Name: computational analysis of the caenorhabditis elegans germline to study the distribution of nuclei, proteins, and the cytoskeleton
Uploaded: 2018-04-19T12:30:00.000+00:00
Duration: 8 min 1 s
Description: Watch this Scientific Journal Video about Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton at JoVE.com

我们感谢显微成像的技术支持。有些菌株是由由 NIH 研究基础设施项目 (P40 OD010440) 资助的秀丽遗传学中心提供的。这项工作得到了一所大学生物医学发现奖学金, 澳洲项目赠款 (GNT1105374), 澳洲高级研究金 (GNT1137645) 和 veski 创新研究金的支持: VIF 23 到罗杰波科克。

1. 制剂和畜牧业
注: 有关所有产品信息, 请参阅材料表。
<ol><li>OP50 大肠杆菌文化: OP50 细菌溶源性汤 (磅) (1% 胰蛋白胨, 0.5% 酵母, 0.5% NaCl, pH 7.0) 隔夜在37°c 没有抗生素. </li>
	<li>种子400µL OP50 细菌的线虫生长培养基 (NGM) 板 (1.5 克氯化钠, 8.5 克琼脂, 1.25 克蛋白胨, 1 毫升1米 CaCl2, 1 毫升5毫克/毫升胆固醇在乙醇, 1 毫升1米 MgSO4, 25 毫升1米 KPO4缓冲器), 空气干燥细菌为 48 h。</li>
	<li>在种子 NGM 板上采摘蠕虫, 在15摄氏度或20摄氏度孵化。<ol>
<li>为分析成年雌雄同体生殖, 选择喂养良好的 L4 蠕虫到细菌草坪和孵化过夜20摄氏度。</li>
		<li>在雌雄同体的精子数量, 孵化 L4 蠕虫在20°c 12 小时, 直到蠕虫达到 L4/adult 蜕皮。要使蠕虫与 L4 阶段同步, 请在漂白溶液中用大量的鸡蛋采摘成虫 (1 米氢氧化钠和漂白剂1:1 的比例) 来准备鸡蛋。让鸡蛋孵化和采摘 L4 动物的实验。</li>
	</ol>
</li>
	<li>通过将25µL 的聚 l-赖氨酸溶液放在滑梯上, 用吸管尖端将溶液涂好, 制备聚四氟乙烯显微镜幻灯片。除去多余的聚 l-赖氨酸与纸巾, 让幻灯片的空气干燥和孵化的幻灯片在65°c 15-20 分钟。</li>
</ol>2. 生殖解剖和染色6,13
<ol><li>斑点10µL 0.01% 四咪唑 (麻醉剂) 在22毫米 x 22 毫米盖玻片并且选择一天老成人蠕虫入它。</li>
	<li>解剖蠕虫在尾部, 因为它成为镇静剂使用注射器 (5 毫升) 和针 (24 "x 1"), 并确保生殖没有损坏。 注意: 解剖必须在蠕虫完全瘫痪之前进行, 这样蠕虫和蠕虫运动中的负压就能帮助生殖的弹射。对于精子的评分, 必须采取预防措施, 以防止在解剖过程中蝗受精囊针的损伤。避免接触生殖与针, 除了解剖点后, 蝗受精囊。如果生殖车厢内有断裂或生殖的任何排出物被观察到, 则生殖不应用于分析。</li>
	<li>将倒置的盖玻片放在聚 l-赖氨酸涂层的滑动上, 使解剖生殖保持在幻灯片上。</li>
	<li>将纸塔放在盖玻片的一个角上, 将盖玻片下的多余液体移开, 然后在液氮中放置1分钟的滑动. 用针与幻灯片上的 germlines 快速移除盖玻片。</li>
	<li>将幻灯片转移到-20 °c 甲醇 30-六十年代在一个房间里, 然后孵化30分钟在新配制的固定溶液 (1x 磷酸盐缓冲盐水 (PBS) 含0.08 米 HEPES pH 6.9, 1.6 毫米 MgSO4, 0.8 毫米乙二醇-双 (β氨基乙基醚)-n, n, n ', n-'-四乙酸四酸 (EGTA), 和3.7% 多聚甲醛) 室温。</li>
	<li>通过放置在包含 1x PBS 的房间, pH 值7.4 与 0.1% Tween-20 10 分钟, 洗涤幻灯片. 重复此步骤一次。</li>
	<li>将湿纸巾放在室温下的塑料盒中, 用30µL 的 germlines (30% 的山羊血清, 在1700年µL 的蒸馏 H2O 和300µL 10x PBS) 在潮湿的房间里, 在室温下放置30分钟。</li>
	<li>在30% 山羊血清中添加50µL 的 REC-8 抗体溶液, 每种样品的比值1:300。在4°c 一夜间孵化。</li>
	<li>在装有 1x PBS 的腔室中放置 0.1% Tween-20 10 分钟, 将其冲洗干净. 重复步骤一次, 通过在幻灯片的拐角处放置纸巾来去除多余的液体。</li>
	<li>通过稀释绿色荧光 (488 nm 发射波长) 共轭二级抗体 (1:1000), 罗丹明 (肌动蛋白染色, 1:4000) 和 DAPI (DNA 染色, 1:4000) 在30% 正常山羊血清中制备二级抗体溶液。</li>
	<li>在幻灯片中添加50µL 二级抗体溶液。</li>
	<li>室温下孵育1小时。</li>
	<li>在装有 1x PBS 的腔室中放置两次幻灯片, 0.1% Tween-20 10 分钟。</li>
	<li>将30µL 的固定试剂添加到幻灯片上, 并将12毫米2 x 1.5 µm 盖玻片放在顶部, 并在4°c 一夜之间干燥幻灯片。</li>
</ol>3. 共焦显微术
<ol><li>打开共焦显微镜、激光器、共振扫描仪和共焦显微镜软件。将带有染色 germlines 的幻灯片放在63X 目标上方的幻灯片架上。</li>
	<li>找到一个生殖, 聚焦在生殖的顶端, 并用共焦显微镜软件标记它。同样专注于底部的生殖和标记它建立总生殖厚度。</li>
	<li>图像的整个生殖通过定义的厚度, 每片0.5 µm. 标记完整的生殖并开始获取。扫描每个切片8次, 并平均值以提高图像质量。共振扫描仪将允许快速扫描, 以避免在成像过程中漂白。如果显微镜没有共振扫描仪, 将扫描次数减少到四, 以避免漂白。</li>
</ol>4. 核数和分布的后成像分析
注: 有关使用的软件工具和按钮的屏幕截图, 请参阅辅助图 1 。
<ol><li>将图像导入 Imaris 8.4.1 或更高版本的软件。</li>
	<li>通过确定各区域核形态, 定义生殖的有丝分裂区、过渡区、减数分裂区、卵母细胞区和蝗受精囊。</li>
	<li>使用软件的 "曲面函数" 按钮 (辅助图 1A) 选择感兴趣的区域。</li>
	<li>取消自动曲面创建向导, 并手动从三维堆栈中绘制图像的第一个和最后一个切片所感兴趣的区域。</li>
	<li>单击创建曲面。 注意: 该软件将自动开发堆栈之间的第一和最后一栈。</li>
	<li>通过单击掩码通道按钮 (辅助图 1 b-C), 屏蔽除 DAPI 之外的所有通道。</li>
	<li>使用 "现场功能" 按钮 (辅助图 1A) 定义核尺寸参数。对于有丝分裂区域, 定义一个 XY 直径为2µm, 同时留下 Z 直径未定义。对于过渡区域, 将2µm 和 Z 直径的 XY 直径定义为1.5 µm. (补充图 1D)。 注: 根据显微镜的放大倍数和规格, 对直径进行故障诊断。对于当前的协议, 使用的目标是 63X, 在成像过程中提供额外的1.70 倍放大倍数。如果目标改变, 例如 40X, 直径应该相应地改变。应使用 wildtype germlines 执行故障排除以建立正确的参数。野生型生殖臂的有丝分裂区有大约250个细胞核。应修改直径以达到此值。使用至少 15 germlines 来确定直径的最佳值。对过渡区 (150-170 核) 和减数分裂区 (600-700 核) 进行标定时, 应采用类似的方法。</li>
	<li>通过定义最小阈值 (辅助图 1E) 来限制对斑点的检测。使用 DAPI 染色的野生型 germlines, 通过增加最小三维渲染阈值来建立最小阈值, 直到第一个点出现在生殖之外。</li>
	<li>保存图像并获取由软件自动生成的表中的核数。将图像导出为 TIF 文件。</li>
</ol>5. 评分精子和染色体编号的后成像分析
<ol><li>通过选择蝗受精囊作为感兴趣的区域来执行三维渲染。要检测每个精子, 请在 X 和 Y 轴之间定义 0.75-1.0 µm 之间的光斑直径, 而 Z 直径仍未定义。使用 "斑点函数" 按钮, 如辅助图 1所示。</li>
	<li>通过滴答作响的背景减法框减去背景, 并使用软件计算的背景校正值 (辅助图 1D)。 注: Imaris 软件首先选取高强度点, 因此背景的影响是最小的, 只要感兴趣区域与背景有显著差异。第二种方法是手动定义背景校正, 在这里可以为背景提供适当的值。如果独立样本的强度需要比较, 手动背景校正将是适当的。</li>
	<li>调整三维渲染阈值以检测蝗受精囊中所有 DAPI 染色的精子(辅助图 1E)。</li>
	<li>对于染色体计数, 在开发三维模型之前, 定义 X 和 Y 轴的光斑直径 < 0.75 µm。</li>
	<li>保存图像并导出为 TIF 文件。</li>
</ol>6. 生殖骨架重建后的影像学分析
<ol><li>通过单击掩码通道按钮, 标识生殖中感兴趣的区域, 并屏蔽除罗丹明之外的所有通道。</li>
	<li>使用 "曲面函数" 按钮 (辅助图 1) 定义0.25 µm 的曲面细节。 注: 这意味着每0.25 µm 将被渲染成三维模型。表面细节可以是恒定的任何区域的生殖, 三维表面模型将创建每0.25 µm。然而, 对于卵母细胞区域, 由于该地区有明确的肌动蛋白纤维, 表面细节可以增加到 0.35-0.5 µm。</li>
	<li>通过滴答作响的背景减法框减去背景, 并使用软件计算的背景校正值 (辅助图 1D)。</li>
	<li>根据需要分析的结构调整三维渲染阈值 (辅助图 1E)。</li>
	<li>保存已开发的三维图像并导出为 TIF 文件。</li>
</ol>

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作者声明没有利益冲突。

该协议的目标是提高准确性, 减少生殖分析所需的时间。在解剖 germlines 的标准制备后, 通过计算渲染, 制备了一个三维生殖核模型。在允许观测生殖核在空间中的分布时, 三维渲染计算了生殖特定区域的核数目。我们的方法的关键方面是准确定义的大小和形状参数的原子核。这取决于染色的清晰度和图像的放大倍数。通过与已发布的手动分析数据1进行比较, 我们确认了该方法的正...

与哺乳动物的信号通路守恒使线虫成为研究多个生物过程的优秀模型1,2。在实验室中, 我们使用线虫生殖研究干细胞的发育、凋亡和基因表达。虽然生殖是一个三维结构, 但由于三维分析的耗时和劳动密集型性质, 许多研究是二维的。二维分析极有可能会在生殖中歪曲体内事件。C. 线虫成体雌雄同体有两个生殖臂, 每个手臂都有一个躯体远端尖端细胞 (DTC), 在未分化状态3,4中维持远端生殖细胞。这些生殖细胞开始分化, 因为它们离开 DTC, 逃避其影响, 并成为卵母细胞和精子, 因为它们到达生殖的近端。在此过程中, 生殖细胞核发生有丝分裂, 然后过渡到减数分裂5,6。精子生产由发育的幼虫阶段 4 (L4) 完成, 之后卵母细胞在成年期间产生。精子储存在蝗受精囊卵母细胞中, 以生成胚胎。
有多种遗传和环境因素可以影响线虫中的生殖发展, 导致细胞核数量、凋亡事件数、染色体动态、蛋白质表达和/或定位的变化7 ,8,9,10,11。对这些事件的分析需要确定基于核形态和分布的各个分化阶段。要用大量的样本量手动准确地分析这些参数, 是劳动密集型和耗时的。为了规避这些缺点, 并使分析的一致性, 我们开发了一种自动化的方法, 三维检查的线虫生殖核计数, 核分布, 蛋白质表达, 和骨架结构。将共焦显微术与三维渲染相结合, 生成了粒度和形状参数, 用于鉴定生殖细胞分化的各个阶段。此外, 这种方法可以计数的生殖细胞核和精子加评分的染色体数在每个卵母细胞。
生殖的一个关键结构是骨架, 它提供了生殖室的稳定性, 艾滋病细胞质流和保护生殖核12。利用计算渲染, 我们对生殖细胞骨架进行了三维重建, 并在生殖中发现了明显的骨架特征。在这里, 我们描述了一步一步的协议, 以说明计算分析如何结合共焦成像, 使对C. 线虫生殖的综合分析。
我们提出了一个快速的方法, 三维分析线虫生殖 (图 1)。利用三维分析, 可以研究生殖核的三维分布 (图 2和图 3), 自动计数单元格 (图 2), 重建生殖骨架 (图 3), 蛋白质的分布 (图 4), 并评分卵母细胞中蝗受精囊和染色体中的精子数量(图 5)。该方法不仅能方便准确地量化生殖, 而且能识别生理上相关的表型。

<table><tbody><tr><td>&lt;em&gt;C. elegans&lt;/em&gt; strains: wild type (N2, Bristol), &lt;em&gt;rnp-8(tm2435) I/hT2[bli-4(e937) let-?(q782) qIs48] (I;III), cpb-3(bt17) I, glp-1 (e2141) III&amp;nbsp;&lt;/em&gt;</td><td>Caenorhabditis Genetics Center (CGC)</td><td></td><td></td></tr><tr><td>OP50 &lt;em&gt;Escherichia coli&lt;/em&gt; bacteria</td><td>Homemade</td><td></td><td></td></tr><tr><td>Nematode Growth Media (NGM) plates</td><td>Homemade</td><td></td><td></td></tr><tr><td>polyclonal rabbit anti-REC-8&amp;nbsp;</td><td>SDIX</td><td>29470002</td><td></td></tr><tr><td>Alexa 488 conjugated antibody raised in goat</td><td>Thermofisher Scientific</td><td>A-21236</td><td></td></tr><tr><td>Cytoskeletal dye phalloidin&amp;nbsp;</td><td>Thermofisher Scientific</td><td>A-12380</td><td></td></tr><tr><td>DAPI&amp;nbsp;</td><td>Thermofisher Scientific&amp;nbsp;</td><td>62248</td><td></td></tr><tr><td>Poly-L-lysine&amp;nbsp;</td><td>Sigma Aldrich</td><td>P5899</td><td></td></tr><tr><td>Tetramisol&amp;nbsp;</td><td>Sigma Aldrich</td><td>P5899</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>M7506</td><td></td></tr><tr><td>1M HEPES buffer, pH 7.4&amp;nbsp;</td><td>Sigma Aldrich</td><td>G0887</td><td></td></tr><tr><td>10X PBS pH 7.4&amp;nbsp;</td><td>Thermofisher Scientific</td><td>AM9625</td><td></td></tr><tr><td>Tween-20&amp;nbsp;</td><td>Sigma Aldrich</td><td>P1389</td><td></td></tr><tr><td>EGTA</td><td>Sigma Aldrich</td><td>E3889</td><td></td></tr><tr><td>37% Paraformaldehyde solution</td><td>Merck Millipore</td><td>1040031000</td><td></td></tr><tr><td>Normal goat serum</td><td>Sigma Aldrich</td><td>G9023</td><td></td></tr><tr><td>Fluoroshield fixing reagent&amp;nbsp;</td><td>Sigma Aldrich</td><td>F6182</td><td></td></tr><tr><td>Ethanol&amp;nbsp;</td><td>Millipore&amp;nbsp;</td><td>1009832511</td><td></td></tr><tr><td>Methanol</td><td>Sigma Aldrich</td><td>34860</td><td></td></tr><tr><td>20&amp;deg;C &amp;amp; 25&amp;deg;CIncubator&amp;nbsp;</td><td>Any brand</td><td></td><td></td></tr><tr><td>Light microscope</td><td>Any brand</td><td></td><td></td></tr><tr><td>Confocal microscope&amp;nbsp;&amp;nbsp;</td><td>Any brand (Leica, Zeiss)</td><td></td><td></td></tr><tr><td>Computer equipped with Imaris suit 8.4.1 or later version, full licence to use the software and Matlab software.</td><td>Bitplane</td><td></td><td></td></tr><tr><td>Phospho buffered saline, pH 7.4</td><td>Homemade</td><td></td><td></td></tr><tr><td>Teflon microscope slides&amp;nbsp;</td><td>Tekdon&amp;nbsp;</td><td>&amp;nbsp;941-322-8288</td><td></td></tr></tbody></table>

computational analysis of the caenorhabditis elegans germline to study the distribution of nuclei, proteins, and the cytoskeleton

秀丽线虫(C. 线虫) 生殖用于研究几个生物学上重要的过程, 包括干细胞发育、细胞凋亡和染色体动力学。虽然生殖是一个很好的模型, 分析往往是二维由于时间和劳动力需要的三维分析。这些研究的主要读数是生殖中细胞核和蛋白质分布的数量/位置。在这里, 我们提出了一个方法来执行生殖的自动分析使用共焦显微镜和计算方法, 以确定核在每个区域的生殖的数量和位置。我们的方法还分析了生殖蛋白的分布, 使三维检测的蛋白质表达在不同的遗传背景。此外, 我们的研究显示在生殖的不同区域骨架体系结构的变化, 可以适应特定的空间发展要求。最后, 我们的方法可以自动计数的精子在每个生殖的蝗受精囊。结合起来, 我们的方法实现了对C. 线虫生殖的快速和可重现的表型分析。

图 1表示三维生殖分析所需的时间。L4 雌雄同体孵化20摄氏度被解剖, 以隔离 germlines 和染色 DAPI, 罗丹明和抗体对生殖蛋白。Germlines 是用共焦显微镜成像的。染色和共焦显微术需要大约24小时的完整生殖计算分析需要 10-15 分钟来计算细胞核的数量和位置, 确定蛋白质分布, 分析骨架结构, 并评分精子数量。完整的手动分析要求每生殖超过2小时, 并受操作...

Watch this Scientific Journal Video about Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton at JoVE.com

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

我们为秀丽线虫生殖提供三维重建的自动方法.我们的方法确定生殖中每个核的数量和位置, 并分析生殖蛋白质分布和骨架结构。

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

computational-analysis-caenorhabditis-elegans-germline-to-study

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

6.2K 次观看.  Monash University. 我们为秀丽线虫生殖提供三维重建的自动方法.我们的方法确定生殖中每个核的数量和位置, 并分析生殖蛋白质分布和骨架结构。

视频: Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a&#160;model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism&#39;s genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analy

The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level.

C. 线虫小肠作为细胞间腔形态发生和体内极化膜生物单细胞水平的模型: 抗体染色、rna 干扰功能分析和成像标记

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

科研

发育生物学

C. elegans Gonad Dissection and Freeze Crack for Immunofluorescence and DAPI Staining

The C. elegans germline makes an excellent model for studying meiosis, in part due to the ease of conducting cytological analyses on dissected animals. Whole mount preparations preserve the structure of meiotic nuclei, and importantly, each gonad arm contains all stages of meiosis, organized in a temporal-spatial progression that makes it easy to identify nuclei at different stages. Adult hermaphrodites have two gonad arms, each organized as a closed tube with proliferating germline stem cells at the distal closed end and cellularized oocytes at the proximal open end, which join in the center at the uterus. Dissection releases one or both gonad arms from the body cavity, allowing the entirety of meiosis to be visualized. Here, a common protocol for immunofluorescence against a protein of interest is presented, followed by DAPI staining to mark all chromosomes. Young adults are immobilized in levamisole and quickly dissected using two syringe needles. After germline extrusion, the sample is fixed before undergoing a freeze crack in liquid nitrogen, which helps permeabilize the cuticle and other tissues. The sample can then be dehydrated in ethanol, rehydrated, and incubated with primary and secondary antibodies. DAPI is added to the sample in the mounting medium, which allows reliable visualization of DNA and makes it easy to find animals to image under a fluorescent microscope. This technique is readily adopted by those familiar with handling C. elegans after a few hours spent practicing the dissection method itself. This protocol has been taught to high-schoolers and undergraduates working in a research lab and incorporated into a course-based undergraduate research experience at a liberal arts college.

C. elegans Gonad Dissection and Freeze Crack for Immunofluorescence and DAPI Staining

This is a commonly used method for C. elegans gonad dissection followed by freeze crack, which produces germline samples for immunofluorescence via antibody staining, or for simple DAPI staining to visualize DNA. This protocol has been successful for undergraduates in a research lab and in a course-based undergraduate research experience.

秀丽隐杆线虫 用于免疫荧光和DAPI染色的性腺解剖和冷冻裂缝

The C. elegans germline makes an excellent model for studying meiosis, in part due to the ease of conducting cytological analyses on dissected animals. ...

遗传学

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases of the cell cycle, or the incorporation of nucleoside analogs. During S-phase, DNA polymerases incorporate thymidine analogs such as EdU or BrdU into chromosomal DNA, marking the cells for analysis. For C. elegans, the nucleoside analog EdU is fed to the worms during regular culture and is compatible with immunofluorescent techniques. The germline of C. elegans is a powerful model system for the studies of signaling pathways, stem cells, meiosis, and cell cycle because it is transparent, genetically facile, and meiotic prophase and cellular differentiation/gametogenesis occur in a linear assembly-like fashion. These features make EdU a great tool to study dynamic aspects of mitotically cycling cells and germline development. This protocol describes how to successfully prepare EdU bacteria, feed them to wild-type C. elegans hermaphrodites, dissect the hermaphrodite gonad, stain for EdU incorporation into DNA, stain with antibodies to detect various cell cycle and developmental markers, image the gonad and analyze the results. The protocol describes the variations in the method and analysis for the measurement of S-phase index, M-phase index, G2 duration, cell cycle duration, rate of meiotic entry, and rate of meiotic prophase progression. This method can be adapted to study the cell cycle or cell history in other tissues, stages, genetic backgrounds, and physiological conditions.

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

An imaging-based method is described that can be used to identify S-phase and analyze cell cycle dynamics in the C. elegans hermaphrodite germline using the thymidine analog EdU. This method requires no transgenes and is compatible with immunofluorescent staining.

种系线虫与胸苷模拟教育的细胞周期分析

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

半高通量成像方法和数据可视化工具包，用于分析C. elegans胚胎发育

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes such as development are affected. The Caenorhabditis elegans germline is a great model system for studying PPIs that are related to the regulation of stem cells, meiosis, and development. There are a variety of well-developed techniques that allow proteins of interest to be tagged for recognition by standard antibodies, making this system advantageous for proximity ligation assay (PLA) reactions. As a result, the PLA is able to show where PPIs occur in a spatial and temporal manner in germlines more effectively than alternative approaches. Described here is a protocol for the application and quantification of this technology to probe PPIs in the C. elegans germline.

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

This protocol demonstrates use of the proximity ligation assay to probe for protein-protein interactions in situ in the C. elegans germline.

使用接近连接测定在C.elegans Germline中对核糖蛋白复合体装配的实地检测

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

成像 C。线虫胚胎使用啶显微镜和开放源码软件

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

生物学

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has proven to be an invaluable model organism in helping to tackle this challenge, from inspiring technological approaches,&#160;building the human brain connectome, to actually shedding light on the specific molecular drivers of basic functional patterns. The bulk of the behavioral studies in C. elegans have been performed on solid substrates. In liquid, animals exhibit behavioral patterns that include movement at a range of speeds in 3D, as well as partial body movements, such as a posterior curl without anterior shape change, which introduce new challenges for quantitation. The steps of a simple procedure, and use of a software that enables high-resolution analysis of C. elegans swim behavior, are presented here. The software, named CeleST, uses a specialized computer program that tracks multiple animals simultaneously and provides novel measures of C. elegans locomotion in liquid (swimming). The measures are mostly grounded in animal posture and based on mathematics used in computer vision and pattern recognition, without computational requirements for threshold cut-offs. The software tool can be used to both assess overall swimming prowess in hundreds of animals from combined small batch trials and to reveal novel phenotypes even in well-characterized genetic mutants. The preparation of specimens for analysis with CeleST is simple and low-tech, enabling wide adaptation by the scientific community. Use of the computational approach described here should therefore contribute to the greater understanding of behavior and behavioral circuits in the C. elegans model.

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

An efficient and simple methodology for computer-based analysis of nematode swimming behavior in liquid is described. The method requires little to no investment for C. elegans laboratories. The hardware used is standard, and the computer software for the behavioral analysis (CeleST) is an open source one.

的自动分析<em&gt;℃。线虫</em&gt;游泳行为使用天书软件

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has ...

神经科学

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

跟踪和量化的发育过程<em&gt;℃。线虫</em&gt;使用开源工具

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein subcellular localization changes; in particular, a nuclear translocation under a heat stress condition. Proteins react correspondingly to external and internal signals. A common mechanism is to change its subcellular localization. This article describes a protocol to track protein localization that does not require an antibody, radioactive labeling, or a confocal microscope. In this article, GFP is used to tag the target protein EXL-1 in C. elegans, a member of the chloride intracellular channel proteins (CLICs) family, including mammalian CLIC4. An integrated translational exl-1::gfp transgenic line (with a promoter and a full gene sequence) was created by transformation and &#947;-radiation, and stably expresses the gene and gfp. Recent research showed that upon heat stress, not oxidative stress, EXL-1::GFP accumulates in the nucleus. Overlapping the GFP signal with both the nuclei structure and the DAPI signals confirms the EXL-1 subcellular localization changes under stress. This protocol presents two different fixation methods for DAPI staining: ethanol fixation and acetone fixation. The DAPI staining protocol presented in this article is fast and efficient and preserves both the GFP signal and the protein subcellular localization changes. This method only requires a fluorescence microscope with Nomarski, a FITC filter, and a DAPI filter. It is suitable for a small laboratory setting, undergraduate student research, high school student research, and biotechnology classrooms.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

This protocol demonstrates how to track a protein nuclear translocation under heat stress by using a green fluorescence protein (GFP) fusion protein as a marker and 4',6-diamidino-2-phenylindole (DAPI) staining. The DAPI staining protocol is fast and preserves the GFP and protein subcellular localization signals.

秀丽线虫绿色荧光蛋白标记和 4 '、6-Diamidino-2-苯基吲哚染色检测蛋白质亚细胞定位

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

研究卡埃纳哈布迪炎细胞结构和细胞形态学的定量方法

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

摘要

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C. 线虫小肠作为细胞间腔形态发生和体内极化膜生物单细胞水平的模型: 抗体染色、rna 干扰功能分析和成像标记

秀丽隐杆线虫用于免疫荧光和DAPI染色的性腺解剖和冷冻裂缝

种系线虫与胸苷模拟教育的细胞周期分析

半高通量成像方法和数据可视化工具包，用于分析C. elegans胚胎发育

使用接近连接测定在C.elegans Germline中对核糖蛋白复合体装配的实地检测

成像 C。线虫胚胎使用啶显微镜和开放源码软件

的自动分析

跟踪和量化的发育过程

秀丽线虫绿色荧光蛋白标记和 4 '、6-Diamidino-2-苯基吲哚染色检测蛋白质亚细胞定位

研究卡埃纳哈布迪炎细胞结构和细胞形态学的定量方法

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

摘要

探索更多视频

此视频中的章节

C. 线虫小肠作为细胞间腔形态发生和体内极化膜生物单细胞水平的模型: 抗体染色、rna 干扰功能分析和成像标记

秀丽隐杆线虫 用于免疫荧光和DAPI染色的性腺解剖和冷冻裂缝

种系线虫与胸苷模拟教育的细胞周期分析

半高通量成像方法和数据可视化工具包，用于分析C. elegans胚胎发育

使用接近连接测定在C.elegans Germline中对核糖蛋白复合体装配的实地检测

成像 C。线虫胚胎使用啶显微镜和开放源码软件

的自动分析

跟踪和量化的发育过程

秀丽线虫绿色荧光蛋白标记和 4 '、6-Diamidino-2-苯基吲哚染色检测蛋白质亚细胞定位

研究卡埃纳哈布迪炎细胞结构和细胞形态学的定量方法

秀丽隐杆线虫用于免疫荧光和DAPI染色的性腺解剖和冷冻裂缝