">Association of ribosomes with in vitro assembled microtubules.</a> Cell Motil. Cytoskeleton. 14, 401-415 (1002).</li><li>Rebagliati, M. R., Weeks, D. L., Harvey, R. P., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+cloning+of+localized+maternal+RNAs+from+Xenopus+eggs.">Identification and cloning of localized maternal RNAs from Xenopus eggs.</a> Cell. 42, 769-777 (1985).</li></ol>

No conflicts of interest declared.

In this report we have described a simple method to produce CSF-arrested egg extracts from X. tropicalis19and use this extract to study microtubule-associated RNAs7. The basic procedure for producing CSF-arrested egg extracts from X. tropicalis is the same as used for X. laevis with a few key differences. One of the most challenging aspects to working with X. tropicalis frogs is obtaining enough high quality eggs to make an extract with microtubule nucleation or spindle assembly activity comparable to X. laevis egg extracts. To achieve optimal egg laying conditions while preventing slippage from meiosis II cell cycle arrest, the interval between hormone injections for X. tropicalis is shorter than that used for X. laevis, and the timing from the third hCG injection to the beginning of egg laying is also much shorter. With X. laevis the timing from the hCG injection to the beginning of egg laying is such that it is convenient and efficient for eggs to be laid overnight into buffer. However, because of the shorter time between hCG injection and egg laying with X. tropicalis it is frequently necessary to manually express the eggs from frogs. Another significant difference between making egg extract from the two different frogs is the dejellying step. With X. laevis the eggs are so large that it is easy to determine when the jelly coat has dissolved by observing how closely the eggs are spaced in the beaker. As the dejellying reaction commences, the eggs begin to pack more densely. However, X. tropicalis eggs are much smaller and it can be quite difficult to determine when the jelly coat has dissolved by egg packing density alone. We have found that the most reliable method to determine when the jelly coat has dissolved is to monitor the orientation of the animal (black) and vegetal (white) poles. When all the vegetal poles orient toward the bottom of the beaker the jelly coat has been removed enough to proceed with the extract. Finally, whereas X. laevis egg extract can be stored at cool temperatures (4-12 &deg;C) we have observed that it is critical to maintain X. tropicalis egg extract at room temperature (20-25 &deg;C) during preparation and experimental manipulations to preserve biochemical activity. Because of the differences in ease of use we prefer to use X. laevis frogs for the production of egg extract. However, for experiments that require or are facilitated by an organism with a sequenced genome, X. tropicalis is an excellent alternative system.
The method that we have described in this report uses taxol as a microtubule-stabilizing agent to induce microtubule polymerization. We chose this method because taxol is a robust microtubule-stabilizing agent that facilitates the large-scale isolation of purified microtubules. The method that we described could likely be improved by comparing the proteins and RNAs associated with microtubules using alternative microtubule polymerization methods. Alternatives could include polymerization using GTP-induced polymerization (a classic technique),22 or using Ran-GTP as a microtubule polymerizer to mimic the microtubules induced by chromatin-driven spindle assembly23. Finally, use of purified sperm nuclei to induce microtubule polymerization would be the closest mimic to the types of microtubules that are nucleated during mitosis (centrosome, chromatin, and kinetochore mediated). Drawbacks to these alternative sources of microtubule nucleation are that the nucleating agents are not as readily available as taxol and they do not nucleate or stabilize microtubules as efficiently as taxol. Therefore, each of these methods would be more difficult to use for large-scale purifications. The advantage of comparing multiple different types of microtubule nucleators is that it could be possible to identify proteins and/or RNAs that are specific to each pathway of microtubule nucleation.
The method that we have described here takes advantage of cytoplasmic extracts of amphibians. However, this approach could be extended to the use of extract system from other organisms. Mitotic extracts have been described from synchronized human tissue culture cells24 that faithfully recapitulate many aspects of microtubule assembly. We have successfully used these extracts to identify microtubule-associated RNAs from HeLa cells5. Similar microtubule purification schemes have been described for many different organisms25,26, although the microtubule associated RNAs have not been examined. The approach described here could be used with any organism that can produce a concentrated cytoplasmic extract capable of nucleating microtubules.
Finally, although the approach that we describe here discusses the purification of microtubules and associated proteins and RNAs, this approach could be generalized to other subcellular structures. While most localized mRNAs have not been identified using biochemical methods the recent advances in DNA and RNA sequencing technologies make this approach an attractive method to identify localized RNAs. In this approach any subcellular or sub-embryo structure of interest could be isolated or purified. Then the associated proteins and RNAs can be identified on a genome wide scale. RNAs can then be compared to the RNA content of the total cell or embryo to identify enriched localized RNAs. This approach could be used with whole eggs (animal and vegetal separation, similar to the approach that identified the first localized RNAs in Xenopus27), actin associated RNAs, ER-associated RNAs, mitochondria-associated RNAs, or to any subcellular structure that can be purified with associated RNAs intact. Based on our work on microtubule-associated RNA we predict that this would be an excellent method to discover new proteins that function at a given location. Furthermore, identification of the location and extent of all localized RNAs will provide insight into how cells and embryos use mRNA localization to control gene expression.

Spatial and temporal control of gene expression is important for all cells, and is especially important for the control of early embryonic pattering1. Spatial control of gene expression is achieved through the active localization of mRNAs to specific destinations within cells or embryos. In many very large cell types, (e.g. oocytes, embryos, and neurons) mRNA localization is used to restrict protein expression to the site of action of the coded protein. Since a localized mRNA can catalyze many rounds of protein production it is more efficient to localize an mRNA than to localize individual protein molecules. Localized mRNAs are typically translationally repressed until they reach their destination, which serves to further limit the localization of the coded protein2. In addition to the many well-documented cases of RNA localization to control embryonic patterning, several studies have documented mRNAs that are localized to the site of action of the encoded protein. Prominent examples include localization of the &beta;-actin3 and Arp2/34 mRNAs to the leading edge of motile fibroblasts and localization of the mRNAs for many mitotic regulators to meiotic and mitotic spindles5-7.
Many of the classic examples of localized mRNAs were identified through genetic screens for maternal effect mutations and were later determined to encode localized RNAs. However, recent genome-wide studies have begun to provide broader insight into the scope of localized RNAs. A recent in situ hybridization screen in Drosophila embryos demonstrated that ~70% of all mRNAs have a specific localization, including many novel destinations8. Purification of pseudopodia from mouse fibroblasts identified a diverse group of localized mRNAs9. Work from our group using biochemical purification of microtubules from meiotic Xenopus egg extracts identified hundreds of mRNAs that copurify with the spindle5,7. Our work showed that the majority of microtubule-localized mRNAs encode proteins that function in control of mitosis, supporting the idea that mRNAs are localized to the site of action of the coded protein. Furthermore, the ability to detect mRNA enrichment in a subcellular fraction by biochemical purification highlights the power of this approach for identification of localized mRNAs.
Most localized RNAs use active transport on the cytoskeleton, either actin or microtubules, to achieve transport to their final destination10. To gain a better understanding of the extent and types of RNAs that are localized to specific destinations using a biochemical approach it is necessary to have an in vitro system that can recapitulate cytoskeletal processes. One of the premier systems for studying cytoskeletal biology is egg extracts produced from unfertilized eggs from the frog Xenopus laevis. X. laevis egg extracts have been used for decades to study a wide array of cytoskeletal processes and have contributed much to our understanding of the mechanisms and molecules that control cytoskeletal assembly and dynamics11. Furthermore, X. laevis egg extracts are amenable to large-scale purifications of microtubules and associated proteins12,13 and there are well-designed methods for the production of various types of egg extracts14-16. However, for genomic studies there are several drawbacks to the use of X. laevis as a model system.
For decades Xenopus laevis frogs have been a powerful system for the study of developmental and cell biology, owing to the large oocyte size and robust external development17. Furthermore, the development of egg extract systems that can recapitulate many cellular processes in a test tube has made this frog a powerful experimental model. However, Xenopus laevis has been hampered by the lack of a complete genome sequence, which has been slowed by the allotetraploid nature of the genome.In contrast, a closely related species, Xenopus tropicalis, has a diploid genome that was sequenced in 201018. While X. tropicalis is not as experimentally tractable as X. laevis17the availability of a sequenced genome makes it an attractive model system to perform genome wide analyses.
In this report we describe a method to make meiosis II-, cytostatic factor-arrested extracts (CSF) from X. tropicalis19. We then describe a simple method to purify microtubules and associated RNAs from this extract. The RNAs can then be converted into libraries amenable to sequencing using recently developed high throughput sequencing technologies. Once the libraries are sequenced they can be aligned to the genome of the frog to identify specific mRNAs that are enriched in the microtubule sample compared to total extract. This provides a powerful method to detect microtubule-targeted mRNA localization on a genome-wide scale. In addition to being able to detect localized mRNAs, the use of high-throughput sequencing and a sequenced genome offer the possibility of discovering novel transcripts that are not currently present in public database annotations.

<table><tbody><tr><td colspan="4">&lt;strong&gt;REAGENTS&lt;/strong&gt;</td></tr><tr><td>&lt;em&gt;Xenopus tropicalis &lt;/em&gt;</td><td>NASCO</td><td>LM00823MX</td><td></td></tr><tr><td>human Chorionic Gonadotropin</td><td>Sigma-Aldrich</td><td>CG10</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H4034</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E5134</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S3014</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9541</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C8106</td><td></td></tr><tr><td>sucrose</td><td>Sigma-Aldrich</td><td>S0389</td><td></td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>S5881</td><td></td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>E3889</td><td></td></tr><tr><td>Leupeptin</td><td>Sigma-Aldrich</td><td>L9783</td><td></td></tr><tr><td>Pepstatin</td><td>Sigma-Aldrich</td><td>P5318</td><td></td></tr><tr><td>Chymostatin</td><td>Sigma-Aldrich</td><td>C7268</td><td></td></tr><tr><td>Cytochalasin D</td><td>Sigma-Aldrich</td><td>C8273</td><td></td></tr><tr><td>Gelatin, porcine skin</td><td>Sigma-Aldrich</td><td>G1890</td><td></td></tr><tr><td>PIPES</td><td>Sigma-Aldrich</td><td>P6757</td><td></td></tr><tr><td>Taxol</td><td>Sigma-Aldrich</td><td>T7191</td><td></td></tr><tr><td>Nocodazole</td><td>Sigma-Aldrich</td><td>M1404</td><td></td></tr><tr><td>Trizol</td><td>Invitrogen</td><td>15596-026</td><td></td></tr><tr><td>L-Cysteine, free base</td><td>USB Corporation</td><td>14030</td><td></td></tr><tr><td>Cichlid Lake Salt</td><td>Seachem</td><td>47894</td><td></td></tr><tr><td>Marine salt</td><td>Seachem</td><td>SC7111</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S6014</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td></tr><tr><td>1 ml syringes</td><td>BD Biosciences</td><td>309659</td><td></td></tr><tr><td>18 gauge needles</td><td>BD Biosciences</td><td>305195</td><td></td></tr><tr><td>30 gauge needles</td><td>BD Biosciences</td><td>305106</td><td></td></tr><tr><td>Rubbermaid Plastic bucket</td><td>Amazon</td><td>6306</td><td></td></tr><tr><td>Beckman Polyallomer 2 x &amp;frac12; inch Ultracentrifuge tubes</td><td>Beckman</td><td>326819</td><td></td></tr><tr><td>15 ml round-bottomed glass centrifuge tubes</td><td>Fisher Scientific</td><td>45500-15</td><td></td></tr><tr><td>Rubber adapter sleeves for 15 ml tubes</td><td>Kimble-Chase</td><td>45550-15</td><td></td></tr><tr><td>5 &amp;frac34; inch glass Pasteur pipettes</td><td>Fisher Scientific</td><td>13-678-20A</td><td></td></tr><tr><td>14 ml polypropylene round-bottom tube</td><td>BD Biosciences</td><td>352059</td><td></td></tr><tr><td>Sorvall HB-6 rotor</td><td>Thermo Scientific</td><td>11860</td><td></td></tr><tr><td>Sorvall RC-6 centrifuge</td><td>Thermo Scientific</td><td>46910</td><td></td></tr></tbody></table>

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.

To identify X. tropicalis transcripts associated with microtubules, we prepare a cytosolic extract from unfertilized eggs arrested in metaphase of meiosis II (CSF). Treatment of this extract with taxol allows the formation of stable microtubules that can be purified by sedimentation through a glycerol cushion (Figure 1A). Coomassie gel analysis confirms that &alpha;/&beta;-tubulin sediments in a taxol-dependent manner, and represents the major protein species recovered in these preparations (Figure 1B). Lower levels of other proteins are also present in the taxol pellet, but not in preparations treated with the microtubule depolymerizing drug nocodazole, indicating that proteins in the taxol fraction specifically associate with microtubules (MAPs).
An Agilent Bioanalyzer is used to examine general RNA composition in all X. tropicalis extract fractions (Figure 1C). Both rRNA and tRNA species are present in CSF extract and the microtubule-containing taxol pellet, consistent with previous findings that translation occurs on microtubules and spindles in X. laevis egg extract5,21. A line trace of the gel projection reveals the mRNA signal is markedly lower in the microtubule-containing taxol pellet, most notably in the region migrating above 28S rRNA, indicating that a subset of mRNAs cosediment with microtubules in X. tropicalis. RNA isolated in this manner is suitable for RNA-seq experiments using commercially available reagents.
<img src="/files/ftp_upload/50434/50434fig1.jpg" alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50434/50434fig1hires.jpg" /> 
Figure 1. Purification of MT-RNA for RNA-seq. (A) Purification scheme to isolate MT-RNA. Eggs are harvested from female X. tropicalis frogs. After preparation of a cytoplasmic extract, taxol is added to induce microtubule polymerization. Microtubules and MT-RNA are purified by sedimentation through a glycerol cushion. (B) Coomassie gel analysis of proteins isolated using the scheme described in (A). Total CSF extract compared to proteins sedimented in the presence of taxol or nocodazole. (C) Bioanalyzer gel analysis of RNA isolated using the scheme described in (A). RNA isolated from CSF extract compared to RNA sedimented in the presence of taxol or nocodazole. Both the gel projection and the line traces are shown. Reprinted with permission from Sharp, et al., (2011). <a href="https://www.jove.com/files/ftp_upload/50434/50434fig1hires.jpg">Click here to view larger image</a>.

Watch this Scientific Journal Video about Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs at JoVE.com

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.

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 ...

production-xenopus-tropicalis-egg-extracts-to-identify-microtubule

Research

JoVE Journal

Biology

13.6K Views.  Massachusetts General Hospital. 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.

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

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.

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

Developmental Biology

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.

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

Biochemistry

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 ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

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 ...

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.

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 ...

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

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic development 1, 2. One of the signaling pathways involved in this regulation is the Wnt pathway, which is used many times during embryogenesis and critical for human disease3, 4, 5. Multiple molecular components of this pathway coordinately regulate signaling in a spatially-restricted manner, but the underlying mechanisms are not fully understood. Xenopus embryonic epithelial cells is an excellent system to study subcellular localization of various signaling proteins. Fluorescent fusion proteins are expressed in Xenopus embryos by RNA microinjection, ectodermal explants are prepared and protein localization is evaluated by epifluorescence. In this experimental protocol we describe how subcellular localization of Diversin, a cytoplasmic protein that has been implicated in signaling and cell polarity determination6, 7 is visualized in Xenopus ectodermal cells to study Wnt signal transduction8. Coexpression of a Wnt ligand or a Frizzled receptor alters the distribution of Diversin fused with red fluorescent protein, RFP, and recruits it to the cell membrane in a polarized fashion 8, 9. This ex vivo protocol should be a useful addition to in vitro studies of cultured mammalian cells, in which spatial control of signaling differs from that of the intact tissue and is much more difficult to analyze.

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

Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity. An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells. This protocol will help address questions related to spatial control of signaling.

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

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

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.

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

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

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 scales with the size of the cell, notably during embryogenesis when dramatic reductions in both cell and nuclear sizes occur. Mechanisms of nuclear size regulation are largely unknown and may be relevant to cancer where altered nuclear size is a key diagnostic and prognostic parameter. In vivo approaches to identifying nuclear size regulators are complicated by the essential and complex nature of nuclear function. The in vitro approach described here to study nuclear size control takes advantage of the normal reductions in nuclear size that occur during Xenopus laevis development. First, nuclei are assembled in X. laevis egg extract. Then, these nuclei are isolated and resuspended in cytoplasm from late stage embryos. After a 30 - 90 min incubation period, nuclear surface area decreases by 20 - 60%, providing a useful assay to identify cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. A major advantage of this approach is the relative facility with which the egg and embryo extracts can be biochemically manipulated, allowing for the identification of novel proteins and activities that regulate nuclear size. As with any in vitro approach, validation of results in an in vivo system is important, and microinjection of X. laevis embryos is particularly appropriate for these studies.

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.

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 ...

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

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

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

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

Preparation and Fractionation of Xenopus laevis Egg Extracts

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

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

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

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

Xenopus laevis as a Model to Identify Translation Impairment

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

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