Name: a cell free assay to study chromatin decondensation at the end of mitosis
Uploaded: 2015-12-19T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis at JoVE.com

Diese Arbeit wurde von der Deutschen Forschungsgemeinschaft und dem ERC (AN377 / 3-2 und 309.528 CHROMDECON zu WA) und ein PhD Fellowship of des Boehringer Ingelheim Fonds unterstützt werden, um AKS Abbildung 1 und 2 werden von Developmental Cell 31 (3), Magalska nachgedruckt et al., RuvB artigen ATPasen Funktion in Chromatindekondensation am Ende der Mitose, 305-318, 2014, mit freundlicher Genehmigung von Elsevier.

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The authors have nothing to disclose.

Xenopus laevis Eiextrakten sind ein sehr nützliches Werkzeug, um originalgetreu zu reproduzieren zellulären Prozessen in vitro, und dieses System wurde erfolgreich für die Charakterisierung einer Zellzyklus und Zellteilung Ereignisse 2, 3,5,6,17 verwendet. Aufgrund großer speichert der Kernkomponenten in dem Ei während der Oogenese sequestriert sind Eiextrakten eine ausgezeichnete Quelle für zelluläre Bestandteile. Im Vergleich zu anderen Ansätzen wie RNAi auf Säugergewebezelllinien oder Genmanipulation und bietet mehrere Vorteile: Der zellfreie System erlaubt das Studium zelluläre Prozesse, bei denen die zelluläre Lebensfähigkeit sonst Einschränkung darstellen. Außerdem Einzelschritte von komplexen Prozessen in einfachen Tests untersucht werden. Die hier vorgestellte Dekondensation Assay ermöglicht das Studium molekularer Mechanismen der postmitotischen Dekondensation ohne Störungen durch andere mitotische Ereignisse auf. Xenopus-Ei-Extrakte sind leicht zu manipulieren durch Verarmung bestimmter Proteineund die Zugabe von Inhibitoren oder mutierte Proteine ​​8. Zum Beispiel 2 zeigt das Ergebnis der Addition der nicht hydrolysierbare ATP oder GTP-Analoga ATPγS und GTP &amp; ggr; zu dem Dekondensation Assay. Durch Verdünnung und differentielle Zentrifugation von Xenopus Eiern Komponenten wie Membranen und Cytosol getrennt 16. In Abbildung 3 zeigt die Dekondensation Assay in Gegenwart oder Abwesenheit von Membranen durchgeführt. Schließlich können die zellfreien Assay auch verwendet, um neuartige Faktoren z identifizieren, durch eine Fraktionierung Ansatz. Unter Verwendung einer solchen Strategie, die wir identifiziert haben, die AAA + -ATPasen RuvBL1 / RuvBL2 als entscheidend Dekondensation Faktoren 8. In-vitro-Systeme auf der Basis X. laevis Eier wurden mit verschiedenen DNA-Matrizen verwendet wurden. Forbes et al zeigten, dass die Injektion von Phage λ-DNA in unbefruchteten X. laevis Eiern induzierte die Montage von Chromatin auf naked Phage λ-DNA. Als Injektion der viralen DNA aktiviert das Ei wurde die Anordnung von Chromatin durch Bildung eines Kerns artige Struktur 18 und in ähnlicher λ-Phagen-DNA, gefolgt kann in Kombination mit Eiextrakten zum Kern artige Strukturen in vitro 19 zu erzeugen. Magnetische Kügelchen mit DNA beschichtet wurden zur chromatinization von DNA 20 und die Rekrutierung von Kernmembran 21 sowie Montage einer Kernhülle zu studieren und Porenkomplexe 22, obwohl es offen bleibt, in welchem ​​Umfang dies glich einem gutgläubigen nuklearen Wiedermontageprozess . Die hier vorgestellte Protokoll ermöglicht Dekondensation isolierter mitotischen Chromatin-Cluster aus HeLa-Zellen unter Verwendung von Extrakt aus aktiviert Xenopus Eier erzeugt. Sie rekonstruiert gründlich Ereignisse, die zu einer Reformation eines interphasic Kern 8. Gegenüber dem weit angewendet Kernbaugruppe Reaktion verwendet, um die Bildung der Kernhülle zu studieren unddie Kernporenkomplexe am Ende der Mitose der mitotischen HeLa Dekondensation Assay Chromatin Clustern statt Sperma-DNA verwendet. Sperma-DNA kann in mitotischen Chromatin oder sogar einzelne Chromosomen nach der Inkubation mit dem Extrakt aus unbefruchteten und nicht aktivierten Eiern 3 hergestellt montiert werden. Wir entschieden uns, mitotische Cluster als Chromatin Quelle zu verwenden, um das Verfahren zu vereinfachen und Störungen von Chromatin-Kondensation zu vermeiden. Darüber hinaus wird die Herstellung des Eiextrakt geringfügig modifiziert: Bei der Chromatindekondensation niedriger Geschwindigkeit Extrakt durch zwei Hochgeschwindigkeitszentrifugation Schritte in Festwinkelrotoren gelöscht verwendet. Niedrige Drehzahl Extrakt kann für bis zu 6 Monate in flüssigem Stickstoff, ohne ihre Aktivität zu verlieren, gespeichert werden. Im Gegensatz dazu ist in den Kernbaugruppe Reaktionen Cytosol und schwebte Membranen werden aus niedriger Drehzahl Extrakte durch Verdünnung und Differenz Hochgeschwindigkeitszentrifugation vor möglichen Einfrieren erzeugt (siehe Eisenhardt et al. 16 für eine detaiLED-Protokoll). In unserem Testsystem, Zugabe von Membranen ermöglicht die Bildung einer geschlossenen Kernhülle einschließlich Kernporenkomplexe. Die resultierenden Kerne sind für die nukleare Import und Export 8 kompetent. So unterstützt das System sowohl Chromatindekondensation und Kernhülle Reformation. Interessanterweise ist Chromatindekondensation auch in Abwesenheit von Membranen (Abbildung 3). Allerdings Zugabe von Membranen führt zu etwas größeren Kernen. Höchstwahrscheinlich ist die Reformation der Kernhülle induziert einen Sekundär Dekondensation Schritt noch nicht definierten Mechanismen, die auf Kernimport abhängig ist. Zur Isolierung von Chromatin mitotischen Clustern aus HeLa-Zellen wurde eine modifizierte Version des von Gasser und Laemmli 15 etablierten Protokoll verwendet. Synchronisiert mitotischen Zellen werden in einem Puffer, der nicht-ionischen Detergens Digitonin und durch mechanische Kräfte lysiert. Das Chromatin ist als Cluster, die enthalten isoliertalle Chromosomen von einem Kern. Der entscheidende Unterschied gegenüber einzelnen Chromosom Isolationsprotokolle ist die Tatsache, dass die Zellen nicht hypotonisch gequollen, kühlt auf 4 ° C vor der Lyse. Dies verhindert die Trennung der einzelnen Chromosomen 15, 23. Im Vergleich zu der von JR Paulson 23 veröffentlichten Protokoll, die den Vorteil der Isolierung des gesamten Chromatin Cluster erkannt Gasser &amp; Laemmli verwendet EDTA enthaltenden Polyamin Puffer anstelle von Mg 2+ Puffern auf, die Aktivität von Kinasen, Nukleasen, Proteasen und Phosphatasen zu reduzieren und dadurch verringern die Menge von Protein und DNA-Modifikationen während des Isolationsverfahrens 15 auftritt. Zusätzlich mit einem kolloidalen Siliciumdioxidteilchen Gradienten während differentielle Zentrifugation reduziert stark zytoplasmatischen Verunreinigungen. Das Protokoll kann auch verwendet werden, um mitotische Chromatin Cluster aus Ovarien chinesischer Hamster und Maus-Zellen 15 zu isolieren. <pclass = &quot;jove_content&quot;&gt; Insgesamt unserem Protokoll getreu rekonstruiert Chromatindekondensation, wie es am Ende der Mitose geschieht. Die ATP-Abhängigkeit der in vitro Chromatindekondensation kann zumindest in der Beteiligung der RuvBL1 / 2, sondern erklärte auch ein weiterer Teil AAA + -ATPase, p97, die die mitotische Kinase Aurora B vom Chromatin während der mitotischen Ausfahrt 24 entfernt. Warum erfordert das Verfahren die GTP-Hydrolyse ist eine der offenen Fragen, die wir beantworten wollen mit diesem Setup.

Xenopus laevis Eiextrakt ist ein leistungsfähiges und breit angelegten Werkzeug komplizierte zelluläre Ereignisse in der Einfachheit eines zellfreien Assay zu studieren. Seit ihrer ersten Beschreibung durch Lohka &amp; Masui 1 wurden sie ausgiebig genutzt, um mitotische Prozesse wie Chromatinkondensation 2, Spindelanordnung 3, Kernhülle Aufteilung 4, aber auch nukleozytoplasmatischen Transport 5 oder DNA-Replikation 6 studieren. Die Veranstaltungen finden am Ende der Mitose für die Reformation der interphasic Kern erforderlich, wie beispielsweise Kernhülle Reformation und Kernporenkomplex Remontage sind viel weniger verstanden, den frühen mitotischen Ereignissen verglichen, sondern kann auf ähnliche Weise unter Verwendung von Xenopus Eiextrakt 7 untersucht werden. Wir haben vor kurzem einen Test basierend auf Xenopus-Ei-Extrakt zu Chromatindekondensation am Ende der Mitose 8 studieren gegründet, ein untersuchten Prozess, den ich erwartetts detaillierte Charakterisierung. In Metazoen, Chromatin ist stark an mitotischen Eintrag, um treu Trennung des genetischen Materials durchzuführen kondensiert. Um sicherzustellen, dass das Chromatin während der Interphase ist für die Genexpression und die DNA-Replikation zugänglich ist, muss es bei Ende der Mitose De-verdichtet werden. Bei Wirbeltieren ist Chromatin bis zu fünffach während der Mitose kompakteren gegenüber Interphase 9, in Gegensatz zu Hefen, wo der mitotischen Verdichtung ist in der Regel viel geringer, beispielsweise nur zwei-fach in S. cerevisiae 10. Vertebraten Chromatindekondensation wurde hauptsächlich im Zusammenhang mit der Spermien DNA Reorganisation nach Eibefruchtung sucht. Ein molekularer Mechanismus, bei dem Nucleoplasmin, eine reichliche Oozyte Protein tauscht spermienspezifische Protamine Histone H2A und H2B im Ei gespeichert. Dieses Verfahren wurde auch unter Verwendung von Xenopus-Ei-Extrakt 11, 12 erläutert. Jedoch bedeutet der Ausdruckvon Nucleoplasmin an Oozyten 13 und mitotischen Chromatin beschränkt nicht diese Spermien spezifische Protamine enthalten. Daher Chromatindekondensation am Ende der Mitose unabhängige Nucleoplasmin 8. Für die in vitro Dekondensation Reaktions beschäftigen wir Auszug aus aktiviert X. laevis Eiern und Chromatin-Cluster aus synchronisierten HeLa-Zellen isoliert generiert. Die Behandlung der Eier mit einem Calcium-Ionophor ahmt die Calcium-Freisetzung in die durch Spermien Eintrag bei der Befruchtung erzeugten Eizelle. Das Calciumwelle triggert den Zellzyklus wieder aufgenommen und das Ei, in der zweiten Metaphase der Meiose fest schreitet zu dem ersten Zwischenphase 14. Daher Ei Extrakte hergestellt Form aktiviert Eier stellen die mitotische exit / Interphasezustand und sind befugt, die spezifisch für mitotische Ausfahrt wie Chromatindekondensation, Kernhülle Ereignisse induzieren und Porenkomplex Reformation. Zur Isolierung von mitotischen chromatin Cluster verwendeten wir eine leicht modifizierte Version des von Gasser &amp; Laemmli 15, in dem Chromosom Cluster werden durch Lyse aus HeLa-Zellen in der Mitose synchronisiert und in Polyamin enthaltenden Puffern getrennt durch eine Gradienten-Zentrifugationen erfolgten Veröffentlichung Protokoll.

<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&amp;nbsp;</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&amp;sup2; 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>&amp;nbsp;Oak Ridge Centrifuge Tubes, polycarbonate (50 ml)</td><td>Nalgene</td><td>3118-0050</td><td></td></tr><tr><td>100 &amp;micro;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&quot;)</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 H&lt;sub&gt;2&lt;/sub&gt;0</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&amp;nbsp;</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>&quot;Cell-Saver&quot; (tips with wide opening, 1,000 &amp;micro;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>&quot;Cell-Saver&quot; (tips with wide opening, 200 &amp;micro;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.

Zeitabhängigkeit der Dekondensation Reaktions Figur 1 zeigt einen typischen zeitlichen Verlauf der Dekondensation Assay. Die Gruppe von Chromosomen zu Beginn der Reaktion sichtbar decondenses und geht in einem einzigen, runden und glatten Kern. Wenn das Ei-Extrakt wird durch Saccharose ersetzt puffern das Chromosom Cluster bleibt Kondenswasser, die vorschlagen, dass Dekondensation Aktivität im Ei-Extrakt vorhanden ist. Chromatindekondensation ist ein energieabhängiger Prozess Die in vitro Dekondensation Reaktion kann bequem manipuliert werden kann zum Beispiel durch Zusatz von Inhibitoren. In der in Figur 2 gezeigte Experiment die nicht hydrolysierbare ATP oder GTP-Analoga oder ATPγS GTPyS wurden zu der Reaktion gegeben. Beide hemmen die Dekondensation zeigt, dass es ein ATP und GTP abhängig, aktiver Prozess (Abbildung 2). Die Dekondensation Assay wurde in Gegenwart oder Abwesenheit von Membranen (3) durchgeführt. Bitte beachten Sie, dass in beiden Bedingungen Chromatin erfährt Dekondensation jedoch Zugabe von Membranen führt zu größeren Kernen. Wahrscheinlich Reformation der Kernhülle induziert einen Sekundär Dekondensation Schritt von einem weiteren Mechanismus abhängig Kerntransport. <img alt="Abbildung 1" src="/files/ftp_upload/53407/53407fig1.jpg" /> Abbildung 1. Zeitlicher Verlauf der th e in vitro Dekondensation Reaktion. Mitotische Chromatin-Cluster aus HeLa-Zellen wurden mit interphasic Xenopus-Ei-Extrakt inkubiert. Proben wurden zu den angegebenen Zeitpunkten mit 4% PFA und 0,5% Glutaraldehyd fixiert, mit DAPI gefärbt und durch Co analysiert<html> nfocal Mikroskopie. Wieder gedruckt von Magalska et al. 8. Maßstabsleiste ist 5 um. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53407/53407fig1large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 2" src="/files/ftp_upload/53407/53407fig2.jpg" /> Abbildung 2 Chromatindekondensation erfordert ATP und GTP-Hydrolyse. Chromatindekondensation wurde in Gegenwart von 10 mM ATPγS, 10 mM GTP &amp; ggr; oder Kontrollpuffer durchgeführt. Proben wurden mit 4% PFA und 0,5% Glutaraldehyd bei angegebenen Zeitpunkten fixiert und durch konfokale Mikroskopie analysiert. Wieder gedruckt von Magalska et al. 8. Maßstabsleiste ist 5 um. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53407/53407fig2large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> Abbildung 3 &quot;src =&quot; / files / ftp_upload / 53.407 / 53407fig3.jpg &quot;/&gt; Abbildung 3 Chromatindekondensation in Gegenwart und in Abwesenheit von Membranen. Chromatindekondensation wurde in Abwesenheit (A) oder Gegenwart (B) der Flotation gereinigten Membranen für 120 min durchgeführt. Proben wurden mit 4% PFA und 0,5% Glutaraldehyd fixiert und durch konfokale Mikroskopie analysiert. Chromatin mit DAPI, Membranen mit DiIC 18 (1,1&#39;-Dioctadecyl-3,3,3 &#39;, 3&#39;-tetramethylindocarbocyanin Perchlorat) gefärbt. Maßstabsleiste ist 5 um. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53407/53407fig3large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a>

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.

Ein zellfreien Assay zu Chromatindekondensation Studieren am Ende der Mitose

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

a-cell-free-assay-to-study-chromatin-decondensation-at-end

Nanyang Technological University

Research

JoVE Journal

Biology

10.2K Aufrufe.  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.

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

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

Chromatin verteilt die Vorbereitungen für die Analyse der Maus Eizelle Progression von Prophase, Metaphase II

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for ...

Forschung

Genetik

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

Langfristige Leben-Cell Imaging zu Zelle Schicksal als Reaktion auf Paclitaxel bewerten

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

Krebsforschung

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

Chromatin Immunoprecipitation Assay mit Mikrokokken-Nukleasen in Säugetierzellen

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Live Cell Imaging der Segregation der Chromosomen während der Mitose

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Entwicklungsbiologie

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

Chromatin Immunopräzipitation (ChIP) zur Assay Dynamische Histonmodifikationen in Activated Gene Expression in menschlichen Zellen

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Biologie

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

Chromatin Interaction Analysis gepaart mit End-Tag-Sequenzierung (Chia-PET) zur Abbildung von Chromatin-Wechselwirkungen zu verstehen und Transkriptionsregulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

Studieren Proteolyse von Cyclin B an der Single Cell Level in Whole Cell Populationen

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

Zwei-und dreidimensionale Live Cell Imaging der DNA-Reparatur-Proteine

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

Isolierung von spezifischen genomischen Regionen und Identifizierung von assoziierten Moleküle durch enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often not seen by imaging fixed cells. Budding yeast has proved to be an important model organism to study meiotic chromosome segregation because many meiotic genes are highly conserved. Time-lapse microscopy of meiosis in budding yeast allows the monitoring of different meiotic mutants to show how the mutation disrupts meiotic processes. However, many proteins function at multiple points in meiosis. The use of loss-of-function or meiotic null mutants can therefore disrupt an early process, blocking or disturbing the later process and making it difficult to determine the phenotypes associated with each individual role. To circumvent this challenge, this protocol describes how the proteins can be conditionally depleted from the nucleus at specific stages of meiosis while monitoring meiotic events using time-lapse microscopy. Specifically, this protocol describes how the cells are synchronized in prophase I, how the anchor away technique is used to deplete proteins from the nucleus at specific meiotic stages, and how time-lapse imaging is used to monitor meiotic chromosome segregation. As an example of the usefulness of the technique, the kinetochore protein Ctf19 was depleted from the nucleus at different time points during meiosis, and the number of chromatin masses was analyzed at the end of meiosis II. Overall, this protocol can be adapted to deplete different nuclear proteins from the nucleus while monitoring the meiotic divisions.

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae

Time-lapse microscopy is a valuable tool for studying meiosis in budding yeast. This protocol describes a method that combines cell-cycle synchronization, time-lapse microscopy, and conditional depletion of a target protein to demonstrate how to study the function of a specific protein during meiotic chromosome segregation.

Einsatz von Zeitraffer-Mikroskopie und stadienspezifischer Kerndepletion von Proteinen zur Untersuchung der Meiose bei S. cerevisiae

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often ...