Questo lavoro è stato sostenuto dalla Fondazione tedesca per la ricerca e l&#39;ERC (AN377 / 3-2 e 309.528 CHROMDECON a WA) e un PhD Fellowship della Boehringer Ingelheim Fonds a AKS Figura 1 e 2 sono ristampati da Developmental Cell 31 (3), Magalska , RuvB-come la funzione ATPasi nella cromatina decondensazione alla fine della mitosi, 305-318, 2014 et al., gentile concessione di Elsevier.

<ol>
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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. 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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. 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The authors have nothing to disclose.

Xenopus laevis estratti a base di uova sono uno strumento molto utile per riprodurre fedelmente i processi cellulari in vitro, e questo sistema è stato utilizzato con successo nella caratterizzazione degli eventi del ciclo cellulare e la divisione cellulare 2, 3,5,6,17. A causa di grandi negozi di componenti nucleari sequestrati nell&#39;uovo durante oogenesi, estratti a base di uova sono una fonte eccellente di componenti cellulari. Rispetto ad altri approcci come RNAi su linee cellulari di mammifero tessuto o manipolazione genetica, che offre diversi vantaggi: Il sistema non cellulare permette lo studio dei processi cellulari in cui la vitalità cellulare sarebbe altrimenti una limitazione. Inoltre singole fasi dei processi complessi possono essere analizzati in saggi semplici. Il test decondensazione qui presentato permette lo studio dei meccanismi molecolari di decondensazione postmitotici senza interferenze da altri eventi mitosi, rispettivamente. Estratti di uova di Xenopus sono facili da manipolare per esaurimento delle proteine ​​specifichee aggiunta di inibitori o proteine ​​mutate 8. Ad esempio, la Figura 2 mostra il risultato della somma dei non idrolizzabili ATP o GTP analoghi, ATPγS e GTPγS al dosaggio decondensazione. Per diluizione e centrifugazione differenziale di Xenopus uova componenti come membrane e citosol possono essere separati 16. La Figura 3 mostra il test decondensazione eseguita in presenza o assenza di membrane. Infine, il saggio acellulare può anche essere utilizzato per identificare nuovi fattori per esempio, da un approccio frazionamento. Usando questa strategia abbiamo individuato la AAA + -ATPasi RuvBL1 / RuvBL2 come decondensazione cruciale fattori 8. Nei sistemi in vitro sulla base di X. laevis uova sono stati impiegati con differenti modelli di DNA:. Forbes et al hanno dimostrato che l&#39;iniezione di fago λ DNA in X. unfertilized uova laevis indotto l&#39;assemblaggio della cromatina su nakDNA ed fago λ. Come iniezione di DNA virale attivato l&#39;uovo, l&#39;assemblaggio della cromatina è stata seguita da formazione di una struttura nucleo-like 18 e analogamente λ-fago DNA può essere utilizzato in combinazione con estratti uovo per generare nucleo come strutture in vitro 19. Perline magnetiche rivestite con DNA sono stati utilizzati per studiare chromatinization di DNA 20 e il reclutamento di membrane nucleari 21 e assemblaggio di una membrana nucleare e pori complessi 22, anche se rimane aperta fino a che punto questo assomigliava un bona fide processo riassemblaggio nucleare . Il protocollo presentato qui permette decondensazione di isolati gruppi cromatina mitosi dalle cellule HeLa utilizzando estratto generato da uova di Xenopus attivati. Si ricostruisce accuratamente eventi che hanno portato a una riforma di un nucleo interfasico 8. Rispetto al gruppo reazione nucleare ampiamente applicato utilizzata per studiare la formazione della membrana nucleare evengono utilizzati complessi pori nucleari alla fine della mitosi, nel saggio decondensazione HeLa mitotico cromatina cluster invece di sperma DNA. DNA dello sperma può essere montato nella cromatina mitotico o anche cromosomi individuali su incubazione con estratto preparato da uova non fecondate e non attivati ​​3. Abbiamo deciso di usare i cluster mitotico come fonte di cromatina di semplificare la procedura ed evitare interferenze da condensazione della cromatina. Inoltre, la preparazione dell&#39;estratto dell&#39;uovo è leggermente modificata: Per la decondensazione cromatina estratto bassa velocità controllata da due fasi di centrifugazione ad alta velocità in rotori ad angolo fisso vengono utilizzati. Estratto a bassa velocità può essere conservato per un massimo di sei mesi in azoto liquido senza perdere la sua attività. Al contrario, nelle reazioni nucleari assemblaggio, citosol e membrane galleggiavano sono generati da estratti bassa velocità di diluizione e centrifugazione differenziale ad alta velocità prima possibile congelamento (vedi Eisenhardt et al. 16 per una detaiprotocollo led). Nel nostro sistema di analisi, aggiunta delle membrane permette la formazione di una membrana nucleare chiusa compresi complessi del poro nucleare. I nuclei risultanti sono competenti per l&#39;importazione e l&#39;esportazione nucleare 8. Così, questo sistema supporta sia cromatina decondensazione e busta riforma nucleare. È interessante notare, decondensazione cromatina è possibile anche in assenza di membrane (figura 3). Tuttavia l&#39;aggiunta di membrane risultati in nuclei leggermente più grandi. Molto probabilmente, la riforma della membrana nucleare induce un passo decondensazione secondario da meccanismi ancora non definite, che dipende importazione nucleare. Per l&#39;isolamento di cluster cromatina mitotiche da cellule HeLa, è stata utilizzata una versione modificata del protocollo stabilito dalla Gasser e Laemmli 15. Cellule mitotiche sincronizzati vengono lisate in un tampone contenente il detersivo digitonina non ionico e da forze meccaniche. La cromatina è isolato come cluster che contengonotutti i cromosomi di un nucleo. La differenza fondamentale rispetto ai protocolli di isolamento singoli cromosoma è il fatto che le cellule non sono hypotonically gonfio ma raffreddato a 4 ° C prima di lisi. Ciò impedisce la disconnessione dei singoli cromosomi 15, 23. Rispetto al protocollo pubblicato da JR Paulson 23 che ha riconosciuto il vantaggio di isolamento di interi cluster cromatina, Gasser &amp; Laemmli usati buffer-EDTA contenente buffer poliamminiche invece di Mg 2+ based per ridurre l&#39;attività di chinasi, nucleasi, proteasi e fosfatasi e da questo diminuire la quantità di proteine ​​e DNA modificazioni che si verificano durante il processo di isolamento 15. Inoltre, utilizzando un gradiente di silice colloidale di particelle durante la centrifugazione differenziale altamente riduce la contaminazione citoplasmatica. Il protocollo può anche essere utilizzata per isolare i cluster cromatina mitotiche dall&#39;ovaia criceto cinese e cellule di topo 15. <pclass = &quot;jove_content&quot;&gt; Nel complesso, il nostro protocollo ricostituisce fedelmente cromatina decondensazione come accade alla fine della mitosi. La dipendenza della cromatina ATP decondensazione in vitro può essere almeno in parte spiegato con il coinvolgimento di RuvBL1 / 2 ma anche un&#39;altra AAA + -ATPasi, p97, che rimuove la chinasi mitotica Aurora B dalla cromatina durante uscita 24 mitosi. Perché il processo richiede GTP idrolisi è una delle questioni aperte che intendiamo rispondere con questa configurazione.

Xenopus laevis estratto uovo è uno strumento potente e ampiamente applicato per studiare eventi cellulari complicati nella semplicità di un test cell-free. Fin dalla loro prima descrizione da Lohka &amp; Masui 1 che sono stati ampiamente utilizzati per studiare i processi mitotici quali la condensazione della cromatina 2, del fuso 3, rottura membrana nucleare 4, ma anche il trasporto nucleocytoplasmic 5 o 6 replicazione del DNA. Gli eventi che si svolgono al termine della mitosi, necessari per la riforma del nucleo interfasico quali riforma membrana nucleare e poro nucleare complesso rimontaggio sono molto meno inteso rispetto ai primi eventi mitotico ma possono essere studiati in modo simile utilizzando Xenopus estratto uovo 7. Abbiamo recentemente stabilito un saggio basato su estratto uovo Xenopus per studiare cromatina decondensazione alla fine della mitosi 8, un processo sotto-indagato che attende its caratterizzazione dettagliata. In metazoi, cromatina è altamente condensato all&#39;entrata mitotica per eseguire fedelmente la segregazione del materiale genetico. Per garantire che la cromatina è accessibile per l&#39;espressione genica e la replicazione del DNA durante interfase, deve essere de-compattato alla fine della mitosi. Nei vertebrati, cromatina è fino a cinquanta volte più compatta durante la mitosi rispetto interfase 9, in contrasto con lieviti dove la compattazione mitotico è generalmente molto inferiore, ad esempio, solo due volte nella S. cerevisiae 10. Vertebrato cromatina decondensazione è stata per lo più studiata nel contesto della riorganizzazione del DNA spermatico dopo la fecondazione delle uova. Un meccanismo molecolare, in cui nucleoplasmin, una proteina ovocita abbondante, scambia protamine specifici sperma di istoni H2A e H2B memorizzati nel uovo. Questo processo è stato anche chiarito utilizzando Xenopus estratto uovo 11, 12. Tuttavia, l&#39;espressionedi nucleoplasmin è limitata a ovociti 13 e cromatina mitotico non contiene queste protamine specifici sperma. Pertanto cromatina decondensazione alla fine della mitosi è nucleoplasmin indipendente 8. Per la reazione decondensazione in vitro impieghiamo estratto generato da attivi X. laevis uova e cluster cromatina isolate da cellule HeLa sincronizzate. Il trattamento di uova con un ionoforo del calcio imita il rilascio del calcio nel citoplasma dell&#39;ovocita generato da ingresso dello spermatozoo durante la fecondazione. L&#39;onda di calcio innesca la ripresa del ciclo cellulare e l&#39;uovo, arrestato nella seconda metafase della meiosi, progredisce alla prima interfase 14. Pertanto, gli estratti di uova preparati forma attivata uova rappresentano lo stato uscita / interfase mitosi e sono competenti per indurre eventi specifici per l&#39;uscita mitosi come cromatina decondensazione, membrana nucleare e poro riforma complessa. Per l&#39;isolamento di ch mitoticaromatin cluster abbiamo usato una versione leggermente modificata del protocollo pubblicato da Gasser &amp; Laemmli 15, dove i cluster cromosomiche sono rilasciate dai lisi da cellule HeLa sincronizzate in mitosi e isolati in poliammine contenenti buffer da centrifugazioni gradiente.

<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.

Ora la dipendenza della reazione decondensazione La figura 1 mostra un tipico andamento temporale del dosaggio decondensazione. Il cluster di cromosomi visibili all&#39;inizio della reazione decondenses e si fonde in un unico, rotondo e liscio nucleo. Quando l&#39;estratto uovo è sostituito dal saccarosio tampone cluster cromosoma rimane condensata, che suggeriscono che l&#39;attività decondensazione è presente nell&#39;estratto uovo. Decondensazione cromatina è un processo a carico di energia La reazione decondensazione in vitro può essere convenientemente manipolato per esempio, per aggiunta di inibitori. Nell&#39;esperimento mostrato in Figura 2, l&#39;ATP non idrolizzabile o analoghi GTP, ATPγS o GTPγS, sono stati aggiunti alla reazione. Entrambi inibiscono la proiezione decondensazione, che è un ATP e GTP dipendente, processo attivo (Figura 2). Il dosaggio decondensazione è stata eseguita in presenza o assenza di membrane (figura 3). Si prega di notare che in entrambe le condizioni cromatina subisce decondensazione, ma l&#39;aggiunta di membrane risultati nei nuclei più grandi. Molto probabilmente, riforma della membrana nucleare induce un passo decondensazione secondario da un altro meccanismo dipendente dal trasporto nucleare. <img alt="Figura 1" src="/files/ftp_upload/53407/53407fig1.jpg" /> Figura 1. Naturalmente il tempo di th e in vitro decondensazione reazione. Mitotic cromatina ammassi di cellule HeLa sono state incubate con interfasico estratto uovo Xenopus. I campioni sono stati fissati in momenti indicati con il 4% PFA e 0,5% glutaraldeide, colorate con DAPI e analizzati dal co<html> microscopia nfocal. Ri-stampata dal Magalska et al. 8. Barra della scala è 5 micron. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53407/53407fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53407/53407fig2.jpg" /> Figura 2. cromatina decondensazione richiede ATP e GTP idrolisi. Decondensazione cromatina è stata eseguita in presenza di 10 mM ATPγS, 10 mM GTPγS o buffer di controllo. I campioni sono stati fissati con 4% PFA e 0,5% glutaraldeide in momenti indicati e analizzati mediante microscopia confocale. Ri-stampata dal Magalska et al. 8. Barra della scala è 5 micron. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53407/53407fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> Figura 3 &quot;src =&quot; / files / ftp_upload / 53407 / 53407fig3.jpg &quot;/&gt; Figura 3. cromatina decondensazione in presenza ed in assenza di membrane. Decondensazione cromatina è stata eseguita in assenza (A) o presenza (B) di galleggiamento purificato membrane per 120 min. I campioni sono stati fissati con 4% PFA e 0,5% glutaraldeide e analizzate mediante microscopia confocale. La cromatina è macchiato con DAPI, membrane con DiIC 18 (1,1&#39;-diottadecil-3,3,3 &#39;, 3&#39;-tetramethylindocarbocyanine perclorato). Barra della scala è 5 micron. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53407/53407fig3large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</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.

Un Free Cell Assay per studiare cromatina decondensazione alla fine del mitosi

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 Views.  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.

Video: 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.

Cromatina diffusa i preparativi per l'analisi della progressione di ovocita Mouse dalla profase alla metafase II

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

Ricerca

Genetica

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

In tempo reale analisi del fattore di trascrizione associazione, trascrizione, traduzione e fatturato per visualizzare eventi globali durante l'attivazione cellulare

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

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.

A lungo termine Live cell Imaging per valutare il destino delle cellule in risposta a 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 ...

Ricerca sul cancro

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.

Cromatina Immunoprecipitazioni Analisi Utilizzando Nucleasi Micrococal nelle cellule di Mammaliana

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 della segregazione cromosomica durante la mitosi

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

Biologia dello sviluppo

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.

Immunoprecipitazione della cromatina (ChIP) per analisi modifica dinamica Histone nell&#39;espressione genica attivata nelle cellule umane

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

Biologia

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.

Cromatina Analisi Interazioni con Tag Sequencing appaiati-End (Chia-PET) per la mappatura Interazioni cromatina e comprensione del regolamento Trascrizione

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

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

Cromosoma Preparazione da cellule in coltura

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

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.

L&#39;isolamento di regioni genomiche specifici e l&#39;identificazione di molecole da Associated 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.

Uso della microscopia time-lapse e della deplezione nucleare stadio-specifica delle proteine per studiare la meiosi 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 ...

Un Free Cell Assay per studiare cromatina decondensazione alla fine del mitosi

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