Name: combining mitotic cell synchronization and high resolution confocal microscopy to study the role of multifunctional cell cycle proteins during mitosis
Uploaded: 2017-12-05T14:00:00
Description: Watch this Scientific Journal Video about Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis at JoVE

我们感谢日本东北大学的 Dr. 三, 他们分享了稳定表达 mCherry 组蛋白 H2B 和 GFP-α-蛋白的 HeLa 细胞。这项工作得到了一笔对 DV (R00CA178188) 和西北大学启动资金的支持。

1. 双胸苷阻滞和释放: 试剂制剂
<ol><li>使500毫升 Dulbecco 的改良鹰培养基 (DMEM) 培养基补充 10% FBS, 青霉素, 和链霉素。</li>
	<li>使100毫米的胸苷在无菌水和储存在等分在-80 ° c。</li>
</ol>2. 有丝分裂进展的固定细胞成像协议 (图 1A)
<ol><li>在1天, 种子〜 2 x 105 HeLa 细胞到一个6井板的井盖滑动 (用70% 乙醇和紫外线辐射灭菌) 和2毫升的 DMEM 培养基。在37° c ?...

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双胸苷同步的最关键的优点是它在短时间内提供了一个增加的有丝分裂细胞的样本大小, 其中许多细胞进入有丝分裂一致, 从而也使分析染色体对齐, 两极纺锤体形成, 染色体分离, 效率更高。
许多调控蛋白复合物和信号通路致力于确保正常进展通过有丝分裂和解除管制这一过程可能导致肿瘤。动态细胞成像的 HeLa 细胞稳定表达 mCherry-H2B 和 GFP-蛋白和释放从双胸苷块显示, 大多?...

在细胞周期中, 细胞经历了一系列高度调控和世俗控制的事件, 以精确复制他们的基因组和增殖。在哺乳动物中, 细胞周期由相间和 M 相组成。在相间, 由三个阶段-G1, S 和 G2, 细胞复制它的基因组并且经历成长是必要的为正常细胞周期进展1,2。在 M 阶段, 由有丝分裂 (前期, 中期, 中期, 后期, 和末期) 和胞, 父母细胞产生两个基因上相同的子细胞。在有丝分裂中, 姊妹染色的复制基因组是凝聚 (前期) 和捕获在他们的 kinetochores 通过微管组装的有丝分裂纺锤 (中期), 驱动他们的对准在中期板块 (中期), 其次是他们的当姐妹染色被分裂对和被运输对相反纺锤杆 (后期) 时, 相等的离析。两个子细胞的物理分离由肌动蛋白为基础的收缩环 (末期和胞) 的活动。粒是一种专门的蛋白质结构, 在染色的丝区域装配, 作为主轴微管的附着点。它的主要功能是驱动染色体捕获, 对齐, 并帮助纠正不正确的主轴微管附着, 同时调解主轴组件检查点, 以维护染色体分离的保真度3,4。
细胞同步技术是了解细胞周期进展过程中的分子和结构事件的理想工具。这一方法已被用来丰富细胞群体在特定阶段的各种类型的分析, 包括基因表达谱, 细胞生化过程分析, 和检测蛋白质的亚单位定位。同步哺乳动物细胞不仅可以用于单个基因产品的研究, 也可用于涉及整个基因组分析的方法, 包括基因表达的微阵列分析5, miRNA 表达式模式6,平移调节7, 蛋白质组蛋白分析的修改8。同步也可以用来研究基因表达或蛋白质敲掉或敲出, 或化学物质对细胞周期进展的影响。
细胞可以在细胞周期的不同阶段同步。物理和化学方法都被广泛用于细胞同步。细胞同步的最重要的标准是同步应该是 noncytotoxic 和可逆的。由于药物对细胞同步的潜在不良影响, 化学依赖性方法对研究关键细胞周期事件是有利的。例如, 羟基、amphidicolin、含羞草和洛伐他汀可用于 G1/S 阶段的细胞同步, 但由于它们对它们抑制的生物化学通路的影响, 它们激活了细胞周期检查点机制, 并杀死了一个重要单元格的分数9,10。另一方面, 通过添加胸苷对生长介质的反馈抑制 DNA 复制, 称为 "胸苷阻滞", 可以在某些点11,12,13中逮捕细胞周期。在 G2/M 阶段也可以通过处理 nocodazole 和 RO-3306914来同步单元格。Nocodazole, 防止微管组装, 具有较高的细胞毒性。此外, nocodazole 细胞可以通过有丝分裂的滑移回到相早熟。双胸苷阻滞细胞在 G1/S 阶段和释放后的块, 细胞被发现进行同步通过 G2 和有丝分裂。在高分辨率的共聚焦显微镜下, 通过细胞固定或活体成像, 可以观察胸苷块释放的细胞周期的正常进展。当细胞进入并通过双胸苷块释放后有丝分裂时, 可以特别研究有丝分裂蛋白的摄动效应。Cdt1 是一种多功能的蛋白质, 在 G1 阶段参与 DNA 复制起源许可, 并且在有丝分裂过程中也需要粒微管附着体 (15。为了研究 Cdt1 在有丝分裂过程中的作用, 我们需要采取一种方法, 在 G1 阶段避免其损耗对复制许可的影响, 同时在 G2/M 阶段只会影响其损耗。在这里, 我们提出了详细的协议的基础上的双胸苷块, 以研究的有丝分裂作用的蛋白质执行多个功能在不同阶段的细胞周期的固定和活细胞成像。

<table>
	<tbody>
		<tr>
			<td>DMEM (1X)</td>
			<td>Life Technologies</td>
			<td>11965-092</td>
			<td>Store at 4 degree C</td>
		</tr>
		<tr>
			<td>DPBS (1X)</td>
			<td>Life Technologies</td>
			<td>14190-144</td>
			<td>Store at 4 degree C</td>
		</tr>
		<tr>
			<td>Leibovitz&#8217;s (1X) L-15 medium</td>
			<td>Life Technologies</td>
			<td>21083-027</td>
			<td>Store at 4 degree C</td>
		</tr>
		<tr>
			<td>Serum reduced medium (Opti-MEM)</td>
			<td>Life Technologies</td>
			<td>319-85-070</td>
			<td>Store at 4 degree C</td>
		</tr>
		<tr>
			<td>Penicillin and streptomycin</td>
			<td>Life Technologies</td>
			<td>15070-063 (Pen Strep)</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Dharmafect2</td>
			<td>GE Dharmacon</td>
			<td>T-2002-02</td>
			<td>Store at 4 degree C</td>
		</tr>
		<tr>
			<td>Thymidine</td>
			<td>MP Biomedicals LLC</td>
			<td>103056</td>
			<td>Dissolved in sterile distiled water</td>
		</tr>
		<tr>
			<td>Cdt1 siRNA</td>
			<td>Life Technologies</td>
			<td></td>
			<td>Ref 10</td>
		</tr>
		<tr>
			<td>Hec1 siRNA</td>
			<td>Life Technologies</td>
			<td></td>
			<td>Ref 13</td>
		</tr>
		<tr>
			<td>HeLa cells expressing GFP-H2B</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa cells expressing GFP-&#945;-tubulin and mCherry H2B</td>
			<td>Generous gift from Dr. Kozo Tanaka of Tohoku university, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Sigma-Aldrich Corporation</td>
			<td>F8775</td>
			<td>Toxic, needs caution</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich Corporation</td>
			<td>D9542</td>
			<td>Toxic, needs caution</td>
		</tr>
		<tr>
			<td>Mouse anti-&#945;-tubulin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>Sc32293</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Rabbit ant-Zwint1</td>
			<td>Bethyl</td>
			<td>A300-781A</td>
			<td>1:400 dilution</td>
		</tr>
		<tr>
			<td>Mouse anti-phospho-&#947;H2AX (Ser139)</td>
			<td>Upstate Biotechnology</td>
			<td>05-626, clone JBW301</td>
			<td>1:300 dilution</td>
		</tr>
		<tr>
			<td>Alexa 488</td>
			<td>Jackson ImmunoResearch</td>
			<td></td>
			<td>1:250 dilution</td>
		</tr>
		<tr>
			<td>Rodamine Red-X</td>
			<td>Jackson ImmunoResearch</td>
			<td></td>
			<td>1:250 dilution</td>
		</tr>
		<tr>
			<td>BioLite 6 well multidish</td>
			<td>Thermo Fisher Scientific</td>
			<td>130184</td>
			<td></td>
		</tr>
		<tr>
			<td>35MM Glass bottom dish</td>
			<td>MatTek Corporation</td>
			<td>P35GCOL-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse TiE inverted microscope</td>
			<td>Nikon Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning disc for confocal</td>
			<td>Yokagawa</td>
			<td>CSU-X1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra 888 EM-CCD Camera</td>
			<td>Andor</td>
			<td>iXon Ultra EMCCD</td>
			<td></td>
		</tr>
		<tr>
			<td>4 wave length laser</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubation System for Microscopes</td>
			<td>Tokai Hit</td>
			<td>TIZB</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-elements software</td>
			<td>Nikon Instruments</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

combining mitotic cell synchronization and high resolution confocal microscopy to study the role of multifunctional cell cycle proteins during mitosis

研究细胞周期的各种调控事件, 以相依赖的方式提供了一个明确的理解细胞生长和分裂。在细胞周期的特定阶段, 细胞数量的同步被发现在这种实验中非常有用。与毒性相对较小的化学物质进行治疗时, 细胞的同步化可以在药物抑制剂的使用上获得好处, 用于研究相应的细胞周期事件, 并获取特定的有丝分裂阶段的丰富度。在这里, 我们描述了在细胞周期的不同阶段同步人类细胞的协议, 包括 S 相和 M 相的双胸苷块和释放程序, 以研究有丝分裂蛋白在染色体排列中的功能和隔离。本协议对于研究具有已建立的相间功能的多功能蛋白的有丝分裂作用非常有用。在我们的例子中, Cdt1 的有丝分裂作用, 一个关键的蛋白质复制原产地许可在 G1 阶段, 可以有效地研究, 只有当 G2/M-specific Cdt1 可以耗尽。我们用双胸苷同步描述了 G2/M-specific Cdt1 损耗的详细协议。我们还解释了细胞固定的协议, 和活细胞成像使用高分辨率共聚焦显微镜后, 胸苷释放。该方法也有助于分析有丝分裂蛋白在生理和扰动条件下的功能, 如 Hec1, Ndc80 复合物的一个组成部分, 因为它使一个获得大样本大小的有丝分裂细胞的固定和活细胞分析, 我们在这里展示。

双胸苷块释放后细胞有丝分裂进展及微管稳定性研究 Cdt1 在 G1 阶段参与了 DNA 复制起源的许可。它在 S 相时被降解, 但在 G2/M 相中重新积累。为了研究其在有丝分裂中的作用, 内源性 Cdt1 需要用最合适的细胞同步技术, 即双胸苷块15, 在 G/M 相的特定阶段被耗尽。细胞在 2nd胸苷块释放后立即转染 siRNAs, 当细胞处于 S 阶段, 而在 G1 中复?...

Watch this Scientific Journal Video about Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis at JoVE

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

我们提出一个协议的双胸苷同步 HeLa 细胞后, 分析使用高分辨率共聚焦显微镜。这种方法是获得大量的细胞, 同步进行从 S 期到有丝分裂的关键, 使研究的有丝分裂作用的功能蛋白, 也具有相间的作用。

有丝分裂细胞同步与高分辨共聚焦显微镜联合应用研究细胞周期蛋白在有丝分裂过程中的作用

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

The overall goal of this procedure is to use double thymidine synchronization and high-resolution confocal microscopy to study the mitotic roles of multifunctional proteins that may possess critical interphase functions. This method can help answer key questions in the cell biology field, about how to delineate the normal functions of proteins involved in cell cycle and mitosis. The main advantages of this technique are that, the cells can maintain their normal physiological behavior and that this method is also very easy to perform.Demonstrating this procedure will be Dr.Mohammed Amin, a post-doc from my laboratory, who is an expert in the use of this method. For fixed cell imaging of mitotic cell progression, begin by seeding approximately two times 10 to the fifth HeLa cells into each well of a six-well plate, containing a 70%ethanol and UV sterilized cover slip and two milliliters of DMEM medium. After 24 hours in a cell culture incubator, block the cells with two milliliters of freshly diluted thymidine and fresh medium per well, and return the plate to the incubator for another 18 hours.The next day, wash the cells two times with two milliliters of PBS, and one time with fresh 37 degree Celsius medium. Return the cells to the incubator for nine hours to release the cells from the block, followed by the addition of another two milliliters of thymidine-supplemented medium per well. After the second blocking incubation, wash the cells two times with two milliliters of PBS, and one time with two milliliters of fresh 37 degree Celsius medium, and add 100 nanomolar of freshly prepared siRNA to the appropriate wells.After nine to 10 hours, aspirate the supernatant and fix the cells onto the cover slips with four percent paraformaldehyde for 20 minutes at room temperature. After washing with PBS, permeabilize the fixed cells with 0.5%detergent for 10 minutes at room temperature, followed by two five-minute washes in PBS. Block the cells with one percent BSA in PBS for one hour at room temperature.Then label the cells with 50 microliters of the primary antibodies of interest for one hour at 37 degrees Celsius. At the end of the incubation, wash the cells three times in PBS, and label them with 50 microliters of the appropriate secondary antibodies of interest. After one hour at room temperature, wash the cells two times in PBS for five minutes each wash, and label the cells with DAPI for five minutes at room temperature.Follow the DAPI staining with two five-minute washes in PBS and use the appropriate mounting medium to mount the cover slips on individual clear microscope slides. Then, using 60 or 100 X 1.4 numerical aperture plan apochromatic DIC oil immersion objectives mounted on an inverted high resolution confocal microscope equipped with an appropriate camera, acquire images of the immunostained proteins in Z stacks of 0.2 micrometer thickness. For live cell imaging of mitotic cell progression, seed approximately 0.5 to one times 10 to the fifth HeLa cells stably expressing mCherry H2B and GFP alpha-tubulin into 35-milliliter glass-bottom dishes containing 1.5 milliliters of DMEM medium.Grow the cells in a cell culture incubator for 24 hours. Then, thymidine block the cells two times as just demonstrated, this time washing the cells two times with two milliliters of PBS and one time with one milliliter of pre-warmed DMEM medium at the end of each thymidine block and treating the cells with siRNA after the first block during the first thymidine washout. Grow the cells in fresh pre-warmed Leibovitz's medium, supplemented with 10%FBS and 20 millimolar HEPES for eight hours to release the cells from the second block.Then place the first plate in the temperature-controlled chamber of a high-resolution confocal microscope, and use the 60 X objective and bright field imaging to bring the cells into focus. When the cells are visible, manually adjust the plate position to the region of choice and use the image acquisition software to set the laser power and exposure, image acquisition parameters, and experiment duration period. Select the appropriate GFP and mCherry filters and acquire pulsed transmitted light and fluorescence images every 10 minutes for up to 16 hours, to obtain time-lapse images of the mitosis process.Nine hours after release from the double thymidine block, acquire the images at 12 one-micrometer separated z-planes. Then analyze the images by tracking individual mitotic cells and use the appropriate source software to assemble a corresponding movie. Although most of the control and Cdt1 siRNA cells are at the metaphase stage at nine hours post-release from double thymidine block, fixation at 10 hours reveals that most of the Cdt1 siRNA-treated cells are still arrested in late prometaphase, while the control cells enter anaphase and segregate their chromosomes as expected.Cold treatment nine hours after release from double thymidine block facilitates the retention of stable kinetochore microtubules which are relatively less robust in Cdt1-depleted cells compared to those within control-depleted cells. DNA replication origin licensing is not perturbed in Cdt1-or control-depleted cells during the G2-M phase, as these cells were not found to induce the accumulation of phosphoralated gamma H2AX, a marker for DNA damage, during the subsequent G2 phase. Cdt1 siRNA transfection of asynchronous cultures, however, induces the accumulation of phospho gamma H2AX positive foci possibly due to the DNA damage induced by improper DNA replication licensing.After nuclear envelope breakdown, normal cells enter mitosis and undergo anaphase onset to exit from mitosis within about 30 to 60 minutes. RNAi-mediated knockdown of Hec1, a key kinetochore protein required for robust kinetochore microtubule attachment formation, however, delays the normal mitotic progression to anaphase onset or exit from mitosis even after several hours of mitosis delay. Once mastered, this technique can be completed in three to four days if it is performed properly.While attempting the procedure, it's important to remember to dissolve the thymidine completely after you thaw it out from the freezer. Following this procedure, other methods like western blotting can be performed to answer additional questions like, what are the expression patterns of proteins of interest during mitosis compared to interphase? After its development, this technique paved the way for researchers in the field of cell biology to explore cancer biogenesis in human cell culture models, and to use high-resolution confocal microscopy to identify the cell cycle related functions of proteins of interest.Don't forget that working with reagents like paraformaldehyde and DAPI can be extremely hazardous and that precautions such as wearing gloves and a lab coat, should always be taken while performing this procedure.

Research

JoVE Journal

Biology

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基于荧光测定活细胞中的钙进入商店经营：从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

科研

生物学

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

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从长期来看，高解析度激光共聚焦时间流逝成像<em&gt;拟南芥子叶</em&gt;表皮萌发过程中

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Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

使用4D共聚焦显微镜分析颅面形态的斑马鱼

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

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结合母细胞和波动测试磁分选，以分析基因组不稳定性的过程中有丝分裂细胞衰老<em&gt;酿酒酵母</em

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

同步<em&gt;新月柄杆菌</em&gt;对细菌细胞周期的研究

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

细胞周期进程的时间跟踪使用流式细胞仪，无需同步

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...

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

无细胞化验，研究染色质解聚在有丝分裂的结束

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

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

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Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking

基于显微镜的测定，用于使用SNARE运输研究和分析回收内体

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in ...

Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy

使用活细胞显微镜测量扩散过程中的细胞边缘突起动力学

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the ...