1。酵母制剂<ol><li>变换酵母菌株的质粒携带GAL1-GFP-Y68L（UBC9 TS）磁带。 </li><li>成长酵母合成培养基中含有2％的棉子糖24小时和16小时或O / N稀释至2％的半乳糖包含媒体孵育细胞在30℃，同时以200rpm振摇。 </li><li>第二天早上，淡化查询应变OD 600 = 0.2。振摇4-6小时，在30℃（200rpm）下，直到培养物达到OD 600 = 0.8-1.0。 </li><li>压制表达式（以便只监视已翻译和折叠的池的GFP-Ubc9时日ts），变介质2％的葡萄糖（SD）的前30分钟，在成像的合成培养基。 </li><li>治疗 - 热休克细胞20分钟，在37℃下诱导错误折叠，或连续地在显微镜培养箱遵循蛋白聚集在热休克条件下。注 - 如果您使用的是显微镜以上RT，在任何温度下预热FOR 2小时左右，沿着身体的显微镜和目标以平衡温度（见下文）。 </li></ol> 2。板\幻灯片的准备工作<ol><li>选择适当的板。主要考虑的是图像采集过程中保持注意力集中的能力。以下几点是关键4D成像，并没有为2D的时间失效或3D成像。 <ol><li>多井板的不同的井的底部可能不是均匀的（ 即井将在不同的高度相对于目标）。这将使得难以维持焦点跨XY点和时间点，而不管所使用的自动对焦技术。 </li><li>幻灯片材料：玻璃与塑料。玻璃底的幻灯片是更多的Z-同质井之间，但更昂贵。 </li><li>厚度板的底部：我们通常使用的盖玻片底板的折射率为1.5，但根据目标指数也是可以接受的。 </li></ol></li><li>冠状病毒呃板底部\幻灯片用刀豆蛋白A（0.25毫克/毫升）10分钟。刀豆蛋白A用于粘附细胞的滑动，从而使一个单一的细胞随着时间的推移之后。 </li><li>删除ConA和孵化幻灯片中的化学罩，使多余的刀豆蒸发。 </li><li>板200微升酵母样品（OD 600 = 0.5）到ConAed井。 </li><li>孵育15分钟，以使细胞粘到板的表面。 </li><li>提取介质，并与新媒体洗脸三次，一层细胞。注：计划如果很长的时间间隔，种子细胞人烟稀少，使新芽不会填写，并中断该地区的利益。 </li></ol> 3。显微镜的准备工作<ol><li>我们使用一个尼康A1Rsi的共聚焦显微镜一些非标准的修改。用于酵母成像，我们使用了4激光器（405nm处，50毫瓦CUBE激光457-514 nm的氩离子激光，65毫瓦的561纳米，50毫瓦蓝宝石激光器;和640 nm，40毫瓦CUBE激光），和高达4个光电倍增管配备过滤器。我们的成像是一个绿色的过滤器设置为EGFP，mCherry和tdTomato和红色过滤器设置。 GFP和mCherry是几乎没有bleedthrough的，因此我们经常使用的同步扫描。然而，行扫描也可以被使用，以防止bleedthrough当它发生。我们的共聚焦还配备了一个PInano压电阶段（MCL），它可以使在z 3毫秒的步骤，使2-3的z栈（30-50节）每秒。该系统还具有的光谱检测器，完美的焦点（激光偏移量），和两台扫描器 - 电流计扫描器和一个谐振扫描器。 </li><li>选择合适的目标。主要考虑： <ol><li>水/油/气目标：主要考虑的是成像介质与介质的样品的折射率。 <ol><li>油客观优势： <ol><li>石油的目标可以有更高的数值孔径（油断比水轻多了，因此，更多的光子，回去的目的）。石油的目标最多可以有到NA 1.49，相比对水至1.27。 （然而，这是不实际的巨大差异分辨率）。 </li><li>油的折射率与玻璃的折射率匹配，因此没有光子从样品都将丢失，透过玻璃的目标。 （注 - 选择浸油的折射率是适合你的目标和你的盖玻片）。 </li><li>石油可以轻松自由的很长一段时间流逝，因为它没有蒸发。 </li></ol>油浸物镜的缺点： <ol><li>由高NA的油目标启动，更高的分辨率的下降很快，如果光具有穿越的水性介质（诸如细胞）。 </li><li>图片通常有更多的球面像差和“伸出”的3D效果。 </li><li>石油是粘性的，杂乱的，和目标的危险。 </li></ol></li><li>水目标的优势<ol><li>细胞本身是水性的，因此有较少的在z方向的扭曲，一个第二，决议仍然很高深的水样。 </li><li>清理和用户友好。水目标缺点：迅速蒸发，因此不适合长期连续泵水的目的没有特别安排电影。 </li></ol></li><li>空中目标的优势：1。无重大蒸发过程中丢失/多点收购或长的电影。 2。清理和用户友好。缺点：低分辨率和低信号的灵敏度。 </li></ol></li><li>室和孵化温度：温度变化会影响物质属性，和在精密系统改变焦点的位置。温度应调整才选择收购点。在采集过程中改变温度会破坏成像，将导致失去焦点。我们让我们的显微镜系统成像之前在所需温度下平衡2小时。 </li><li>修正衣领：许多目标来校正环使对于一个给定的盖玻片的厚度进行调整的目标。我们建议调整使用荧光珠的同时进行可视化的点扩散函数的校正铤。这将确保每个样本的正确的设置。 </li><li>稳定的样品持有人：我们发现，大部分市售样品持有人是最弱的一环，在显微镜。他们往往摇摆不定和不直。这是一个灾难，高分辨率，多点4D成像。我们设计我们自己的样品架，直到舞台上，并紧密贴合。它们也可以用螺栓固定在需要的时候。 </li><li>多点采集：我们的成像系统配备了尼康“完美的对焦系统，这基本上是一种基于激光自动对焦功能。然而，自动对焦不一定必要，4D成像，特别是在系统不漂移的特定的。 </li></ol></li></ol> 4。成像<ol><li>打开系统：激光，舞台，控制器，摄像头和COMPU后的软件。 </li><li>舞台上支架现浇板，安全和稳定。 </li><li>使用眼部端口的酵母，以确定的位置和取向。 </li><li>使用明场，评估细胞的健康与活力，根据形状和质地。 </li><li>打开啶光根据相关荧光基团（ 如 FITC过滤器GFP），专注于细胞显示的表型，这是您的研究。有很强的荧光，在一个以上的波长的细胞，这些细胞可能是死亡，因此，自体荧光。可视化的核心与一个tdTomato的融合到SV40免入息审查贷款计划的信号（NLS-TFP）的荧光。 TFP是GFP的大小的两倍，因此，上面的细胞核内的扩散限制，因此它工作得很好作为核标记。我们也可以与绿色（488 nm）的激光为GFP同时激发的TFP，但收集的绿色和红色的光谱分辨率分成两个单独的光电倍增管的排放量。 </li><li>调整以下设置，以减少噪音过饱和： <ol><li>激光功率 - 漂白和光毒性与图像的亮度应予以考虑。 </li><li>增益：影响摄像机的灵敏度，从而信噪比。 </li><li>应调节针孔的直径 - 根据与更短的波长的激光。针孔的直径确定成像光学部的厚度（高度）。因此，开放的针孔会收集更多的光线让更多的光子，但会降低分辨率在Z（少共焦）。 </li></ol></li><li>有用的提示： <ol><li>如果不同的荧光发射波长一致的，使用光谱检测功能，可让虚拟过滤器的选择。请记住，比普通的光电倍增管光谱探测器是较不敏感。 </li><li>如果不同的荧光基团很高兴在相同的激光，使用逐行扫描功能。 </li><li>电流扫描仪能够更灵敏，但具有较高的风险漂白。共振扫描仪能够提供更快的一个cquisition，从而降低了漂白的风险，但较不敏感。 </li><li>在2和16之间的图像的平均值，它增强信噪比，但使得采集慢，当然，所涉及的样品的激光曝光。 </li><li>收购的持续时间取决于生物学问题（ 如 JUNQ和IPOD形成约需2小时）。我们已成像的酵母与谐振扫描器长达30小时，在三维的时间推移。 </li><li>时间流逝的时间间隔更小的间隔将创建一个更为连贯的电影，但可能导致的光漂白，因此，信号损失。 </li></ol></li></ol>

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	<li>Gershenson, A., Gierasch, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+folding+in+the+cell:+challenges+and+progress.">Protein folding in the cell: challenges and progress.</a> Current opinion in structural biology. 21, 32-41 (2011).</li><li>Aguzzi, A., Calella, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prions:+protein+aggregation+and+infectious+diseases.">Prions: protein aggregation and infectious diseases.</a> Physiological reviews. 89, 1105-1152 (2009).</li><li>Morimoto, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteotoxic+stress+and+inducible+chaperone+networks+in+neurodegenerative+disease+and+aging.">Proteotoxic stress and inducible chaperone networks in neurodegenerative disease and aging.</a> Genes &amp; development. 22, 1427-14 (2008).</li><li>Cohen, E., Dillin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+insulin+paradox:+aging,+proteotoxicity+and+neurodegeneration.">The insulin paradox: aging, proteotoxicity and neurodegeneration.</a> Nature reviews. Neuroscience. , (2008).</li><li>Hershko, A., Ciechanover, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin+system.">The ubiquitin system.</a> Annual review of biochemistry. 67, (1998).</li><li>Tyedmers, J., Mogk, A., Bukau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+strategies+for+controlling+protein+aggregation.+Nature+reviews.">Cellular strategies for controlling protein aggregation. Nature reviews.</a> Molecular cell biology. 11, 777-788 (2010).</li><li>Treusch, S., Cyr, D. M., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+deposits:+Protection+against+toxic+protein+species?.">Amyloid deposits: Protection against toxic protein species?.</a> Cell cycle (Georgetown, Tex.). 8, 1668-1674 (2009).</li><li>Spokoini, R., 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=Confinement+to+Organelle-Associated+Inclusion+Structures+Mediates+Asymmetric+Inheritance+of+Aggregated+Protein+in+Budding+Yeast.">Confinement to Organelle-Associated Inclusion Structures Mediates Asymmetric Inheritance of Aggregated Protein in Budding Yeast.</a> Cell Rep. , (2012).</li><li>Weisberg, S. 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=Compartmentalization+of+superoxide+dismutase+1+(SOD1G93A)+aggregates+determines+their+toxicity.">Compartmentalization of superoxide dismutase 1 (SOD1G93A) aggregates determines their toxicity.</a> Proc Natl Acad Sci U S A. 109, 15811-15816 (2012).</li><li>Kaganovich, D., Kopito, R., Frydman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Misfolded+proteins+partition+between+two+distinct+quality+control+compartments.">Misfolded proteins partition between two distinct quality control compartments.</a> Nature. 454, 1088 (2008).</li><li>Betting, J., Seufert, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+yeast+Ubc9+mutant+protein+with+temperature-sensitive+in+vivo+function+is+subject+to+conditional+proteolysis+by+a+ubiquitin-+and+proteasome-dependent+pathway.">A yeast Ubc9 mutant protein with temperature-sensitive in vivo function is subject to conditional proteolysis by a ubiquitin- and proteasome-dependent pathway.</a> The Journal of biological chemistry. 271, 25790 (1996).</li><li>Tongaonkar, P., Beck, K., Shinde, U. P., Madura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+temperature-sensitive+mutant+of+a+ubiquitin-conjugating+enzyme+and+its+use+as+a+heat-inducible+degradation+signal.">Characterization of a temperature-sensitive mutant of a ubiquitin-conjugating enzyme and its use as a heat-inducible degradation signal.</a> Analytical biochemistry. 272, 263 (1999).</li></ol>

没有利益冲突的声明。

我们关于生化过程的直觉来自台式实验中，在烧杯中，充分混合的溶液，反应物和产物的是，允许达到平衡。在这样的设置中，一个给定的化学物种的浓度可以表示为一个单一的数字，这是分子的摩尔量的比率到一个宏观体积。我们所知道的有关蛋白质的结构和功能源于反映的经典之作，散装的反应图片：西方墨点法，离心，从整个细胞群的匀浆提取物进行分光光度测量的使用方法。 在放大镜下看细胞的技术，我们提高了长足的发展，它变得越来越清晰， 体内生化反应发生的条件只承担一点相似之处的经典台式情况下。不仅是内部的大公LA密集的环境中，在拥挤效应显着改变各种反应物的活动，它是完全相反的混合。这解释了频繁的差距在体外和体内的效率的范围广泛的复杂的高分子反应。 现在，即使是直觉源于经典的体外生化实验， 在体内的折叠，错误折叠和聚集的蛋白质有关的问题，更容易产生误导。而研究蛋白质化学在散装反应的治疗作为一个简单的是或否的问题对于一个给定的蛋白质折叠的问题，任何试图在活细胞中的动态跟踪整个人口的大分子必须是敏感的整体分布的可能可用的多肽链的构象的结果，特别是错误折叠的和聚集的风险。例如，我们可能会检查一个体细胞LY沙爹凝集蛋白通过Western印迹，并确定该蛋白主要是不溶性的，而不是泛素化。然而，在活细胞中的一个离散的子种群的蛋白质，很难检测到平均超过许多细胞时，可以是在一个特定的室，其中的物种是极其高的局部浓度可溶性和泛素化。在后一种情况下，可能有更重要的影响的细胞的生存能力都比较大批量的亚群体。此外，而的伴侣显示各种体外的多效性的行为和功能，它是越来越明显，在细胞中的离散函数空间和时间上的局限。 在新兴的范式对于理解生物化学，浓度每一个特定的纳米环境中的细胞变成了一个可变的财产，基础生物过程的分子事件，必须测定不仅在时间，但在SPACE。建模，4D成像这里介绍的方法，使敏感的在活细胞中蛋白质的错误折叠，虽然它可以用于任何数量的其他生物过程研究以及它们是如何调节的空间，时间和环境条件的变化。在本文中，我们使用的UBC9 TS折叠式传感器，从而有效地演示了处理的蛋白质聚集在细胞质中的发病阶段和选项。除了 ​​示出聚集的质量控制的细 ​​胞生物学，这种方法可以作为一个强大的工具用于解密特定的扰动或基因突变在proteostasis（例如Ubc9时ts可以被用于测量响应氧化蛋白质折叠的应力的影响，一种有毒的聚集体，或突变的质量控制途径）的表达。 4D成像也是必不可少的，为准确地确定在两个不同的蛋白的蛋白定位或共存，检测现象，这可能是短暂的，但重要的。举例来说，尤其是在小球状生物如酵母，它可能会出现的情况下的结构或总有juxtanuclear的的本地化，而4D成像显示，这简直是神器的角度检查。 在示例实验中，我们在这里，我们表明，使用模型错误折叠的蛋白质，Ubc9时TS，跟随在细胞质中的时间和空间聚集的质量控制。在允许温度，Ubc9时日ts在细胞核和细胞质中被折叠和扩散。当热引起的错误折叠，初步形成快速扩散的总应力小疫源地处理蛋白酶体降解。当蛋白酶体部分受到抑制，这些应力的焦点，被转换到JUNQ和IPOD夹杂物的过程约2小时。如果泛素介导的降解是不可用作为质量控制选项，UBC9 TS是立即重新路由到IPOD的保护性聚集列入。这些工具提供了令人难以置信的机会，发现新的遗传因素，参与汇聚质量控制，并探讨其空间和时间的调节在细胞内。

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MG132</td>
			<td>Mercury</td>
			<td>mbs474790</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>con A</td>
			<td>Sigma</td>
			<td>C2010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass bottom plates</td>
			<td>ibidi</td>
			<td>ibd81158</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>4D Fluorescence Imaging of Protein Aggregation 
			Confocal 3D movies were acquired using a Nikon A1R-si microscope equipped with a PInano Piezo stage (MCL), using a 60X water objective NA 1.27, 0.3 micron slices, 0.5% laser power (from 65 mW 488 laser and 50 mW 561 laser). z-stacks were acquired every 5 min for 90 min. Each z-series was acquired with 0.5 micron step size and 30 total steps. Image processing was performed using NIS-Elements software.</td>
		</tr>
	</tbody>
</table>

4d imaging of protein aggregation in live cells

的任何活细胞的主要任务之一是保持适当的折叠新合成的蛋白质在面对不断变化的环境条件和细胞内环境，紧凑，粘，及有害蛋白质的稳定性。以动态的平衡蛋白的生产，折叠和降解的能力，需要高度专业化的质量控制机制，其绝对的必要性时，观察到的最佳故障。如ALS，阿尔茨海默氏症，帕金森氏症，囊性纤维化和某些形式的疾病有直接的联系蛋白质折叠的质量控制组件2，和治疗因此，未来的发展需要一个基本的了解的基本过程。我们的实验面临的挑战是了解细胞的损伤信号集成和安装的反应，根据不同的情况。 最主要的原因蛋白质的错误折叠的生存THRE在该单元格的不正确折叠的蛋白质聚集的倾向，从而造成全球性的扰动的拥挤和细腻的细胞内折叠环境1。折叠健康，或的“proteostasis，”细胞蛋白质组的维护，甚至老化，压力和氧化损伤的胁迫下，通过不同的机械单位的协调行动，在一个精心制作的质量控制系统3,4。一个专门的机器，分子伴侣可以绑定非原生的多肽和促进其折叠到原有的状态1，他们的目标降解的泛素-蛋白酶体系统5，或引导他们保护的聚集夹杂物6-9。 在真核生物中，胞浆聚集质量控制负载两个舱室8-10：的juxtanuclear质量控制的室（JUNQ）和不溶性蛋白质存款（IPOD）（ 图1 -模型）之间分配。由蛋白质折叠的质量控制机械的泛素化的蛋白质，它们被传递到JUNQ，在那里被处理，被蛋白酶体降解。错误折叠的蛋白质，不泛素化的转移到iPod，他们正在积极地聚集在一个保护仓。 直到此时，活细胞的荧光显微镜观察的方法范式已经在很大程度上被标记蛋白，其在细胞中的位置和跟踪在特定的时间点，并通常在两个维度。随着新技术已经开始给予实验者获得了前所未有的亚微米尺度在活细胞的细胞质中，动态架构映入眼帘的一个具有挑战性的新前沿的实验表征。我们提出了一种方法，用于快速监测多个荧光标记的蛋白质的三维空间分布的，随着时间的推移在酵母细胞质。三维间隔拍摄（4D成像）不仅是一个技术上的挑战，大鼠她，它也有利于的概念框架，用于分析细胞的结构发生了巨大变化。 我们利用蛋白质在活酵母细胞内的折叠式传感器的可视化截然不同的命运，错误折叠的蛋白质在细胞聚集的质量控制，使用快速4D荧光成像。 Ubc9两种蛋白10-12（Ubc9时TS）的温度敏感突变体既作为一个传感器蜂窝proteostasis，和用于跟踪聚合质量控制的生理模型是非常有效的。与大多数TS蛋白，Ubc9时TS是完全折叠，在许可下，由于活跃的细胞分子伴侣的功能。 30°C以上，或当细胞面临的错误折叠压力，UBC9 TS错误折叠，并遵循原生球状蛋白质的错误折叠的突变，热变性，或氧化损伤的命运。通过熔化它GFP或其他的荧光基团，它可以在三维跟踪，因为它形成胁迫佛慈，或者是二rected到JUNQ或iPod。

<html><img alt="图1" fo:content-width="4.5in" fo:src="/files/ftp_upload/50083/50083fig1highres.jpg" src="/files/ftp_upload/50083/50083fig1.jpg" /> 图1。型号：亚细胞条块分割的错误折叠的蛋白质质量控制的机械指示到不同的车厢不同的功能：可溶性蛋白，有针对性地进行降解错误折叠的蛋白质，经过多聚泛素被送到朱XTA-N uclear 品质无损控制盒（JUNQ ）。不能被泛素化的不溶性蛋白质，用于保护封存发送的 I nsoluble 镨oteinðeposit（IPOD），相邻的液泡，这里它们经受活性聚合。 es.jpg“SRC =”/ files/ftp_upload/50083/50083fig2.jpg“/&gt; 图2。蛋白质的错误折叠与GFP-Ubc9时TS建模 。 在正常情况下，GFP-Ubc9时TS（绿色）本身折叠，和本地化弥漫在细胞核和细胞质中。细胞核标记的NLS-TFP（红色）。 Ubc9时TS的表达被关闭，另外的2％的葡萄糖，然后成像在所有的实验。 <a href="https://www.jove.com/files/ftp_upload/50083/50083fig2large.jpg" target="_blank">点击此处查看大图</a> 。 <ol><li>当温度变化到37°C，的GFP-Ubc9时TS（绿色）错误折叠和形成胞内应力疫源地。细胞核标记的NLS-TFP（红色）。 </li><li>在23℃下，从热休克恢复后的热变性的GFP-Ubc9时ts是退化，如荧光水平下降所指示。 </li><li>当温度37°C和蛋白酶抑制转移到80毫米MG132，GFP-U<html> BC9 TS错误折叠和处理到JUNQ和IPOD夹杂物。细胞核标记的NLS-TFP（红色）。 </li><li>当温度换档至37°C和泛素化的抑制，GFP-Ubc9时日ts错误折叠的和处理到IPOD列入。细胞核标记的NLS-TFP（红色）。泛素蛋白酶4（Ubp4）的过度表达，阻止UBC9 TS泛素化。 </li></ol><img alt="图3" fo:content-width="6in" fo:src="/files/ftp_upload/50083/50083fig3highres.jpg" src="/files/ftp_upload/50083/50083fig3.jpg" /> 图3。时间流逝的JUNQ和IPOD的形成。当温度漂移至37°C和80毫米MG132，蛋白酶体抑制GFP-UBC9 TS应力疫源地处理到JUNQ和IPOD夹杂物。细胞核标记的NLS-TFP（红色）。在4分钟的时间间隔三维图像被收购。 （看电影）。com/files/ftp_upload/50083/50083fig3large.jpg“目标=”_blank“&gt;点击这里查看大图。

Watch this Scientific Journal Video about 4D Imaging of Protein Aggregation in Live Cells at JoVE.com

4D Imaging of Protein Aggregation in Live Cells

细胞存活率依赖于蛋白质的错误折叠的及时和有效地管理。在这里，我们描述了一种方法，可视化的错误折叠的蛋白质复性，退化，或夹杂物封存在不同的潜在的命运。我们演示了如何使用的折叠式传感器，UBC9<sup&gt; TS</sup，为的监控proteostasis和使用4D显微镜的活细胞聚集的质量控制。

在活细胞中蛋白质聚集的4D成像

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

4d-imaging-of-protein-aggregation-in-live-cells

Research

JoVE Journal

Biology

17.2K 次观看.  Hebrew University of Jerusalem. 细胞存活率依赖于蛋白质的错误折叠的及时和有效地管理。在这里，我们描述了一种方法，可视化的错误折叠的蛋白质复性，退化，或夹杂物封存在不同的潜在的命运。我们演示了如何使用的折叠式传感器，UBC9 TS

视频: 4D Imaging of Protein Aggregation in Live Cells

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

科研

生物学

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

成像蛋白质 - 蛋白质相互作用在体内</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

研究蛋白质 - 蛋白质相互作用活细胞使用生物发光共振能量转移

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

体外聚集测定使用tau蛋白的蛋白质

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

共聚焦显微镜通过图像相关谱揭示细胞表面受体聚集

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

酵母的4D显微镜

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with F&#246;rster resonance energy transfer (FRET) provide accurate information about PPIs in live cells. FLIM-FRET relies on measuring the fluorescence lifetime decay of a FRET donor at each pixel of the FLIM image, providing quantitative and accurate information about PPIs and their spatial cellular organizations. We propose here a detailed protocol for FLIM-FRET measurements that we applied to monitor PPIs in live Pseudomonas aeruginosa in the particular case of two interacting proteins expressed with highly different copy numbers to demonstrate the quality and robustness of the technique at revealing critical features of PPIs. This protocol describes in detail all the necessary steps for PPI characterization - starting from bacterial mutant constructions up to the final analysis using recently developed tools providing advanced visualization possibilities for a straightforward interpretation of complex FLIM-FRET data.

We describe here a protocol to characterize protein-protein interactions between two highly-differently expressed proteins in live Pseudomonas aeruginosa using FLIM-FRET measurements. The protocol includes bacteria strain constructions, bacteria immobilization, imaging and post-imaging data analysis routines.

活细菌中蛋白质-蛋白质相互作用的FLIM-FRET测量。

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with F&#246;rster ...