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).

All Xenopus procedures and studies were conducted in compliance with the NRC Guide for the Care and Use of Laboratory Animals 8th edition. Protocols were approved by the University of Wyoming Institutional Animal Care and Use Committee (Assurance # A-3216-01).
1. Preparation of X. laevis Egg Extract (adapted from27,28)
<ol>
	<li>Prime female&#160;X. laevis&#160;frogs a minimum of three days and a maximum of two weeks before egg collection with a single 100 IU injection of pregnant mare serum gonadotropin (PMSG).</li>
	<li>One day before the experiment, inject primed frogs with 800 IU of human chorionic gonadotropin (HCG) and place at 16 &#176;C in 1 L per frog of 1/3x Marc&#39;s modified ringers (MMR). See Table 1 for all buffer compositions. 
	&#8203;Note: Some frogs will not lay eggs or will lay poor quality eggs. Typically 2 - 3 frogs are injected to ensure at least one frog will lay sufficient numbers of good quality eggs. The average frog will lay enough eggs to produce at least 1 ml of egg extract.</li>
	<li>Prepare 1 L 1/3x MMR with cold distilled deionized water (ddH2O), 500 ml buffer B, 1 L buffer C, 500 ml buffer D, and 100 ml buffer E.</li>
	<li>Transfer laid eggs to a glass crystallizing dish (150 x 75 mm). Using a plastic Pasteur pipet with the tip cut off, farm out white puffy activated eggs or lysed eggs. Only retain eggs with distinct and equal dark animal poles and white vegetal poles.</li>
	<li>Prepare 13 x 51 mm centrifuge tubes with 1 ml of Buffer E supplemented with 355 &#181;M cytochalasin D added just prior to use in step 1.7.</li>
	<li>Dejelly and wash the eggs.
	<ol>
		<li>Wash eggs briefly with cold 1/3x MMR in an appropriately sized beaker, changing the buffer 3 - 4 times.</li>
		<li>Remove the jelly coats from the eggs by incubation with Buffer B. This will take anywhere from 3 - 10 min. As the cloudy jelly coats are released from the eggs, change the buffer. 
		&#8203;Note: Dejellying is complete when the eggs appear tightly packed in the corner of the dish when tilted and vegetal poles are oriented downwards. Eggs are much more fragile after dejellying, so do not incubate eggs with Buffer B for longer than necessary and subsequent washes need to be performed very carefully.</li>
		<li>Wash eggs briefly with 3 - 4 changes of Buffer C.</li>
		<li>Wash eggs briefly with 2 changes of Buffer D.</li>
		<li>Wash eggs briefly with 2 changes of Buffer E.</li>
	</ol>
	</li>
	<li>Fill centrifuge tubes with eggs using a wide bore glass pipette or plastic dropper. Aspirate off excess buffer. To pack the eggs and remove as much buffer as possible, centrifuge in swinging bucket rotor at 212 x g for 60 sec and then at 478 x g for 30 sec. Aspirate off excess buffer. 
	&#8203;Note: It is important to remove as much excess buffer as possible at this step to ensure the egg extract is minimally diluted.</li>
	<li>In a swinging bucket rotor, ultracentrifuge under vacuum for 15 min at 12,000 x g and 4 &#176;C to crush eggs open and separate into different layers, with the cytoplasm being the amber-colored layer in the middle. Remove centrifuge tubes and place immediately on ice.</li>
	<li>Collect the extract.
	<ol>
		<li>Using an 18 gauge needle and syringe, puncture the centrifuge tube just above the dark pigmented layer at the bottom of the tube and remove the middle amber-colored cytoplasmic layer. Do not collect any of the top lipid layer. Collect the cytoplasm in an appropriately sized tube. 
		Note: Generally, 1 frog produces at least 1 ml of extract.</li>
		<li>Supplement cytoplasm with 1/1,000 the volume of 19.7 mM cytochalasin D, 1/1,000 the volume of LPC stock, and 1/50 the volume of 50x energy mix. Gently invert the tube to mix. Do not excessively pipet the extract.</li>
		<li>Keep the extract on ice and use it within 3 hr of preparation for best results.</li>
	</ol>
	</li>
</ol>
2. Preparation of Demembranated X. laevis Sperm (adapted from29)
Note: The procedure volumes presented here are for up to 8 testes.
<ol>
	<li>Remove 0.05% benzocaine (10% benzocaine stock prepared in ethanol and diluted to 0.05% in frog tank water) from 4 &#176;C and warm to room temperature. Anesthetize and sacrifice male X. laevis frogs in benzocaine solution at room temperature for 20 - 30 min. Ensure death by absence of heartbeat by examining and feeling the chest area, by lack of response to mechanical stimulation, and/or by decapitation.</li>
	<li>Make a U-shaped incision along the abdomen. Remove the tubulated mass of yellow fat bodies. At the top of the kidneys, locate the testes, which are oval-shaped pale pink masses about 1 cm in length29. Excise the testes and roll them on blotting paper to remove blood and other tissue.</li>
	<li>Wash testes in Buffer T. Use a tight fitted pestle to mince testes with 1 ml of Buffer T in a 1.5 ml tube until homogenous (10 strokes or more). Centrifuge 5 - 10 sec in a mini centrifuge and collect the supernatant, removing large pieces of tissue. Repeat the centrifugation once more and collect the supernatant.</li>
	<li>Transfer the sperm-containing solution to a 15 ml plastic tube. Increase the volume to a total of 2 ml with Buffer T. Centrifuge at 1,411 x g for 10 min at 16 &#176;C to pellet the sperm.</li>
	<li>Remove the supernatant and resuspend the pellet in 500 &#181;l of Buffer T. Centrifuge at 1,411 x g for 10 min at 16 &#176;C. Repeat this step once or twice as needed to clean the pellet of somatic and red blood cells.</li>
	<li>Resuspend the pellet in 100 &#181;l Buffer T and 300 &#181;l Buffer S. Incubate at room temperature for 5 min. 
	Note: Buffer S contains lysolecithin, which is responsible for demembranation.</li>
	<li>Add three volumes of Buffer R (prepared fresh before use). Invert tube exactly once. Centrifuge at 1,411 x g for 15 min at 16 &#176;C.</li>
	<li>Resuspend the pellet in 400 &#181;l Buffer R and then add an additional 2 ml of Buffer R. Centrifuge at 1,411 x g for 15 min at 16 &#176;C.</li>
	<li>Resuspend the pellet in 50 &#181;l Buffer T.</li>
	<li>Dilute 1 &#181;l demembranated sperm nuclei with 9 &#181;l Buffer T. Apply this dilution to a hemocytometer. Count the number of thin S-shaped sperm nuclei in one large 4 x 4 grid, and multiply that number by 100 to obtain the concentration of the stock in sperm nuclei per &#181;l.</li>
	<li>Dilute the stock to 100,000 sperm nuclei per &#181;l with Buffer T, resulting in a 200x stock. Prepare 3 - 5 &#181;l aliquots. Flash freeze in liquid nitrogen and store at -80 &#176;C.</li>
</ol>
3. Nuclear Assembly
<ol>
	<li>Supplement 100 &#181;l of egg extract with 1.5 &#181;l 35.5 mM cycloheximide (final concentration ~ 0.5 mM), 6 &#181;l 20x calcium stock solution (final concentration ~ 0.6 mM), and 0.7 &#181;l 200x sperm (final concentration ~ 700 sperm nuclei/&#181;l). Scale the reaction volume up or down as necessary. Invert or gently tap to mix. 
	&#8203;Note: Extract cycled from metaphase into interphase provides a more robust and reliable platform for nuclear assembly.</li>
	<li>Incubate in a water bath at 16 - 20 &#176;C for 90 min. Mix by gently tapping every 15 min to ensure nuclei remain suspended in the extract.</li>
	<li>Monitor progress of nuclear assembly at 45 min by preparing a squash as follows.
	<ol>
		<li>Pipet 2 &#181;l of extract onto a glass slide and add 2 &#181;l nucleus fix.</li>
		<li>Overlay with a 22 mm2 coverslip. Image on an epifluorescence microscope using water, oil, or air 20 - 100X objectives and a DAPI filter. 
		&#8203;Note: Nuclei can be visualized by bright-field imaging, but are much easier to visualize by fluorescence.</li>
		<li>Use nuclei immediately or flash freeze aliquots in liquid nitrogen and store at -80 &#176;C. 
		Note: Adding 4% glycerol to the nuclei prior to freezing can help to maintain their integrity. Frozen nuclei are generally still viable and capable of nuclear import, however the freezing process can lead to disrupted or more structurally fragile nuclei. Nuclei up to a year after freezing can be used.</li>
	</ol>
	</li>
</ol>
4. Preparation of X. laevis Embryo Extract
<ol>
	<li>Induce female X. laevis frogs to lay eggs as described in 1.1 and 1.2.</li>
	<li>Isolate testes from male X. laevis frogs as described in 2.2. Submerge testes in L15 media supplemented with 50 IU/ml of penicillin and streptomycin in a glass 35 x 10 mm Petri dish. Store at 4 &#176;C for up to two weeks.</li>
	<li>Collect and fertilize eggs.
	<ol>
		<li>Collect freshly laid eggs by holding frogs over a glass reusable Petri dish and promote egg laying by applying gentle pressure to the lower back near the cloaca.&#160;Squeezing the frog too hard can result in injury, leading to a bloated abdomen and requiring euthanasia29.</li>
		<li>Prop the dish at an approximate 45&#176; angle, allow eggs to collect in the edge of the dish, remove all excess buffer, and add enough 1/3x MMR to barely cover the eggs. Fertilize eggs within 15 min of collection.</li>
		<li>Use a tightly fitted pestle to macerate and homogenize 1/4 of a testis in a 1.5 ml tube with 500 &#181;l of high salt Modified Barth&#39;s Saline (High Salt MBS). Add macerated testes to the eggs and allow for fertilization to occur at room temperature for 15 - 20 min, then flood eggs with 1/3x MMR. 
		&#8203;Note: Effectiveness of testes decreases with storage time, therefore more testes tissue may be required for successful fertilization when using testes that have been stored for more than one week.</li>
		<li>Confirm fertilization by checking for contraction of the dark pigmented animal pole and by rotation of the embryos with their animal poles facing up, usually about 20 - 30 min after addition of crushed testes. Expect the first cell cleavage to occur within 90 min.</li>
		<li>Using a wide bore glass pipette diligently remove dead (white and puffy or uneven distribution of yolk and pigment), lysed, or unfertilized eggs (i.e., not cleaved), as they will rapidly induce death in neighboring embryos.</li>
		<li>Anywhere from 1 - 2.5 hr post-fertilization, dejelly embryos in 2 - 3% cysteine dissolved in 1/3 x MMR and adjusted to pH 7.9 with 10 N KOH. Perform two buffer changes that are each 3 - 4 min. Thoroughly wash the embryos with 6 - 10 brief changes of 1/3x MMR to remove all traces of cysteine. 
		Note: Dejellied embryos have a vitelline membrane and are not as fragile as dejellied eggs.</li>
	</ol>
	</li>
	<li>Using a wide bore glass pipette, transfer healthy fertilized (i.e., cleaved) embryos to a Petri dish containing fresh 1/3x MMR and allow them to develop to the desired stage at RT or, if slower development is preferred, 16 &#176;C. Continue to remove dead or lysed embryos indicated by lack of first division or white puffy appearance.</li>
	<li>Verify the stage of the embryos by checking for nearly complete closure of the blastopore and by comparing to drawings of staged embryos available in reference materials24. 
	Note: Embryos cultured at 16 &#176;C will reach stage 11.5 - 12 in approximately 12 hr.</li>
	<li>Incubate stage 11.5 - 12 embryos in fresh 1/3x MMR supplemented with 0.5 mM cycloheximide for 1 hr at RT, to arrest embryos in late interphase.</li>
	<li>Transfer with wide bore glass or plastic pipette a minimum of 15 (preferably 30 or more) interphase-arrested embryos to a microcentrifuge tube. 
	Note: 15 embryos are sufficient to produce approximately 20 &#181;l of extract. Scale up as needed.</li>
	<li>Add 1 ml egg lysis buffer (ELB) supplemented with 1 &#181;l of LPC stock. Gently invert to wash embryos, allow embryos to fall to the bottom of tube, and remove the buffer. Repeat this wash step two more times.</li>
	<li>Resuspend embryos in 500 &#181;l of ELB plus 0.5 &#181;l of LPC stock, 5 &#181;l of 19.7 mM cycloheximide, and 5 &#181;l of 35.5 mM cytochalasin D (to inhibit actin polymerization). Centrifuge at 200 x g for 1 min in a tabletop microcentrifuge.</li>
	<li>Remove excess buffer and use a pestle to thoroughly crush the embryos. Centrifuge in a swinging bucket rotor for 10 min at 10,000 x g and 16 &#176;C.</li>
	<li>Puncture the lipid layer from the top with a 200 &#181;l pipet tip. With a clean 200 &#181;l pipet tip, remove the middle cytoplasmic amber-colored layer to an appropriate sized tube.</li>
	<li>Supplement embryo extract with 1/50 the volume of 50x energy mix (to provide an ATP regeneration system), 1/100 the volume of 35.5 mM cyclohexamide, 1/500 the volume of 19.7 mM cytochalasin D, and 1/1,000 the volume of LPC stock.</li>
	<li>Prepare a squash as described in 3.3 to visualize endogenous embryonic nuclei in the extract. 
	Note: Embryo extract can be frozen with the endogenous nuclei at this point. Aliquot to reduce freeze/thaw cycles and store at -80 &#176;C.</li>
	<li>To remove nuclei from the embryo extract, dilute the extract with an equal volume of ELB containing 1/50 the volume of 50x energy mix, 1/100 the volume of 35.5 mM cyclohexamide, 1/500 the volume of 19.7 mM cytochalasin D, and 1/1,000 the volume of LPC stock. Centrifuge in a swinging bucket rotor at 17,000 x g for 15 min at 16 &#176;C.</li>
	<li>Collect the supernatant being careful to avoid any remaining lipid at the top. Do not disturb the pelleted nuclei at the bottom of the tube. Prepare a squash as described in 3.3 to ensure that most nuclei have been removed. Use the extract fresh or aliquot and store at -80 &#176;C.</li>
	<li>To heat inactivate embryo extract for control experiments, heat 30 - 100 &#181;l aliquots of extract at 56 &#176;C for 30 min using a thermal cycler. Allow heat inactivated extract to return to room temperature prior to use.</li>
</ol>
5. Nuclear Shrinking Assay and Immunofluorescence
<ol>
	<li>Isolate nuclei assembled in egg extract by diluting 25 - 150 &#181;l of pre-assembled nuclei in 1 ml ELB in a 1.5 ml tube. Centrifuge at 1,600 x g for 3 min at 4 &#176;C in a tabletop microcentrifuge. Remove buffer, being careful not to disturb the fragile pellet of nuclei at the bottom of the tube.</li>
	<li>Resuspend nuclei in a volume of embryo extract equal to the original volume of egg extract used in 5.1. Use ELB or heat inactivated extract for control reactions, as appropriate.</li>
	<li>Gently tap the tube to break up the pellet and resuspend the nuclei. Incubate at room temperature for 90 min, gently tapping the tube to mix every 15 - 30 min. Note: Most of the nuclear shrinking occurs within the first 30 min.</li>
	<li>Monitor progress of nuclear shrinking by preparing a squash.
	<ol>
		<li>Pipet 2 &#181;l of extract onto a glass slide and add 2 &#181;l nucleus fix.</li>
		<li>Overlay with a 22 mm2 coverslip. Image on an epifluorescence microscope.</li>
	</ol>
	</li>
	<li>Tap the tube to resuspend the nuclei just prior to adding 500 &#181;l of fixative consisting of ELB, 15% glycerol, and 2.6% paraformaldehyde. Invert immediately, and place the tube on a rotator for 15 min at room temperature.</li>
	<li>Prepare spin down tubes by outfitting 15 ml round bottom glass tubes with round bottom plastic inserts (design and schematics available upon request). Add 5 ml of nuclear cushion buffer. Drop a 12 mm circular coverslip into the tube, being sure it lays flat on top of the plastic insert.</li>
	<li>Gently layer the solution containing fixed nuclei on top of the nuclear cushion using a wide-bore pipet tip. Centrifuge in a swinging bucket rotor for 15 min at 1,000 x g and 16 &#176;C.</li>
	<li>Use an aspirator to remove all of the buffer and pull the plastic insert to the top of the tube. Remove the coverslip with a pair of fine forceps, being careful to note the side of the coverslip onto which the nuclei were spun. Post-fix in cold methanol (stored at -20&#176; C) for 5 min at RT.</li>
	<li>Transfer the coverslip nucleus side up onto a sheet of plastic paraffin film lining a large plastic petri dish. Place wet disposable wipes along the side of the dish to prevent dehydration.</li>
	<li>Rehydrate nuclei on the coverslip with 500 &#181;l of PBS-NP40 (1x PBS plus 0.1% NP40) and aspirate after 5 - 10 sec.</li>
	<li>Carefully layer 75 &#181;l of PBS-3% Bovine Serum Albumin (BSA) onto the coverslip. Allow to block 1 hr at room temperature or overnight at 4 &#176;C.</li>
	<li>Remove blocking solution, and incubate with primary antibody (e.g., mAb414 against the nuclear pore complex, 1:1,000) diluted in PBS-3% BSA for 1 hr at room temperature or overnight at 4 &#176;C. Wash with five immediate changes of PBS-NP40.</li>
	<li>Incubate with secondary antibody diluted in PBS-3% BSA for 1 hr at RT. Wash with five immediate changes of PBS-NP40.</li>
	<li>Incubate with 10 &#181;g/ml Hoechst diluted in PBS-3% BSA for 5 min at RT. Wash five times with PBS-NP40. Remove all excess buffer.</li>
	<li>Mount the coverslip onto a slide with 5 &#181;l antifade mounting medium. Seal with clear nail polish. Image immediately using an epifluorescence microscope with 20 - 100x objectives or store at 4 &#176;C for later imaging. 
	Note: Perform quantification as previously described23.</li>
</ol>

<ol>
	<li>Conklin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+size+and+nuclear+size.">Cell size and nuclear size.</a> J. Exp. Embryol. 12, 1-98 (1912).</li><li>Wilson, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Cell in Development and Heredity. , 727-733 (1925).</li><li>Levy, D. L., 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=Nuclear+size+is+regulated+by+importin+alpha+and+Ntf2+in+Xenopus.">Nuclear size is regulated by importin alpha and Ntf2 in Xenopus.</a> Cell. 143 (2), 288-298 (2010).</li><li>Chan, Y. H., Marshall, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaling+properties+of+cell+and+organelle+size.">Scaling properties of cell and organelle size.</a> Organogenesis. 6 (2), 88-96 (2010).</li><li>Walters, A. D., Bommakanti, A., Cohen-Fix, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaping+the+nucleus:+Factors+and+forces.">Shaping the nucleus: Factors and forces.</a> J Cell Biochem. 113 (9), 2813-2821 (2012).</li><li>Webster, M., Witkin, K. L., Cohen-Fix, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sizing+up+the+nucleus:+nuclear+shape,+size+and+nuclear-envelope+assembly.">Sizing up the nucleus: nuclear shape, size and nuclear-envelope assembly.</a> J Cell Sci. 122 (Pt 10), 1477-1486 (2009).</li><li>Edens, L. J., White, K. H., Jevtic, P., Li, X., Levy, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+size+regulation:+from+single+cells+to+development+and+disease.">Nuclear size regulation: from single cells to development and disease.</a> Trends Cell Biol. 23 (4), 151-159 (2013).</li><li>Jevtic, P., Edens, L. J., Vukovic, L. D., Levy, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sizing+and+shaping+the+nucleus:+mechanisms+and+significance.">Sizing and shaping the nucleus: mechanisms and significance.</a> Curr Opin Cell Biol. 28, 16-27 (2014).</li><li>Levy, D. L., 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=Mechanisms+of+intracellular+scaling.">Mechanisms of intracellular scaling.</a> Annu Rev Cell Dev Biol. 28, 113-135 (2012).</li><li>Vukovic, L. D., Jevtic, P., Edens, L. J., Levy, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+mechanisms+and+functions+of+nuclear+size+regulation.">New insights into the mechanisms and functions of nuclear size regulation.</a> Int Rev Cell Mol Biol. 322, 1-59 (2016).</li><li>Jevtic, P., Levy, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+size+scaling+during+Xenopus+early+development+contributes+to+midblastula+transition+timing.">Nuclear size scaling during Xenopus early development contributes to midblastula transition timing.</a> Curr Biol. 25 (1), 45-52 (2015).</li><li>Hara, Y., Iwabuchi, M., Ohsumi, K., Kimura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranuclear+DNA+density+affects+chromosome+condensation+in+metazoans.">Intranuclear DNA density affects chromosome condensation in metazoans.</a> Mol Biol Cell. 24 (15), 2442-2453 (2013).</li><li>Zink, D., Fischer, A. H., Nickerson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+structure+in+cancer+cells.">Nuclear structure in cancer cells.</a> Nat Rev Cancer. 4 (9), 677-687 (2004).</li><li>Jevtic, P., Levy, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+nuclear+size+regulation+in+model+systems+and+cancer.">Mechanisms of nuclear size regulation in model systems and cancer.</a> Adv Exp Med Biol. 773, 537-569 (2014).</li><li>Chow, K. H., Factor, R. E., Ullman, 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=The+nuclear+envelope+environment+and+its+cancer+connections.">The nuclear envelope environment and its cancer connections.</a> Nat Rev Cancer. 12 (3), 196-209 (2012).</li><li>Blom, J. H., Ten Kate, F. J., Schroeder, F. H., van der Heul, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphometrically+estimated+variation+in+nuclear+size.+A+useful+tool+in+grading+prostatic+cancer.">Morphometrically estimated variation in nuclear size. A useful tool in grading prostatic cancer.</a> Urol Res. 18 (2), 93-99 (1990).</li><li>Dey, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+nucleus:+morphology+and+beyond.">Cancer nucleus: morphology and beyond.</a> Diagn Cytopathol. 38 (5), 382-390 (2010).</li><li>Cross, M. K., Powers, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+about+cancer+from+frogs:+analysis+of+mitotic+spindles+in+Xenopus+egg+extracts.">Learning about cancer from frogs: analysis of mitotic spindles in Xenopus egg extracts.</a> Dis Model Mech. 2 (11-12), 541-547 (2009).</li><li>Voeltz, G. K., Prinz, W. A., Shibata, Y., Rist, J. M., 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=A+class+of+membrane+proteins+shaping+the+tubular+endoplasmic+reticulum.">A class of membrane proteins shaping the tubular endoplasmic reticulum.</a> Cell. 124 (3), 573-586 (2006).</li><li>Chan, R. C., Forbes, D. I. <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+study+of+nuclear+assembly+and+nuclear+import+using+Xenopus+egg+extracts.">In vitro study of nuclear assembly and nuclear import using Xenopus egg extracts.</a> Methods Mol Biol. 322, 289-300 (2006).</li><li>Edens, L. J., Levy, D. L., Kloc, M., Kubiak, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Xenopus Development. , 325-345 (2014).</li><li>Levy, D. L., 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=Biological+Scaling+Problems+and+Solutions+in+Amphibians.">Biological Scaling Problems and Solutions in Amphibians.</a> Cold Spring Harb Perspect Biol. 8 (1), a019166 (2016).</li><li>Edens, L. J., Levy, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cPKC+regulates+interphase+nuclear+size+during+Xenopus+development.">cPKC regulates interphase nuclear size during Xenopus development.</a> J Cell Biol. 206 (4), 473-483 (2014).</li><li>Nieuwkoop, P. D., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal Table of Xenopus laevis (Daudin). , (1967).</li><li>Newport, J., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+major+developmental+transition+in+early+Xenopus+embryos:+II.+Control+of+the+onset+of+transcription.">A major developmental transition in early Xenopus embryos: II. Control of the onset of transcription.</a> Cell. 30 (3), 687-696 (1982).</li><li>Newport, J., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+major+developmental+transition+in+early+Xenopus+embryos:+I.+characterization+and+timing+of+cellular+changes+at+the+midblastula+stage.">A major developmental transition in early Xenopus embryos: I. characterization and timing of cellular changes at the midblastula stage.</a> Cell. 30 (3), 675-686 (1982).</li><li>Maresca, T. J., 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=Methods+for+studying+spindle+assembly+and+chromosome+condensation+in+Xenopus+egg+extracts.">Methods for studying spindle assembly and chromosome condensation in Xenopus egg extracts.</a> Methods Mol Biol. 322, 459-474 (2006).</li><li>Hannak, E., 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=Investigating+mitotic+spindle+assembly+and+function+in+vitro+using+Xenopus+laevis+egg+extracts.">Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts.</a> Nat Protoc. 1 (5), 2305-2314 (2006).</li><li>Murray, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+extracts.">Cell cycle extracts.</a> Methods Cell Biol. 36, 581-605 (1991).</li><li>Khan, M. 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=Quantitative+alterations+in+nuclear+structure+predict+prostate+carcinoma+distant+metastasis+and+death+in+men+with+biochemical+recurrence+after+radical+prostatectomy.">Quantitative alterations in nuclear structure predict prostate carcinoma distant metastasis and death in men with biochemical recurrence after radical prostatectomy.</a> Cancer. 98 (12), 2583-2591 (2003).</li><li>Tan, P. H., Goh, B. B., Chiang, G., Bay, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+nuclear+morphometry+with+pathologic+parameters+in+ductal+carcinoma+in+situ+of+the+breast.">Correlation of nuclear morphometry with pathologic parameters in ductal carcinoma in situ of the breast.</a> Mod Pathol. 14 (10), 937-941 (2001).</li><li>Zeimet, A. G., 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=DNA+ploidy,+nuclear+size,+proliferation+index+and+DNA-hypomethylation+in+ovarian+cancer.">DNA ploidy, nuclear size, proliferation index and DNA-hypomethylation in ovarian cancer.</a> Gynecol Oncol. 121 (1), 24-31 (2011).</li><li>Theerthagiri, G., Eisenhardt, N., Schwarz, H., Antonin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nucleoporin+Nup188+controls+passage+of+membrane+proteins+across+the+nuclear+pore+complex.">The nucleoporin Nup188 controls passage of membrane proteins across the nuclear pore complex.</a> J Cell Biol. 189 (7), 1129-1142 (2010).</li><li>Wuhr, M., 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=Evidence+for+an+upper+limit+to+mitotic+spindle+length.">Evidence for an upper limit to mitotic spindle length.</a> Curr Biol. 18 (16), 1256-1261 (2008).</li><li>Loughlin, R., Wilbur, J. D., McNally, F. J., Nedelec, F. J., 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=Katanin+contributes+to+interspecies+spindle+length+scaling+in+Xenopus.">Katanin contributes to interspecies spindle length scaling in Xenopus.</a> Cell. 147 (6), 1397-1407 (2011).</li><li>Wilbur, J. D., 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=Mitotic+spindle+scaling+during+Xenopus+development+by+kif2a+and+importin.">Mitotic spindle scaling during Xenopus development by kif2a and importin.</a> eLife. 2, e00290 (2013).</li><li>Kieserman, E. 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=Mitotic+chromosome+size+scaling+in+Xenopus.">Mitotic chromosome size scaling in Xenopus.</a> Cell Cycle. 10 (22), 3863-3870 (2011).</li><li>Maresca, T. J., Freedman, B. S., 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=Histone+H1+is+essential+for+mitotic+chromosome+architecture+and+segregation+in+Xenopus+laevis+egg+extracts.">Histone H1 is essential for mitotic chromosome architecture and segregation in Xenopus laevis egg extracts.</a> J Cell Biol. 169 (6), 859-869 (2005).</li><li>MacCallum, D. E., Losada, A., Kobayashi, R., Hirano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ISWI+remodeling+complexes+in+Xenopus+egg+extracts:+identification+as+major+chromosomal+components+that+are+regulated+by+INCENP-aurora+B.">ISWI remodeling complexes in Xenopus egg extracts: identification as major chromosomal components that are regulated by INCENP-aurora B.</a> Mol Biol Cell. 13 (1), 25-39 (2002).</li><li>Goehring, N. W., Hyman, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle+growth+control+through+limiting+pools+of+cytoplasmic+components.">Organelle growth control through limiting pools of cytoplasmic components.</a> Curr Biol. 22 (9), R330-R339 (2012).</li><li>Du Toit, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development:+Honey,+I+shrunk+the+nucleus.">Development: Honey, I shrunk the nucleus.</a> Nat Rev Mol Cell Biol. 15 (10), 633 (2014).</li><li>Buchner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+protein+kinase+C+in+the+regulation+of+cell+growth+and+in+signalling+to+the+cell+nucleus.">The role of protein kinase C in the regulation of cell growth and in signalling to the cell nucleus.</a> J Cancer Res Clin Oncol. 126 (1), 1-11 (2000).</li><li>Mochly-Rosen, D., Das, K., Grimes, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C,+an+elusive+therapeutic+target.">Protein kinase C, an elusive therapeutic target.</a> Nat Rev Drug Discov. 11 (12), 937-957 (2012).</li></ol>

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...

The sizes of cellular organelles typically scale with the size of the cell, and this has been perhaps best documented for the scaling of nuclear size with cell size1-10. This is particularly true during embryogenesis and cell differentiation, when dramatic reductions in both cell and nuclear size are often observed11,12. Furthermore, altered nuclear size is a key parameter in cancer diagnosis and prognosis13-17. Mechanisms that contribute to nuclear size regulation are largely unknown, in part due to the complexity and essential nature of nuclear structure and function. The method described here was developed as an in vitro assay for nuclear size scaling that is amenable to biochemical manipulation and elucidation of mechanisms of nuclear size regulation.
Xenopus laevis egg extract is a well-established system to recapitulate and study complex cellular processes in an in vitro context. These extracts have revealed new fundamental information about several cellular processes including the assembly and function of the mitotic spindle, endoplasmic reticulum, and nucleus18-22. One key advantage to the extract system is that X. laevis egg extracts represent a nearly undiluted cytoplasm whose composition can be easily altered, for instance through addition of recombinant proteins or immunodepletion. Furthermore, one is able to manipulate essential processes by employing treatments that might otherwise be lethal in an in vivo context. Modifications of the egg extract procedure allow for isolation of extracts from X. laevis embryos rather than eggs, and these embryo extracts are equally amenable to biochemical manipulation23. During X. laevis development, the single-cell fertilized embryo (~1 mm diameter) undergoes a series of twelve rapid cell divisions (stages 1 - 8) to generate several thousand 50 &#181;m diameter and smaller cells, reaching a developmental stage termed the midblastula transition (MBT) or stage 824-26. The MBT is characterized by the onset of zygotic transcription, cell migration, asynchronous cell divisions, acquisition of gap phases, and establishment of nuclear steady-state sizes rather than continual nuclear expansion as in the pre-MBT embryo. From stage 4 to gastrulation (stages 10.5 - 12), the volume of individual nuclei decreases by more than 10-fold11.
Here, the goal is to identify mechanisms responsible for these reductions in nuclear size during developmental progression. The approach is to first assemble nuclei in X. laevis egg extract and to isolate those nuclei from the egg cytoplasm/extract. These nuclei are then resuspended in cytoplasm from late gastrula stage embryos. After an incubation period, the nuclei from egg extract become smaller in late stage embryo extract. We reasoned that this would be a useful assay for identifying cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. Using this assay, coupled with in vivo validation, we demonstrated that protein kinase C (PKC) contributes to developmental reductions in nuclear size in X. laevis23.

<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

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.

Assembly of Nuclei in Egg Extract
The first steps of this protocol are to prepare X. laevis egg extract (Protocol 1) and demembranated sperm nuclei (Protocol 2). These reagents are then used to assemble nuclei de novo (Protocol 3). Figure 1 shows some representative data. Addition of calcium drives the meiotically arrested egg extract into interphase, and the cycloheximide keep...

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.

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 ...

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Biology

8.3K Views.  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.

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

Developmental Biology

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.

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.

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 ...

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.

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.

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 ...

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.

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.

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.

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

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