The authors have no Acknowledgements.

1.细胞的制备<ol><li> 生长的细胞 <ol><li> D.成长菌细胞在组织培养板在光照下在23℃的AX介质。避免将电池在高密度的75％以上汇合。 注意:为获得最佳的反应热应力，细胞需要在指数生长期和不应该达到稳定期。 </li><li>成像前一天，分裂细胞进入低荧光介质（LFM）至1×10 4个细胞/ ml。 注:该步骤是必要的，以减少所造成的对AX介质背景荧光。培养时间可以减少到最低限度2小时。然而，在AX中占据囊泡仍然可以产生2小时后的背景信号。 </li></ol></li><li> 准备细胞显微镜 <ol><li>成像前，从在LFM组织板收获细胞并转移细胞的足够数量的成玻璃底菜。 注:通常情况下，1×10 5个细胞/ ml就足够了;然而，如果该实验装置需要长的摄像周期（超过8小时以上），细胞数需要仔细调整，如D.菌细胞将过渡到发育周期，并开始流和聚集体，如果细胞数是高而营养素是低的。 </li><li>使细胞沉降20分钟。通过用新鲜培养基小心替换使用介质去除未结算的细胞。 </li></ol></li></ol> 2.影像学检查注:该协议可在宽视场的系统，以及在共聚焦显微镜的设置中使用。覆盖的目标温度控制的培育箱和显微镜载物台的使用是有益的，但对于温度控制不是必须的。如果进行热应力孵育箱的温度设置为在实验中， 例如 ，所使用的最高温度，以40℃。否则，将温度设定为256℃。温度的变化，应提前作出允许系统适应，作为成像期间的温度变化可影响焦点。 注意:在成像时，温度是使用冷却室进行控制。用于成像非应激细胞默认的温度为23℃。对热应力的条件下，将温度相应地增加至期望的水平。在冷却室中的压电元件可以非常快速响应温度变化。然而，图像的焦点会略微改变（它可以偏离到最多20微米的取决于温度移）。因而手动重聚焦是图像采集的第一分钟内需要，除非所使用的显微镜​​配备有硬件自动对焦。 <ol><li> 图像无压力条件下采集 <ol><li>在23℃下非胁迫条件下的图像的细胞，获得最小的视图6字段（视场）的。 注:非应激条件可以是visualiz编辑或者通过记录代表剧照很短的时间推移（5-30分钟）或收购。对于每一个条件或细胞系获得最低的视图6场（视场）的。 注:如果单个细胞是感兴趣，定位各个单元中的视场的中部，并记录一个3×3瓦系列用含有感兴趣的细胞在中间视场D.。菌细胞是能动的，并可能迁移出FOV的。然而，3×3瓦系列增加了由感兴趣的细胞潜在的探索区域，并为〜6小时的时间推移周期保持追踪。 </li><li>获得的最大为7μm的z堆叠在1-0.25微米步骤，捕获细胞的整个体积。 </li><li>获得一个基准亮场图像，以评估细胞的总数。 </li></ol></li><li> 图像采集热压力 <ol><li>根据制造商的说明调整冷却室的温度升高到30℃。&lt;/ LI&gt; <li>温度显示已达到所需的值后，手动重新聚焦在明场通道和启动时间推移前检查所有记录的视场。 </li><li>开始的时间推移对于具有5-10分钟的时间点之间的时间间隔的热应力的4小时。检查收购前两个时间点的过程中正确的对焦。 </li></ol></li><li> 恢复过程中图像采集 <ol><li>热应激后，降低温度回到23°C，等待温度显示调节和手动重新调整。 </li><li>记录时间推移电影在10-20分钟的时间间隔8-10个小时的恢复阶段。检查收购第一时间点的过程中正确的对焦。 </li></ol></li></ol> 3.图像分析注意:为了量化细胞窝藏胞质灶的数目，评估细胞的总数和细胞与每次在胞质灶的数目帧。由于来自亮场图像分割细胞的难度，量化需要手动完成。在下文中，数据分析是针对游离的图像处理包（（http://fiji.sc/Fiji），它需要的插件"细胞计数"（http://fiji.sc/Cell_Counter）中所述。 <ol><li> 评估细胞总数 <ol><li>通过点击"文件"和"打开"加载亮场图像堆栈（XYT）。 </li><li>打开"细胞计数"插件（"插件" - "细胞计数"）。通过点击"初始化"加载图像堆栈。 </li><li>勾选相应的方框选择合适的类型计数器。 注:作为计数器细胞总数和2型作为计数器与胞浆灶细胞利用类型1。算上所有的细胞。 </li><li>通过点击"保存标记"保存标记。 </li></ol></li><li> 评估细胞的胞浆与病灶数 <ol> &lt;李&gt;加载荧光图像hyperstack（XZYT）（"文件" - "打开"），并使用"Z-投影机"（图/堆栈）的最大强度投影。 </li><li>打开"细胞计数"插件。通过点击"初始化"加载生成的图像堆栈（XYT）。 </li><li>通过点击"加载标记"加载标记。 注意:参考图像和荧光栈的文件名必须相同。如果有必要，初始化图像堆栈前据此命名。 </li><li>选择适当的标记类型（参见3.1.3节）和计数与胞浆灶细胞。现有的标记物可以用作单个单元的位置的引导。 </li><li>通过点击"结果"检索每片计数的数量和其他地方保存的计数进一步计算。 </li></ol></li></ol>

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The authors have nothing to disclose.

这里所描述的协议可以用来研究响应于热诱导应力感兴趣的特定蛋白质的行为。温度上升至30℃已报道触发热应激反应和D的在这些条件下的生存能力菌被显着地降低了。 修改 该协议可以被修改，以比较同胁迫条件下不同的蛋白质的行为。为此，表达相同荧光标记不同的蛋白质细胞转移到多井菜肴，如四腔菜（第1.3.1节）。该协议也可以在细胞共表达不同的标记物，如GFP或RFP蛋白施加。这可以是例如用于监测蛋白质的质量控制（PQC）系统的不同组件的行为。表达GFP标记聚集的标记和不同的RFP标记PCQ组件细胞可以使用多井观察抛出成像。这确保相同的应力条件下（温度上升/下降，温度上升/下降的持续时间速度），并允许进行比较研究。 此外，该协议可以用于研究PQC系统对热应激反应的影响。各组分的活性可以通过改变使用的遗传工具，例如敲除或过度表达水平或通过使用商购的特异性抑制剂6进行调制。蛋白酶可以通过添加MG132（100μM）或乳胞素（10μM）的生长培养基中被抑制。伴侣Hsp90的可使用格尔德霉素（6微米）或根赤壳菌素（10μM）被抑制。伴侣热休克蛋白70可与VER-155088抑制，虽然我们无法证实抑制的功效在我们的实验设置为止。抑制剂应成像之前添加一天，将细胞温育14小时。 Criti<html>该议定书内校准步骤 为热诱导扰动的评估关键步骤是先于成像实验的细胞的状态。在酵母中的研究表明，细胞在固定相13获得的各种环境胁迫的抵抗。我们还观察到最小响应施加热应激，如果D.菌细胞已经成像之前达到稳定期。因此，保持恒定的细胞数&lt;5×10 5个细胞/ ml是至关重要的。 此外，高细胞数量可以从D的生长周期诱导转型菌的发育周期，从而引发饥饿的途径，这可能会导致不同的响应热应激。如果高细胞数在热应力，流后的恢复阶段达到和凝集细胞可以与数据分析干扰随着细胞移出焦点（ 见图4）。 内容"&gt;的技术限制 重点的损失是协议的瓶颈。在温度改变期间，焦平面可以显着移位，这需要在一个时间周期手动再聚焦改变温度之后。在聚焦跳有时可能太极端通过软件自动聚焦可以克服，特别是如果时间点之间的时间是高的。但是，如果使用显微镜配备有硬件自动对焦，这个瓶颈可以规避。 相对于现有方法的技术意义 在与现有的设置，如使用的空调房，温度控制的培育箱覆盖的目标和显微镜载物台或温度控制的阶段和客观衣领比较，使用冷却室提供了环境的一个更精确的控制温度。在冷却室中的珀耳帖元件可以保持恒定和均匀的统023在整个实验装置。在古典设置中，局部温度差异可能导致不同的观察结果。此外，它可以迅速和快速响应的温度引起的变化，而经典的设置适应非常缓慢地引起的温度变化，特别是降低温度。在冷却室中的珀耳帖元件也可以覆盖一个更宽的温度范围（15-40℃）比传统的设置，与到达温度如15℃或40℃下是很困难的。 粘菌是光毒性特别敏感。以前的研究使用抗坏血酸作为清除剂，以减少光毒性。然而，长期的成像时间需要额外补充。此外，使用抗坏血酸限于其中感兴趣的机构不受抗氧化剂补充研究。我们建议，温度控制的成像可被用来作为一种替代APPRoach以最小化光毒性和可与加成抗坏血酸被组合以进一步减少毒性。 光毒性效果可以通过使用冷却室中提供的23℃的恒定和均匀的温度最小化。使用温度控制的成像细胞显示较少的光毒性，如细胞变圆的迹象，在较长的时间段。这也使得成像具有更高的强度，更小的时间间隔或视（视场）更多的领域。 一般应用 在更高温度下的热应力已经显示出触发不同热应激反应14。因此，温度上升到34℃或37℃可能诱导不同的响应。除了所应用的温度应力，一个特定的压力的情况下的持续时间可以被修改以研究立即响应或长期适应热应力。 在一般情况下，所描述的协议可以是扩大到一系列广泛的应用。由于精确的温度控制大大降低光毒性作用，该协议可以在需要高的曝光时间和/或较高的激发光强度， 例如 ，对于具有低表达水平可视化蛋白设置来使用，或者在短的时间间隔组合成像， 例如 ，延时成像期间之间。它也可以是用于成像跨越整个细胞的对象是有利的， 例如 ，微管，因为这些设置需要大量的光学Z截面的。

粘菌是独居生活的土壤变形虫，关于细菌和其他微生物，这是由吞噬占用饲料。它具有独特和卓越的生命周期，因为它的发现1已成为研究的主要领域。在多细胞发展2和趋3的分子基础早期兴趣很快被研究集中于细胞运动性，细胞极性，先天免疫补充。此外，D。菌是作为生物医学研究4,5模型系统。 最近，我们成立D.菌作为一个新的系统来研究蛋白质的质量控制（PQC）系统6,7。其蛋白质在聚集倾向朊状蛋白质，这对以蛋白质量控制8个挑战富集。调查是否D.菌开发了特殊的分子机制，以控制其高度股份公司gregation容易发生蛋白质组，我们研究了两个在正常生长条件和应力期间聚集倾向标记蛋白的行为。应力的条件下，如热应力，可用于增加蛋白质错误折叠9的速率。因此，我们寻求的系统，其中，我们能诱导的温度变化，同时按照标记蛋白的行为。为了这个目的，我们结合使用热阶段插入件（冷却室）珀耳帖控制的加热活细胞成像。该方法使我们能够保持恒定和均匀的温度，以及以诱导快速而精确的温度变化。 活细胞成像是用于研究各种D中的生物过程的菌 。然而，这种方法面临两个主要限制。首先，将细胞显示高运动性和倾向于迁移出的视场，从而跟踪单个细胞通常需要大面积成像。细胞迁移率可以通过琼脂覆盖10被降低，然而，在这些条件不适合长期成像由于存活率下降。其次，D.菌细胞显示到光毒性，这导致细胞变圆和有丝分裂停滞11特别高的灵敏度。以前的协议通过加入抗坏血酸作为自由基清除剂和减少曝光时间12解决了这个问题。如果感兴趣的蛋白以低水平表达，并显示弱荧光信号，后者可以是至关重要的。作者还建议要么通过在空调房间成像或通过使用温度控制孵化箱，覆盖的目标和显微镜载物台12，以提供21℃的恒定温度。 这里，我们通过使用冷却室设置在23℃下描述了具有改进的温度控制的方法。在成像过程中我们设置了显著增强了光毒性的阻力。它允许更高的曝光时间和更高的激励光强度的用法。这是特别重要的时间推移成像过程中，当时间间隔需要针对成像位置的数量和所使用的曝光时间仔细地平衡。增加成像位置的数量的可能性还允许更宽成像区域的覆盖范围，并促进个体细胞的跟踪在一段较长的时间。

<table><tbody><tr><td>AX Medium</td><td>ForMedium</td><td>AXM0102</td><td></td></tr><tr><td>LoFlo medium</td><td>ForMedium</td><td>LF1001</td><td></td></tr><tr><td>MG132</td><td>Sigma</td><td>C2211</td><td></td></tr><tr><td>Lactacystin</td><td>Sigma</td><td>L6785</td><td></td></tr><tr><td>Geldanamycin</td><td>Santa Cruz</td><td>sc-200617</td><td></td></tr><tr><td>Radicicol</td><td>Santa Cruz</td><td>sc-200620</td><td></td></tr><tr><td>MatTek disch 35 mm</td><td>MatTek corporation</td><td>P35G-1.5-14-C</td><td>glass bottom imaging dish</td></tr><tr><td>CellViell cell culture dish</td><td>Greiner</td><td>627870</td><td>4-compartment glass-bottom imaging dish</td></tr><tr><td>Thermal Stage Heater/Cooler Insert</td><td>Warner Istruments</td><td>TB-3/CCD</td><td></td></tr><tr><td>Bipolar temperature controller</td><td>Warner Istruments</td><td>CL-100</td><td></td></tr><tr><td>Liquid cooling System</td><td>Warner Instruments</td><td>LCS-1</td><td></td></tr></tbody></table>

studying the protein quality control system of d. discoideum using temperature-controlled live cell imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

所描述的成像协议可以用于分析在变形虫D.聚集倾向朊状蛋白质的行为热应激菌 。 图1示出了GFP标记多聚蛋白Q103的细胞正常生长条件（ 图1A）根据在30℃（ 图1B）和6小时应力恢复之后和分配2小时热胁迫之后生长在正常生长条件（ 图1C）。在从23°C的温度上升到30℃时，Q103-GFP标记从弥漫性分布聚结，点状结构。在应力释放和生长在常温下，这些结构溶解。 Q103-GFP的再分配和形成热应力诱发的胞质灶可以利用图像分析来定量。 图2A示出了使用斐济和插件"C个人视场的分析。埃尔计数器" 图2B显示了热应激反应的量化。这表明在数量胞质Q103-GFP疫源地增加与持续热应激。在热应激，蛋白质的质量控制（PQC）系统不堪重负，从而在一起的汇聚易发生蛋白如朊状蛋白质Q103不能以可溶状态在细胞内病灶进行维护和日益集合体。细胞也展现出特征性修圆， 如图1B中所示胞质灶的应力除去后的减少表明，聚集蛋白质溶解。 成像质量是对初始细胞密度敏感。 图3示出了应力恢复后的细胞的一个例子，比较合适的初始细胞密度（ 图3A）和高的初始细胞密度（ 图3B）。如果初始细胞密度过高，将细胞星如在图3b中描绘泰德以形成流。这其中规定对后续量化的细胞困难移出焦平面。 所描述的方法可以用作上述监测温度引起的变化。它也可用于在生理生长温度成像时降低光毒性。 图4显示细胞在冷却室（ 图4A），在23℃成像和细胞在空调房间没有温度控制成像的对比，设置至25℃（ 图4B）。在恒定暴露于激发光，将细胞围捕时温度控制的条件下不成像。这种改变细胞形状正指示应力。 <img alt="图1" src="/files/ftp_upload/54730/54730fig1.jpg" /> 图1: <s仲&gt;聚集倾向的朊病毒样蛋白热应激过程中改变自己的细胞分布。GFP标记的多聚谷氨酰胺丰富，亨廷顿外显子1（Q103）在AX2-214 D.组成型表达菌细胞（在倒灰度查询表中描绘的GFP信号）。 （a）根据在23℃，Q103-GFP正常生长条件被漫分布在细胞质中。 （B）后在30℃下2小时的热应力，Q103-GFP凝聚成胞质灶（红色箭头）。这些细胞表现出典型的形状变化和围捕。 （c）在应力释放和增长在23°C，胞浆灶溶解。 6小时后，没有胞质灶可以观察到。比例尺= 10微米。 <a href="http://ecsource.jove.com/files/ftp_upload/54730/54730fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图2: 热诱导细胞内病灶形成的定量 （A）使用免费的图像处理包斐济和插件"细胞计数"细胞分析。细胞的总金额（类型1，蓝计数器标记）在亮场图像来确定。标记被加载在GFP通道，并与细胞质灶的细胞（2型，红色计数器标记）被确定的数目。 （B）灶的量化后在30℃热应激3小时，并在23℃搅拌3小时紧张的恢复。（N =来看，误差棒= SD 4场）， <a href="http://ecsource.jove.com/files/ftp_upload/54730/54730fig2large.jpg" target="_blank">请点击这里查看这个大版本数字。</a> <img alt="图3" src="/files/ftp_upload/54730/54730fig3.jpg" /> <stron<html> g&gt;的图3: 在长期的时间推移成像单元密度的影响初始细胞密度&lt;10 5个细胞/毫升成像（A）细胞。在30℃热应激3小时，并在23℃，压力恢复6小时后，细胞仍然停留在生长周期和鸿沟。只有少数细胞退出焦平面。 （B）中的细胞以初始细胞密度&gt; 10 6个细胞/ ml的成像。成像（设置在A）的9小时后，将细胞已进入发育周期，并开始流和聚集。在聚集体的细胞不能明确区分。细胞的很大一部分离开焦平面，不能进行分析。比例尺= 10微米。 <a href="http://ecsource.jove.com/files/ftp_upload/54730/54730fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <html> .JPG"/&gt; 成像影响光毒作用的温度控制（A）的细胞10分钟，用温度控制在23℃:图4。图，Z堆叠（间距0.5微米，样品厚度8微米），时间推移的6个字段（总时间10分钟，经过1分钟），使用GFP通道（0.07毫秒的曝光时间，10％激光强度），RFP通道（0.1毫秒的曝光时间，10％激光强度）。细胞没有显示与光毒性应激相关的形态学改变的迹象。成像（B）的细胞为不带温度控制的10分钟（相同的条件A）。细胞表现出光毒性诱发的舍入的迹象，表明应力。比例尺= 10微米。将细胞成像基于与100×/ 1.4数值孔径（NA）物镜的显微镜的系统上。该图像用CCD照相机作为使用1×1像素合并1024x1024个像素文件中收集。4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging at JoVE.com

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

研究蛋白质的质量控制体系<em&gt;Ð。菌</em&gt;使用温控活细胞成像

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

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Biology

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

7.0K 次观看.  Max-Planck Institute for Molecular Cell Biology and Genetics. The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

视频: Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

基于细菌选择和生长的盘状细胞基因工程

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

科研

遗传学

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

应用活细胞成像和低温电子断层扫描解决军团菌肺瘤点/Icm分泌系统的时空特征

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

免疫与感染

4D Imaging of Protein Aggregation in Live Cells

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 conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

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 ...

生物学

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

耦合检测监控蛋白复性<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

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 ...

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

裂殖酵母性生命周期的显微镜

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

对急性机械刺激的网柄反应的评估

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

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 ...

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 ...

Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins

Membrane protein trafficking regulates the incorporation and removal of receptors and ion channels into the plasma membrane. This process is fundamentally important for cell function and cell integrity of neurons. Drosophila photoreceptor cells have become a model for studying membrane protein trafficking. Besides rhodopsin, which upon illumination becomes internalized from the photoreceptor membrane and is degraded, the transient receptor potential-like (TRPL) ion channel in Drosophila exhibits a light-dependent translocation between the rhabdomeral photoreceptor membrane (where it is located in the dark) and the photoreceptor cell body (to which it is transported upon illumination). This intracellular transport of TRPL can be studied in a simple and non-invasive way by expressing eGFP-tagged TRPL in photoreceptor cells. The eGFP fluorescence can then be observed either in the deep pseudopupil or by water immersion microscopy. These methods allow detection of fluorescence in the intact eye and are therefore useful for high-throughput assays and genetic screens for Drosophila mutants defective in TRPL translocation. Here, the preparation of flies, the microscopic techniques, as well as quantification methods used to study this light-triggered translocation of TRPL are explained in detail. These methods can be applied also for trafficking studies on other Drosophila photoreceptor proteins, for example, rhodopsin. In addition, by using eGFP-tagged rhabdomeral proteins, these methods can be used to assess the degeneration of photoreceptor cells.

Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins

Here, non-invasive methods are described for localization of photoreceptor membrane proteins and assessment of retinal degeneration in the Drosophila compound eye using eGFP fluorescence.

使用eGFP标记的蛋白质研究 果蝇 光感受器细胞中的膜蛋白运输

Membrane protein trafficking regulates the incorporation and removal of receptors and ion channels into the plasma membrane. This process is fundamentally ...

研究蛋白质的质量控制体系

摘要

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基于细菌选择和生长的盘状细胞基因工程

应用活细胞成像和低温电子断层扫描解决军团菌肺瘤点/Icm分泌系统的时空特征

在活细胞中蛋白质聚集的4D成像

耦合检测监控蛋白复性

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

裂殖酵母性生命周期的显微镜

对急性机械刺激的网柄反应的评估

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

酵母的4D显微镜

使用eGFP标记的蛋白质研究果蝇光感受器细胞中的膜蛋白运输

研究蛋白质的质量控制体系

摘要

探索更多视频

基于细菌选择和生长的盘状细胞基因工程

应用活细胞成像和低温电子断层扫描解决军团菌肺瘤点/Icm分泌系统的时空特征

在活细胞中蛋白质聚集的4D成像

耦合检测监控蛋白复性

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

裂殖酵母性生命周期的显微镜

对急性机械刺激的网柄反应的评估

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

酵母的4D显微镜

使用eGFP标记的蛋白质研究 果蝇 光感受器细胞中的膜蛋白运输

使用eGFP标记的蛋白质研究果蝇光感受器细胞中的膜蛋白运输