Name: imaging replicative domains in ultrastructurally preserved chromatin by electron tomography
Uploaded: 2022-05-20T14:00:00
Description: Watch this Scientific Journal Video about Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography at JoVE.com

这项工作得到了RSF（拨款#17-15-01290）和RFBR（拨款#19-015-00273）的部分支持。作者感谢莫斯科罗蒙诺索夫国立大学发展计划（PNR 5.13）和别洛泽尔斯基物理化学生物学研究所的尼康相关成像卓越中心对成像仪器的使用。

该方案针对贴壁细胞进行了优化，并在HeLa，HT1080和CHO细胞系上进行了测试。
1. 细胞标记和固定
<ol><li>在3厘米培养皿中酸性清洁的盖玻片上板细胞。将推荐用于细胞系的培养基中的细胞生长至70%汇合度。</li>
	<li>从10mM储备液中加入EdU（5-乙炔基-2'-脱氧尿苷）至10μM终浓度，并将细胞置于培养箱中10分钟或更长时间（取决于实验目的）。对于较短的脉冲（长达2分钟...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+factories+and+nuclear+bodies:+the+ultrastructural+characterization+of+replication+sites+during+the+cell+cycle.">Replication factories and nuclear bodies: the ultrastructural characterization of replication sites during the cell cycle.</a> Journal of Cell Science. 107 (8), 2191-2202 (1994).</li><li>Deng, X., Zhironkina, O. A., Cherepanynets, V. D., Strelkova, O. S., Kireev, I. I., Belmont, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytology+of+DNA+replication+reveals+dynamic+plasticity+of+large-scale+chromatin+fibers.">Cytology of DNA replication reveals dynamic plasticity of large-scale chromatin fibers.</a> Current Biology. 26 (18), 2527-2534 (2016).</li><li>Salic, A., Mitchison, T. 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与以前发布的协议相比，此处描述的方法具有几个优点。首先，使用点击化学标记复制的DNA消除了用抗体检测BrdU的DNA变性先决条件的必要性，从而更好地保留了染色质超微结构。 
其次，利用生物素作为戊二醛固定和未结合醛基团适当淬火后产生的二级配体，最大限度地减少了靶标的化学修饰，从而提高了标记效率，同时减少了内源性生物素引起的背景。生物素 - 链霉亲和素...

DNA复制是在细胞分裂过程中忠实复制和传递遗传信息所需的基本生物学过程。在高等真核生物中，DNA复制受到严格的时空调节，这表现为复制起源1的顺序激活。同步触发的相邻复制源形成 replicon2 的簇。在光学显微镜水平上，正在进行的DNA复制的位点被检测为各种数量和大小的复制焦点。复制病灶显示细胞核内空间分布的特定模式，这取决于标记的DNA3，4的复制时间，这反过来又与其基因活性密切相关。由于明确定义的DNA复制序列，在空间和时间上严格排序，复制标记是一种强大的精确DNA标记方法，不仅用于研究复制过程本身，还用于区分具有定义的转录活性和压实水平的特定DNA亚级分。复制染色质的可视化通常通过检测DNA复制机制的主要蛋白质成分（通过免疫染色或通过荧光蛋白标签5，6的表达）或通过掺入修饰的DNA合成前体7，8，9，10进行.其中，只有基于将修饰的核苷酸掺入新复制的DNA中的方法才能在复制过程中捕获染色质的构象变化，并在复制完成后跟踪复制结构域的行为。
在高等真核生物中，DNA包装成染色质为基本遗传功能（转录，复制，修复等）的调节增加了另一个层次的复杂性。染色质折叠影响DNA对调节反式因子的可及性和模板合成所需的DNA构象变化（双螺旋放卷）。因此，人们普遍认为，细胞核中DNA依赖性的合成过程需要染色质的结构转变，从其浓缩的抑制状态到更容易获得的开放构象。在细胞学上，这两种染色质状态被定义为异染色质和真染色质。然而，关于细胞核中DNA折叠的模式仍然没有达成共识。假设的范围从“聚合物熔体”模型11，其中核小体纤维表现为随机聚合物，其堆积密度由相分离机制控制，到分层折叠模型假设连续形成厚度增加12，13的染色质纤维样结构。分层折叠模型最近获得了基于 原位 DNA-DNA接触分析的分子方法的支持（染色体构象捕获，3C），证明了染色质结构域层次结构的存在14。重要的是要注意，复制单元与这些染色质结构域15的相关性非常好。对这些模型的主要批评是基于样品制备程序引起的潜在的人工染色质聚集，例如细胞膜的透化和非染色质成分的去除，以改善超微结构研究的染色质对比度，同时提高染色质对各种探针（例如抗体）的可及性。最近通过DNA结合荧光团介导的二氨基联苯胺光氧化（ChromEMT6）进行选择性DNA染色电子显微镜检查的技术进展已经消除了这一障碍。然而，同样的考虑也适用于复制DNA17，18的电子显微镜可视化。在这里，我们描述了一种技术，该技术允许在完整的醛交联细胞中同时对新合成的DNA和总染色质进行高分辨率超微结构图谱。该技术通过点击化学检测EdU标记的DNA与生物素化探针和链霉亲和素纳米金以及ChromEMT相结合。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5-ethynyl-2`-deoxyuridine (EdU)</td>
			<td>Thermo Fisher</td>
			<td>A10044</td>
			<td></td>
		</tr>
		<tr>
			<td>2-(4-Morpholino)ethane Sulfonic Acid (MES)</td>
			<td>Fisher Scientific</td>
			<td>BP300-100</td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 555-azide</td>
			<td>Termo Fisher</td>
			<td>A20012</td>
			<td></td>
		</tr>
		<tr>
			<td>biotin-azide</td>
			<td>Lumiprobe</td>
			<td>C3730</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumine</td>
			<td>Boval</td>
			<td>LY-0080</td>
			<td></td>
		</tr>
		<tr>
			<td>DDSA</td>
			<td>SPI-CHEM</td>
			<td>26544-38-7</td>
			<td></td>
		</tr>
		<tr>
			<td>DMP-30</td>
			<td>SPI-CHEM</td>
			<td>90-72-2</td>
			<td></td>
		</tr>
		<tr>
			<td>DRAQ5</td>
			<td>Thermo Scientific</td>
			<td>62251</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy resin monomer</td>
			<td>SPI-CHEM</td>
			<td>90529-77-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde (25%, EM Grade)</td>
			<td>TED PELLA, INC</td>
			<td>18426</td>
			<td></td>
		</tr>
		<tr>
			<td>Gum arabic</td>
			<td>ACROS Organics</td>
			<td>258850010</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Panreac</td>
			<td>141396.1209</td>
			<td></td>
		</tr>
		<tr>
			<td>NaBH4</td>
			<td>SIGMA-ALDRICH</td>
			<td>213462</td>
			<td></td>
		</tr>
		<tr>
			<td>NMA</td>
			<td>SPI-CHEM</td>
			<td>25134-21-8</td>
			<td></td>
		</tr>
		<tr>
			<td>N-propyl gallate</td>
			<td>SIGMA-ALDRICH</td>
			<td>P3130</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>MP Biomedicals</td>
			<td>2810305</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver lactate</td>
			<td>ALDRICH</td>
			<td>359750-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-AlexaFluor 488 conjugate</td>
			<td>Termo Fisher</td>
			<td>S11223</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-Nanogold conjugate</td>
			<td>Nanoprobes</td>
			<td>2016</td>
			<td></td>
		</tr>
		<tr>
			<td>tetrachloroauric acid</td>
			<td>SIGMA-ALDRICH</td>
			<td>HT1004</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane (Tris)</td>
			<td>CHEM-IMPEX INT&#39;L</td>
			<td>298</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fluka Chemica</td>
			<td>93420</td>
			<td></td>
		</tr>
		<tr>
			<td>Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon Coater</td>
			<td>Hitachi</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Copper single slot grids</td>
			<td>Ted Pella</td>
			<td>1GC10H</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy5 fluorescence filter set (Ex620/60 DM660 Em700/75)</td>
			<td>Nikon</td>
			<td>Cy5 HQ</td>
			<td>Alternatives: Zeiss, Leica, Olympus</td>
		</tr>
		<tr>
			<td>Diamond knife Ultra Wet 45o</td>
			<td>Diatome</td>
			<td>DU</td>
			<td>Alternatives: Ted Pella</td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Nikon</td>
			<td>Ti-E</td>
			<td>Alternatives: Zeiss, Leica, Olympus</td>
		</tr>
		<tr>
			<td>High-tilt sample holder</td>
			<td>Jeol</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotator</td>
			<td>Biosan</td>
			<td>Multi Bio RS-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission electron microscope operating at 200 kV in EFTEM mode, with high-tilt goniometer</td>
			<td>Jeol</td>
			<td>JEM-2100</td>
			<td>Alternatives: FEI, Hitachi</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Ted Pella</td>
			<td>523</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>Leica</td>
			<td>UltraCut-E</td>
			<td>Alternatives: RMC</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image acquisition</td>
			<td>Open Source</td>
			<td>SerialEM (https://bio3d.colorado.edu/SerialEM/)</td>
			<td></td>
		</tr>
		<tr>
			<td>Image processing</td>
			<td>Open Source</td>
			<td>IMOD (https://bio3d.colorado.edu/imod/)</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging replicative domains in ultrastructurally preserved chromatin by electron tomography

细胞核中DNA折叠的原理及其在基本遗传功能（转录，复制，分离等）实现过程中发生的动态转化仍然知之甚少，部分原因是缺乏实验方法来高分辨率地可视化结构上保存的细胞核中的特定染色质位点。在这里，我们提出了一种方案，用于 原位单层细胞培养中复制结构域的可视化，通过将新合成DNA的EdU标记与随后的标记检测与纳米金颗粒的Ag扩增和染色质的ChromEM染色相结合。该协议允许高对比度，高效率的预包埋标记，与传统的戊二醛固定兼容，为室温样品处理提供染色质的最佳结构保存。预嵌入标记的另一个优点是可以预先选择感兴趣的单元格进行切片。这对于分析异质细胞群，以及与电子断层扫描方法的兼容性，以高分辨率3D分析复制位点的染色质组织，以及分析复制后染色质重排和相间姐妹染色单体分离尤为重要。

哺乳动物细胞核中的复制病灶根据S期进展在细胞核内显示出不同的分布模式。这些模式与被复制的位点的转录活性相关。由于这里介绍的方法使用相当强的固定程序，因此使用复制脉冲标记来特异性检测各种转录状态下的染色质位点是相当简单的，即使在提供通过室温化学固定获得的染色质的最佳结构保存的条件下也是如此。
质量控制步骤旨在确保成功完成该协议中的关键?...

Watch this Scientific Journal Video about Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography at JoVE.com

Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography

该协议提出了一种在结构上保存的染色质 中原位 复制位点的高分辨率映射技术，该技术采用预包埋EdU-链霉亲和素 - 纳米金标记和ChromEMT的组合。

通过电子断层扫描对超构造保存的染色质中的复制结构域进行成像

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, ...

我们提出了一种方案，通过EdU标记新合成的DNA和标记检测，通过纳米金颗粒的银增强和染色质的ChromEM染色进行标记检测，对细胞中的复制域进行电子显微镜可视化。该方案使高对比度的预包埋标记与传统的戊二醛固定，为室温样品处理提供了染色质的最佳结构保存。该方案针对生长在培养皿中盖玻片上的粘附细胞进行了优化。将EdU加入10微摩尔终浓度，并将细胞放入培养箱中以达到所需的标记时间。用2.5%葡萄糖醛酸酐在100毫摩尔二甲酸钠溶液中固定标记的细胞一小时。较长的固定时间可能会对EdU标记产生不利影响。通过用补充五毫摩尔氯化镁的PBS冲洗样品来去除戊二醛，进一步被视为PBS星。洗涤三次，每次 10 分钟。在PBS星中用1%的Trione X-100溶液渗透质膜，每次进行两次更改，每次五分钟。这种溶液又称PBS星T.用PBS星光广泛洗涤样品。五次更改，每次五分钟。用PBS星中20毫摩尔甘氨酸淬灭残留的醛基，两次10分钟。将样品置于PBS星光中，含1%BSA，持续半小时。在BSA中阻断时，准备用于EdU检测的反应鸡尾酒。组件添加的顺序很重要。EdU生物素 - 叠氮化物偶联在潮湿的腔室中进行，以最大限度地减少反应混合物中的蒸发和浓度变化。准备潮湿的腔室。将盖玻片放在对映膜上，使细胞朝上，并将50至100微升的反应混合物层到盖玻片上。室温下反应需要30分钟。通过在PBS星T中洗涤样品来停止反应，每次五次，每次五分钟。在PBS明星T中再次用1%BSA阻止半小时。在PBS星T中制备链霉亲和素 - 纳米金溶液与1%BSA。将样品与链霉亲和素在新制备的潮湿室中以加四度孵育过夜。该过程与EdU生物素 - 叠氮化物偶联相同。停止反应并洗涤样品，每次5次，每次10分钟，每片用PBS星T.稳定生物素链霉亲和素复合物，通过另外固定将1%戊二醛PBS星形固定半小时。用去离子水强洗，去除戊二醛。用每毫升一毫克硼氢化钠淬灭残余醛基。再次彻底清洗。下一步是通过在黄金路线上沉积银来增加纳米金颗粒的尺寸。准备预混料以最大限度地减少暗室活动。用洗涤缓冲液平衡样品并进入暗室。在暗室中混合组分以制备洗涤溶液并将其应用于样品。金合欢粉溶液是高度粘稠的。因此，在混合组分时，慢慢摇动管子，以避免气泡和泡沫的形成。反应需要两到五分钟。我们建议执行测试运行以确定最佳反应时间。较长的孵育时间可能导致非特异性银沉积。为了停止反应，用三到五次变化的去离子水快速冲洗样品。随后再进行三次更改，每次更改五分钟。为了保护银纳米颗粒不被四氧化锇溶解，将样品在0.05%四氯金酸中在黑暗中孵育两分钟。用去离子水彻底清洗。在此阶段，向含有DNA的材料添加额外的对比度。用DRAQ5 DNA染色剂浸透样品。向样品中加入二氨基联苯胺溶液并孵育一分钟。将盖玻片移入玻璃底培养皿中，并将其放在倒置显微镜的载物台上。用640纳米红光照射多个视场。左侧面板显示了 DRAQ5 的完整照片漂白。在右侧面板上，相同的视场在显示DAB沉淀物的透射光中成像。用去离子水洗涤样品。下一阶段应在通风橱下进行。制备部分还原的四氧化锇溶液。小心。该物质具有高挥发性和毒性。将准备好的溶液涂在样品上。在此阶段，还原的锇化合物与二氨基联苯胺沉积反应并增加DNA的对比度。根据您当地的法规处理含锇溶液，并清洗样品三次，每次五分钟。样品在一系列增加的乙醇浓度中进一步脱水，然后用乙醇树脂混合物浸润细胞。用新鲜制备的纯树脂代替醇树脂混合物。用新鲜制备的树脂填充适当尺寸的包埋模具。将盖玻片与一个细胞面朝下放在树脂填充的腔体上。树脂在37度下固化24小时。然后在60度下至少两天。从盖子的底面上取下树脂。将板坯放入液氮中，然后转移到沸水中。玻璃应分离。如果需要，可重复上述步骤。由于二氨基联苯胺沉积和 DAB 染色，照射视野应清晰可见。使用钢锯或任何其他合适的仪器从楼板上切出感兴趣的区域。将切口放入超微量离心机简单支架中。设置超微量切片机以进行最终的块修剪。使用剃须刀片，准备金字塔形的皮球。金字塔顶部平坦区域的大小应约为400×200微米。将准备好的金字塔安装到超微球臂中并安装刀。准备各节。截面厚度可以调整以适应实验要求。我们的目标是进行EM断层扫描。因此，我们制备了厚度为250纳米的切片，也称为半薄切片。从刀刃上取下这些部分，然后将其安装在槽栅上。我们用了一个槽格，它们周围是一毫米的圆形，一毫米的侧面开口。小开口尺寸提供了更好的截面稳定性。让样品在网格上干燥。此时，各部分已准备好进行观察。将网格放入电子显微镜样品架中。将样品装入电子显微镜。获取倾斜系列。哺乳动物细胞核中的复制病灶根据S期进展显示出不同的分布模式。通过与荧光标记的叠氮化物的点击反应确认的成功EdU标记在顶部固定戊二醛后在异质细胞群中显示出典型的复制模式。链霉亲和素标记可受戊二醛影响。所以首先检查荧光链霉亲和素染色在与链霉亲和素纳米金相同的条件下在底部。银增强手术的良好结果是一些细胞核的深棕色染色和非常微弱的黄色细胞体染色，如底部所示。预嵌入标记的优点是可以选择用于切片的单元格。在这个阶段是可能的，因为标记图案在明场显微镜下变得可见。适当的标记会产生边界清楚的复制站点。箭头显示DAB染色的染色质纤维。右侧的背景标记仅限于每平方微米几个纳米颗粒。半薄切片可用于断层扫描，不需要添加金纳米颗粒。所描述的方法用于研究具有启用复制的位点的染色质组织。用于分析染色质的复制后重排，包括染色质分离的高分辨率成像。它还用于大染色体结构域的复制标记以及高阶染色质折叠和异染色质的研究。这种成像技术作为其他非微观方法生成的数据的参考点也很重要。该方法还可以用于相关的显微研究，包括超分辨率技术。这为研究染色质高阶结构及其在细胞各种生理状态下的动力学开辟了新的视野。

Research

JoVE Journal

Biology

Chromatin Isolation by RNA Purification (ChIRP)

Chromatin Isolation by RNA Purification ChIRP

RNA纯化的染色质隔离（CHIRP）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

科研

生物学

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

ATP合酶二聚体线粒体通过冷冻电子断层扫描可视化

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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

具体的基因组区域及相关分子鉴定enChIP隔离

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

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

准备厚生物样品的观察扫描透射电子断层扫描

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

透射电镜下体的样品制备与成像

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography

3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography

连续切片电子断层扫描显示的 3D果蝇间接飞行肌的线粒体超微结构研究

Mitochondria are cellular powerhouses that produce ATP, lipids, and metabolites, as well as regulate calcium homeostasis and cell death. The unique ...

Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography

高压低温电子层析成像对蓝藻 DNA 压实的可视化研究

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

高压冷冻、微波辅助对比度增强和最小树脂嵌入在体积成像中的生物样品制备

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

超结构保存细胞内膜蛋白定位电子显微镜分析的CryoAPEX方法

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...