This work was supported by a grant from the US National Institutes of Health National Institute for Arthritis and Musculoskeletal and Skin Diseases (AR-054342) to NJS. CJG was funded by a Doctoral training studentship funded by the University of Nottingham. JJB was funded by an MRC Doctoral training award (J500495). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

1株，文化和显微镜<ol><li> 秀丽隐杆线虫的处理和维护的菌株，如前所述19。菌株可从线虫遗传学中心（CGC）。 注意:在这些实验中使用的菌株是CB5600（ccIs4251（Pmyo-3 :: Ngfp-lacZ启动; Pmyo-3 :: Mtgfp）我;他-8（e1489Ⅳ））与定位于线粒体和核GFP融合蛋白; PJ727（jls01（肌醇3 :: GFP，ROL-6（su1006））; UNC-54 :: lacZ的 V）与局部的收缩设备和PD55（ccIs55（SUP-7（ST5）GFP融合蛋白; unc- 54 :: lacZ启动）Ⅴ）与定位于胞质溶胶β半乳糖苷酶融合蛋白。 </li><li>如前面的几代RNAi实验13说明，并获得RNA干扰细菌的序列完整的RNAi实验证实了马克·维达尔等 12构建ORF-的RNAi文库。 </li><li>构建含有的菌株&lt;EM&gt; CCIS 4251，jls01或CCIS 55使用标准技术20。使用这些转基因创建的菌株，以评估线粒体的网络结构，肌球蛋白组织或proteostasis的状态下，分别。交叉菌株进入一个突变，其中在看这些亚细胞区室中的一个在每个动物是需要的。 </li><li>用磷脂酰肌醇-3-激酶抑制剂LY-294002（LY），如前所述21。简言之，将添加LY-294002，在160μM的干OP50种子NGM板的顶端的最终浓度。 </li><li>使用GFP的滤波器，用于基于GFP的实验并以三倍通滤波器的显微镜，其同时允许红，绿和兰通道的成像来评估在体内的膜电位与C 32 H 32 Cl 2中N 2 O的捕捉所有的图像，通常销售作为MitoTracker红CMXRos。进行图像分析和人物制剂中的图像处理软件。 </li></ol><ol><li>最少1天之前需要，使M9的缓冲区- 22.04毫米KH 2 PO 4，42.27毫米的Na 2 HPO 4，85.56毫米的NaCl（终浓度），并通过高压灭菌消毒。暂时冷却至45℃，高压灭菌后加灭菌硫酸镁至1mM，以防止沉淀22。允许M9与在4℃下与等份冷却到50ml试管中。 </li><li>在实验当天，添加3毫升M9缓冲到一个单一9厘米板具有高数的L1幼虫。通过吸出的M9液体和移液对以一定角度保持在板的琼脂表面反复洗涤板。 </li><li>从平板上洗L1的，然后用移液管M9的转移到10ml的试管中。 </li><li>离开2.5分钟，以允许较大的成人和存货幼虫阶段的动物下降到试管的底部，而较小的L1动物保持靠近吨运M9的缓冲区。 </li><li>在用吸管试管中取出前500微升M9的。然后，吸管含M9 L1幼虫到一个新的NGM板下的解剖范围接种500微升OP50。 </li><li>通常情况下，目的是转移〜每盘200-300 L1幼虫。每日从成年监测板，如果有必要，挑选的动物上，以新鲜OP50 NGM板，以防止OP50食物源被消耗。 注意:这通常是1-3滴的M9使用P1000的移液管，虽然这变化很大程度上取决于L1的对原板的数目。 </li><li>如果动物个体的前瞻性研究需要，动物一旦长大成人，挑选个别的动物分开种子NGM板。转移动物每天24-48小时，从动物的幼虫分开。 </li></ol> 3.运动测定19 <ol><li>实验开始前至少2小时，取出50毫升等分M9，并允许以平衡至室温erature。 </li><li>挑一个成年动物到50微升M9缓冲液在显微镜载玻片上。 </li><li>计数10秒，其中，一个向左和1向右弯曲等于一个笔划的左和右身弯曲的数目。 </li><li>重复体笔画的计数，得到总共至少5次测量，从该平均可以采取。六乘以这些数字给每分钟的移动速度。 </li><li>使用移液管，传送回该动物的皮氏培养皿。此方法使得能够移动的时间测量整个C中elegan S中的整个寿命。 </li></ol><img alt="图1" fo:content-width="6in" src="/files/ftp_upload/52043/52043fig1highres.jpg" width="600" /> 图1:运动检测。一旦蠕虫被挑入缓冲区，1左（A）和一个向右（B）弯曲相加，得到一个完整的运动（中风）。 体壁肌肉4.成像线粒体网络和肌节<ol><li>注:相同的协议同时适用于线粒体和肌节的成像。 </li><li>如先前所描述的（第1），并在20℃下生长48-60小时时将达到青年期同步蠕虫。 </li><li>挑20蠕虫（代表整个盘的）从平板到20微升的M9在显微镜载玻片上。 </li><li>应用盖玻片，用下一个GFP过滤器（460-500纳米，蓝光激发）的荧光显微镜放大40倍进行图像。 </li></ol> 5.评估膜电位用体内累积试验与MitoTracker红CMXRos（C 32 H 32 Cl 2中N 2 O） <ol><li>同步CB5600（第1部分），所有的标记有绿色荧光蛋白在肌肉的线粒体中，并且在20℃下生长至成年早期对NGM含有OP50为48-60小时。 </li><li>加入100微升DMSO中的C 32 H 32 Cl 2中N 2 O的50微克等分试样进行的940.692微米的储备液。 </li><li>取1微升的940.692微米原液，重悬到199微升M9创造的4.70微米（4.70346微米），一个可行的解决方案。准备这个棕色1.5毫升管，因为C 32 H 32 Cl 2中N 2 O是感光。 </li><li>准备4.70微米工作液（每20微升等分20成虫）的20微升等分。 </li><li>挑选20只成年动物成20微升4.70微米C 32 H 32 Cl 2中N 2 O的棕色1.5毫升管。孵育在20℃下搅拌1小时。 </li><li>继1小时培养，加入1 ml M9的棕色1.5毫升管含有蠕虫，离心机在2000×g离心10秒，然后取出上清液。重新悬浮颗粒蠕虫和转移，其余20μ之前重复此清洗步骤升（包含蠕虫），以滑动以进行成像。 </li><li>取线粒体的图像与用三通滤波器，它同时既允许红，蓝和绿通道的可视40X放大倍数。使用三重通滤波器的成像显示共定位的C 32 H 32 Cl 2中N 2 O的用GFP定位于显示为橙色的线粒体。 </li><li>已经采取了橙色线粒体的图像，光漂白的动物使用与去除所有筛选条件的最大设定光波长二十五分之五百四十毫微米为10秒。这将漂白线粒体内的红色，揭示了线粒体GFP单独确认共定位于细胞线粒体。 </li></ol>在秀丽隐杆线虫的肌肉蛋白质降解6.评估<ol><li>同步到NGM板PD55s（或含有lacZ的任何转基因株）接种OP50（第1节）。长到成年早期为48-60小时，在20℃。 </li><li>挑选20成虫为20微升DDH 2 O的显微镜载玻片上。小心尝试并挑选只蠕虫，并与不挑任何细菌。标签铅笔显微镜载玻片。 </li><li>放置在干燥器中30分钟，干燥开裂的动物的表皮。确保使用围绕密封润滑脂的干燥器的紧密密封。 </li><li>检查干燥一直在解剖显微镜（ 图2）有效。 </li><li>淹没在冰冷的丙酮显微镜载玻片为3.5分钟，然后离开在纸巾上的显微镜载玻片上，在室温下搅拌15分钟，使载玻片上的丙酮蒸发。在这一点上解冻在X-gal和氧化缓冲器，并允许平衡到室温。 </li><li>加入2μl的X-gal的100微升氧化缓冲和涡轻轻地保证这是均匀的。这是X-GAL /氧化缓冲液母液。 </li><li>加入20μl的X-GAL /氧化缓冲主结构的每一个O20蠕虫riginal区。根据解剖范围检查，以确保主结构完全覆盖所有的动物完全。轻轻倾斜显微镜载玻片移动预混过的地方的动物都没有涉及。 </li><li>放置在湿度室中的载玻片为1-2.5小时，在20℃下。当一个深蓝色的颜色是存在于对照动物的体壁肌肉图​​像动物。下解剖范围评估的对照动物每15分钟，以确定何时应当将动物成像。 </li></ol><img alt="图2" fo:content-width="6in" src="/files/ftp_upload/52043/52043fig2highres.jpg" width="600" /> 图2:使用β半乳糖苷酶测定法肌肉蛋白降解的评价。 （A）含有lacZ的转基因动物是从培养皿回升至20微升DDH 2 O的一个显微镜载玻片（B）。动物是干燥的，并成为破碎<s仲&gt;（C）。动物然后洗涤在丙酮（D）中 。一旦丙酮蒸发X-GAL /氧化缓冲液母液加入（E）。蓝颜色中的对照动物的发展进行评估，在从t = 0分钟（F），每间隔15分钟（佛罗里达州）的整个动物（L）的长度而得到的深蓝色为止。蠕虫的照片在佛罗里达州采取2.5倍的放大倍率在AE和8X。放大C，D，F，G和L分别为4X原来的放大倍数。

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</ol>

The authors have nothing to disclose.

这些方法允许肌肉的来自移动macrophysiology的探索，以确定亚细胞缺陷足以造成一个运动缺陷。当结合这些方法让肌肉进行详细分析。的洞察力使肌肉化的假设，涉及到一般的生理学，病理生理学和老化的进一步测试。 测定运动 一个运动缺陷指示正常神经肌肉功能的破坏。这可能是特定于神经或肌肉或它可以是几个共同的组织之间。完成运动检测中最困难的阶段被选择的蠕虫具有代表性的人口和/或治疗，并且还确保实验者时不转移至M9的伤害的动物。具有大的样本大小，以获得运动的代表性和准确的评估是重要的。当测定高度笔etrant的效果，例如作为频繁出现在突变体中，10只动物的样品大小各评估运动10次，足以发现统计学显著差异相对于野生型。然而，运动检测和易于培养线虫的简单性意味着上百蠕虫可以迅速，以确保定量健壮的结果进行评估。当测定显示存在于一个种群中只有一部分的效果，以确定哪些人口的比例似乎受到影响以及缺陷在受影响最严重的动物的严重程度是很重要的。在这样的情况下更大的动物的数量应，以便提供最小的数据为人口受到影响的比例来评价。双方频繁的药物和RNAi治疗会产生严重的运动缺陷人口的一小部分。在某些情况下，增加了治疗和或递送的方法的剂量会增加财务司的比例ected动物和运行的剂量反应曲线可以是在确定药物或RNAi的最佳浓度是有用的。这种运动分析的第二个限制是该蠕虫被操纵，以评估运动。因此，蠕虫都刺激并进行某种程度的渗透胁迫时挑入液体。渗透胁迫的程度可以通过使用的M9或BU相对于水的缓冲器19来限定。有些限制是通过测量运动习惯在平板上使用市售的跟踪系统34或免费插件为ImageJ的35缓解。测量动物的运动速率可以提供关于整个肌肉健康的重要信息，包括可以在整个生命过程进行测量和非侵袭性这种方法的关键是对肌肉功能的预期寿命长的研究变化的功能。 收缩设备的成像 &lt;P类="jove_content"&gt;这里使用的图像收缩装置结构的蠕虫病毒株PJ727 36表示的是局部肌节的体壁肌肉中的肌凝蛋白GFP融合蛋白。使用该菌株使肌节结构的可视化在活的动物。类似于上面描述的移动检测，使用绿色荧光蛋白标记的肌节，以评估肌节结构的一个主要优点是，该方法是相对非侵入性的，并因此可用于前瞻性地跟随肌节结构中各个动物在整个生命过程。最大的困难是这种方法是，那个被成像的虫子还活着，所以移动，这使得它很难得到良好的聚焦图像。几种方法可用于减少运动和使成像更简单;这其中就包括麻醉或通过压，吸气，或使用高粘度的解决方案瘫痪蠕虫和固定。每个immobiliz这些方法ATION的缺点是，它本身可以改变神经肌肉功能。因此，至关重要的是在分析中包括适当的未处理的对照组。它也可以是谨慎地利用固定化的至少两个独立的方法来确认的结果是不是由于问题的固定化方法，这取决于如何广泛使用所选择的固定方法为所研究的问题。在这里，我们使用简单的压力从盖玻片上的蠕虫病毒诱导减少运动。放置盖玻片后，盖玻片下方的液体会蒸发，盖玻片将因此开始时的蠕虫病毒，产生更大的压力，通常这需要2-5分钟，以产生足够的运动减少，使容易成像。 重要的是要监视蠕虫密切后盖玻片已引入作为压力最终会增加足以引起蠕虫从过度的压力破裂，共后通常10-15分钟verslip加法。附加缓冲器可以与周围的盖玻片边缘一个枪头的帮助下被引入，以避免挤压伤到的蠕虫。指甲涂剂可用于密封所述盖片的边缘和防止蒸发，虽然这可防止动物的检索从盖玻片下，如果需要的话。同样，在该溶液中使用的有机硅珠粒可以帮助减少压力的量上的蠕虫盖玻片的地方。 正如评价的运动缺陷，考虑效果的外显率是很重要的。外显率可以考虑，如果高80-100％的动物显示在NGM板的表型，如果人口显示屏≤20％的部分影响，如果21-79％低。对于高渗透效果，更小的样本大小可以用于产生对肌缺陷显著定量数据。如遇有影响具有低外显率，选择的动物，在应对毒品显示清晰的运动缺陷或RNAi治疗可能是测定在大多数受处理动物肌缺陷很有用。然而，它不一定的情况下的治疗作用，对运动和亚细胞结构的程度是相同的。因此，可以期望以检查存在于人口众多的缺陷的范围内，例如100只动物。这使得缺陷在整个人群中分布情况的了解。它也可能希望检查缺陷的程度，在最严重的影响动物和/或检查的肌节缺损程度和运动缺陷程度之间的相关性。潜在的困难之外，这种方法比评估肌节结构的其它方法更快和更容易。这个速度和方便，然而，保留一个关键性的限制，含有GFP融合蛋白肌节是不完全正常37，因此，评估破坏肌节的应用附加的劳动密集的方法来确认。这些方法包括使用偏振光38，鬼笔环肽染色39和间接免疫荧光40。这些方法中，只有偏振光是相对非侵入性的;其它方法需要固定，因此不能在动物个体的前瞻性研究中使用，而它们只能被用于确认，横截面，从这些研究的结果。 线粒体网络影像 用于线粒体成像实验的蠕虫菌株是CB5600 7其表达定位于线粒体和定位于细胞核GFP的β半乳糖苷酶融合蛋白GFP融合蛋白，无论是在体壁肌肉。与使用PJ727用于成像肌节的，使用该菌株的使线粒体网络结构的活体动物的可视化。因此，该菌株可用于评估所发生的动态变化，例如减少mitochondrial融合和/或增加线粒体裂变41。另外，作为成像肌节，最大的困难是这种方法是被成像蠕虫还活着动人，使得很难得到良好的聚焦图像。而几种方法，如上面更详细讨论的，可以使用以降低运动，线粒体出现特别敏感的干预，诱导降低的运动。例如，最常用的线粒体呼吸链的麻醉药目标成分，因此必须谨慎在正常线粒体的结构和功能的研究中。同样，大多数麻痹剂必须谨慎在正常线粒体的结构和功能的研究中所用的，因为大多数麻痹剂引起较少信号通过神经肌肉接头和肌肉的功能性去神经是足以诱导线粒体碎片。在本研究的实验中采用的压力来固定动物s但其他已经成功地使用左旋咪唑42，虽然它是必不可少的图像立即动物。 最后，各种细菌污染的培养，例如枯草芽孢杆菌，可引起线粒体碎片;因此，至关重要的是在分析中包括适当的未处理的对照组。对于高渗透效果，更小的样本大小可以用于产生对线粒体网络缺陷显著定量数据。然而，由于在线粒体网络较小缺陷的发生率，这少数动物很可能是两次所需的肌节的结构的评估的数目。在情况下，作用具有低的渗透率，选择动物响应于药物或RNAi治疗清楚地显示运动缺陷的可能是在测定中大多数受治疗动物的线粒体缺陷是有用的。然而，如上所述，这不是必需的情况，该的治疗效果上的运动和亚细胞结构的程度是相同的。因此，可以期望以检查存在于人口众多的缺陷的范围内，例如150-200的动物，以及破坏在最严重的影响动物的程度和破坏的程度的同程度的存在或不存在运动缺陷。其它菌株的荧光标记的线粒体可用于确认在CB5600获得的结果，然而，使用不同的荧光蛋白可以影响线粒体的基线联网。例如，使用一个TOMM-20的RFP融合蛋白的形象化线粒体似乎导致增加基线线粒体碎片与使用线粒体本地化的GFP 17。而其它的方法，如免疫荧光显微镜和电子显微镜可用于评估线粒体结构，它们不允许在体内评估线粒体动力学的，因为一个固定步骤是需要ð。其它方法，如使用的线粒体局部染料可用于无固着，因此，也可以代替使用线粒体本地化的GFP的使用。如在下一节中所讨论的，使用这种染料也可以在体内线粒体功能的初步评估。 评估线粒体膜电位的体内线虫 各种染料可用于标记线粒体28。这些染料提供在活线虫可视线粒体网络结构的另一种方法。许多这些染料也允许的线粒体功能的体内粗的评估，因为它们积累在基于所述线粒体膜电位的线粒体。在这里，我们使用C 32 H 32 Cl 2中N 2 O是通过摄入消化道，经由因为通过溶解在0.5％DMSO中，得到透的角质层一些附加地进入蠕虫。下面进入细胞C 32 H 32 Cl 2中的N 2 O被氧化并通过它结合含有氨基酸（ 例如甲硫氨酸和半胱氨酸）硫线粒体隔离。这些染料-肽复合物的形成是指C 32 H 32 Cl 2中N 2 O的残留在一次标记28中的线粒体。与使用线粒体染料的一个重要难题是它们将标记所有线粒体中的动物。因此，我们在CB5600，上述用于评估肌肉线粒体网络结构相同的应变进行标记。通过使用红色线粒体染料，蠕虫在肌肉线粒体表达GFP的菌株，和一个三通滤波器，这是相对简单的图像通过发现线粒体即分别为邻肌肉只有线粒体范围内的红色染料和GFP标记线粒体共定位。在执行此共存的方法是最困难的一步是成像，因为染料是光漂白下高强度荧光灯秒之内。这种效果可以通过保持在低功率荧光直到实验者准备的图像，然后相应地调整荧光强度的限制。一旦一个是经历了与使用染料的另一种方法是利用共聚焦显微镜带Z堆叠图像仅在正确的组织线粒体;相比于其它组织中肌线粒体网络是相当明显的。不幸的是，C 32不能H 32 Cl 2中N 2 O的离开线粒体28指的是，与肌节和线粒体成像技术上面讨论的，它不能被用于前瞻性地跟随随时间的线粒体功能的损失，除非C 32 H 32 CL 2 N 2 O在离散的时间点被添加到分开的动物种群。这种限制可以通过使用染料如JC-10，其执行后膜电位43的崩离开线粒体被克服。线粒体也可以从C分离30 线虫为后续生化分析。这样的提取允许线粒体膜电位的定量通过定量的使用流式细胞术的亲脂性阳离子染料的吸收或荧光板读数器44的速率。在确定受损的线粒体膜电位，但也可以，以确定是否质子梯度的损失转化为ATP产生的使用敏感bioluminometric荧光素酶为基础的方法45的损失。 评价蛋白降解C.线虫 这里用于评估肌肉蛋白降解的蠕虫菌株是PD55，它包含一个肌肉特异性β半乳糖苷酶，不间断地在整个开发合成，直到成年期达到并保持稳定的细胞质在未来72-96小时19。因此，在开发过程中β半乳糖苷酶的水平的测量主要是表示干预对合成的肌球蛋白启动子的影响，而β半乳糖苷酶的成年时期的损耗表示干预触发增加胞质蛋白降解19。在评估β半乳糖苷酶活性的关键步骤是确保有效的干燥。如果不能有效地变干的动物造成规定在动物体壁的肌肉X-gal的衬底和蓝色，因此穷人的穷人。为了确保良好的干燥对干燥机的密封应进行检查和样品应在5-10分钟的间隔进行检查整个干燥评估进展情况（ 即 20微升样品应在10分钟内，剩下的20分钟干为确保全面干燥）。的β半乳糖苷酶测定法，因此活性测定法的适当控制是必不可少的。此外，降低的成年人染色处理条件相对于对照，并不证明降解发生;相反，它表示响应于所述治疗降低酶活性。为了证实降解，更多的劳动力密集型Western blot分析，必须完成对β半乳糖苷酶13，这使得定量的蛋白质降解在计算虫小种群。免疫印迹条带密度可以使用ImageJ 46进行量化。这种方法的另一个局限是，利用转基因蛋白质的不告知以降解的生理指标，并为这样的答案蛋白质组和/或靶向假设驱动的蛋白质印迹必须采用13。最后，在这些研究中所用的转基因蛋白的合成在成年早期19关闭</suP&gt;，其他的方法都必须希望检查肌肉蛋白的合成改建全面发展C.时，可以利用线虫的肌肉;例如，稳定或放射性同位素的方法。

肌肉是一种多功能的组织，以及赞赏其在运动，体位支撑和新陈代谢的作用。健康肌肉的发育和维持需要多种细胞过程和部件的协调。无论是通过损伤或疾病，失调可以发生，导致改变肌肉结构和/或功能。肌肉功能与年龄相关的下降提出的医疗保健预算在西方世界一个显著的负担，因为肌肉块1,2，特别是肌肉的力量和耐力3-5负与死亡率相关。相关的恶化肌肉功能，如改变的线粒体功能或肌节的破坏方面，测量可以允许支撑整体肌肉发育和健康的机制的认识。 Çaenorhabditis线虫 （ 线虫 ）是一个微观线虫BES吨已知的，因为它是第一个多细胞生物体有其整个基因组的测序6，第一个拥有基因的RNAi使用7沉默，唯一的后生动物有它的整个细胞系8和9神经解剖学确定。 秀丽隐杆线虫是第一次提出作为一款型号为1974的行为的遗传控制由悉尼·布雷10和这方面的重要性和后续工作的研究是公认的第一三个诺贝尔奖授予了线虫的研究人员。约35％的C的线虫基因已经在人类中鉴定直向同源物，与秀丽隐杆线虫是用来理解肌肉发育11的遗传控制的关键的有机体。几乎是在秀丽隐杆线虫基因组中每个基因可以通过RNA干扰7,12撞倒。因此，通过撞倒基因​​在发育完全的肌肉，遗传因素有助于重新肌肉健康gulation可以用相对简单13进行调查。 使用RNAi技术，突变体，和化合物的结合能力，使线虫的首要系统探索肌肉结构和功能的基因调控7,14,15与16岁和在疾病模型17。基因敲除突变，和/或暴露于经肌肉结构和/或功能的化学化合物的影响，可通过多个方面来衡量。这里我们简要地描述用于评估线虫肌肉结构和功能，其中有许多在肌肉健康的前瞻性研究可以使用简单的技术。 绿色荧光蛋白的发展（GFP）的18，使特定蛋白的两个局部性和细胞器在秀丽隐杆线虫的一般结构的可视化。在这里，我们将介绍如何GFP表达特定fically内体壁肌肉线粒体，细胞核，或肌节可用于确定这些亚细胞结构的营养不良是本。作为结构并不总是可靠的替代品的功能，我们还说明了如何在体内使用的阳离子染料允许在肌肉受损的线粒体膜电位的评价。当染料在用GFP组合使用时，它们允许体系结构和功能的整个生命周期的时间方式的研究。最后，我们还说明了如何内肌肉蛋白质稳态可以评估和如何将所有这些措施可用于通过使用运动检测的相关性的变化在整个动物肌肉健康。这些技术中，结合的基因敲除，突变，和/或化学化合物提供了一个强大的军火库用于研究特定基因或化合物如何影响肌肉健康体内 。 秀丽隐杆线虫和之间在结构，代谢和肌肉的总功能的相似之处哺乳动物的意思是秀丽隐杆线虫是一种优秀的模式生物的理解高等生物，包括人类肌肉的调节。

<table><tbody><tr><td>MitoTracker Red CMXRos (C&lt;sub&gt;32&lt;/sub&gt;H&lt;sub&gt;32&lt;/sub&gt;Cl&lt;sub&gt;2&lt;/sub&gt;N&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Invitrogen</td><td>M-7512</td><td>Red mitochondrial dye which accumulates based on a negative membrane potential</td></tr><tr><td>DMSO</td><td>Thermo Scientific</td><td>67-63-5</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>7778-77-0</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>BDH</td><td>301584L</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>7647-14-5</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>M-7506</td><td></td></tr><tr><td>Microscopy Slides</td><td>Starfrost</td><td>K220</td><td></td></tr><tr><td>Microscopy Cover Slips</td><td>VW2 International</td><td>631-0124</td><td></td></tr><tr><td>1.5 ml Eppendorph Tubes</td><td>Fisher Scientific</td><td>FB74031</td><td></td></tr><tr><td>Dessicator</td><td>Nalgene</td><td>D2672</td><td></td></tr><tr><td>Nikon Microscope</td><td>Nikon</td><td>H600L</td><td>Nikon H600L microscope with proprietary software</td></tr><tr><td>Nikon Camera</td><td>Nikon</td><td>DS-Fi1</td><td>Nikon Digital Sight DS-Fi1 digital camera&amp;nbsp;</td></tr><tr><td>Zeiss Microscope</td><td>Zeiss</td><td>Ax10</td><td>Zeiss AX10 microscope with an Axiocam MRC digital camera and Axiovision LE software and a triple-pass filter</td></tr><tr><td>Plates 9 cm</td><td>Starstedt</td><td>82-1473</td><td></td></tr><tr><td>Plates 6 cm</td><td>Gosselin</td><td>BP53-01</td><td></td></tr><tr><td>Potassium Ferrocyanide</td><td>Sigma-Aldrich</td><td>14459-95-1&amp;nbsp;</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>7786-30-3</td><td></td></tr><tr><td>Na&lt;sub&gt;3&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>7601-54-9</td><td></td></tr><tr><td>5-Bromo-4-chloro-3-indolyl &amp;beta;-D-galactopyranoside</td><td>Sigma-Aldrich</td><td>7240-90-6&amp;nbsp;</td><td></td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-dimethylformamide</td><td>Sigma-Aldrich</td><td>68-12-2&amp;nbsp;</td><td></td></tr></tbody></table>

methods to assess subcellular compartments of muscle in c. elegans

肌肉是动态组织响应的变化，营养，运动，和疾病状态。肌肉质量和功能与疾病和年龄的损失是显著公共卫生负担。目前，我们了解到一些关于肌肉的健康基因调控与疾病或年龄。线虫秀丽隐杆线虫是一种建立的模型用于理解所关注的生物过程的基因组调节。此蠕虫的体壁肌肉显示了很大程度的同源性较高的后生动物物种的肌肉。因为线虫是一种透明的生物，绿色荧光蛋白的线粒体和肌节的定位使得这些结构体内的可视化。类似地，饲养动物的阳离子染料，其中积攒基于对线粒体膜电位的存在，使得在体内的线粒体功能的评估。这些方法中的肌蛋白homeosta，以及评估虫病，被结合的整体动物肌肉功能评估，在运动检测的形式，以允许与肌肉性能的功能性措施亚细胞缺陷的相关性。因此， 线虫（C.elegans）提供了有力的平台，用以评估突变，基因敲除和/或化学化合物的经肌肉结构和功能的影响。最后，如绿色荧光蛋白，阳离子染料，以及运动检测被评估非侵入性地，肌肉结构和功能的前瞻性研究可以在整个生命过程中进行，这在目前还不能很容易地分析体内任何其他生物体。

运动测定量化运动缺陷 运动跌幅死亡的C.一个很好的预测线虫 16，以减小速率指示问题的神经肌肉系统。变化的运动的评估，运动检测，可导致RNA干扰对一个特定基因或药物治疗（ 图3-5），或在突变体动物13,23。在运动的下降可以从例如改变发展导致以下发育敲复杂的编码I，III，IV或V 24电子传递下来的基因。此外，具体的干预措施，可以只成年线虫 ，以调查在后生理发育的影响进行测试。例如，动物对UNC-112，它编码RNA干扰治疗的线虫 Kindlin-1同源基因被定位于整合包含肌肉附着复合物（ 图3和图4），或气体-1，其编码的电子传递链（ 图4）的复合物I的组成部分，则显示一条逐步减少在运动中对从48-72小时后的成年控制。这可以指示问题的肌肉生理。类似地，从24移动的损失- - 72小时响应于治疗发育正常成人与PI3K抑制剂LY-294002（ 图5）治疗的观察。另外，也可以进行测试的第二RNAi的治疗，突变，或在恢复中的动物显示在响应降低移动到初始的干预动作的药物的效果。例如，unc- 112突变体显示一个可量化的减少在运动相比，被衰减的第二突变的存在在暗淡-1 13的野生型动物。因此，运动试验可以鉴别这两种干预措施，改变neuromuscu拉尔发育和/或功能，以及那些改善和/或恢复神经肌肉功能。 肌节中断的评价 在确定了一个运动缺陷，通常期望以确定的运动缺陷的原因可以由神经，肌肉，或两者中的问题进行说明。肌节是肌肉内的多蛋白复合物，允许进行收缩，从而迫使产生和移动。通过利用动物表达定位于收缩装置球蛋白GFP融合蛋白，中断对肌球蛋白丝和肌节的程度可以通过开发和/或成年期可以观察到相对非侵入性和前瞻性动物个体。野生型动物显示出现在每个体壁肌肉内为多个绿色平行的直线（ 图3）排列的肌节。破坏这些正常的阵列可能是相对较小的同排列的肌节出现合并，而不是留在平行。这种表型，用的unc-112的RNAi可见在t = 24小时或48小时（ 图3），是相当于Z线流的哺乳动物肌动蛋白的附着位点。同样，阵列的一小部分可以折叠或者它们可以完全消失，如治疗用的unc-112的RNAi在t = 48小时或叔= 72小时（ 图3）。肌节的缺陷是显示运动缺陷的动物的肌肉内的存在（ 图3）就足以占到运动缺陷，但不一定排除其他问题肌肉，神经，和/或其它组织响应于介入。 线粒体网络结构评估 中的运动缺陷的存在，有时希望以检查肌肉对其它缺陷可能导致或有助于运动日FECT。线粒体提供大量细胞能量的和下降的运动可以是降低的ATP供应的结果，减少了可用于肌肉收缩的能量。因此线粒体的结构可以被检查异常。它不麻醉动物，因为它快速诱导线粒体碎片叠氮化钠是很重要的;动物在这些实验中由盖片施加的压力固定。在野生型线虫 体壁肌肉，线粒体包含有组织的网络内（ 图4）和线粒体存在和功能的网25。线粒体的网络中断呈现作为网络（ 气体-1的RNAi， 图4B）26的碎片。碎片可能会变得严重，以至于没有外表有组织的网络仍然如使用UNC-112的RNAi（ 图4B和C）13治疗。随着无线网络个中断的肌节结构，即显示一个运动缺陷在肌肉线粒体结构中的缺陷的动物可以足以占到运动缺陷，但这并不一定排除其他问题肌肉，神经，和/或其他组织。例如， 的unc-112突变体13和治疗用的unc-112的RNAi（ 图3和4）显示既破坏肌节和破坏线粒体结构。 线粒体功能的体内评估 线粒体膜电位确定线粒体创建质子动力，并产生ATP的27，因此评估体内线粒体膜电位的能力的能力表明，动物产生ATP的能力。作为线粒体结构破坏可能或可能不改变线粒体功能的能力，这可能是德斯黎rable以评估在动物中显示改变的线粒体结构线粒体功能。 C 32 H 32 Cl 2中N 2 O的累加所有类型的细胞，其中，膜电位的存在和污渍线粒体28（ 图4C）的线粒体中。具体评估肌肉线粒体功能，我们可以使用C 32 H 32 Cl 2中N 2 O和GFP标记的细胞线粒体证明干预特别在肌肉线粒体（ 图4C）的影响。例如，具有的unc-112的RNAi线粒体膜电位以下治疗的损失防止C 32 H 32 Cl 2中的N 2 O与输入矩阵。因此，线粒体显示很小，如果除了线粒体的GFP标记（ 图4C）的线粒体中任红染色。光漂白也可以用于确认所观察到的线粒体是特别是那些在肌肉（ 图4C）。容量降低的识别产生ATP足以占据一个观察到的运动缺陷，但不内肌肉，神经排除其他的问题，和/或其他组织。无论如何，按照还原的ATP生产能力在人体29和老化30与疾病状态相关，识别响应于介入这样的缺陷提供了一个平台，用于筛选能够提高ATP的生产能力和这些可再化合物和/或RNAi治疗进一步探索治疗潜力。 蛋白质降解的评估 中的一个运动缺陷和正常肌节和线粒体中的存在，有时希望以检查肌肉为其它缺陷，这些缺陷可能导致或促成的运动缺陷。维持蛋白质稳态的能力是一个签名，也不发作状况，而在蛋白质合成和/或增加的蛋白质降解的减少是老化31和多种疾病状态32,33的结果。因此，可能希望检查动物的肌肉与改变的蛋白质稳态运动缺陷。这可以通过胞质内肌蛋白水平的评估来实现。利用转基因编码的蛋白质可以让肌肉特异性proteostasis的评估。例如，使用含β半乳糖苷酶，融合于肌球蛋白的N-末端部分的动物，这是一个肌球蛋白启动子的控制下合成和增强剂允许的胞质肌蛋白含量19评估。蓝染色野生型成人存在表明活性β半乳糖苷酶的存在和不存在病理性细胞溶质蛋白降解。染色的响应于治疗甲损失表明病理蛋白降解发生（ 图5 </strong&gt;），如β半乳糖苷酶是稳定的72 - 96小时后的成年野生型成人13,14,19。例如，未经处理的野生型动物显示强蓝色染色72小时成年后，而LY-249002治疗的动物显示缺乏染色的（ 图5）。如在肌节结构和线粒体功能的缺陷，病理肌肉蛋白降解的存在足以占观察到的运动缺陷，但不一定排除其他问题肌肉，神经，和/或其他组织。 <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/52043/52043fig3highres.jpg" width="600" /> 图3:（A）UNC-112的RNAi导致在t = 72小时的显著运动缺陷（B）在体壁的肌肉摆着一个带结构的评估。在瓦特T，A频段的肌肉出现在成年早期（T = 0小时）的直线和基本保持一致，直到超过72小时成年后（见重量变焦）。UNC-112的RNAi导致肌节失去建筑的小面积肌肉（T = 24小时），倒塌，丢失的肌节（T = 48小时），聚集（T = 72小时前面板和缩放）和肌节的线性度（T = 72小时底部面板和缩放）的损失。比例尺代表25微米和变焦是2.5倍。 <img alt="图4" fo:content-width="6in" src="/files/ftp_upload/52043/52043fig4highres.jpg" width="600" /> 图4:（A）中 的unc-112或气体-1的RNAi导致运动缺陷（B）的内体壁肌肉线粒体结构的评估。线粒体的肌肉出现在成年早期（重量）作为网络的直线。动物与UNC-112的RNAi治疗结果mitochondrIAL碎裂如前24小时和72小时的成年（变焦和箭头）13之间变化。类似地，RNAi的反气-1导致线粒体网络的碎片如先前观察到26。这些存在于72小时（见变焦和箭头）。 在体内线粒体膜电位（C）评估在T = 24小时，进步的线粒体网络普遍混乱小的差距。在动物中还含有绿色荧光蛋白定位于线粒体，C 32 H 32 Cl 2中N 2 O的累积在体壁肌肉线粒体和出现在从年轻成年性（t = 0小时）的三通滤波器，直到72小时后的成年橙色。治疗UNC-112的RNAi导致膜电位的逐渐丧失，如图72小时，其中剩余的线粒体显示了C 32少积累H 32 Cl 2中N 2 O与重量。漂白10 SEc。使用二十五分之五百四十纳米的光漂白剂从MitoTracker红色，并显示局部肌肉线粒体（+照片漂白剂）的GFP。比例尺代表25微米和放大的2.5倍。 <img alt="图5" fo:content-width="6in" src="/files/ftp_upload/52043/52043fig5highres.jpg" width="600" /> 图5:用LY-294002的动物（A）的处理诱导运动缺陷蛋白质降解的（B）中使用的是β半乳糖苷酶测定法评估。 年龄（重量）的L1幼虫同步传送到NGM之前生长至成年早期，在20℃（t = 0的小时）接种OP50（重量对照）或NGM与LY-294002（PI3K抑制剂处理），在20℃接种24小时。在A，大约20-30动物染色在t = 0小时β半乳糖苷酶活性（蓝色）和24小时，48小时和72小时后。

Watch this Scientific Journal Video about Methods to Assess Subcellular Compartments of Muscle in C. elegans at JoVE.com

评估线虫肌肉亚细胞区室的方法生物学 |视频

Methods to Assess Subcellular Compartments of Muscle in C. elegans

骨骼肌的运动是必不可少的，是机构的主要蛋白质商店。 秀丽隐杆线虫中的肌肉健康测量进行了描述。有意改变肌肉的结构和功能使用本地化的GFP和阳离子染料进行评估。

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 ...

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Methods to Assess Subcellular Compartments of Muscle in C. elegans

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16.6K 次观看.  University of Nottingham. 骨骼肌的运动是必不可少的，是机构的主要蛋白质商店。 秀丽隐杆线虫中的肌肉健康测量进行了描述。有意改变肌肉的结构和功能使用本地化的GFP和阳离子染料进行评估。 

视频: Methods to Assess Subcellular Compartments of Muscle in C. elegans

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

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生物化学

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 ...

发育生物学

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of diverse human diseases. Worms are particularly well-suited for high-throughput genetic and drug screens to gain deeper insight into therapeutic targets and therapies by exploiting their fast development cycle, large brood size, short lifespan, microscopic transparency, low maintenance costs, robust suite of genomic tools, mutant repositories, and experimental methodologies to interrogate both in vivo and ex vivo physiology. Worm locomotor activity represents a particularly relevant phenotype that is frequently impaired in mitochondrial disease, which is highly heterogeneous in causes and manifestations but collectively shares an impaired capacity to produce cellular energy. While a suite of different methodologies may be used to interrogate worm behavior, these vary greatly in experimental costs, complexity, and utility for genomic or drug high-throughput screens. Here, the relative throughput, advantages, and limitations of 16 different activity analysis methodologies were compared that quantify nematode locomotion, thrashing, pharyngeal pumping, and/or chemotaxis in single worms or worm populations of C. elegans at different stages, ages, and experimental durations. Detailed protocols were demonstrated for two semi-automated methods to quantify nematode locomotor activity that represent novel applications of available software tools, namely, ZebraLab (a medium-throughput approach) and WormScan (a high-throughput approach). Data from applying these methods demonstrated similar degrees of reduced animal activity occurred at the L4 larval stage, and progressed in day 1 adults, in mitochondrial complex I disease (gas-1(fc21)) mutant worms relative to wild-type (N2 Bristol) C. elegans. This data validates the utility for these novel applications of using the ZebraLab or WormScan software tools to quantify worm locomotor activity efficiently and objectively, with variable capacity to support high-throughput drug screening on worm behavior in preclinical animal models of mitochondrial disease.

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

This study presents protocols for two semi-automated locomotor activity analysis approaches in C. elegans complex I disease gas-1(fc21) worms, namely, ZebraLab (a medium-throughput assay) and WormScan (a high-throughput assay) and provide comparative analysis among a wide array of research methods to quantify nematode behavior and integrated neuromuscular function.

线粒体疾病脑 膜炎基因 模型中量化动物活动的实验方法比较分析

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of ...

行为学

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

生物学

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed orientation. Here we describe methods to label, image, and analyze the microtubule dynamics and growth during the development and regeneration of touch neurons in C. elegans. Using genetically encoded fluorescent reporters of microtubule tips, we imaged the axonal microtubules. The local changes in microtubule behavior that initiates axon regeneration following axotomy can be quantified using this protocol. This assay is adaptable to other neurons and genetic backgrounds to investigate the regulation of microtubule dynamics in various cellular processes.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

A protocol for imaging the dynamic microtubules in vivo using fluorescently labeled End binding protein has been presented. We described the methods to label, image, and analyze the dynamic microtubules in the Posterior lateral microtubule (PLM) neuron of C. elegans.

在体内卡诺哈布德炎电子神经元微管动力学和方向评估

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed ...

神经科学

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 ...

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 ...

Hand Dissection of Caenorhabditis elegans Intestines

Comprised of only 20 cells, the Caenorhabditis elegans intestine&#160;is the nexus of many life-supporting functions, including digestion, metabolism, aging, immunity, and environmental response. Critical interactions between the C. elegans host and its environment converge within the intestine, where gut microbiota concentrate. Therefore, the ability to isolate intestine tissue away from the rest of the worm is necessary to assess intestine-specific processes. This protocol describes a method for hand dissecting adult C. elegans intestines. The procedure can be performed in fluorescently labeled strains for ease or training purposes. Once the technique is perfected, intestines can be collected from unlabeled worms of any genotype. This microdissection approach allows for the simultaneous capture of host intestinal tissue and gut microbiota, a benefit to many microbiome studies. As such, downstream applications for the intestinal preparations generated by this protocol can include but are not limited to RNA isolation from intestinal cells and DNA isolation from captured microbiota. Overall, hand dissection of C. elegans intestines affords a simple and robust method to investigate critical aspects of intestine biology.

Hand Dissection of Caenorhabditis elegans Intestines

The present protocol describes a procedure for isolating intestines from adult Caenorhabditis elegans nematode worms by hand for input in genomics, proteomics, microbiome, or other assays.

秀丽隐杆线虫肠的手部解剖

Comprised of only 20 cells, the Caenorhabditis elegans intestine&#160;is the nexus of many life-supporting functions, including digestion, metabolism, ...

<meta charset="utf8"></head><body>分析秀丽隐杆线虫的线粒体形态

Imaging and Quantifying Mitochondrial Morphology in C. elegans During Aging

Mitochondria, important cellular organelles found in most eukaryotic cells, are major sites of energy production through aerobic respiration. Beyond this well-known role as the 'cellular powerhouse,' mitochondria are also involved in many other essential cellular processes, including the regulation of cellular metabolism, proliferation, immune signaling, and hormonal signaling. Deterioration in mitochondrial function during aging or under mitochondrial stress is often characterized by distinct changes in mitochondrial morphology and volume. The nematode C. elegans is an ideal model for studying these changes due to its transparent body and short lifespan, which facilitate live microscopy throughout its lifetime. However, even within the C. elegans field, numerous transgenic constructs and methods for mitochondrial imaging are available, each with its own limitations. Here, single-copy, matrix-localized GFP constructs are presented as a robust and reliable method for imaging mitochondrial morphology in C. elegans. This study specifically focuses on experimentally controllable factors to minimize errors and reduce variability between replicates and across studies when performing mitochondrial imaging during the aging process. Additionally, mitoMAPR is recommended as a robust method to quantify changes in mitochondrial morphology across tissue types during aging.

<meta charset="utf8"></head><body>衰老过程中 秀丽隐杆线 虫线粒体形态的成像和定量

This protocol provides a standardized approach for imaging mitochondrial morphology in multiple tissues of C. elegans during aging.

衰老过程中 秀丽隐杆线 虫线粒体形态的成像和定量

Mitochondria, important cellular organelles found in most eukaryotic cells, are major sites of energy production through aerobic respiration. Beyond this ...

方法来评估肌肉的亚细胞中

摘要

探索更多视频

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

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

线粒体疾病脑膜炎基因模型中量化动物活动的实验方法比较分析

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

在体内卡诺哈布德炎电子神经元微管动力学和方向评估

体内神经元钙处理的亚细胞成像

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

秀丽隐杆线虫肠的手部解剖

衰老过程中秀丽隐杆线虫线粒体形态的成像和定量

方法来评估肌肉的亚细胞中

摘要

探索更多视频

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

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

线粒体疾病脑 膜炎基因 模型中量化动物活动的实验方法比较分析

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

在体内卡诺哈布德炎电子神经元微管动力学和方向评估

体内神经元钙处理的亚细胞成像

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

秀丽隐杆线虫肠的手部解剖

衰老过程中 秀丽隐杆线 虫线粒体形态的成像和定量

线粒体疾病脑膜炎基因模型中量化动物活动的实验方法比较分析

衰老过程中秀丽隐杆线虫线粒体形态的成像和定量