本研究使用的C. 线虫菌株取自秀丽遗传学中心, 由国立卫生研究院 (P40 OD010440) 提供支持。这项工作得到了 NIH 的支持: 1R03AG048578-01, 纽约市立大学-CCRG 1501 至俊良, 和 PSC-纽约市立大学 66184-00 44 和 67248-00 45 至俊良。我们感谢凯西野蛮-邓恩亲切地分享她的实验室空间。所有荧光图像都收集在皇后学院的核心设施。我们感谢威廉 j. 赖斯对手稿的评论。

1. 解决方案
<ol><li>
NGM 板
	<ol>
<li>在2升锥形烧瓶中, 加入3克氯化钠、17克琼脂、2.5 克蛋白胨和975毫升的 dH2o 盖上铝箔的烧瓶口。将烧瓶蒸压50分钟, 冷却20–30分钟。</li>
		<li>然后添加灭菌的解决方案: 1 毫升的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 (pH 6.0) 缓冲。漩涡的解决方案, 以混合良好。</li>
		<li>使用液体分配器, 将 NGM 溶液分配给60毫米的培养板。把2/3 装满琼脂的盘子填满。将它们存放在4摄氏度的气密容器中, 供将来使用。</li>
	</ol>
</li>
	<li>
1 M KPO4缓冲 (pH 6.0)
	<ol>
<li>在 dH2O 中溶解10.83 克的2PO4和3.56 克的 K4HPO2 , 然后将总容积调整为100毫升。蒸压釜解决方案为15分钟。</li>
	</ol>
</li>
	<li>
M9
	<ol>
<li>溶解3克的2PO4, 6 克的 Na2HPO4, 和 5 g 的氯化钠在 dH2O, 添加1毫升1米 MgSO4, 并调整总容积为1升。</li>
	</ol>
</li>
	<li>
10µg/毫升 DAPI
	<ol>
<li>添加1µL 10 毫克/毫升 DAPI 到999µL 的 dH2O。</li>
	</ol>
</li>
	<li>
95% 酒精 (v/v)
	<ol>
<li>测量95毫升100% 酒精和增加蒸馏水调整容量到100毫升。</li>
	</ol>
</li>
	<li>
30% 丙酮 (v/v)
	<ol>
<li>将30毫升丙酮添加到 dH2O, 并将总容积调整为100毫升。</li>
	</ol>
</li>
</ol>2. 热应力
<ol><li>用目标基因9,10的平移结构生成染色体外阵列线。或者, 从秀丽遗传学中心 (CGC) 获得这样的线, 这是一个公共资源的线虫研究, 或从以前发表的研究实验室。</li>
	<li>通过将蠕虫暴露到 3800 Rad γ辐射9, 将染色体外线整合到基因组中。然后, 选择稳定表达的线条, 使每个动物表达一个标记蛋白, 如 GFP 或滚筒。</li>
	<li>为了消除辐射过程中产生的突变, outcross 至少2x。利用这一转基因线检测在压力下蛋白质表达模式的变化。</li>
	<li>如步骤1.1 所述, 准备 NGM 蠕虫板材。OP50 克隆和培养的细菌在液体 LB 汤过夜。用液体 OP50 培养种子 NGM 板, 在一夜之间孵化37摄氏度孵化皿中的盘子, 种植细菌草坪作为蠕虫的食物来源。在使用前, 在室温下冷却30分钟以上的板材。</li>
	<li>在热应激试验前至少2代, 在20摄氏度不挨饿的情况下种植转基因线。</li>
	<li>选择8–10年轻的妊娠成人 (子宫充满鸡蛋) 到一个新的蠕虫板块, 然后让他们产卵约 3–5 h 和删除所有的成年人从盘子里。</li>
	<li>为了同步蠕虫, 让卵孵化和生长幼虫大约48小时到第四幼虫 (L4) 阶段。检查他们的外阴结构 (半月结构), 以确定 L4 阶段 (图 1, 箭头)11。</li>
	<li>将蠕虫板块与 L4 幼虫放在一个35摄氏度孵化箱中, 2–5 h 以达到热应力。将温度计放在孵化器中, 准确测量温度。</li>
</ol>3. DAPI 染色
<ol><li>
乙醇固定
	<ol>
<li>在玻璃滑梯的中心添加10µL 的 M9 缓冲器。</li>
		<li>从步骤2.8 中选取热休克的蠕虫, 并将它们放入玻璃滑梯上的 M9 缓冲区中。<ol>
<li>或者, 用 M9 把盘子洗掉, 在室温下将其离心 1000 x克1 分钟, 然后丢弃上清。将蠕虫添加到玻璃幻灯片中。</li>
		</ol>
</li>
		<li>用软组织排出多余的液体。在解剖显微镜下观察这一步。</li>
		<li>在蠕虫上添加10µL 95% 的酒精, 让它们风干。观察解剖显微镜下的过程, 不要让动物变得太干燥。乙醇从动物体内蒸发后立即进行下一步。 注: 乙醇蒸发很快。</li>
		<li>重复步骤3.1.4 两次, 以便修复蠕虫。 注意: 这是正常的动物看起来更暗和扭曲。</li>
		<li>添加10µL 的 DAPI (10 µg/毫升) 的蠕虫。立即用盖玻片盖上, 用透明指甲油凝胶封住幻灯片。</li>
		<li>在荧光显微镜下观察10分钟左右的动物。</li>
	</ol>
</li>
	<li>
丙酮固定 (另一种方法)
	<ol>
<li>用 M9 从步骤2.8 中冲洗热震板, 在室温下将其离心 1000 x克, 并丢弃上清液。用1毫升 dH2O 清洗颗粒, 收集颗粒, 并丢弃上清液。</li>
		<li>在室温下添加400µL 30% 丙酮, 15 分钟离心机, 常温下1分钟, 并丢弃上清液。使用500µL 的 dH2O 洗涤动物2x。放弃上清。</li>
		<li>添加200µL 的 DAPI (10 µg/毫升), 或4x 的总量蠕虫的体积, 在管15分钟。</li>
		<li>离心机在 1000 x g 1 分钟, 并丢弃上清。用500µL 洗净颗粒 2x2o 将蠕虫放在玻璃滑梯上, 用盖玻片覆盖, 用透明指甲油凝胶封住幻灯片, 并在荧光显微镜下观察动物。</li>
	</ol>
</li>
</ol>4. 显微成像
<ol><li>打开紫外线光源和荧光显微镜。打开连接到显微镜的计算机。</li>
	<li>在荧光显微镜上加载幻灯片。使用低功耗物镜 (10X) 定位蠕虫, 增加放大功率到 400X, 并专注于标本。</li>
	<li>打开显微镜提供的软件。点击菜单中的 "获取";点击 "相机", 选择相机连接到显微镜;这允许选定的照相机与软件通信。 注: 不同的显微镜在操作上可能有所不同。</li>
	<li>在相同的 "获取" 下, 单击 "多维获取"。这将打开一个新的 "多维获取" 导航窗口。单击 "多声道" 按钮, 所有可用的频道都会出现。</li>
	<li>选择蓝色、绿色和 DIC (差异干扰对比度) 观察标本。在 "多维采集" 导航窗口中, 留下 "蓝 DAPI"、"绿 GFP"、"灰 DIC"、"Nomarski" 频道;右键单击其他通道, 如 "红色罗丹明" 和 "白光场", 以关闭它们。</li>
	<li>
首先, 测量每个通道的曝光时间:
	<ol>
<li>左键点击 "蓝 DAPI" 频道选择它, 然后点击 "测量", 以采取在 DAPI 通道下的实时图像与自动曝光时间。将出现一个具有实时图像的新窗口。</li>
		<li>在 "实时图像" 窗口的左侧, 如果需要, 手动调整曝光时间以使图像达到预期效果。</li>
		<li>最后, 在 "实时图像" 窗口中单击"确定"。这将设置 DAPI 通道下的曝光时间。</li>
	</ol>
</li>
	<li>重复步骤 4.6, 为其他渠道, "绿色 GFP" 和 "灰色 DIC", "Nomarski", 设置的曝光时间, 这些渠道。</li>
	<li>在 "多维采集" 导航窗口中, 单击 "开始" 以根据设定的特定曝光时间自动收集3不同通道下的3幅图像 (见上文)。</li>
	<li>要保存文件, 请转到主菜单, 单击 "文件" 然后在 "另存为"。输入文件名和文件夹名称。将文件保存为默认文件格式, 以保留详细的图像信息以供将来参考。</li>
	<li>
以其他格式导出文件:
	<ol>
<li>转到 "文件", 然后单击 "导出"。这将打开 "导出设置" 窗口。</li>
		<li>设置文件名和文件夹。检查 "创建项目文件夹" 以更好地组织数据。该软件还允许用户指定其他4选项: "生成合并图像"、"使用通道图像的颜色"、"使用通道名称" 和 "仅合并图像"。</li>
		<li>从下拉菜单中选择所需的文件类型, 如 "TIF"、"BMP"、"PSD" 或 "JPG" 等类型。然后单击 "开始" 导出。</li>
	</ol>
</li>
</ol>

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J., Shen, Z. Z., Cunha, A., Emmons, S. W., Leroi, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+polyploidization+and+cellular+proliferation+drive+body+size+evolution+in+nematodes.">Somatic polyploidization and cellular proliferation drive body size evolution in nematodes.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (10), 5285-5290 (2000).</li><li>Nagamatsu, Y., Ohshima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+for+the+control+of+body+size+by+a+G-kinase+and+a+downstream+TGFbeta+signal+pathway+in+Caenorhabditis+elegans.">Mechanisms for the control of body size by a G-kinase and a downstream TGFbeta signal pathway in Caenorhabditis elegans.</a> Genes to Cells. 9 (1), 39-47 (2004).</li></ol>

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本文提出了一种快速有效的方法来验证蛋白质亚细胞变化从细胞质到细胞核。蛋白表达由 GFP 融合显示, 而核结构由 DAPI 染色验证 (图 3)。由于染色线虫蛋白具有挑战性, 大多数C. 线虫蛋白亚细胞定位的特点是标记它们与标记蛋白, 如 GFP, 拉扎, mCherry, 和其他18,19,20,21. 在本文中, gfp被用来标记目标基因exl-1 , 以显示其蛋白质细胞定位。为确定核定位, 采用了 DAPI 染色法。提出了两种不同的 DAPI 染色方法。两者都有效地显示了在一个体面的工作时间框架 (少于1小时) 蠕虫的强核。乙醇固定适用于小样本收集, 如质量观察 (收集少于50蠕虫)。防止蠕虫完全干燥是关键。一旦95% 酒精从蠕虫中蒸发, 立即进行下一步。丙酮固定适合大规模的样本收集, 如定量分析 (收集超过50动物)。然而, 由于洗涤样品的多个步骤, 一些标本将丢失。因此, 强烈建议仔细清除上清, 并保留尽可能多的蠕虫。一旦密封, 由丙酮固定产生的幻灯片可以储存一个星期在4摄氏度。
该协议可以调整到其他感兴趣的蛋白质跟踪亚细胞定位变化。除了这里提出的热应力外, 还可以评估其他的应力检测方法, 如氧化应激、重金属胁迫、饮食限制等。蛋白质亚细胞定位的变化也可以通过改变兴趣蛋白的遗传背景来检验。例如, 转基因线可以交叉成一个不同的突变体的背景, 从而可以确定新的基因相互作用。rna 干扰也可以应用于特定基因的敲除, 并研究产生的蛋白质亚细胞定位变化。这些应用程序对识别新的管理组件非常感兴趣。对于未知的蛋白质, 应测试各种实验条件, 如检测试剂浓度、应力时间的长短以及蠕虫的特定发育阶段。在我们的研究中, 我们尝试了不同的工期, 从30分钟到5小时, 以及不同的热应力温度。热休克在35°c 为 3 h 清楚地显示蛋白质核易位。
DAPI 染色的其他应用包括检查蠕虫的减数分裂和有丝分裂过程, 特别是在生殖细胞22、23、24、25和一个计数的核基因突变体的背景20,26,27。

蛋白质亚细胞定位的变化是反应热应激、饥饿、氧化应激、细胞凋亡、蛋白质磷酸化等内部或外部信号的一种常见机制。例如, 热应力诱导一个 foxo 蛋白成员 DAF-16 核易位1,2和亲凋亡 BCL-2 蛋白出价 translocates 的线粒体后, 接受死亡信号3,4。可以使用各种技术来检测这些更改。西方印迹和生化分离亚细胞结构 (如线粒体或细胞核) 的组合可以很好地实现目标3。然而, 它需要一个特定的抗体对抗蛋白质的兴趣。因此, 一个完善的抗体成为成功的关键。另一种方法是标签不同的亚细胞结构或细胞器与各种标记, 如绿色荧光蛋白 (GFP), 红色荧光蛋白 (RFP), 黄色荧光蛋白 (YFP), 和 mCherry, 同时标签的蛋白质与其他标记的兴趣。然后, 观察他们在共聚焦显微镜下, 以本地化目标5,6。放射性同位素是标记靶蛋白, 然后检测其亚细胞定位7的替代选择。但是, 这种方法需要对放射性废料进行适当的训练和处理。在诸如缺乏特定抗体、缺少适当的标记或 scarceness 等设备的情况下, 需要考虑一种替代方法。为了确定蛋白质核易位, 它是有吸引力的仅标记目标蛋白质与标记和染色核与化学试剂 4 ', 6-diamidino-2-苯基吲哚 (DAPI), 因为这只需要常规荧光显微镜。
Immunolabeling线虫具有抗体是挑战, 由于低渗透性的蛋壳或胶原角质层周围的动物。同时, 由于线虫蛋白与脊椎动物的 orthologs 有明显的差异, 一些商业公司提供特定产品的c. 线虫。小实验室很难为自己生成线虫抗体。社区的研究人员经常使用标记蛋白作为标记来显示蛋白质定位或基因表达。本文以 EXL-1::GFP 为例, 在热应力作用下跟踪蛋白质核易位8。将一个集成的平移exl-1::gfp到动物基因组中, 用于稳定地表达gfp融合的基因。研究表明, exl-1在肠道、体壁肌和其他亚细胞结构中表达8。该协议演示如何将蠕虫同步到第四幼虫 (L4) 阶段, 进行热应激实验, 并在常规荧光显微镜下进行 DAPI 染色、乙醇固定、丙酮固定和成像。

<table><tbody><tr><td>DAPI</td><td>Biotium</td><td>40043</td><td>both Hoechst 33258 and Hoechst 33342 also works well</td></tr><tr><td>OP50</td><td>CGC</td><td>OP50</td><td>https://cgc.umn.edu/</td></tr><tr><td>Caenorhabditis Genetics Center (CGC)</td><td></td><td></td><td>https://cgc.umn.edu/</td></tr><tr><td>Kim Wipe</td><td>Kimberly-Clark Corporation</td><td></td><td>soft tissue</td></tr><tr><td>Zeiss&amp;nbsp; AX10</td><td>Zeiss</td><td></td><td>fluorescence microscope</td></tr><tr><td>Axiovision Rel 4.8</td><td>Zeiss</td><td></td><td>microscope software</td></tr><tr><td>AxioCam MR Rev3</td><td>Zeiss</td><td></td><td>digital camera</td></tr><tr><td>Incubator&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Micro centrifuge</td><td></td><td></td><td></td></tr><tr><td>Transparent nail polish gel</td><td></td><td></td><td></td></tr><tr><td>60 mm petri dishes</td><td></td><td></td><td></td></tr><tr><td>Glass slides</td><td></td><td></td><td></td></tr><tr><td>Glass coverslips</td><td></td><td></td><td></td></tr><tr><td>1-20 &amp;micro;L pipettor</td><td></td><td></td><td></td></tr><tr><td>20-200 &amp;micro;L pipettor</td><td></td><td></td><td></td></tr><tr><td>200-1000 &amp;micro;L pipettor</td><td></td><td></td><td></td></tr><tr><td>1-200 &amp;micro;L pipet tips</td><td></td><td></td><td></td></tr><tr><td>200-1000 &amp;micro;L pipet tips</td><td></td><td></td><td></td></tr><tr><td>1.5 mL microcentrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>Platinum wire worm pick</td><td></td><td></td><td></td></tr></tbody></table>

detecting protein subcellular localization by green fluorescence protein tagging and 4',6-diamidino-2-phenylindole staining in caenorhabditis elegans

本协议采用绿色荧光蛋白 (GFP) 融合蛋白和 4 '、6-diamidino-2-苯基吲哚 (DAPI) 染色法跟踪蛋白质亚细胞定位变化;特别是在热应力条件下的核易位。蛋白质的反应与外部和内部信号相应。一种常见的机制是改变其亚细胞定位。本文介绍了一种跟踪蛋白质定位的协议, 不需要抗体, 放射性标签, 或共焦显微镜。在这篇文章中, GFP 是用来标记的目标蛋白 EXL-1 在C. 线虫, 一个成员的氯离子胞内通道蛋白 (CLICs) 家族, 包括哺乳动物 CLIC4。采用转化与γ辐射相结合的法, 建立了一种集成的转化exl-1::gfp转基因线 (具有启动子和全基因序列), 并稳定地表达了该基因和gfp。最近的研究表明, 在热应力, 而不是氧化应激, EXL-1::GFP 积累在细胞核。将 GFP 信号与细胞核结构和 DAPI 信号重叠, 证实了 EXL-1 亚细胞在应力作用下的定位变化。本协议提出两种不同的固定方法 DAPI 染色: 乙醇固定和丙酮固定。本文提出的 DAPI 染色协议具有快速、高效的功效, 同时保留了 GFP 信号和蛋白质亚细胞定位的变化。此方法只需要使用 Nomarski、FITC 过滤器和 DAPI 过滤器的荧光显微镜。适用于小型实验室设置、本科生研究、高中学生研究、生物技术教室。

氯离子胞内通道蛋白 (点击) 是一种在12种高度保守的多功能蛋白质。许多研究表明, CLICs 调节细胞的压力, 自噬, 凋亡, 癌变, 血管生成和巨噬细胞先天免疫反应在哺乳动物系统13,14,15,16 ,17。线虫中有两个点击同系物: exl-1和exc-4。我们最近的研究表明, exl-1调控动物的压力管理8。我们将一个平移exl-1::gfp结构整合成一个动物基因组, 并创建了转基因线, 它以 gfp 作为标记稳定地表达基因exl-1 (图 2A和2B)。在热应激下, EXL-1::GFP 在肠道内的细胞核积聚, 因为强烈的 GFP 信号与细胞核结构重叠 (图 2C-E)。为了确认亚细胞定位, DAPI 染色作为一种染色细胞核的方法。用乙醇固定 DAPI 染色显示动物体内有清晰的细胞核 (图 3B)。丙酮固定也达到类似的结果 (图 3E)。重叠的 GFP 和 DAPI 信号证实 EXL-1::GFP 确实在肠道内的细胞核中积聚 (图 3C, F)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57914/57914fig1.jpg"> 图 1: 在不同的放大功率下, 第四幼虫 (L4) 级线虫的图像。(A) 63X、 (B) 115X 和(C) 400X 放大功率。箭指向 L4 蠕虫的外阴;注意半月结构。缩放栏 = 所有图像中的100µm。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57914/57914fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57914/57914fig2.jpg"> 图 2: 在热应力下, EXL-1::GFP 积聚在肠道的细胞核中.(A)热休克前肠道区域的 Normaski 图像。(B)在热休克之前, instestinal 地区发现了强烈的 GFP 信号。(C)热休克后肠道区域的 Normaski 图像 (箭头指向肠道区域的细胞核)。(D)热休克后, 强烈的 GFP 信号积聚。(E) GFP 和 Normaski 的重叠图像。所有图片都采用400X 放大功率。缩放栏 = 所有图像中的50µm。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57914/57914fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57914/57914fig3.jpg"> 图 3: DAPI 染色证实了 EXL-1::GFP 核易位.(丙)用乙醇固定法 DAPI 染色。(D F)用丙酮固定法 DAPI 染色。在A和D, 绿色颜色显示 GFP 信号在肠道区域。在B和E中, 蓝颜色通过 DAPI 染色显示细胞核。C和F显示了 GFP、DAPI 和 Normaski 的重叠图像。缩放栏 = 所有图像中的50µm。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57914/57914fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
补充数字:丙酮固定保存标本和60天后, 丙酮固定, GFP 和 DAPI 信号仍然可见。绿色是用于 GFP 的。(B)蓝色用于 DAPI 信号。(C) GFP、DAPI 和 DIC 的重叠图像。所有图像都是在400X 放大功率下拍摄的。刻度条 = 50 微米。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57914/supplemental_figure.pdf">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans at JoVE.

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

该协议演示了如何在热应激下跟踪蛋白质核易位, 方法是使用绿色荧光蛋白 (GFP) 融合蛋白作为标记, 4 ', 6-diamidino-2-苯基吲哚 (DAPI) 染色。DAPI 染色协议快速, 保留了 GFP 和蛋白质亚细胞定位信号。

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 ...

detecting-protein-subcellular-localization-green-fluorescence-protein

Universidad San Sebastian

Research

JoVE Journal

Biology

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

9.5K 次观看.  Borough of Manhattan Community College/CUNY. 该协议演示了如何在热应激下跟踪蛋白质核易位, 方法是使用绿色荧光蛋白 (GFP) 融合蛋白作为标记, 4 ', 6-diamidino-2-苯基吲哚 (DAPI) 染色。DAPI 染色协议快速, 保留了 GFP 和蛋白质亚细胞定位信号。

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

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

C. 线虫排泄管作为细胞内腔形态发生和体内极化膜生物在单个细胞中的模型: GFP-融合、rna 干扰界面和成像的标记

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended ...

科研

发育生物学

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

固定化的线虫线虫分析神经元内细胞的转运

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

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

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

C.在体内神经元钙成像技术线虫

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

神经科学

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological conditions. Post-mitotic neurons represent a major cell type affected by aging. Although multiple mammalian models of neuronal aging exist, they are challenging and expensive to establish. The roundworm Caenorhabditis elegans is a powerful model to study neuronal aging, as these animals have short lifespan, an available robust genetic toolbox, and well-cataloged nervous system. The method presented herein allows for seamless isolation of specific cells based on the expression of a transgenic green fluorescent protein (GFP). Transgenic animal lines expressing GFP under distinct, cell type-specific promoters are digested to remove the outer cuticle and gently mechanically disrupted to produce slurry containing various cell types. The cells of interest are then separated from non-target cells through fluorescence-activated cell sorting, or by anti-GFP-coupled magnetic beads. The isolated cells can then be cultured for a limited time or immediately used for cell-specific ex vivo analyses such as transcriptional analysis by real time quantitative PCR. Thus, this protocol allows for rapid and robust analysis of cell-specific responses within different neuronal populations in C. elegans.

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

Here, we present a protocol for simple isolation of specific groups of live neuronal cells expressing green fluorescent protein from transgenic Caenorhabditis elegans lines. This method enables a variety of ex vivo studies focused on specific neurons and has the capacity to isolate cells for further short-term culturing.

特定神经元种群与圆虫卡埃纳哈布迪炎的分离

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

生物学

RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a tool to identify and visualize microbes in nematodes from the wild or to experimentally characterize and quantify infection in nematodes infected with microbes in the lab. FISH probes, labeling the highly abundant small subunit ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. Probes designed to target conserved regions of ribosomal RNA common to many species can detect a broad range of microbes, whereas targeting divergent regions of the ribosomal RNA is useful for narrower detection. Similarly, probes can be designed to label viral RNA. A protocol for RNA FISH staining with either paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal for nematodes associated with bacteria, microsporidia, and viruses, whereas acetone fixation is necessary for the visualization of microsporida spores. Animals were first washed and fixed in paraformaldehyde or acetone. After fixation, FISH probes were incubated with samples to allow for the hybridization of probes to the desired target. The animals were again washed and then examined on microscope slides or using automated approaches. Overall, this FISH protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

RNA Fluorescence in situ Hybridization FISH to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

Intestinal microbes, including extracellular bacteria and intracellular pathogens like the Orsay virus and microsporidia (fungi), are often associated with wild Caenorhabditis nematodes. This article presents a protocol for detecting and quantifying microbes that colonize and/or infect C. elegans nematodes, and for measuring pathogen load after controlled infections in the lab.

RNA荧光 原位 杂交（FISH）可视化 秀丽隐杆线虫 肠道中的微生物定植和感染

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as ...

免疫与感染

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

方法来评估肌肉的亚细胞中<em&gt;温度。线虫</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

体内神经元钙处理的亚细胞成像

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

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

摘要

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此视频中的章节

C. 线虫排泄管作为细胞内腔形态发生和体内极化膜生物在单个细胞中的模型: GFP-融合、rna 干扰界面和成像的标记

固定化的线虫线虫分析神经元内细胞的转运

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

C.在体内神经元钙成像技术线虫

特定神经元种群与圆虫卡埃纳哈布迪炎的分离

时间推移显微镜早期胚胎发育线虫

RNA荧光原位杂交（FISH）可视化秀丽隐杆线虫肠道中的微生物定植和感染

方法来评估肌肉的亚细胞中

体内神经元钙处理的亚细胞成像

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

摘要

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此视频中的章节

C. 线虫排泄管作为细胞内腔形态发生和体内极化膜生物在单个细胞中的模型: GFP-融合、rna 干扰界面和成像的标记

固定化的线虫线虫分析神经元内细胞的转运

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

C.在体内神经元钙成像技术线虫

特定神经元种群与圆虫卡埃纳哈布迪炎的分离

时间推移显微镜早期胚胎发育线虫

RNA荧光 原位 杂交（FISH）可视化 秀丽隐杆线虫 肠道中的微生物定植和感染

方法来评估肌肉的亚细胞中

体内神经元钙处理的亚细胞成像

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

RNA荧光原位杂交（FISH）可视化秀丽隐杆线虫肠道中的微生物定植和感染