无细胞化验，研究染色质解聚在有丝分裂的结束
Summary
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.
Abstract
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.
Introduction
非洲爪蟾卵提取物是一个强大的和广泛应用的工具，研究在无细胞的测定法的简单复杂细胞事件。由于通过Lohka＆升井1首次描述它们已经被广泛地用于研究的有丝分裂过程，如染色质缩合2，主轴组件3，核膜破裂4，而且核质运输5或DNA复制6。发生在有丝分裂的末端的事件，所必需的interphasic核的改造如核膜改革与核孔复合重组小得多理解相比早期有丝分裂事件，但可使用爪蟾卵提取物7被类似研究。最近，我们建立了基于爪蟾卵提取物研究染色质解聚在有丝分裂8月底的检测，欠调查过程，等待着我TS细致的刻画。
在后生动物，染色质是高度会聚在进入有丝分裂，以便执行的遗传物质的忠实偏析。以确保该染色质是用于基因表达和DNA复制访问间期，它需要被解压缩在有丝分裂的末端。在脊椎动物中，染色质是高达50倍相比相间9有丝分裂期间更紧密，而相比之下，酵母，其中有丝分裂压实通常要低得多， 例如，只有两个倍S.酵母 10。脊椎动物的染色质解聚已大部分研究精子DNA重组的卵子受精后的环境。的分子机制，其中nucleoplasmin，一种丰富的卵母细胞的蛋白质，交换精子特异性精蛋白组蛋白H2A和H2B存储在蛋。这个过程也阐明用爪蟾卵提取11，12。然而，表达nucleoplasmin的仅限于卵母细胞13和有丝分裂的染色体不包含这些精子特异性鱼精蛋白。因此，在有丝分裂的结束染色质解聚为nucleoplasmin 独立的8。
对于体外解聚反应，我们采用的激活爪蟾卵和染色质集群从同步的HeLa细胞中分离产生的提取物。鸡蛋与钙离子载体治疗模仿的钙释放到受精过程中精子的进入所产生的卵母细胞。钙波触发细胞周期的恢复和卵子在减数分裂的第二个中期逮捕，发展到第一相间14。因此，准备形式激活卵卵提取物所代表的有丝分裂退出/间期状态，并有能力来诱导特异性的有丝分裂的退出状染色质解聚，核膜事件和孔复合体改造。对于有丝分裂通道的隔离romatin簇我们使用加塞＆的Laemmli 15，其中染色体簇从HeLa细胞中有丝分裂的同步和通过梯度离心分离在含有多胺缓冲器释放裂解发布的协议的一个略加修改的版本。
Protocol
Representative Results
Discussion
非洲爪蟾卵提取物是忠实地再现体外细胞过程一个非常有用的工具，并且该系 统被成功地在细胞周期和细胞分裂事件2，3,5,6,17表征使用。由于隔离在卵子发生在卵核部件的大型商店，卵提取物的细胞成分的极好来源。相对于其他方法，如RNA干扰对哺乳动物组织的细胞系或遗传操作，它提供了几个优点：无细胞系统允许研究细胞过程中细胞生存力将是否则限制。?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了德国研究基金会和ERC（AN377 / 3-2和309528 CHROMDECON到华盛顿州）和勃林格殷格翰全宗的博士研究生奖学金，以AKS图1和2是从的Developmental Cell 31（3），Magalska转载等人 ，RuvB样ATP酶在有丝分裂，305-318，2014年年底的功能染色质解聚，与爱思唯尔许可。
Materials
| spermine tetrahydrochloride | Fluka analytical | 85610-25G | |
| spermidine trihydrochloride | Sigma | S2501-5G | |
| high-purity digitonin | Millipore | 300410-1GM | toxic |
| PMSF | Applichem | A0999,0100 | toxic |
| thymidine | Calbiochem | 6060 | |
| nocodazole | Calbiochem | 487928 | toxic |
| 37 % formaldehyde solution | Roth | 7398-1 | toxic |
| trypan blue solution (0.4%) | Sigma | T8154 | toxic |
| 1,4-dithiothreitol (DTT) | Roth | 6908.2 | |
| AEBSF hydrochloride | Applichem | A1421,0001 | |
| pepstatin | Roth | 2936.1/2/3 | |
| leupeptin | Roth | CN334 | |
| aprotinin | Roth | A162.3 | |
| Percoll (colloidal silica particles solution) | GE Healthcare | 17-0891-01 | |
| glutamine | Gibco | 25030-024 | |
| Penicillin-Streptomycin | Gibco | 15140-122 | |
| 75 cm² tissue culture flasks | Greiner Bio-one | 658175 | |
| heat-inactivated fetal bovine serum (FBS) | Gibco | 10500-064 | |
| Homogenizer (40 mL tissue grinder) | Wheaton | 357546 | |
| Neubauer chamber | Assistent | 441/1 | |
| Oak Ridge Centrifuge Tubes, polycarbonate (50 ml) | Nalgene | 3118-0050 | |
| 100 µm cell strainer, nylon | BD Falcon | 352360 | |
| cytochalasin B | Applichem | A7657,0010 | toxic |
| cycloheximide | Roth | 8682.3 | toxic |
| L-cystein | Merck | 1,028,381,000 | |
| hCG available as Ovogest | MSD | 1431593 | |
| PMSG available as Intergonan | MSD | 1431015 | |
| A23187 (mixed calcium-magnesium-salt) | Enzo | ALX-450-002-M010 | toxic |
| syringe needles (1.20 x 40 mm, 18 G x 1 1/2") | Braun | 4665120 | |
| ATP | Serva | 10920.03 | |
| GTP, 2 Na x 3 H20 | Roth | K056.1/2/3/4 | |
| creatine phosphat disodium salt | Calbiochem | 2380 | |
| creatine phosphokinase | Sigma | C3755-35KU | |
| DMAP | Sigma | D2629-1G | |
| DAPI | Roche | 10236276001 | |
| PFA | Sigma | P-6148 | toxic |
| centrifugation tubes for TLA 100 (7 x 10 mm, 5/16 x 13/16 in.) | Beckman Coulter | 343775 | |
| "Cell-Saver" (tips with wide opening, 1000 µL) | Biozym | 729065 | |
| 50 % glutaraldehyde solution, grade I | Sigma alderich | G7651-10 mL | toxic |
| 0.1 % (w/v) poly-L-lysine solution | Sigma | P8920-100 mL | |
| flat-bottom tubes (6 mL, 16.0/55 mm) | Greiner Bio-one | 145211 | |
| Vectashield mounting medium | Vector laboratories | H1000 | |
| tubes for TLA120 (11 x 34 mm, 7/16 x 1 3/8 in.) | Beckman Coulter | 343778 | |
| "Cell-Saver" (tips with wide opening, 200 µL) | Biozym | 729055 | |
| 12 mm coverslips | Thermo Scientific | 0784 #1 |
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