体内生物传感器跟踪非凋亡胱天蛋白酶的活动在果蝇
Summary
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。
Abstract
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.
Introduction
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 。<SUP> 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
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| CONSUMABLES AND REAGENTS | |||
| Vectashield | Vector Products | H-1000 | Mounting medium |
| Forceps | Ted Pella | #505 (110mm, #5) | Dumont tweezer biology grade, stainless steel |
| Hanging Drop Slides | Fisher Scientific | 12-565B | Glass slides |
| Hoechst 33342 | Molecular Probes | H1399 | DNA stain |
| Alexa Fluor 633 Phalloidin | Molecular Probes | A22284 | Actin stain |
| Rat-Elav-7E8A10 anti-elav antibody | Developmental Studies Hybridoma Bank (DSHB) | Antibody Registry ID: AB_528218 | Stain for Drosophla pan-neuronal ELAV |
| Cleaved caspase-3 (Asp175) antibody | Cell Signaling Technology | #9661 | Stain for active fragment of caspase-3 |
| ProLong Gold antifade reagent | Life Technologies | P36934 | to preserve fluorophores |
| ProLong Diamond Antifade Mountant | Life Technologies | P36961 | to preserve fluorophores |
| SylGard 182 Silicone Elastomer Kit | Dow Corning | Product code: 0001023934 | for dissection plates |
| EQUIPMENT | |||
| LSM780 confocal microscope | Carl Zeiss | N/A | Imaging |
| Carl Zeiss Stereomicroscope Stemi 2000 | Carl Zeiss | N/A | Drosophila dissection |
| AmScope Fiber Optic Dual Gooseneck Microscope Illuminator, 150W | AmScope | WBM99316 | Light source |
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