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

<ol>
	<li>Bonner, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+the+Formation+of+Cell+Aggregates+by+Chemotaxis+in+the+Development+of+the+Slime+Mold+Dictyostelium+Discoideum.">Evidence for the Formation of Cell Aggregates by Chemotaxis in the Development of the Slime Mold Dictyostelium Discoideum.</a> Journal of Experimental Zoology. 106 (1), 1-26 (1947).</li><li>Loomis, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+signaling+during+development+of+Dictyostelium.">Cell signaling during development of Dictyostelium.</a> Developmental Biology. 391 (1), 1-16 (2014).</li><li>Nichols, J. M. E., Veltman, D., Kay, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+of+a+model+organism:+progress+with+Dictyostelium.">Chemotaxis of a model organism: progress with Dictyostelium.</a> Current Opinion in Cell Biology. 36, 7-12 (2015).</li><li>Williams, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dictyostelium+finds+new+roles+to+model.">Dictyostelium finds new roles to model.</a> Genetics. 185 (3), 717-726 (2010).</li><li>M&#252;ller-Taubenberger, A., Kortholt, A., Eichinger, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+system+-+substantial+share:+The+use+of+Dictyostelium+in+cell+biology+and+molecular+medicine.">Simple system - substantial share: The use of Dictyostelium in cell biology and molecular medicine.</a> European Journal of Cell Biology. 92 (2), 45-53 (2013).</li><li>Malinovska, L., Palm, S., Gibson, K., Verbavatz, J. M., Alberti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dictyostelium+discoideum+has+a+highly+Q/N-rich+proteome+and+shows+an+unusual+resilience+to+protein+aggregation.">Dictyostelium discoideum has a highly Q/N-rich proteome and shows an unusual resilience to protein aggregation.</a> Proc Natl Acad Sci U S A. 112 (20), E2620-E2629 (2015).</li><li>Malinovska, L., Alberti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+misfolding+in+Dictyostelium:+Using+a+freak+of+nature+to+gain+insight+into+a+universal+problem.">Protein misfolding in Dictyostelium: Using a freak of nature to gain insight into a universal problem.</a> Prion. 9 (5), 339-346 (2015).</li><li>Schneider, K., Bertolotti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surviving+protein+quality+control+catastrophes+-+from+cells+to+organisms.">Surviving protein quality control catastrophes - from cells to organisms.</a> Journal of Cell Science. 128 (21), 3861-3869 (2015).</li><li>Dobson, C. 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+and+misfolding.">Protein folding and misfolding.</a> Nature. 426 (6968), 884-890 (2003).</li><li>Fukui, Y., Yumura, S., Yumura, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agar-overlay+immunofluorescence:+high-resolution+studies+of+cytoskeletal+components+and+their+changes+during+chemotaxis.">Agar-overlay immunofluorescence: high-resolution studies of cytoskeletal components and their changes during chemotaxis.</a> Methods in cell biology. 28, 347-356 (1987).</li><li>Graf, R., Rietdorf, J., Zimmermann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+spinning+disk+microscopy.">Live cell spinning disk microscopy.</a> Microscopy Techniques. 95, 57-75 (2005).</li><li>Samereier, M., Meyer, I., Koonce, M. P., Graef, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Cell-Imaging+Techniques+for+Analyses+of+Microtubules+in+Dictyostelium.">Live Cell-Imaging Techniques for Analyses of Microtubules in Dictyostelium.</a> Methods in cell biology. 97, 341-357 (2010).</li><li>Herman, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stationary+phase+in+yeast.">Stationary phase in yeast.</a> Current opinion in microbiology. 5 (6), 602-607 (2002).</li><li>Loomis, W. F., Wheeler, S., Wheeler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+shock+response+of+Dictyostelium.">Heat shock response of Dictyostelium.</a> Developmental Biology. 79 (2), 399-408 (1980).</li></ol>

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="https://ecsource-jove-com.bdigitaluss.remotexs.co/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="https://ecsource-jove-com.bdigitaluss.remotexs.co/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="https://ecsource-jove-com.bdigitaluss.remotexs.co/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 ...

studying-protein-quality-control-system-d-discoideum-using

Universidad San Sebastian

Research

JoVE Journal

Biology

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

6.2K 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

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

长期的实时成像<em&gt;果蝇</em&gt;眼睛光盘

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

科研

发育生物学

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development.
Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis.
Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers&#8217; circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (&#62;50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications.

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins.

使用培养的果蝇卵室延时电影对局部细胞和组织尺度的actomyosin动力学的分析

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

半高通量成像方法和数据可视化工具包，用于分析C. elegans胚胎发育

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy. Moreover, the fast reproductive cycle and short lifespan make the nematode a suitable model to screen for genes and molecules that modulate this process.
However, the quantification of aggregate load in living animals is poorly standardized, typically performed by manual inclusion counting under a fluorescence dissection microscope at a single time point. This approach can result in high variability between observers and limits the understanding of the aggregation process. In contrast, amyloid-like protein aggregation in vitro is routinely monitored by thioflavin T fluorescence in a highly quantitative and time-resolved fashion.
Here, an analogous method is presented for the unbiased analysis of aggregation kinetics in living C. elegans, using a high-throughput confocal microscope combined with custom-made image analysis and data fitting. The applicability of this method is demonstrated by monitoring inclusion formation of a fluorescently labeled polyglutamine (polyQ) protein in the body wall muscle cells. The image analysis workflow allows the determination of the numbers of inclusions at different timepoints, which are fitted to a mathematical model based on independent nucleation events in individual muscle cells. The method described here may prove useful to assess the effects of proteostasis factors and potential therapeutics for protein aggregation diseases in a living animal in a robust and quantitative manner.

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Here, a method is presented for the analysis of protein aggregation kinetics in the nematode Caenorhabditis elegans. Animals from an age-synchronized population are imaged at different time points, followed by semiautomated inclusion counting in CellProfiler and fitting to a mathematical model in AmyloFit.

使用秀丽隐杆线虫中的自动包涵体计数监测体内蛋白质聚集动力学

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis ...

生物化学

Real-Time Imaging of Germline Stem Cells in Drosophila Ex Vivo Germaria Culture

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to explore the dynamics of a myriad of developmental processes, such as cell division, stemness, differentiation, migration, apoptosis, autophagy, cellular adhesion, etc., over time. Recently, we have implemented an extended ex vivo culture and live imaging of the female Drosophila GSC niche. Using a Drosophila line harboring a GFP::Par-1 transgene as an example, this method allows the visualization of the GSCs&#39; asymmetric division within their niche and the description of the changes in the spectrosome morphology along the cell cycle. Here, we present a detailed protocol for the ex vivo culture of Drosophila germaria, enabling prolonged visualization of the female GSC niche. Importantly, this protocol is broadly applicable to live imaging GSCs with multiple fluorescently tagged proteins of interest that are available in stock centers and/or in the Drosophila research community.

Here, we provide a detailed protocol to perform live imaging of the asymmetric division of germline stem cells (GSCs) in the Drosophila ovarian niche. We use a transgenic line that ubiquitously expresses a green fluorescent protein (GFP) fusion of the spectrosome protein Par-1.

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to ...

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. The molecular clock within each pacemaker neuron generates circadian rhythms in gene expression, which underlie the rhythmic neuronal functions essential to the operation of the circuit. Investigation of the properties of the individual molecular oscillators in different subclasses of pacemaker neurons and their interaction with neuronal signaling yields a better understanding of the circadian pacemaker circuit. Here, we present a time-lapse fluorescent microscopy approach developed to monitor the molecular clockwork in clock neurons of cultured Drosophila larval brain. This method allows the multi-day recording of the rhythms of genetically encoded fluorescent circadian reporters at single-cell resolution. This setup can be combined with pharmacological manipulations to closely analyze real-time response of the molecular clock to various compounds. Beyond circadian rhythms, this multipurpose method in combination with powerful Drosophila genetic techniques offers the possibility to study diverse neuronal or molecular processes in live brain tissue.

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The goal of this protocol is to establish ex vivo Drosophila larval brain culture optimized to monitor circadian molecular rhythms with long-term fluorescence time-lapse imaging. The application of this method to pharmacological assays is also discussed.

单细胞分辨荧光活成像在幼虫脑培养中的果蝇昼夜节律时钟

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...

神经科学

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

在Live哺乳动物细胞中质粒复制多代荧光显微镜监测

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

生物学

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

单分子成像的基因调控<em在体内</em使用转位激活裂解（CoTrAC）

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many biological and biomedical applications. However, long-term time-lapse SRS imaging of live cells has not been widely adopted. SRS microscopy often uses a high numerical aperture (NA) water-immersion objective and a high NA oil-immersion condenser to achieve high-resolution imaging. In this case, the gap between the objective and the condenser is only a few millimeters. Therefore, most commercial stage-top environmental chambers cannot be used for SRS imaging because of their large thickness with a rigid glass cover. This paper describes the design and fabrication of a flexible chamber that can be used for time-lapse live-cell imaging with transmitted SRS signal detection on an upright microscope frame. The flexibility of the chamber is achieved by using a soft material - a thin natural rubber film. The new enclosure and chamber design can be easily added to an existing SRS imaging setup. The testing and preliminary results demonstrate that the flexible chamber system enables stable, long-term, time-lapse SRS imaging of live cells, which can be used for various bioimaging applications in the future.

We report a stage-top, flexible environmental chamber for time-lapse imaging of live cells using upright stimulated Raman scattering microscopy with transmitted signal detection. Lipid droplets were imaged in SKOV3 cells treated with oleic acid for up to 24 h with a 3 min time interval.

用于使用受激拉曼散射显微镜进行延时活细胞成像的柔性腔室

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many ...

生物工程

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

研究蛋白质的质量控制体系

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