We thank Cindy Voisine and Yoko Shibata for helpful discussion and critical comments on the manuscript. We acknowledge the High Throughput Analysis Laboratory (HTAL) and the Biological Imaging Facility (BIF) at Northwestern University for their assistance. This work was funded by grants from the National Institutes of Health (NIGMS, NIA, NINDS), the Ellison Medical Foundation, and the Daniel F. and Ada L. Rice Foundation (to R.I.M.). C.I.N.-K. was supported by the Deutsche Forschungsgemeinschaft (KR 3726/1-1).

1.监控跨细胞扩散的朊病 ​​毒样蛋白在体内时间推移成像注：成长C.线虫野生型（WT）（N 2）和转基因品系根据标准方法，并仔细控制培养温度47。 <ol><li> C.产生的转基因株系线虫表达朊状蛋白质，具有标记的单体红色荧光蛋白（MRFP）。观看此视频，演示了如何使用微量注射48。对于进一步的细节和方法，描述如何将这些外的线路整合，见49。 </li><li>通过根据标准方法50产蛋或漂白准备同步种群。 <ol><li>通过同步产卵<ol><li>转让10 - 20妊娠成年人放在盘子里，让他们产卵1 - 2小时。从板除去成虫，并让后代生长，直到所需的年龄。 </li></ol></li> &lt;LI&gt;同步漂白<ol><li>收集妊娠成人的非同步的人口和与碱性次氯酸盐溶液（250毫NaOH和1:4（V / V）稀释的市售漂白剂的H 2 O）漂白它们。与M9的缓冲区47（21 MMNA 2 HPO 4·两次（218 XG 1分钟）将鸡蛋洗净后7H 2 O，22 mMKH 2 PO 4，86 mMNaCl，1 mMMgSO 4·7H 2 O，加起来的dh 2 O〜1升）。 </li><li>允许它们在M9缓冲孵化温和搅拌O / N在20℃。蠕虫发展将阻止在所述L1阶段在没有食物来源的，留下一个同步的人口。转移到L1S新鲜线虫生长介质（NGM）接种OP50 E.板杆菌细菌并让子代发展，直到所希望的47岁。 </li></ol></li></ol></li><li>在显微镜载玻片上制备2％的琼脂糖垫（在H 2 O）如所述50。 <ol><li>准备两英里croscope幻灯片与标签带放置在其整个长度将用作间隔物​​。把他们之间的第三显微镜载玻片。 </li><li>溶解2％琼脂糖在H 2 O和吸管一滴到干净的幻灯片。 </li><li>将第四幻灯片垂直于琼脂滴顶部的其他三个幻灯片。轻轻按下变平垫以相同厚度作为标记带。 </li><li>让除去间隔物，轻轻拉动滑动分开之前，干燥1分钟。琼脂板将继续坚持其中之一。 </li></ol></li><li>吸管〜10微升麻醉（2毫左旋咪唑在M9缓冲液中）到垫和转让〜使用铂丝挑10只动物。盖上盖玻片（〜22×22毫米），在1小时内拍摄图像。 </li><li>或者，为了获得电影在一段较长的时间，或以进一步降低对动物的任何移动的可能性，使用麻醉剂和珠固定51的组合。 <ol><li>准备10％AGA玫瑰区（在M9缓冲区）所描述的51，并添加蠕虫3微升纳米球的大小标准溶液（聚苯乙烯珠，100纳米），加上3微升麻醉剂（4毫米左旋咪唑M9缓冲）。以盖玻片覆盖平缓。为了避免干燥，密封盖玻片与VALAP（等量的凡士林，羊毛脂和石蜡的混合物）。 </li></ol></li><li>利用共聚焦显微镜图像固定蠕虫。 注意：测试结果是使用一个转盘的AF共聚焦显微镜配备有EM-CCD照相机和显微镜自动化及图像分析软件如的MetaMorph获得和轮廓的细节在下面，但其他类似的共焦成像系统也可以使用。 <ol><li>使用63X或100X / 1.4NA油浸物镜，并将包含蠕虫到显微镜载玻片架​​显微镜载玻片。 </li><li>打开软件。调整激光功率和过滤器MRFP成像。用10％的功率和排放过滤器&gt; 600纳米561纳米的激光。 </li><l我&gt;打开“多维收购”。在“主”选项卡，勾选框“慢速拍摄”和“运行日志”（硬件自动对焦：关闭）。 </li><li>在“保存”选项卡上选择或创建所在的文件保存的目录文件夹。指定一个名称的文件。 </li><li>在“波浪长度”选项卡中选择合适的照明和调整曝光和增益。用“YokoQuad红”（或等同的照明设置为MRFP成像）与100和300毫秒之间，100和300的照相机增益之间的曝光，这取决于个体的样品（使用实时图像评估）。 </li><li>在“时光倒流”选项卡中选择“时间点数量”= 301，“持续时间”= 5分钟，“时间/间隔”= 1秒。 </li><li>在“日志”选项卡中选择杂志：“AFC集合Z HOLD”和“AFC返回到Z HOLD”，类型：＃8220;特别“（两次），初始点：”时间起点“和”结束时间点“。此自动聚焦选项是很重要的图像相同的部分在一段较长的时间。 </li><li>当安装完成后，点击“获取”。在慢速拍摄视频萨法德作为独立的TIFF文件。 </li><li>打开一个给定的时间序列的ImageJ所有.tiff文件。转到“图像”→“栈”→“图像至栈”。或者，调整亮度和对比度，加上大小酒吧等出口“文件”下的电影→“另存为”→“AVI ...”（对于一个例子，看视频1和2对应的图1）。 </li></ol></li></ol> 2.采用折叠式传感器和应力记者调查细胞自主和非细胞自治影响的Proteostasis和毒性<ol><li>产生共表达折叠传感器或压力reporte转基因株系R 3与朊病毒蛋白样蛋白在一起。关于如何建立十字架或产生转基因株系方法见48,49,52。 见表1 C.名单线虫菌株可以使用。 </li><li>准备转基因动物的种群同步成长，直到上述（1.2节）50所描述所需的年龄。 </li><li>利用体视显微镜，检查折叠传感器或压力记者各自的表型。 <ol><li>对于折页传感器，确定动物在同步之后窝藏在每一天的聚集体的数目（对于一个例子，参见图2E和F）。 </li><li>为应力记者，如果测试的朊病 ​​毒样蛋白导致报告的增加的荧光的共表达（对于一个例子，参见图3）。 </li></ol></li></ol> 3.全基因组屏幕为抑制在C.朊病毒引起的毒性线虫<img alt="图4" src="/files/ftp_upload/52321/52321fig4highres.jpg" /> 图所述RNAi筛选方案4.示意图。参见协议部3的各个步骤的详细描述。 <ol><li> C.同步线虫蠕虫和RNAi文库的重复<ol><li>对于RNAi筛选，使用Ahringer的RNAi库（或比达尔的RNAi库）53,54。填充用100μl的LB-amp培养基补充有10％甘油的板（50微克/毫升氨苄青霉素的LB）Rearray所述RNAi文库从原来384孔格式到96孔板，并接种使用96针复制器。生长在37℃CO / N在搅拌并储存在-80℃。 </li><li>维持C.线虫 WT和在20℃下对NGM板的转基因株系接种有大肠杆菌 OP50根据标准方法47 杆菌细菌。 </li><li>通过漂白朊病毒域转基因和WT（控制）线虫同步（见1.2）。 </li><li>就在同一天，该蠕虫被漂白，准备补充50微克/毫升氨苄青霉素的LB培养基。使用自动试剂分配器分配（或吸管手动）65微升的LB-amp培养基中于96孔板的每个孔中。 </li><li>从-80℃取出96 Ahringer的RNAi库板，将他们无菌罩内衬用纸巾。立即端着盘子倒挂，而板块仍然冻结，小心，以避免对板的外冰污染取出密封胶带。 Reinvert板，并让它们解冻约30分钟。 </li><li>浸1无菌96针复制到一个RNA干扰库板，然后到一个新鲜的LB-Amp平板。选用一个新鲜无菌复制每个板块。所有封板的粘合铝箔胶带，并返回库板至-80℃。该复制器可浸泡在漂白，清洗，并高压灭菌，以再次使用。 </li><li>允许重复的RNAi板成长O / N在孵化器设置为37℃和300转。使用HT115 E.杆菌细菌携带空载体（L4440）作为对照，分别生长在一个培养管中，然后吸取65微升用多道移液器培养成四分之一2 96孔板的。 </li></ol></li><li>感应细菌的dsRNA产生和附加蠕虫的<ol><li>稀释异丙基β-D- thiogalactopyranosid（IPTG）至5mM的浓度在双蒸2 O.使用自动化工作站与多通道头分配（或吸管手动）10微升稀释的IPTG到的RNAi的细菌和对照板的每个孔中。重新密封，并放置在板放回孵化器。让他们在37℃摇动3小时。 </li><li>虽然细菌正在动摇，准备蠕虫。混合在M9 /蠕虫悬浮良好，并吸取少量样品（〜5 - 10微升）到玻璃显微镜载片上。算蠕虫在立体显微镜下的数量，并计算每微升的蠕虫数目。 </li><li>使用无菌技术，制备补充的M9（M9 +）的溶液具有以下的最终浓度：0.20毫克/毫升的IPTG，8.0微克/毫升胆固醇，50微克/毫升氨苄青霉素，9.6微克/毫升四环素，0.0835微克/毫升两性霉素B，和每50微升15蠕虫。 <ol><li>使朊病毒域转基因和WT蠕虫单独的解决方案。的需要将依赖于复制（见下文）的RNAi板的数量的M9 +蠕虫溶液的最终体积，加〜30毫升死体积，将留在储，如果一个自动化分配器使用的。 </li></ol></li><li>在3小时的诱导细菌的dsRNA生产后，将板从孵化器，并让它们冷却至室温（约30分钟），以避免热应激反应。 </li><li>分配50μl的M9 +朊病毒域的转基因线虫的解决了RNAi的板的每个孔中，并在空载体对照板之一。使一定要混合各步骤之前蜗杆溶液作为动物倾向于沉淀到容器的底部。 </li><li>分配的WT蠕虫入第二控制板。离开启封，以允许通气板。以防止蒸发的液体培养基，堆栈4 - 5板块一起，敷用湿纸巾和铝箔。孵育以200rpm在20℃下4天。 </li></ol></li><li>进球 注意：之后在孵化4天，所述蠕虫将会在成年期的第二天，并准备屏幕。让这些动物筛选，以确保无干扰抖动之前调整到非振荡的条件进行30分钟。 <ol><li>使用猎鹰4M60相机连接到计算机的监视器，屏幕目视增加抖动，与对照处理的朊病毒域的转基因动物进行比较。 </li><li>编制初步命中名单，以确认使用wrMTrck（见下节）。 </li></ol></li></ol> 4.确认初步的屏幕点击<ol><li>固体板活力测定<ol><li>制备板的NGM补充有100微克/毫升氨苄青霉素，12.5微克/毫升四环素和1mM IPTG，按照标准方法47。如果可能的话，使用的板浇注机，以确保所有的板块有媒体相同的高度，这将允许一个更精简的视频采集过程。 </li><li>生长的不同的RNAi克隆中约1毫升LB培养基+50μg/ ml的氨苄青霉素，O / N在37℃和250转。 </li><li>第二天，诱导dsRNA的表达用1mM IPTG 3小时。种子用150微升每RNAi的细菌克隆，摊成薄层板。让干燥为在黑暗2天在RT的细菌。准备每RNAi技术克隆3板。 </li><li>通过按照标准方法50（参见1.2节）漂白，蠕虫人口同步，让卵孵化O / N在M9媒体。 </li><li>取蜗杆悬浮的样品，并确定NEM的量每微升atodes在体视显微镜。然后，吸管的M9加上蠕虫的正常体积到各试验板，以使其包含25 - 30 L1的蠕虫。成长的线虫在20℃4天，直到蠕虫达到成年的2天。 </li></ol></li><li>虫蠕动的wrMTrck插件ImageJ的定量分析 注：使用立体显微镜，在放大10倍与滨松的Orca-R2数码相机C10600-10B和滨松简单PCI图像处理软件的视频记录。 <ol><li>打开相机和简单PCI成像软件。点击“在线”，以允许调整的成像条件。 </li><li>设置的视频条件如下：增益= 0;光模式=高;速度指数= 1;分级= 2.单击“自动暴露”，然后调整照明条件下，移动显微镜镜和亮度和对比度拨号。 注：视频需要具有高的对比度而不曝光过度osed，从而使动物出现在明亮的背景为黑色的形状。 </li><li>点击“时间扫描”，然后选择一个文件夹和文件名。设置“现场延迟”到20毫秒，“停止在时间”，以30秒。按“在线评论”，以便选择该板的面积来记录（其中，最蠕虫）。酶标板3次或4次在舞台上，快速确认其观点，按“开始”领域的地位。 </li><li>之后，电影拍摄完成后，右键单击图像，然后选择“导出蒙太奇序列”的电影从.cxd格式导出到一个.avi。 </li></ol></li><li>分析蠕动视频<ol><li>打开ImageJ的软件，进入“插件”选项卡，然后在“wrmtrck”，然后选择“wrMTrck批”。选择包含​​的所有文件的目录来进行分析。 </li><li>在wrMTrck_Batch的主输入窗口，如在造型玩具详述加载的输入值Ë5C。对于每个参数的解释可以在附带的插件的说明中找到。点击“OK”，让运动分析运行。 </li><li>为策展的结果，并确认所有检测到的轨道与实际C.线虫和消除文物，打开每个为每部电影创建的.txt文件和复制信息到数据分析软件的文件。打开创建的“* _labels.zip”文件，并运行所产生的“* _labels.tif”手动检查并消除虚假蠕虫轨道。 </li></ol></li></ol>

The authors declare no competing financial interests.

此处所描述的方法有助于说明扩频和朊病毒样蛋白的复杂细胞中自主和非细胞自主毒性。我们最近发现，一个聚集倾向的胞质朊病毒域被吸收到膜结合的囊泡中的自噬相关的过程。这些囊泡的特定子集运内和细胞和组织40之间的朊病 ​​毒域。监测其在活的动物运动的关键是，该蛋白质必须具有标记MRFP，因为只有MRFP标签蛋白均在这些推测酸性囊泡可见。用同样的方法，我们目前正在研究...

许多神经变性疾病，包括阿尔茨海默氏病（AD），帕金森氏病（PD），肌萎缩性侧索硬化症（ALS），和传染性海绵状脑病（海绵状脑病），都与聚集倾向的蛋白质相关联，并且因而是统称为蛋白质错误折叠疾病（PMDS ）。的TSE或朊病毒病构成一类独特的PMD的，因为它们可以是传染性在人类和动物1。在分子水平上，朊病毒复制招募和单体α螺旋丰富的主机编码细胞朊蛋白（PRP C）转换成病理β折叠丰富的朊蛋白构象钪2,3。自我繁殖的蛋白质聚集体已经还确定了真菌，它与哺乳动物朊病毒4,5共享的重要特征。此外，哺乳动物朊病毒能够从细胞至细胞移动及感染幼稚细胞6,7。 而超视距的PMD呃不是传染性海绵状脑病是不会传染的，他们与朊病毒疾病8,9都有一个共同的致病原理。虽然链接到每个所述的PMD的蛋白质的结构或功能是不相关的，它们经由一个结晶状过程中的所有形式的聚集体称为有核或接种聚合;而且蛋白质的种子长出通过招募他们的可溶性亚型2,10,11。效率，以自我传播体内而变化，这取决于蛋白的固有性质，连同另外的细胞因子，如分子伴侣最终确定聚合成核，播种，碎裂和扩频12-15的速率。因此，必须存在这些因素之间，允许蛋白质聚集的有效传播一个微妙的平衡。这可能也解释了为什么只有某些淀粉样蛋白聚集怀有朊病毒的特点，因而并不是所有的PMD都具有传染性。朊病毒似乎代表“顶级表演&#39;O广发自我复制的蛋白质聚集的频谱，这使得他们的强大工具来研究的PMD 8,13。 有趣的是，与疾病相关的聚集相关的毒性经常具有非细胞自主组件16,17。这意味着，它们影响邻近的细胞不表达相应的基因，而相比之下，严格细胞自主的效果，这意味着只有该细胞表达该基因表现出特定的表型。这是令人信服由组织特异性表达证实或敲在神经变性疾病18-26的众多模型的各蛋白质的向下。各种机制被建议作为用于这种非细胞中的PMD自主毒性，包括减少营养供给，失衡神经信号，谷氨酸兴奋毒性，和神经炎症16,27,28的基础。此外，疾病联的聚集体的细胞之间的朊病毒样运动migh吨有助于这方面的29,30。越来越多的证据表明，其它蛋白质夹杂比朊病毒可以从细胞-细胞传递，这可以解释病理学在许多的PMD 30-36观察的特征传播。然而，它尚未确定是否有疾病的蛋白质的细胞间移动和对邻近细胞的毒性效果之间的明确的因果关系。因此，更好地理解所依据的细胞至细胞传播和非细胞自主毒性的细胞途径的是必要的和必不可少的新疗法的发展。然而，朊病毒状扩频和细胞因子的许多方面，影响在后生动物细胞 - 细胞传输错误折叠的蛋白质都没有很好地理解，特别是在有机体的水平。 该线虫具有提供潜力几个优点 发现的朊病毒样spreadi新层面纳克在后生动物17。它是透明的，允许在体内跟踪的荧光标记的蛋白在活的有机体。此外，受疾病的许多细胞和生理过程是从蠕虫到人类的保守，和C.线虫也是适合于各种各样的遗传操作和分子和生物化学分析37-39的。正是959的体细胞组成成人雌雄同体用一个简单的身体的计划，仍然有几个不同的组织类型，包括肌肉，神经元和肠。 要建立C.一个新的朊病 ​​毒模型线虫，我们选择了外源表达的良好表征谷氨酰胺/天冬酰胺（Q / N）的胞质酵母朊病毒Sup35的富朊病毒域NM，因为有在蠕虫4,40没有已知的内源性朊病毒蛋白。酵母朊病毒已经非常宝贵的阐明朊病毒复制41-44的基本机制。此外，NM是杉木已被证明扼要重述朊病毒的整个生命周期中的哺乳动物细胞培养45,46 ST胞质朊状蛋白质。同样地，在C时表示线虫 ，通过非常好，为在后生动物细胞繁殖的不同的要求相比，酵母细胞和朊病毒生物学表现关键特征40。NM聚集了深刻的毒性表型相关联，包括线粒体完整性和外观的破坏的Sup35朊病毒域各种自噬相关囊泡在细胞水平上，以及胚胎和幼虫逮捕，发育迟缓，并在有机体水平的蛋白质折叠的环境的一个普遍的干扰。引人注目的是，朊病毒域表现出细胞中自主和非细胞自主毒性，影响相邻的组织，其中，转基因不表达。此外，内和细胞间的朊病毒域的囊泡运输监测实时&lt;EM&gt;在体内40。 在这里，我们将介绍如何检查朊病毒般传播C.线虫 。我们将解释如何监控含使用时间推移荧光显微镜朊病毒域囊泡内和细胞间的运输。我们将强调使用组织特异性折叠传感器和遍在表达的应力记者，以评估对细胞的健身细胞中自主和非细胞自主效应。最后，我们将描述一个最近执行的全基因组RNA干扰（RNAi）屏幕的方法来识别朊病毒诱导的毒性的新调节剂。总之，这些方法可以帮助捉弄除了参与蛋白质的细胞间运动及其非细胞毒性自主遗传途径。

<table border="0" cellpadding="0" cellspacing="0" width="665">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Reagent</td>
			<td style="width:107px;"></td>
			<td style="width:92px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Nanosphere size standards 100 nm</td>
			<td style="width:107px;">ThermoScientific</td>
			<td style="width:92px;">3100A</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Levamisole</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:92px;">L-9756</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">IPTG</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:92px;">15502-10G</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:211px;">Ahringer RNAi library</td>
			<td style="width:107px;">Source BioScience LifeSciences&#160;</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.lifesciences.sourcebioscience 
			.com/clone-products/non-mammalian/c-elegans/c-elegans-rnai-library/</td>
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			<td height="21" style="height:21px;width:211px;">Equipment</td>
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			<td style="width:255px;"></td>
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		<tr height="51">
			<td height="51" style="height:51px;width:211px;">Sorvall Legend XTR Refrigerated Centrifuge, 120VAC</td>
			<td style="width:107px;">ThermoScientific</td>
			<td style="width:92px;">75004521</td>
			<td style="width:255px;">http://www.coleparmer.com/Product/Thermo_Scientific_Sorvall_Legend_ 
			XTR_Refrigerated_Centrifuge_120 
			VAC/EW-17707-60</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:211px;">96 pin replicator&#160;</td>
			<td style="width:107px;">Scionomix&#160;&#160;</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.scinomix.com/all-products/96-pin-replicator/</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:211px;">HiGro high-capacity, incubating shaker&#160;</td>
			<td style="width:107px;">Digilab</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.digilabglobal.com/higro</td>
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		<tr height="34">
			<td height="34" style="height:34px;width:211px;">Multidrop Combi Reagent Dispenser&#160;</td>
			<td style="width:107px;">Titertrek</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://groups.molbiosci.northwestern.edu/hta/titertek.htm</td>
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			<td height="34" style="height:34px;width:211px;">Biomek FX AP96 Automated Workstation&#160;</td>
			<td style="width:107px;">Beckman Coulter</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://groups.molbiosci.northwestern.edu/hta/biomek_multi.htm</td>
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		<tr height="68">
			<td height="68" style="height:68px;width:211px;">Innova44 shaker</td>
			<td style="width:107px;">New Brunswick</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.eppendorf.com/int///index.php?sitemap=2.3&#38;pb=d78efbc05310ec 
			04&#38;action=products&#38;contentid=1&#38; 
			catalognode=83389</td>
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			<td height="85" style="height:85px;width:211px;">M205 FA&#160;</td>
			<td style="width:107px;">Leica</td>
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			<td style="width:255px;">http://www.leica-microsystems.com/de/produkte/stereomikroskope-makroskope/fluoreszenz/details/product/leica-m205-fa/</td>
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		<tr height="51">
			<td height="51" style="height:51px;width:211px;">ORCA-R2 C10600-10BDigital CCD camera</td>
			<td style="width:107px;">Hamamatsu</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.hamamatsu.com/jp/en/community/life_science_camera/product/search/C10600-10B/index.html</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:211px;">Spinning Disc AF Confocal Microscope&#160;</td>
			<td style="width:107px;">Leica</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.leica-microsystems.com/products/light-microscopes/life-science-research/fluorescence-microscopes/details/product/leica-sd-af/</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:211px;">Falcon 4M60 camera&#160;</td>
			<td style="width:107px;">Teledyne Dalsa&#160;</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.teledynedalsa.com/imaging/products/cameras/area-scan/falcon/PT-41-04M60/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Software</td>
			<td style="width:107px;"></td>
			<td style="width:92px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:211px;">MetaMorph Microscopy Automation &#38; Image Analysis Software</td>
			<td style="width:107px;">Molecular Devices</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.moleculardevices.com/products/software/meta-imaging-series/metamorph.html</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:211px;">Hamamatsu SimplePCI Image Analysis Software</td>
			<td style="width:107px;">Meyer Instruments</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://meyerinst.com/imaging-software/hamamatsu/index.htm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">ImageJ</td>
			<td style="width:107px;">NIH</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://rsbweb.nih.gov/ij/download.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">wrMTrck plugin for ImageJ</td>
			<td style="width:107px;"></td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.phage.dk/plugins/wrmtrck.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">C. elegans strains</td>
			<td style="width:107px;"></td>
			<td style="width:92px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:211px;">N2 (WT)</td>
			<td style="width:107px;">Caenorhabditis Genetics Center (CGC)</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.cgc.cbs.umn.edu/strain.php?id=10570</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:211px;">AM815&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; rmIs323[myo-3p::sup35(r2e2)::rfp]</td>
			<td style="width:107px;">Morimoto lab</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">available from our laboratory&#160;</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:211px;">See table 1 for a source for folding sensor and stress reporter strains</td>
			<td style="width:107px;"></td>
			<td style="width:92px;"></td>
			<td style="width:255px;"></td>
		</tr>
	</tbody>
</table>

investigating the spreading and toxicity of prion-like proteins using the metazoan model organism c. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

监测细胞间的朊病 ​​毒样蛋白扩频通过体内延时成像 转基因C.线虫系表达朊病毒域是特别适合的朊病 ​​毒样蛋白， 例如 ，细胞至细胞传播和非细胞自主毒性某些方面的分析。使得能够追踪的荧光动物的透明度从活体内标记的蛋白在生命的各个阶段。趁着此，跨细胞和使用荧光显微镜组织朊状蛋白质运动被可视化。一个63X或100X / 1.4NA油?...

Watch this Scientific Journal Video about Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans at JoVE.com

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

调查的传播和毒性朊病毒样蛋白使用后生动物模式生物<em&gt;温度。线虫</em

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

investigating-spreading-toxicity-prion-like-proteins-using-metazoan

Research

JoVE Journal

Biology

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

15.7K 次观看.  Northwestern University. Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

视频: Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

科研

生物学

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

线虫神经肌肉交界处的解剖和录制

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

生产 C。线虫</em通过重组工程与转基因 galK选择标记

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

创建定义的气体环境研究缺氧对<em&gt; C。线虫</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

使用屏幕在车身尺寸规例中的线虫基因介导的RNA干扰取食策略<em&gt; C.线虫</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

抗体在染色<em&gt; C。线虫</em&gt;使用“冻结开裂”

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

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 ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

测量细菌的影响<em&gt; C。线虫</em&gt;行为使用一个鸡蛋保留含量

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

对缺血/再灌注在缺氧，饥饿型号<em&gt; C。线虫</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.