作者对 Dr. 麻子杉本 (东北大学) 的有益讨论深表谢意。wyIs251是 Dr. 康沈 (斯坦福大学) 的一份慷慨礼物。mnIs17由 CGC 提供, 由 NIH 研究基础设施项目办公室 (P40 OD010440) 出资。这项工作得到了 jsp KAKENHI 赠款 #17H05010 和 #16H06536 和第一三基金会, 脑科学基金会和内基金会的支持。

1. 样品制备 <ol> <li> 使用转基因方法生成转基因的 线虫的兴趣 18 。或者, 从 线虫 遗传学中心获得合适的菌株 (CGC)。为了可视化突触囊泡前体的轴突转运, 请使用转基因线 wyIs251 [ Pmig-13::gfp::rab-3 ] 7 。要观察 IFT, 获得转基因线 mnIs17 [ osm-6::gfp ] 19 。这些菌株在本协议中使用. 
	注: 无论是外阵列, 基因组整合, 甚至 GFP 基因作品。要减少背景信号, 请使用特定于细胞的启动子, 而不是泛神经启动子. 
	注意: 蠕虫的维护在其他协议 20 中有说明。在线虫生长培养基 (NGM) 板 (1.7% (w/v) 琼脂糖上, 在 Escherischia 大肠杆菌 OP50 饲养者的草坪上维持菌株. 50mM 氯化钠, 0.25% (含钒) 蛋白胨, 1 毫米 CaCl 2 , 5 毫克/毫升胆固醇, 25 mm, 2 PO 4 , 1 mm MgSO 4 在 60 mm 直径的塑料碟上) 在20和 #176; C. </li>
	<li> 在20和 #176 中生长蠕虫; C 在 NGM 板上, 到蠕虫的任何生命周期阶段. 
	注意: 在任何阶段都可以观察轴突运输和 IFT。晚 L4 对年轻成人是一个很好的积极控制, 因为多个出版物使用了这些阶段和可视化的贩运活动 14 , 21 , 22 , 23 . </li>
</ol> 2。10% 琼脂糖的制备 注意: 许多供应商提供类似的产品, 如琼脂、琼脂粉和琼脂糖。使用电泳级琼脂糖 (凝胶强度和 #62; 1200 克/厘米 2 )。廉价的琼脂粉不起作用, 因为产生的凝胶不够坚固, 无法固定蠕虫. 
<ol> <li> 在玻璃管 (1.5 cm x 10.5 厘米) 的4毫升蒸馏水 (DW) 中混合0.4 克琼脂糖。在玻璃管上盖上盖子以避免蒸发. </li>
	<li> 将玻璃管放在95和 #176 上; C 热块90分钟。此外, 准备另一玻璃管 (1.5 厘米 x 10.5 厘米) 填充 DW, 并保持在95和 #176; c. 在95和 #176 中放置一个巴斯德吸管; c DW ( 图 1 a )。在琼脂糖管中会出现很多小气泡, 溶液混浊。离开它, 直到解决方案变得清晰。然后, 移混合, 直到琼脂糖溶液变得均匀. 
	注: 微波不能用于制备10% 琼脂糖溶液时, 由 DW 和琼脂糖。微波导致的小气泡阻止了琼脂糖的融化。然而, 固化10% 琼脂糖凝胶可以很容易地融化再次使用微波。含有10% 琼脂糖凝胶的玻璃管可以提前准备, 储存在4和 #176; C 至少6月。如果这样做, 在需要时用微波炉熔化库存, 并从本协议的步骤3开始. 
	注: 琼脂糖在95和 #176 时逐渐失去凝固的能力; C 长时间或反复凝固和熔化后。如果发生这种情况, 准备新的10% 琼脂糖. </li>
</ol> 3。琼脂糖垫的制备 <ol> <li> 使用在步骤2.2 中准备的预热的巴斯德吸管, 在 76 x 22 mm 幻灯片上放置2滴10% 琼脂糖. </li>
	<li> 快速覆盖第二个 76 x 22 mm 2 在琼脂糖滴凝固之前滑动, 如图 1 B 所示, 然后向下推第二张幻灯片以拼合琼脂糖溶液。厚度应为 0.5-1 mm. </li>
	<li> 快速冲洗移95和 #176 的巴斯德吸管; C DW 向上和向下直到琼脂糖溶液被洗掉。否则, 吸管会被琼脂糖凝胶堵塞。将吸管放在95和 #176; C DW. </li>
	<li> 等待1分钟。琼脂糖垫形成并且变得多云。然后, 手动滑动幻灯片 ( 图 1 C ). </li>
</ol> 4。安装蠕虫 注意: 即使是少量的蠕虫运动也会阻碍良好的观察。左旋咪唑传统上用于防止在琼脂糖垫 24 , 25 上的蠕虫移动。然而, 左旋咪唑在 C. 线虫 中抑制神经元受体, 因此可能影响神经元中的贩运事件 15 。使用聚苯乙烯微描述存在一个好的替代 17 . 
<ol> <li> 在装有可镜像透视的立体声显微镜下, 将30转基因蠕虫转移到一个新的 NGM 板上, 用铂丝镐来表示不带细菌的适当标记. 
	注: 白金线镐是由铂丝和巴斯德吸管 (5 英寸), 铂金丝的尖端被夷为平地。在 Wormbook (http://www.wormbook.org) 中描述了铂丝采摘和处理这些蠕虫的准备工作. </li>
	<li> 离开盘子1分钟. 细菌会自动从蠕虫的表面移除, 因为蠕虫在无细菌的 NGM 琼脂糖凝胶上移动. </li>
	<li> 将 0.5-1.0 和 #181; 将 2.6% 100 nm 直径的聚苯乙烯微在水中放在琼脂垫上 ( 图 1 D )。将10-20 蠕虫从无菌 NGM 板转移到微。使用铂线拾取来除去和传播蠕虫, 以避免蠕虫体的重叠 ( 图 1 E ). </li>
	<li> 应用 22 x 40 mm 2 片 ( 图 1 F )。轻轻推, 以消除气泡, 但避免粉碎蠕虫。该示例现在已准备好进行成像. 
	注: 由于不进行麻醉, 蠕虫只由琼脂垫、聚苯乙烯微和片 17 产生的摩擦力来固定。饲养细菌的存在和/或过多的溶液会使摩擦力减弱. 
	注意: 不同大小的片 ( 例如 , 22 x 22 mm 2 , 18 x 18 mm 2 ) 工作。然而, 更大的片可以防止浸入水或油从物镜泄漏到样品期间的观察. </li>
</ol> 5。观察 注意: 对于每个显微镜系统和照相机, 适当的成像参数 (激光功率、增益、分、 等 ) 将有所不同。在这里, 一个 widefield 显微镜配备了旋转磁盘共焦扫描仪和数字 CCD 相机使用. 
<ol> <li> 将舞台温度控制设置为20和 #176; c, 或将室温调整为20和 #176; c. </li>
	<li> 将样本幻灯片放在显微镜阶段。使用 100x (数值孔径 = 1.3 或更好) 物镜. </li>
	<li> 查找 图 2 A (用于 wyIs251 和轴突传输) 或 图 2 B (用于 mnIs17 和 IFT) 作为正控制的区域.可以分析其他区域以及 22 . </li>
	<li> 设置参数 (CCD 增益 = 200, 分 = 1 或 2, 激光功率在 488 nm = 1.0-1.3 兆瓦)。执行4帧/秒的时间间隔记录, 最多1分钟将文件保存为多-TIFF 格式. 
	注意: 如果 GFP 信号消失, 很难继续观察 GFP::RAB-3 或 OSM-6::GFP 三十年代, 激光功率太强。在这种情况下, 减少激光功率。反之, 如果没有记录水泡运动, 激光功率太弱。在这种情况下, 增加激光功率. </li>
	<li> 观察不同的蠕虫并重复5.3 和5.4。观察可以持续多达20分钟, 但每个蠕虫应该只记录一次, 以避免影响 photo 损坏. 
	注意: 蠕虫已被观察至少20分钟没有明显的缺陷 7 , 27 , 28 。更长的观察也许是可能的, 但避风港和 #39; 没有被测试。神经元死亡的一个明显的迹象是神经元形态的变化, 如突起肿胀。由于弱 GFP 信号可以看到在轴突和树突在两个 wyIs251 和 mnIs17 , 突起形态学可以很容易地检查只看在轴突或树突。神经元形态学在 图 2 中显示. </li>
	<li> 生成影片文件. 
	<ol> <li> 使用斐济软件打开多 TIFF 文件。转到文件和 #62; 打开然后选择在步骤5.4 中保存的多 TIFF 文件. 
		注: 斐济可下载 jttps://斐济。图像 J 和插件也可以用来生成 kymographs。如果这样做, 请按照所选插件的相关说明进行操作. #160; </li> <li> 单击 #34; 矩形选择和 #34; 按钮 ( 图 3 , 箭头), 并通过绘制矩形来选择感兴趣的区域使用计算机鼠标 ( 图 3 a 中的黄色矩形)。转到图像和 #62; 堆栈和 #62; 工具和 #62; 使 Substacks 并选择影片中包含的切片编号。生成 substack. </li>
		<li> 通过单击并转到图像和 #62 来激活 substack 窗口; 调整和 #62; 亮度/对比度。一个调整亮度和对比度的窗口出现了。在窗口中, 将顶部 (最小) 向右滑动, 使背景信号消失, 第二个顶栏 (最大) 向左, 以便在背景下可以看到移动的泡和颗粒. </li>
		<li> 转到图像和 #62; 堆栈和 #62; 时间压模。由于影片在步骤5.4 的4帧/秒录制, 时间间隔为0.25 秒。因此, 类型和 #34; 0.25 和 #34; 间隔. </li>
		<li> 从 "文件" 菜单 (影片1和 2) 中选择 "保存为" 和 "#62", 然后生成影片文件. 
		注意: 通过使用适当的插件, 您可以将文件保存为其他格式, 如和 #34; mpeg4 和 #34; 或 #34; #34; 文件. </li>
	</ol> </li> <li> 生成 kymographs. 
	<ol> <li> 再次使用斐济软件打开原始影片文件, 并重复步骤5.6.2 和5.6.3 以调整亮度和对比度. </li>
		<li> 单击和 #34; 分段行和 #34; ( 图 3 B , 箭头)。使用鼠标, 绘制沿纤毛或轴突 (黄线, 在 图 3 B ) 的分段线. </li>
		<li> 转到分析和 #62; 多 Kymograph 和 #62; 多 Kymograph ( 图 3 C )。将出现 "线条宽度" 窗口 ( 图 3 D )。类型和 #34; 3 和 #34; 在 "线宽" 窗口中, 单击 "确定" ( 图 3 D ). </li>
		<li> Kymograph 窗口已创建。以 TIFF 或 JPEG 格式保存图像. </li>
	</ol>
	</li>
</ol>

<ol>
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I., Avery, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+microsurgery+in+Caenorhabditis+elegans.">Laser microsurgery in Caenorhabditis elegans.</a> Methods Cell Biol. 107, 177-206 (2012).</li><li>Kumar, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+Kinesin-3+motor+UNC-104/KIF1A+is+degraded+upon+loss+of+specific+binding+to+cargo.">The Caenorhabditis elegans Kinesin-3 motor UNC-104/KIF1A is degraded upon loss of specific binding to cargo.</a> PLoS Genet. 6 (11), 1001200 (2010).</li><li>Zheng, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vesicle+protein+SAM-4+regulates+the+processivity+of+synaptic+vesicle+transport.">The vesicle protein SAM-4 regulates the processivity of synaptic vesicle transport.</a> PLoS Genet. 10 (10), 1004644 (2014).</li><li>Blacque, O. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+WD+repeat-containing+protein+IFTA-1+is+required+for+retrograde+intraflagellar+transport.">The WD repeat-containing protein IFTA-1 is required for retrograde intraflagellar transport.</a> Mol Biol Cell. 17 (12), 5053-5062 (2006).</li><li>Evans, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+modulation+of+IFT+kinesins+extends+the+sensory+repertoire+of+ciliated+neurons+in+Caenorhabditis+elegans.">Functional modulation of IFT kinesins extends the sensory repertoire of ciliated neurons in Caenorhabditis elegans.</a> J Cell Biol. 172 (5), 663-669 (2006).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nat Methods. 2 (12), 905-909 (2005).</li><li>Bindels, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mScarlet:+a+bright+monomeric+red+fluorescent+protein+for+cellular+imaging.">mScarlet: a bright monomeric red fluorescent protein for cellular imaging.</a> Nat Methods. 14 (1), 53-56 (2017).</li><li>Prevo, B., Mangeol, P., Oswald, F., Scholey, J. M., Peterman, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+differentiation+of+cooperating+kinesin-2+motors+orchestrates+cargo+import+and+transport+in+C.+elegans+cilia.">Functional differentiation of cooperating kinesin-2 motors orchestrates cargo import and transport in C. elegans cilia.</a> Nat Cell Biol. 17 (12), 1536-1545 (2015).</li><li>Robison, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detyrosinated+microtubules+buckle+and+bear+load+in+contracting+cardiomyocytes.">Detyrosinated microtubules buckle and bear load in contracting cardiomyocytes.</a> Science. 352 (6284), 0659 (2016).</li><li>Hayashi, S., Okada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+superresolution+fluorescence+imaging+with+spinning+disk+confocal+microscope+optics.">Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics.</a> Mol Biol Cell. 26 (9), 1743-1751 (2015).</li></ol>

作者没有什么要透露的。

关于现有方法的限制 本文所描述的方法进行了优化, 以观察快速事件, 如轴突运输和 IFT。因此, 固定化比较长的潜伏期更有优先权。虽然我们已经能够观察贩运活动至少20分钟没有明显的摄动, 这种方法可能并不总是适合观察慢事件需要更长的观察, 如轴突伸长和细胞迁移。对于较长的观测, 需要通过减少琼脂糖的百分比 (即, 增加水以避免干燥和减少可能造成损坏的压力) 来优?...

活细胞成像是分析细胞内转运的重要工具。在神经元细胞生物学中, 用活细胞成像分析轴突转运是了解神经元功能和形态发生1的必要条件。轴突运输缺陷的基础上几个神经退行性疾病2。驱动超家族蛋白和蛋白进行轴突转运 anterogradely 和逆行, 分别为1,2。
纤毛是另一个细胞室, 其中微管网络和贩卖机械是高度发达的3。蛋白质合成机械没有在纤毛中定位, 这意味着纤毛蛋白必须从细胞质输送到纤毛的尖端。纤毛特异性驱动和蛋白, 分别称为 kinesin-2 和细胞质 dynein-2, 运输的成分纤毛4, 在一个现象称为 intraflagellar 运输 (IFT)5。IFT 的损害导致一系列疾病称为 ciliopathies6。因此, 需要对 IFT 机制进行活体细胞成像分析, 以了解纤毛形成的基本机制和发病机理。
线虫线虫(线虫) 是研究轴突传输和 IFT7、8、9的好模型。为了观察 IFT,衣藻已被广泛用作模型生物体56。由于衣藻是一个单细胞生物体, IFT 与衰老、神经元功能和行为的关系将难以分析。此外, 诸如 CRISPR/Cas9 等基本遗传技术尚未应用到衣藻。在高模型生物体中, 如小鼠和果蝇, 轴突转运和 IFT 的中断经常导致致命的表型, 因为轴突转运和 IFT 对动物的形态发生和稳态至关重要10, 11。在小鼠的情况下, 细胞培养和转染一般需要观察轴突运输和 IFT, 这需要很多技能和大量的时间12,13。此外, 许多重要的生理环境可能会失去在培养细胞和细胞系。因为神经系统对于蠕虫的生存来说不是必需的, 所以轴突运输或 IFT 被破坏的线虫突变体通常不是致命的7、9、14。轴突运输和 IFT 可以直接观察在体内没有解剖, 因为C. 线虫是透明的, 因此很容易观察 GFP 标记标记。
有几种协议可以固定C. 线虫, 例如使用微流体设备15、带有麻醉的琼脂糖垫16或微17。麻醉的纳入可能抑制神经元的贩运事件15。微流控装置方法的一个明显的缺点是, 准备一台流控装置并不总是容易的。相反, 用琼脂糖垫和微固定是一种方便和简单的方法来执行时间推移成像在C. 线虫。在这里, 我描述这个基本的协议, 以固定的线虫和可视化轴突运输和 IFT在体内在线虫.与其他方法相比, 这里所描述的方法不需要特殊设备, 而且更便宜、更容易执行。

<table border="0" cellpadding="0" cellspacing="0" width="837">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Slideglass (76&#160; x 26 mm)</td>
			<td style="width:167px;">Matsunami</td>
			<td style="width:139px;">S1111</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Coverglass (22&#160; x 40 mm)</td>
			<td style="width:167px;">Matsunami</td>
			<td style="width:139px;">C024401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Agarose</td>
			<td style="width:167px;">Wako</td>
			<td style="width:139px;">318-01195</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Polystylene microbeads 0.1 micron</td>
			<td style="width:167px;">Polysciences</td>
			<td style="width:139px;">#00876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Heat block</td>
			<td style="width:167px;">TAITEC</td>
			<td style="width:139px;">0063288-000</td>
			<td>CTU-mini</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Microscope</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:139px;">IX-71</td>
			<td>widefield microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Spinning disk Scanner</td>
			<td style="width:167px;">Yokogawa</td>
			<td style="width:139px;">CSU-X1</td>
			<td>spinning disc confocal scanner&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Digital CCD camera</td>
			<td style="width:167px;">Hamamatsu Photonics</td>
			<td style="width:139px;">C10600-10B</td>
			<td>ORCA-R2 degital CCD camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Objective lens (x100, NA1.4)</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:139px;">UPLSAPO 100XO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Pasteur pipette (5 inch)</td>
			<td style="width:167px;">IWAKI</td>
			<td style="width:139px;">IK-PAS-5P</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">Glass tube (1.5 cm diameter x 10.5 cm)</td>
			<td style="width:167px;">IWAKI</td>
			<td style="width:139px;">9820TST15-105NP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">TV9211: wyIs251</td>
			<td style="width:167px;">Laboratory of Kang Shen</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">OTL11: mnIs17</td>
			<td style="width:167px;">Ref. 27, 29</td>
			<td style="width:139px;">N/A</td>
			<td>SP2101 was backcrossed with wild type for 6 times</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Stereo microscope</td>
			<td style="width:167px;">Carl Zeiss</td>
			<td style="width:139px;">435064-9000-000</td>
			<td>STEMI 508&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Mirror transillumination unit</td>
			<td style="width:167px;">Carl Zeiss</td>
			<td>435425-9010-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">platinum wire (0.2 mm)</td>
			<td style="width:167px;">Nilaco Corporation</td>
			<td style="width:139px;">m78483501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 mm plastic dish</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:139px;">#351007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Fiji</td>
			<td style="width:167px;">N/A</td>
			<td style="width:139px;">N/A</td>
			<td>https://fiji.sc/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">nematode growth medium (NGM)&#160;&#160;</td>
			<td style="width:167px;"></td>
			<td style="width:139px;"></td>
			<td>1.7% (w/v) agarose, 50mM NaCl, 0.25% (w/v) Peptone, 1 mM CaCl2, 5 mg/mL Cholesterol, 25 mM KH2PO4, 1 mM MgSO4&#160;</td>
		</tr>
	</tbody>
</table>

immobilization of caenorhabditis elegans to analyze intracellular transport in neurons

轴突运输和 intraflagellar 转运 (IFT) 是轴索和纤毛形态发生和功能的关键。驱动超家族蛋白和蛋白是分别调节顺和逆行转运的分子马达。这些马达使用微管网络作为路轨。线虫线虫(线虫) 是研究轴突转运和 IFT的一个强有力的模型有机体。在这里, 我描述了一个协议, 以观察轴突运输和 IFT 在生活中的C. 线虫。运输货物可以通过使用荧光蛋白 (如绿色荧光蛋白 (GFP)) 标记货物蛋白质来可视化。C. 线虫是透明的, 而 GFP 标记的货物蛋白可以在特定细胞的细胞特异促进剂下表达。活蠕虫可以通过微10% 琼脂糖凝胶固定, 而不会杀死或麻醉蠕虫。在这种情况下, 货物的运动可以直接观察到的轴突和纤毛的生活线虫没有解剖。这种方法可用于观察任何细胞的任何货物分子通过修改目标蛋白和/或他们所表达的细胞。大多数基本的蛋白质, 如分子马达和适配器蛋白, 参与轴突运输和 IFT 是保存在C. 线虫。与其他模型生物体相比, 突变体可以在C. 线虫中更容易获得和维护。将此方法与各种C. 线虫突变体结合, 可以阐明轴突转运和 IFT 的分子机制。

DA9 神经元轴突转运 使用wyIs251线, 顺和逆行轴突运输 GFP::RAB-3 可以同时记录在 DA9 运动神经元。DA9 神经元近端轴突的顺和逆行转运的平均速度分别为1.8 和2.6 微米/秒,22。移动的泡的数量是大约0.03 和0.018 每μ m 轴突每 s。因此, 三十年代观察10μ m DA9 轴突使用100x 透镜, 你能发现大约9和5移动的泡在轴突 (图 4和<strong...

Watch this Scientific Journal Video about Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons at JoVE.com

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

线虫线虫(线虫) 是研究轴突和细胞内转运的良好模型。在这里, 我描述了一个协议的在体内记录和分析轴突和 intraflagellar 运输在C. 线虫。

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 ...

The overall goal of this time lapse recording is to observe the intracellular transport events in Caenorhabditis elegans neurons.This method can help answer key questions in the cell biology field such as axonal transport and interflagellar transport.The main advantage of this technique is that one can observe intracellular transport in C.Elegans without dissection.Start this experiment by growing transgenic C.elegans strain of interest at 20

immobilization-caenorhabditis-elegans-to-analyze-intracellular

Research

JoVE Journal

Developmental Biology

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

7.9K 次观看.  Tohoku University. The overall goal of this time lapse recording is to observe the intracellular transport events in Caenorhabditis elegans neurons.This method can help answer key questions in the cell biology field such as axonal transport and interflagellar transport.The main advantage of this technique is that one can observe intracellular transport in C.Elegans without dissection.Start this experiment by growing transgenic C.elegans strain of interest at 20

视频: Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

生殖系化学毒性的利用线虫综合评估<em&gt;秀丽隐杆线虫</em

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

科研

发育生物学

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

在Active ERK的时空分析<em&gt;℃。线虫</em&gt;生殖系

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods are described here to analyze distinct populations of MTs in the developing neural tube of the zebrafish embryo. While the focus is on neural tissue, this methodology is broadly applicable to other tissues. The procedures are optimized for early to mid-somitogenesis-stage embryos (1 somite to 12 somites), however they can be adapted to a range of other stages with relatively minor adjustments. The first protocol provides a method to assess the spatial distribution of stable and dynamic MTs and perform a quantitative analysis of these populations with image-processing software. This approach complements existing tools to image microtubule dynamics and distribution in real-time, using transgenic lines or transient expression of tagged constructs. Indeed, such tools are very useful, however they do not readily distinguish between dynamic and stable MTs. The ability to image and analyze these distinct microtubule populations has important implications for understanding mechanisms underlying cell polarization and morphogenesis. The second protocol outlines a technique to analyze nascent MTs specifically. This is accomplished by capturing the de novo growth properties of MTs over time, following microtubule depolymerization with the drug nocodazole and a recovery period after drug washout. This technique has not yet been applied to the study of MTs in zebrafish embryos, but is a valuable assay for investigating the in vivo function of proteins implicated in microtubule assembly.

Immunolabeling methods to analyze distinct populations of microtubules in the developing zebrafish brain are described here, which are broadly applicable to other tissues. The first protocol outlines an optimized method for immunolabeling stable and dynamic microtubules. The second protocol provides a method to image and quantify nascent microtubules specifically.

利用 Immunolabeling 分析斑马鱼胚胎中的稳定、动态和初生的微管

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are facilitated by the transparency of the animal, the ability to do forward and reverse genetic assays, the relative ease of generating fluorescently labeled proteins, and the use of fluorescent dyes that can either be microinjected into the early embryo or incorporated into its food (E. coli strain OP50) to label cellular organelles (e.g. 9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#39;H-2,2&#39;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Here, we present the use of a fluorescent pH-sensitive dye that stains intestinal lysosomes, providing a visual readout of dynamic, physiological changes in lysosomal acidity in live worms. This protocol does not measure lysosomal pH, but rather aims to establish a reliable method of assessing physiological relevant variations in lysosomal acidity. cDCFDA is a cell-permeant compound that is converted to the fluorescent fluorophore 5-(and-6)-carboxy-2&#39;,7&#39;-dichlorofluorescein (cDCF) upon hydrolysis by intracellular esterases. Protonation inside lysosomes traps cDCF in these organelles, where it accumulates. Due to its low pKa of 4.8, this dye has been used as a pH sensor in yeast. Here we describe the use of cDCFDA as a food supplement to assess the acidity of intestinal lysosomes in C. elegans. This technique allows for the detection of alkalinizing lysosomes in live animals, and has a broad range of experimental applications including studies on aging, autophagy, and lysosomal biogenesis.

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

A step-by-step guide to probe loss of lysosomal acidity in the intestine of C. elegans using the pH-sensitive vital dye 5(6)-carboxy-2&#39;,7&#39;-dichlorofluorescein diacetate (cDCFDA)

评估溶酶体碱化在活的秀丽线虫中的肠道

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

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

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

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

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

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

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

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented protocol uses this model to determine the pathogenicity caused by the mitis group streptococci via the production of H2O2. The mitis group streptococci are an emerging threat that cause many human diseases such as bacteremia, endocarditis, and orbital cellulitis. Described here is a protocol to determine the survival of these worms in response to H2O2 produced by this group of pathogens. Using the gene skn-1 encoding for an oxidative stress response transcription factor, it is shown that this model is important for identifying host genes that are essential against streptococcal infection. Furthermore, it is shown that activation of the oxidative stress response can be monitored in the presence of these pathogens using a transgenic reporter worm strain, in which SKN-1 is fused to green fluorescent protein (GFP). These assays provide the opportunity to study the oxidative stress response to H2O2 derived by a biological source as opposed to exogenously added reactive oxygen species (ROS) sources.

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an excellent model to dissect host-pathogen interactions. Described here is a protocol to infect the worm with members of the mitis group streptococci and determine activation of the oxidative stress response against H2O2 produced by this group of organisms.

线虫中耳炎群链球菌引起氧化应激的研究

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

亚细胞分辨率的各向同性光片显微镜及细胞自动动力学分析

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial&#160; dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.

线虫生殖道内精子引导和运动的测定

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female ...