我们感谢拾破烂桑托斯和达伦Obbard在图果蝇插图。 2A，马塞洛雅各布 - 洛雷纳使用的JHMRI昆虫饲养的。这项工作是由生命科学研究基金会奖学金（HLT）的支持下，大学教育资助委员会，香港的AoE / B-07/99（MCF），和NIH授予NS096677，NS037402和NS083373（江铃控股）。何林堂是生命科学研究基金会的Shurl和凯库尔奇基金会研究员。

1. CaspaseTracker苍蝇的制备<ol><li>为了准备CaspaseTracker（DQVD）为飞行实验，执行此交叉:UBI-CaspaseTracker×g的-TRACE（UAS-RFP; UAS-FLP;育碧&gt;停止&gt; GFP-NLS），转增7-10处女女（或男性）通过泛素启动子35一起苍蝇携带的caspase生物传感器基底mCD8-DIAP1-Gal4的驱动具有相同数目的男性（或女性）的G-TRACE苍蝇，其具有第二染色体CYO平衡器避免G-的纯合组合的杀伤力TRACE（UAS-RFP，UAS-FLP和育碧&gt;停止&gt; GFP-NLS）34。地方苍蝇与鲜酵母膏作为蛋白质来源新鲜食物小瓶。 </li><li>在18℃孵育文件5至7天（最多2周），然后删除父从小瓶苍蝇，以避免与新的后代人满为患，并成立新的养殖瓶。 </li><li>在18℃的孵化持续直到后代过得E关闭。选择非CYO [非C网址Y翼]后代CaspaseTracker正确的基因型，这是转基因CaspaseTracker和G-微量元素35。 </li><li>同时，产生控制亲非转基因果蝇w118和caspase不敏感（DQVA）苍蝇并行核实具体CaspaseTracker RFP和绿色荧光。 </li><li>进行PCR基因分型，以确认用引物与CaspaseTracker文件:5&#39;-TCCCCCGGGCTGCAGGAATTC，3&#39;- TGGAATTGGGGTACGTCTAGA，产生3,897 bp的产物。对于基因分型的G-跟踪轨迹，使用以下引物GFP（474基点），5&#39;-CAC GAC TTC TTC AAG TCC GCC ATG CCC G，3&#39;-CTT GTA CAG CTC GTC CAT GCC GAG AGT GAT℃;对于RFP（258基点），5&#39;- GGC TGC TTC ATC TAC AAG GTG TTC AAG ATC GG，3&#39;-GAT GTC CAG CTT GGA GTC CAC GTA GTA GTA GC;和Flpase（655基点），5&#39;- CCACCTAAGGTGCTTGTTCGTCAGTTTGTGG，3&#39;-GCC TAC TAA CGC TTG TTG TCT TCT CTG TCA C.对于基因分型Gal4的在pUWR向量（554基点），5&#39;-GAA GCA CAC CTT CGC GCT ATC CAGTCA CGC，3&#39;-TGG AAT TGG GGT ACG TCT AGA。 </li></ol> 2.组织准备，染色与安装<ol><li>对于飞解剖，防止解剖组织粘附到溶解于水的塑料吸头和离心管，外套提示和管以1％牛血清白蛋白（BSA）。 </li><li>解剖上的飞行使用镊子硅胶垫。 <ol><li>为了避免在执行组织解剖时损坏钳提示在坚硬的表面，在硅表面上进行清扫。用于制备有机硅夹层板，根据制造商的说明（材料清单）熔融硅中的商业试剂盒。熔化硅氧烷后，放入3至4毫升到60毫米直径组织培养皿中。硅菜肴可重复使用。 </li><li>麻醉用CO 2的苍蝇，并将其与1毫升冷的PBS转移到硅胶板。引入CO 2用吹箭筒含有苍蝇的小瓶，然后转移麻醉苍蝇ŤOA垫具有CO 2的供应。 </li><li>使用一对镊子保持飞头，以及第二对钳子的拉胸部在相反的方向上从主体覆盖而不损坏磁头和前肠之间的连接断开飞头。 </li><li>使用2对镊子从胸部取出翅膀和腿。 </li><li>用一对镊子保持胸部，和另一对镊子的拉腹部，将其从胸部再次而不损坏前肠和肠之间的连接分离。 </li><li>如以前证明解剖内脏器官包括脑，肠，马氏管和卵巢39-41 </li><li>除去角质层的所有作品和脂肪体用钳子因为这些组织产生强烈的自发荧光。耐心和实践都需要准备一个完整的器官系统，如图所示。 </li></ol></li><li>解剖转移组织对1.5毫升离心管中，并修复组织的Wi个0.5毫升的4％多聚甲醛的PBS（磷酸盐缓冲盐水）在RT 20至30分钟，在黑暗中旋转，以避免在组织漂白RFP和GFP。避免长时间固定会危及随后的染色效果。 </li><li>通过温和移液除去多聚甲醛，用0.5ml PBST（PBS + 0.1％的Triton X-100）在室温下洗涤3次。 </li><li>在4℃下轻轻摇动透用0.5mL PBST组织过夜。较短的孵化可能导致不完整的通透性和妥协染色结果。 <ol><li>如果有必要，以减少背景染色，考虑在0.5毫升的PBST孵育组织用1％BSA，代替PBS中。 </li></ol></li><li>用0.5毫升PBST洗涤组织3次。 </li><li>染色细胞核和丝状肌动蛋白（F-肌动蛋白），应用0.5毫升的PBST以10微克/毫升的Hoechst的33342蓝色核染料和0.3μM的Alexa Fluor 633鬼笔环肽F-肌动蛋白染色和组织同时在室温下孵育瓦特1小时第i个温柔的转动在黑暗中。避免长时间染色，因为这会增加背景。 <ol><li>对于免疫染色，孵育固定和透化组织用0.1％的Triton X-100在0.5毫升PBS中过夜，在4℃下，染色用1:100稀释的抗ELAV抗体或以1:抗caspase-3的200稀释过夜4°C（300μL）。通过稀释1相应的次级抗体如下:100和在室温下孵育1至3小时。请参阅材料清单特异性抗体的信息。 </li></ol></li><li>洗组织3次，每次在0.5毫升的PBST 5分钟以慢速转动。 </li><li>用移液管，取出所有的PBST，然后添加200微升抗漂白安装剂（见材料）完全覆盖组织在RT 4℃1-3小时，或过夜。已充分吸收了安装剂最佳组织会沉到试管底部。 </li><li>预清洁与水或70％乙醇的玻璃片上，并与安装剂加入到GLAS转移组织小号幻灯片。 </li><li>小心地在组织盖玻片。适用凡士林在载玻片和盖玻片，以避免过压缩破坏组织。用纸巾除去多余的固定剂。 </li><li>密封通过施加指甲油所有沿盖玻片的边缘，以避免组织安装剂泄漏盖玻片。 </li></ol> 3.共聚焦显微镜<ol><li>使用倒置共焦落射荧光显微镜。然而，上右显微镜也可使用。使用平铺图像的组织，保持稳定的室温，以避免聚焦和xy平面的偏移漂移由于部件的热膨胀和收缩。环境控制系统和焦点漂移补偿系统有助于避免这个问题由于显微镜系统的热平衡的损失。 </li><li>放置在显微镜阶段的幻灯片。使用低分辨率采取一些测试图像（125×125像素）的ð确定以覆盖感兴趣区域（ROI）的整个区域所需的瓦片的适当数量。 </li><li>设置显微镜扫描系统以捕获与扫描分辨率，如500×500像素的图像。选择目标诸如20X NA 0.8或63X NA 1.4计划复消色差透镜目标为通过盖玻片分析组织中，以便在各方面每个图像（瓷砖）的重叠了20％。 </li><li>从最长的建立图像序列至最短激发波长，作为长波长光会导致低光漂白。从最长至最短激发波长的顺序是:（i）用于鬼笔环肽F-肌动蛋白和抗体来活化胱天633纳米，（ii）用于RFP 561纳米，（ⅲ）的GFP 488纳米，和（iv）405nm处为赫斯特核染色。 </li><li>在成像过程中，成像过程通过降低激光强度，以获得组织的高品质的图像，以避免光致漂白期间最小化细胞的荧光激光激发的曝光。 </li><li>我后法师收集，合并使用显微镜的软件图像。 </li></ol>

The authors have nothing to disclose.

在这里，我们说明CaspaseTracker的建设和内部运作促进健康组织的广泛基础caspase活性的检测。 用于体内检测非凋亡胱天蛋白酶活性的关键步骤是:1）产生苍蝇与生物传感器的转基因，2）验证与适当的控制的具体的caspase-记者功能，3）练解剖技术观察成年果蝇的所有内部器官系统，和4）从自体荧光的工件识别生物传感器的活性。 在作为具体的caspase-传感器天然蛋白?...

胱天蛋白酶是由天门冬氨酸键后残留许多裂解细胞内蛋白介导的细胞凋亡的半胱氨酸蛋白酶。例如，引发剂胱天蛋白酶激活效应器半胱天冬，derepress的DNA核酸酶，裂开细胞骨架元件和改变细胞膜的脂质组合物迅速拆除细胞和由邻近该处置细胞尸体细胞刺激它们的识别和吞噬1-4据估计数十亿的细胞每天死于在人体内，与细胞凋亡是化疗诱发的肿瘤细胞死亡的一个重要机制。5一组不同胱天蛋白酶可以通过不同的非凋亡进程会导致细胞死亡，刺激先天免疫6因此，对半胱天冬大多数研究都集中在其促死亡功能。 有趣的是，在该领域早期证据表明，负责促进细胞死亡同一半胱氨酸蛋白酶也具有非死亡˚Functions。先驱研究表明，胱天蛋白酶涉及健康细胞不同的细胞功能，包括细胞增殖和迁移的胚胎发育过程中的调节。7-9胱天蛋白酶所需的精子细胞个体在果蝇 10,11，用于阻断的替代necroptotic细胞死亡途径小鼠12,13，和用于微RNA加工中C.线虫 14,15在或许是最长寿的细胞，神经元，半胱天冬等凋亡机制在神经元活动的调节通过修剪突触末梢牵连，认为过程是必须加强其他突触学习和记忆。16 18这是可能的半胱氨酸蛋白酶促进由类型没有全细胞死亡微小神经突起的小细胞凋亡的突触修剪。19然而，胱天蛋白酶可具有无关的替代功能凋亡样的事件。20,21双重作用在生活中和死亡不是唯一的胱天蛋白酶; BCL-2家族的蛋白和细胞色素C在健康细胞的细胞能量学的作用，但也芯凋亡途径是受许多类型的细胞应激的激活的一部分。22-25尽管没有证明，逻辑上似乎进化联天 - 工作在同一分子内死亡的作业，以确保不适宜或不良细胞及时消除。 目前，非凋亡胱天蛋白酶活性的分子机制尚不了解，和非凋亡胱天蛋白酶活性的胚胎发育过程中和在成体组织中的程度也没有已知的。一个主要挑战是从半胱氨酸蛋白酶的死亡区分工作一天工作的难度。在对比凋亡和pyroptosis，当caspase活性是由一种蛋白水解级联放大，胱天蛋白酶的天作业，预计通过许多可用TECHN发生在低得多的水平的酶活性，可能低于检测ologies。 在此之前这里介绍的工作，其他人开发出了多种生物传感器的caspase为不同的目的。该生物传感器SCAT（ 例如 ，ECFP-DEVD金星）使用快速检测FRET实时caspase活性在培养细胞和动物组织。26,27经蛋白酶裂解，Apoliner（mCD8-RFP-DQVD-核针对性GFP部分nucGFP）发生时，其细胞膜系绳是由半胱天冬裂解分钟内的亚细胞重新定位。28同样，ApoAlert-pCaspase3传感器（NES-DEVD-YFP-NLS）从胞浆到在蛋白酶裂解细胞核relocalizes。29,30更多最近，在iCasper生色被巧妙地设计的，当由胱天蛋白酶主要在发育性细胞死亡关联裂解，允许生物传感器的活性检测在实时在果蝇胚胎神经元，但发荧光。嗅觉神经元的31胱天蛋白酶依赖的死亡过程中老化demonst通过每次收看生物传感器（ 例如，mCD8-PARP-金星）的胱天蛋白酶切割形式的免疫检测评分，32,33重要的是，在不存在的细胞死亡中的棘检测由敏感免疫染色的caspase-3的活化形式培养的神经元，并在使用核CellEvent报告染料的胱天蛋白酶依赖的荧光，但困难遇到由于光毒性，虽然细胞死亡被推迟，直到脊柱消除后的体细胞。19因此，需要新的胱天蛋白酶的生物传感器，以检测和跟踪与体内基底caspase活性的细胞。 为了克服这些困难，我们产生了新的双色蛋白酶的生物传感器，指定CaspaseTracker。这一策略结合果蝇胱天蛋白酶敏感Apoliner生物传感器28与果蝇的G-TRACE FRT重组酶系统34的修改版本以永久标记和跟踪细胞在体内 。&lt;SUP&gt; 35 Gal4的激活的G-TRACE系统允许胱天蛋白酶的非常低的水平，以激活CaspaseTracker，导致RFP的表达在已经历过caspase活性的任何细胞的细胞质和永久核靶向GFP的表达。35该系统可标记细胞在整个使用果蝇 ，对胱天蛋白酶和细胞死亡研究的易于处理的和广泛使用的模型系统整体动物的生命。36-38

<table><tbody><tr><td colspan="4">&lt;strong&gt;Consumables and Reagents&lt;/strong&gt;</td></tr><tr><td>Vectashield</td><td>Vector Products</td><td>H-1000</td><td>Mounting medium</td></tr><tr><td>Forceps</td><td>Ted Pella</td><td>#505 (110mm, #5)</td><td>Dumont tweezer biology grade, stainless steel</td></tr><tr><td>Hanging Drop Slides</td><td>Fisher Scientific</td><td>12-565B</td><td>Glass slides</td></tr><tr><td>Hoechst 33342</td><td>Molecular Probes</td><td>H1399</td><td>DNA stain</td></tr><tr><td>Alexa Fluor 633 Phalloidin</td><td>Molecular Probes</td><td>A22284</td><td>Actin stain</td></tr><tr><td>Rat-Elav-7E8A10 anti-elav antibody</td><td>Developmental Studies Hybridoma Bank (DSHB)</td><td>Antibody Registry ID:&amp;nbsp; AB_528218&amp;nbsp;</td><td>Stain for Drosophla pan-neuronal ELAV</td></tr><tr><td>Cleaved caspase-3 (Asp175) antibody</td><td>Cell Signaling Technology</td><td>#9661</td><td>Stain for active fragment of caspase-3</td></tr><tr><td>ProLong Gold antifade reagent</td><td>Life Technologies</td><td>P36934</td><td>to preserve fluorophores&amp;nbsp;</td></tr><tr><td>ProLong Diamond Antifade Mountant</td><td>Life Technologies</td><td>P36961</td><td>to preserve fluorophores&amp;nbsp;</td></tr><tr><td>SylGard 182 Silicone Elastomer Kit</td><td>Dow Corning&amp;nbsp;</td><td>Product code: 0001023934</td><td>for dissection plates</td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>LSM780 confocal microscope</td><td>Carl Zeiss</td><td>N/A</td><td>Imaging</td></tr><tr><td>Carl Zeiss Stereomicroscope Stemi 2000</td><td>Carl Zeiss</td><td>N/A</td><td>&lt;em&gt;Drosophila&lt;/em&gt; dissection</td></tr><tr><td>AmScope Fiber Optic Dual Gooseneck Microscope Illuminator, 150&amp;nbsp;W</td><td>AmScope</td><td>WBM99316</td><td>Light source</td></tr></tbody></table>

in vivo biosensor tracks non-apoptotic caspase activity in drosophila

Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by available technologies, apoptosis is generally assumed to occur shortly thereafter. Caspases can cleave many functional and structural components to cause rapid and complete cell destruction within a few minutes. However, accumulating evidence indicates that in normal healthy cells the same caspases have other functions, presumably at lower enzymatic levels. Studies of non-apoptotic caspase activity have been hampered by difficulties with detecting low levels of caspase activity and with tracking ultimate cell fate in vivo. Here, we illustrate the use of an ultrasensitive caspase reporter, CaspaseTracker, which permanently labels cells that have experienced caspase activity in whole animals. This in vivo dual color CaspaseTracker biosensor for Drosophila melanogaster transiently expresses red fluorescent protein (RFP) to indicate recent or on-going caspase activity, and permanently expresses green fluorescent protein (GFP) in cells that have experienced caspase activity at any time in the past yet did not die. Importantly, this caspase-dependent in vivo biosensor readily reveals the presence of non-apoptotic caspase activity in the tissues of organ systems throughout the adult fly. This is demonstrated using whole mount dissections of individual flies to detect biosensor activity in healthy cells throughout the brain, gut, malpighian tubules, cardia, ovary ducts and other tissues. CaspaseTracker detects non-apoptotic caspase activity in long-lived cells, as biosensor activity is detected in adult neurons and in other tissues at least 10 days after caspase activation. This biosensor serves as an important tool to uncover the roles and molecular mechanisms of non-apoptotic caspase activity in live animals.

有允许CaspaseTracker检测正常健康的细胞（ 图1a）caspase活性的两个关键部件。的第一个是对胱天蛋白酶的生物传感器Apoliner（ 图1b）建模的146个氨基酸的caspase-裂解多肽。28，该多肽被从包含被凋亡过程中典型地切割的单一天然存在的半胱天冬站点DIAP1（细胞凋亡的果蝇抑制剂）衍生由胱天蛋白酶DrICE。42,43 DrICE相当于胱天蛋白?...

Watch this Scientific Journal Video about In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila at JoVE.com

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

以检测健康细胞在包含蛋白酶的活性水平低的整体动物， 果蝇生成指定CaspaseTracker高度敏感的生物传感器。依赖性的caspase-生物传感器的活性在长寿命的健康细胞中检测遍布在没有死亡刺激的最佳条件下饲养成年动物的内部器官。

体内生物传感器跟踪非凋亡胱天蛋白酶的活动在果蝇

Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by ...

in-vivo-biosensor-tracks-non-apoptotic-caspase-activity-in-drosophila

Research

JoVE Journal

Biology

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

9.0K 次观看.  Johns Hopkins University Bloomberg School of Public Health. 以检测健康细胞在包含蛋白酶的活性水平低的整体动物， 果蝇生成指定CaspaseTracker高度敏感的生物传感器。依赖性的caspase-生物传感器的活性在长寿命的健康细胞中检测遍布在没有死亡刺激的最佳条件下饲养成年动物的内部器官。 

视频: In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first to be activated in these pathways. This group includes caspase-2, -8, and -9, as well as the inflammatory caspases, caspase-1, -4, and -5. The initiator caspases are all activated by dimerization following recruitment to specific multiprotein complexes called activation platforms. Caspase Bimolecular Fluorescence Complementation (BiFC) is an imaging-based approach where split fluorescent proteins fused to initiator caspases are used to visualize the recruitment of initiator caspases to their activation platforms and the resulting induced proximity. This fluorescence provides a readout of one of the earliest steps required for initiator caspase activation. Using a number of different microscopy-based approaches, this technique can provide quantitative data on the efficiency of caspase activation on a population level as well as the kinetics of caspase activation and the size and number of caspase activating complexes on a per cell basis.

This protocol describes caspase Bimolecular Fluorescence Complementation (BiFC); an imaging-based method that can be used to visualize induced proximity of initiator caspases, which is the first step in their activation.

利用分子荧光互补的方法照亮 Caspase 活化的途径

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first ...

科研

生物化学

Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators and executioners of apoptosis, the most studied form of programmed cell death. Apoptosis occurs during developmental processes and is a necessary event in tissue homeostasis. Pyroptosis is another form of cell death that utilizes caspases and is a critical process in activating the immune system through the activation of the inflammasome, which results in the release of members of the interleukin-1 (IL-1) family. To assess caspase activity, target substrates can be assessed. However, sensitivity can be an issue when examining single cells or low-level activity. We demonstrate how a fluorogenic substrate can be used with a population-based assay or single-cell assay by flow cytometry. With proper controls, different amino acid sequences can be used to identify which caspases are active. Using these assays, the simultaneous loss of the inhibitors of apoptosis proteins upon tumor necrosis factor (TNF) stimulation has been identified, which primarily induces apoptosis in macrophages rather than other forms of cell death.

The present protocol describes two methods to measure caspase activity through a fluorogenic substrate using flow cytometry or a spectrofluorometer.

使用荧光测定法或流式细胞术测量半胱天冬酶活性

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators ...

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

We herein describe a phagocytosis assay using the dispersed embryonic cells of Drosophila. It enables us to easily and precisely quantify in vivo phagocytosis levels, and to identify new molecules required for the phagocytosis of apoptotic cells.

细胞凋亡细胞的吞噬作用<em&gt;果蝇</em&gt;胚胎

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and ...

免疫与感染

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

用 CaspaseTracker 生物传感器检测 Anastasis体内

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

医学

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, innate immune cells perform a number of essential functions, such as fighting infection, clearing necrotic debris, and stimulating matrix deposition. In order to fully understand the diverse signaling events that regulate this immune response, it is crucial to observe the complex behaviors of (and interactions that occur between) multiple cell lineages in vivo, and in real-time, with the high spatio-temporal resolution. The optical translucency and the genetic tractability of Drosophila embryos have established Drosophila as an invaluable model to live-image and dissect fundamental aspects of inflammatory cell behavior, including mechanisms of developmental dispersal, clearance of apoptotic corpses&#160;and/or microbial pathogens, and recruitment to wounds. However, more recent work has now demonstrated that employing a much later stage in the Drosophila lifecycle &#8211; the Drosophila pupa &#8211; offers a number of distinct advantages, including improved RNAi efficiency, longer imaging periods, and significantly greater immune cell numbers. Here we describe a protocol for imaging wound repair and the associated inflammatory response at the high spatio-temporal resolution in live Drosophila pupae. To follow the dynamics of both re-epithelialization and inflammation, we use a number of specific in vivo fluorescent markers for both the epithelium and innate immune cells. We also demonstrate the effectiveness of photo-convertible fluorophores, such as Kaede, for following the specific immune cell subsets, to track their behavior as they migrate to, and resolve from, the injury site.

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene inactivation.

果蝇蛹中炎症细胞动力学的长期体内追踪

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

在体内果蝇前运动神经末端的单分子跟踪

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

神经科学

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  ...

生物学

In Vitro Cleavage Assays using Purified Recombinant Drosophila Caspases for Substrate Screening

Caspases are very specific cell death proteases that are involved in apoptotic and non-apoptotic processes. While the role of caspases during apoptosis has been very well defined and many apoptotic proteolytic substrates of caspases have been identified and characterized, the role of caspases for non-apoptotic processes is not well understood. In particular, few non-apoptotic substrates of caspases have been identified thus far. Here, in order to facilitate the identification and characterization of potential caspase substrates, a protocol that allows the testing of candidate substrates in caspase cleavage assays in vitro is described. This protocol includes the production and purification of recombinant caspase proteins, the production of the candidate substrates either recombinantly or in a cell-free expression system, and the actual in vitro cleavage reaction followed by SDS-PAGE and immunoblotting. This protocol is tailored for the Drosophila caspases Dronc and Drice but can easily be adapted for caspases from other organisms, including mammals.

In Vitro Cleavage Assays using Purified Recombinant Drosophila Caspases for Substrate Screening

Here we present a protocol to express and purify recombinant Drosophila caspases Dronc and Drice, and their use in in vitro cleavage assays.

体外 使用纯化的重组 果蝇 半胱天冬酶进行底物筛选的切割测定

Caspases are very specific cell death proteases that are involved in apoptotic and non-apoptotic processes. While the role of caspases during apoptosis ...

生物工程

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct regulation of inflammatory signaling and is activated by proximity-induced dimerization following recruitment to inflammasomes. Caspase-1 is abundant in the monocytic cell lineage and induces maturation of the pro-inflammatory cytokines interleukin (IL)-1&#946; and IL-18 to active secreted molecules. The other inflammatory caspases, caspase-4 and -5 (and their murine homolog caspase-11) promote IL-1&#946; release by inducing pyroptosis. Caspase Bimolecular Fluorescence Complementation (BiFC) is a tool used to measure inflammatory caspase induced proximity as a readout of caspase activation. The caspase-1, -4, or -5 prodomain, which contains the region that binds to the inflammasome, is fused to non-fluorescent fragments of the yellow fluorescent protein Venus (Venus-N [VN] or Venus-C [VC]) that associate to reform the fluorescent Venus complex when the caspases undergo induced proximity. This protocol describes how to introduce these reporters into primary human monocyte-derived macrophages (MDM) using nucleofection, treat the cells to induce inflammatory caspase activation, and measure caspase activation using fluorescence and confocal microscopy. The advantage of this approach is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complex in living cells. However, careful controls need to be considered to avoid compromising cell viability and behavior. This technique is a powerful tool for the analysis of dynamic caspase interactions at the inflammasome level as well as for the interrogation of the inflammatory signaling cascades in living MDM and monocytes derived from human blood samples.

This protocol describes the workflow to obtain monocytes-derived macrophages (MDM) from human blood samples, a simple method to efficiently introduce inflammatory caspase Bimolecular Fluorescence Complementation (BiFC) reporters into human MDM without compromising cell viability and behavior, and an imaging-based approach to measure inflammatory caspase activation in living cells.

炎症性半月酶诱导的人单核细胞来源巨噬细胞邻近性的可视化

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct ...

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

策略跟踪Anastasis，A细胞存活的现象，细胞凋亡反转

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

体内生物传感器跟踪非凋亡胱天蛋白酶的活动在果蝇

摘要

探索更多视频

此视频中的章节

利用分子荧光互补的方法照亮 Caspase 活化的途径

使用荧光测定法或流式细胞术测量半胱天冬酶活性

细胞凋亡细胞的吞噬作用

用 CaspaseTracker 生物传感器检测 Anastasis体内

果蝇蛹中炎症细胞动力学的长期体内追踪

在体内果蝇前运动神经末端的单分子跟踪

凋亡细胞清除过程中实时成像

体外使用纯化的重组果蝇半胱天冬酶进行底物筛选的切割测定

炎症性半月酶诱导的人单核细胞来源巨噬细胞邻近性的可视化

策略跟踪Anastasis，A细胞存活的现象，细胞凋亡反转

体内生物传感器跟踪非凋亡胱天蛋白酶的活动在果蝇

摘要

探索更多视频

此视频中的章节

利用分子荧光互补的方法照亮 Caspase 活化的途径

使用荧光测定法或流式细胞术测量半胱天冬酶活性

细胞凋亡细胞的吞噬作用

用 CaspaseTracker 生物传感器检测 Anastasis体内

果蝇蛹中炎症细胞动力学的长期体内追踪

在体内果蝇前运动神经末端的单分子跟踪

凋亡细胞清除过程中实时成像

体外 使用纯化的重组 果蝇 半胱天冬酶进行底物筛选的切割测定

炎症性半月酶诱导的人单核细胞来源巨噬细胞邻近性的可视化

策略跟踪Anastasis，A细胞存活的现象，细胞凋亡反转

体外使用纯化的重组果蝇半胱天冬酶进行底物筛选的切割测定