This work was supported by the German Research Foundation and the ERC (AN377/3-2 and 309528 CHROMDECON to W.A.) and a PhD Fellowship of the Boehringer Ingelheim Fonds to A.K.S. Figure 1 &#38; 2 are reprinted from Developmental Cell 31(3), Magalska et al., RuvB-like ATPases function in chromatin decondensation at the end of mitosis, 305-318, 2014, with kind permission from Elsevier.

Mitotic Chromatin Cluster Isolation from HeLa Cells

1. Preparations

<ol>
	<li>Cell Culture Solutions
	<ol>
		<li>Prepare complete Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) by adding 10% fetal calf serum, 100 units/ml penicillin, 100 &#181;g/ml streptomycin and 2 mM glutamine to the DMEM. Prepare Phosphate buffer saline (PBS) containing 2.7 mM KCl, 137 mM NaCl, 10 mM Na2HPO42H2O and 2 mM KH2PO4 i...

<ol>
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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>Galy, 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=A+role+for+gp210+in+mitotic+nuclear-envelope+breakdown.">A role for gp210 in mitotic nuclear-envelope breakdown.</a> J Cell Sci. 121, 317-328 (2008).</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>Gillespie, P. J., Gambus, A., Blow, J. J. <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+use+of+Xenopus+egg+extracts+to+study+DNA+replication+and+chromatin+associated+proteins.">Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins.</a> Methods. 57, 203-213 (2012).</li><li>Gant, T. M., Wilson, K. 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+assembly.">Nuclear assembly.</a> Annu Rev Cell Dev Biol. 13, 669-695 (1997).</li><li>Magalska, 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=RuvB-like+ATPases+function+in+chromatin+decondensation+at+the+end+of+mitosis.">RuvB-like ATPases function in chromatin decondensation at the end of mitosis.</a> Developmental Cell. 31, 305-318 (2014).</li><li>Belmont, A. S. <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+structure+and+condensation.">Mitotic chromosome structure and condensation.</a> Curr Opin Cell Biol. 18, 632-638 (2006).</li><li>Lavoie, B. D., Tuffo, K. M., Oh, S., Koshland, D., Holm, C. <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+condensation+requires+Brn1p,+the+yeast+homologue+of+Barren.">Mitotic chromosome condensation requires Brn1p, the yeast homologue of Barren.</a> Mol Biol Cell. 11, 1293-1304 (2000).</li><li>Philpott, A., Leno, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleoplasmin+remodels+sperm+chromatin+in+Xenopus+egg+extracts.">Nucleoplasmin remodels sperm chromatin in Xenopus egg extracts.</a> Cell. 69, 759-767 (1992).</li><li>Philpott, A., Leno, G. H., Laskey, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+decondensation+in+Xenopus+egg+cytoplasm+is+mediated+by+nucleoplasmin.">Sperm decondensation in Xenopus egg cytoplasm is mediated by nucleoplasmin.</a> Cell. 65, 569-578 (1991).</li><li>Burglin, T. R., Mattaj, I. W., Newmeyer, D. D., Zeller, R., De Robertis, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+nucleoplasmin+from+Xenopus+laevis+oocytes+and+analysis+of+its+developmental+expression.">Cloning of nucleoplasmin from Xenopus laevis oocytes and analysis of its developmental expression.</a> Genes Dev. 1, 97-107 (1987).</li><li>Whitaker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+at+fertilization+and+in+early+development.">Calcium at fertilization and in early development.</a> Physiological reviews. 86, 25-88 (2006).</li><li>Gasser, S. M., Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methods+for+the+isolation+of+individual+and+clustered+mitotic+chromosomes.">Improved methods for the isolation of individual and clustered mitotic chromosomes.</a> Exp Cell Res. 173, 85-98 (1987).</li><li>Eisenhardt, N., Schooley, A., 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=Xenopus+in+vitro+assays+to+analyze+the+function+of+transmembrane+nucleoporins+and+targeting+of+inner+nuclear+membrane+proteins.">Xenopus in vitro assays to analyze the function of transmembrane nucleoporins and targeting of inner nuclear membrane proteins.</a> Methods Cell Biol. 122, 193-218 (2014).</li><li>Wignall, S. M., Deehan, R., 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=The+condensin+complex+is+required+for+proper+spindle+assembly+and+chromosome+segregation+in+Xenopus+egg+extracts.">The condensin complex is required for proper spindle assembly and chromosome segregation in Xenopus egg extracts.</a> J Cell Biol. 161, 1041-1051 (2003).</li><li>Forbes, D. J., Kirschner, M. W., Newport, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+formation+of+nucleus-like+structures+around+bacteriophage+DNA+microinjected+into+Xenopus+eggs.">Spontaneous formation of nucleus-like structures around bacteriophage DNA microinjected into Xenopus eggs.</a> Cell. 34, 13-23 (1983).</li><li>Hartl, P., Olson, E., Dang, T., Forbes, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+assembly+with+lambda+DNA+in+fractionated+Xenopus+egg+extracts:+an+unexpected+role+for+glycogen+in+formation+of+a+higher+order+chromatin+intermediate.">Nuclear assembly with lambda DNA in fractionated Xenopus egg extracts: an unexpected role for glycogen in formation of a higher order chromatin intermediate.</a> J Cell Biol. 124, 235-248 (1994).</li><li>Sandaltzopoulos, R., Blank, T., Becker, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+repression+by+nucleosomes+but+not+H1+in+reconstituted+preblastoderm+Drosophila+chromatin.">Transcriptional repression by nucleosomes but not H1 in reconstituted preblastoderm Drosophila chromatin.</a> EMBO J. 13, 373-379 (1994).</li><li>Ulbert, S., Platani, M., Boue, S., Mattaj, I. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+membrane+protein-DNA+interactions+required+early+in+nuclear+envelope+assembly.">Direct membrane protein-DNA interactions required early in nuclear envelope assembly.</a> J Cell Biol. 173, 469-476 (2006).</li><li>Zhang, C., Clarke, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin-independent+nuclear+envelope+assembly+induced+by+Ran+GTPase+in+Xenopus+egg+extracts.">Chromatin-independent nuclear envelope assembly induced by Ran GTPase in Xenopus egg extracts.</a> Science. 288, 1429-1432 (2000).</li><li>Paulson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+chromosome+clusters+from+metaphase-arrested+HeLa+cells.">Isolation of chromosome clusters from metaphase-arrested HeLa cells.</a> Chromosoma. 85, 571-581 (1982).</li><li>Ramadan, K., 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=Cdc48/p97+promotes+reformation+of+the+nucleus+by+extracting+the+kinase+Aurora+B+from+chromatin.">Cdc48/p97 promotes reformation of the nucleus by extracting the kinase Aurora B from chromatin.</a> Nature. 450, 1258-1262 (2007).</li></ol>

Xenopus laevis egg extracts are a very useful tool to faithfully reproduce cellular processes in vitro, and this system was successfully used in the characterization of cell cycle and cell division events 2,3,5,6,17. Due to large stores of nuclear components sequestered in the egg during oogenesis, egg extracts are an excellent source of cellular components. Compared to other approaches like RNAi on mammalian tissue cell lines or genetic manipulation, it offers several advantages: ...

Xenopus laevis egg extract is a powerful and widely applied tool to study complicated cellular events in the simplicity of a cell-free assay. Since their first description by Lohka &#38; Masui 1 they have been extensively used to study mitotic processes such as chromatin condensation 2, spindle assembly 3, nuclear envelope breakdown 4, but also nucleocytoplasmic transport 5 or DNA replication 6. The events taking place at the end of mitosis, necessary for reformation of the interphasic nucleus such as nuclear envelope reformation and nuclear pore complex reassembly are much less understood compared to the early mitotic events but can be similarly studied using Xenopus egg extract 7. We have recently established an assay based on Xenopus egg extract to study chromatin decondensation at the end of mitosis 8, an under-investigated process that awaits its detailed characterization.
In metazoans, chromatin is highly condensed at mitotic entry in order to perform faithfully segregation of the genetic material. To ensure that the chromatin is accessible for gene expression and DNA replication during interphase, it needs to be de-compacted at the end of mitosis. In vertebrates, chromatin is up to fifty-fold more compacted during mitosis compared to interphase 9, in contrast to yeasts where the mitotic compaction is usually much lower, e.g., only two-fold in S. cerevisiae 10. Vertebrate chromatin decondensation has been mostly studied in the context of sperm DNA reorganization after egg fertilization. A molecular mechanism, in which nucleoplasmin, an abundant oocyte protein, exchanges sperm-specific protamines to histones H2A and H2B stored in the egg. This process was also elucidated using Xenopus egg extract 11,12. However, the expression of nucleoplasmin is limited to oocytes 13 and mitotic chromatin does not contain these sperm-specific protamines. Therefore chromatin decondensation at the end of mitosis is nucleoplasmin independent 8.
For the in vitro decondensation reaction we employ extract generated from activated X.laevis eggs and chromatin clusters isolated from synchronized HeLa cells. Treatment of eggs with a calcium ionophore mimics the calcium release into the oocyte generated by sperm entry during fertilization. The calcium wave triggers the cell cycle resumption and the egg, arrested in the second metaphase of meiosis, progresses to the first interphase 14. Therefore, egg extracts prepared form activated eggs represent the mitotic exit/interphase state and are competent to induce events specific for mitotic exit like chromatin decondensation, nuclear envelope and pore complex reformation. For the isolation of mitotic chromatin clusters we used a slightly modified version of the protocol published by Gasser &#38; Laemmli 15, where chromosome clusters are released by lysis from HeLa cells synchronized in mitosis and isolated in polyamine containing buffers by gradient centrifugations.

<table>
	<tbody>
		<tr>
			<td>spermine tetrahydrochloride</td>
			<td>Fluka analytical</td>
			<td>85610-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>spermidine trihydrochloride</td>
			<td>Sigma&#160;</td>
			<td>S2501-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>high-purity digitonin</td>
			<td>Millipore</td>
			<td>300410-1GM</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Applichem</td>
			<td>A0999,0100</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>thymidine</td>
			<td>Calbiochem</td>
			<td>6060</td>
			<td></td>
		</tr>
		<tr>
			<td>nocodazole</td>
			<td>Calbiochem</td>
			<td>487928</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>37 % formaldehyde solution</td>
			<td>Roth</td>
			<td>7398-1</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>trypan blue solution (0.4%)</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>1,4-dithiothreitol (DTT)</td>
			<td>Roth</td>
			<td>6908.2</td>
			<td></td>
		</tr>
		<tr>
			<td>AEBSF hydrochloride</td>
			<td>Applichem</td>
			<td>A1421,0001</td>
			<td></td>
		</tr>
		<tr>
			<td>pepstatin</td>
			<td>Roth</td>
			<td>2936.1/2/3</td>
			<td></td>
		</tr>
		<tr>
			<td>leupeptin</td>
			<td>Roth</td>
			<td>CN334</td>
			<td></td>
		</tr>
		<tr>
			<td>aprotinin</td>
			<td>Roth</td>
			<td>A162.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll (colloidal silica particles solution)</td>
			<td>GE Healthcare</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>glutamine</td>
			<td>Gibco</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm&#178; tissue culture flasks</td>
			<td>Greiner Bio-one</td>
			<td>658175</td>
			<td></td>
		</tr>
		<tr>
			<td>heat-inactivated fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>10500-064</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer (40 mL tissue grinder)</td>
			<td>Wheaton</td>
			<td>357546</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>Assistent</td>
			<td>441/1</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Oak Ridge Centrifuge Tubes, polycarbonate (50 ml)</td>
			<td>Nalgene</td>
			<td>3118-0050</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;m cell strainer, nylon</td>
			<td>BD Falcon</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>cytochalasin B</td>
			<td>Applichem</td>
			<td>A7657,0010</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>cycloheximide</td>
			<td>Roth</td>
			<td>8682.3</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>L-cystein</td>
			<td>Merck</td>
			<td>1,028,381,000</td>
			<td></td>
		</tr>
		<tr>
			<td>hCG available as Ovogest</td>
			<td>MSD</td>
			<td>1431593</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSG available as Intergonan</td>
			<td>MSD</td>
			<td>1431015</td>
			<td></td>
		</tr>
		<tr>
			<td>A23187 (mixed calcium-magnesium-salt)</td>
			<td>Enzo</td>
			<td>ALX-450-002-M010</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>syringe needles (1.20 x 40 mm, 18 G x 1 1/2&#34;)</td>
			<td>Braun</td>
			<td>4665120</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Serva</td>
			<td>10920.03</td>
			<td></td>
		</tr>
		<tr>
			<td>GTP, 2 Na x 3 H20</td>
			<td>Roth</td>
			<td>K056.1/2/3/4</td>
			<td></td>
		</tr>
		<tr>
			<td>creatine phosphat disodium salt</td>
			<td>Calbiochem</td>
			<td>2380</td>
			<td></td>
		</tr>
		<tr>
			<td>creatine phosphokinase</td>
			<td>Sigma</td>
			<td>C3755-35KU</td>
			<td></td>
		</tr>
		<tr>
			<td>DMAP</td>
			<td>Sigma</td>
			<td>D2629-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI&#160;</td>
			<td>Roche</td>
			<td>10236276001</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>Sigma</td>
			<td>P-6148</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>centrifugation tubes for TLA 100 (7 x 10 mm, 5/16 x 13/16 in.)</td>
			<td>Beckman Coulter</td>
			<td>343775</td>
			<td></td>
		</tr>
		<tr>
			<td>&#34;Cell-Saver&#34; (tips with wide opening, 1000 &#181;L)</td>
			<td>Biozym</td>
			<td>729065</td>
			<td></td>
		</tr>
		<tr>
			<td>50 % glutaraldehyde solution, grade I</td>
			<td>Sigma alderich</td>
			<td>G7651-10 mL</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>0.1 % (w/v) poly-L-lysine solution</td>
			<td>Sigma</td>
			<td>P8920-100 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>flat-bottom tubes (6 mL, 16.0/55 mm)</td>
			<td>Greiner Bio-one</td>
			<td>145211</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield mounting medium</td>
			<td>Vector laboratories</td>
			<td>H1000</td>
			<td></td>
		</tr>
		<tr>
			<td>tubes for TLA120 (11 x 34 mm, 7/16 x 1 3/8 in.)</td>
			<td>Beckman Coulter</td>
			<td>343778</td>
			<td></td>
		</tr>
		<tr>
			<td>&#34;Cell-Saver&#34; (tips with wide opening, 200 &#181;L)</td>
			<td>Biozym</td>
			<td>729055</td>
			<td></td>
		</tr>
		<tr>
			<td>12 mm coverslips</td>
			<td>Thermo Scientific</td>
			<td>0784 #1</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Time dependence of the decondensation reaction
Figure 1 shows a typical time course of the decondensation assay. The cluster of chromosomes visible at the beginning of the reaction decondenses and merges into a single, round and smooth nucleus. When the egg extract is replaced by sucrose buffer the chromosome cluster remains condensed, which suggest that decondensation activity is present in the egg extract.
Chromatin decondensation is an energy dep...

Watch this Scientific Journal Video about A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis at JoVE.com

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

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 ...

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10.2K vistas.  Max Planck Society. 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.