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 和 5% CO2的湿润培养箱中生长细胞24小时。</li>
	<li>1st胸苷块: 在2天, 彻底混合的要求体积的胸苷在新鲜的 DMEM 媒体 (100 毫米的股票, 2 毫米最终浓度)。</li>
	<li>在6井板的每一个井中加入2毫升的胸苷含培养基, 并孵育18小时。</li>
	<li>3天, 吸入培养基, 用2毫升 1x PBS 冲洗两次细胞, 再用新鲜 prewarmed DMEM;在新鲜的 prewarmed DMEM 培养基中培养细胞, 9 h 释放细胞。</li>
	<li>2nd胸苷块: 再次添加2毫升的胸苷含培养基 (2 mM 最终浓度) 的细胞在每个井的 6-井板。</li>
	<li>再孵育18小时</li>
	<li>4天, 吸入培养基, 用2毫升 1x PBS 冲洗细胞两次, 再用新鲜 prewarmed DMEM。</li>
	<li>将 siRNAs (控制和 Cdt1) 和转染试剂分别用于血清自由生长培养基10分钟, 将它们混合在一起, 在 RT 中孵育20分钟。在每一个转染 siRNA 的最终浓度为 100 nM。将反应混合物添加到已被胸苷洗掉的细胞中。</li>
	<li>孵育细胞在37° c 为 9-10 h 释放从堵塞和固定在盖子滑动的细胞与4% 煤灰为 20 min 在 RT。 注意: 粉煤灰是有毒的, 穿戴适当的保护。</li>
	<li>
免疫细胞后, permeabilizing 与 0.5% (v/v) 洗涤剂为10分钟在 RT 和洗涤细胞两次与 1x PBS 5 分钟每。
	<ol>
<li>在 RT 1x PBS 中用 1% BSA (w/v) 在1小时内阻断细胞, 然后用50µL 的原代抗体治疗细胞 (小鼠抗α-蛋白抗体稀释 1:1, 000, 兔 anti-Zwint1 抗体稀释 1:400, 和小鼠抗磷ɣ H2AX 稀释 1:300) 在 1% (w/v)BSA 在 1x PBS 中为1小时, 在 37 oC。</li>
		<li>冲洗后的细胞与 1x pbs 三次, 治疗细胞50µL 的第二抗体为每盖玻璃 (Alexa 488 和罗丹明红色在1:250 稀释 1% (瓦特/v) BSA 在 1x PBS) 为 1 h 在 RT。</li>
		<li>在用 1x pbs 清洗两次细胞后, 每次5分钟, 用 DAPI (0.1µg/毫升在 1x pbs) 治疗细胞, 在 RT 5 分钟。</li>
		<li>在用 1x PBS 清洗两次电池5分钟后, 将盖板滑向适当的安装介质, 在清晰的显微镜下滑动。</li>
	</ol>
</li>
	<li>图像的细胞为 immunostained 蛋白60X 或 100X 1.4 NA 计划-物镜 DIC 油浸没目标安装在一个倒置的高分辨率共焦显微镜配备了适当的相机, 必要的图像质量所需的。</li>
	<li>使用适用于显微镜的软件, 在室温下获得0.2 µm 厚度的 z 栈的图像。</li>
</ol>3. 有丝分裂进展的活细胞成像协议 (图 3A)
<ol><li>在1天, 种子大约 0.5-1 x 105 HeLa 细胞稳定表达 mCherry-H2B 和 GFP-α-蛋白 (根据细胞类型和他们表达的蛋白质略有变化) 到35毫米玻璃底菜与1.5 毫升的 DMEM 培养基和增长他们在湿化孵化器为24小时, 在 37 oC 和 5% CO2。</li>
	<li>1st胸苷块: 在2天, 在盘中的细胞中加入1.5 毫升的胸苷含有的培养基 (100 毫米胸苷, 2 毫米最终浓度,)。</li>
	<li>孵育18小时。</li>
	<li>3天, 吸入含有胸苷的培养基, 三次冲洗细胞, 用2毫升 1x PBS, 一次用新鲜 prewarmed DMEM。</li>
	<li>siRNAs (控制和 Hec1) 和血清自由生长培养基的转染试剂分别为10分钟, 将它们混合在一起, 孵育20分钟。在每一个转染 siRNA 的最终浓度为 100 nM。将反应混合物添加到已被胸苷洗掉的细胞中。</li>
	<li>在1.5 毫升的新鲜 prewarmed DMEM 培养基中培养8小时的细胞, 从块中释放细胞。</li>
	<li>2nd胸苷块: 在盘中的细胞中加入1.5 毫升的胸苷含培养基 (2 mM 最终浓度)。</li>
	<li>再孵育18小时</li>
	<li>4天, 吸入培养基, 三次冲洗细胞, 2 毫升 1x PBS, 一次与新鲜 prewarmed DMEM。然后在新鲜的 prewarmed 莱博维茨的 (L-15) 培养基中生长细胞, 并辅以 10% FBS 和20毫米 HEPES, 在 pH 7.0 为 8 h 释放细胞从块。</li>
	<li>将碟放在高分辨率共聚焦显微镜的温度控制箱中, 在开始成像前已经开启了至少30分钟, 以稳定舞台和实验温度。</li>
	<li>聚焦在明亮的领域与60x 目标, 直到细胞是可见的, 然后手动筛选的舞台上的选择区域。</li>
	<li>建立了光和激光功率/曝光、图像采集参数, 以及实验时间的使用显微镜图像采集软件的选择。使用荧光蛋白 (488 激发; 385 nm 发射) 和 mCherry (561 nm 激发和 385 nm 发射) 的过滤器来获取图像。</li>
	<li>通过分别获取脉冲透射光和荧光图像, 进行时间间隔试验, 每10分钟可达到16小时。</li>
	<li>获取图像作为十二1.0 µm 分离的 z 平面在9小时后, 从双胸苷块使用适当的显微镜的软件释放。</li>
	<li>分析图像跟踪个别细胞有丝分裂进展, 最后组装相应的电影与源软件斐济/ImageJ。</li>
</ol>

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作者声明他们没有竞争的财政利益。

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 &amp;deg;C</td></tr><tr><td>DPBS (1x)</td><td>Life Technologies</td><td>14190-144</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Leibovitz&amp;rsquo;s (1x) L-15 medium</td><td>Life Technologies</td><td>21083-027</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Serum reduced medium (Opti-MEM)</td><td>Life Technologies</td><td>319-85-070</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Penicillin and streptomycin</td><td>Life Technologies</td><td>15070-063 (Pen Strep)</td><td>1:1,000 dilution</td></tr><tr><td>Dharmafect2</td><td>GE Dharmacon</td><td>T-2002-02</td><td>Store at 4 &amp;deg;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-&amp;alpha;-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-&amp;alpha;-tubulin</td><td>Santa Cruz Biotechnology</td><td>Sc32293</td><td>1:1,000 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-&amp;gamma;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>35 mm 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 ...

combining-mitotic-cell-synchronization-high-resolution-confocal

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

14.2K 次观看.  Northwestern University. 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。

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

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

通过两个互补细胞同步方案研究细胞周期调控的基因表达

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

科研

遗传学

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.

有丝分裂过程中染色体分离的活体细胞成像

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

发育生物学

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

生物学

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

3D 基质中哺乳动物细胞分裂的定量共焦反射显微镜观察

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

生物工程

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

龙虎斗动粒蛋白的动态行为与染色体运动，在生活学习有丝分裂关卡<em&gt;果蝇</em&gt;合胞胚胎

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

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.

在整个细胞群在单细胞水平上研究细胞周期蛋白B的蛋白水解

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

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.&#160;pombe)&#160;is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

The fission yeast, Schizosaccharomyces pombe&#160;is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

在裂殖酵母中Cytokinetic活动的时空分析

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

荧光活细胞显微镜对微孔裂变酵母核动力学的检测

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, misunderstood, or misinterpreted by the fixed-cell analysis. While the fixed cell imaging and analysis is robust and sufficient to observe cellular steady-state, it can be limited in defining a temporal order of events at the cellular level and is ill-equipped to assess the transient nature of dynamic processes including mitotic progression. In contrast, live cell imaging is an eloquent tool that can be used to observe cellular processes at the single-cell level over time and has the capacity to capture the dynamics of processes that would otherwise be poorly represented in fixed cell imaging. Here we describe an approach to generate cells carrying fluorescently labeled markers of chromatin and microtubules and their use in live cell imaging approaches to monitor metaphase chromosome alignment and mitotic exit. We describe imaging-based techniques to assess the dynamics of spindle formation and mitotic progression, including the identification of cells at various stages in mitosis, identification&#160;and tracking of mitotic defects, and analysis of spindle dynamics and mitotic cell fate following the treatment with mitotic inhibitors.

Here we present a protocol to assess the dynamics of spindle formation and mitotic progression. Our application of time-lapse imaging enables the user to identify cells at various stages of mitosis, track and identify mitotic defects, and analyze spindle dynamics and mitotic cell fate upon exposure to anti-mitotic drugs.

活细胞成像，以评估在斜接主轴扰动后元相定时和细胞命运的动态

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, ...

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.

使用延时显微镜和蛋白质的阶段特异性核耗竭来研究酿酒酵母的减数分裂

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