测量生活中的斑马鱼胚胎蛋白质稳定性使用荧光衰减光转化后（FDAP）
Summary
在细胞和组织中的蛋白水平通常紧紧蛋白质生产和清除的平衡调节。使用荧光衰减光转化（FDAP）后，蛋白质的间隙动力学可以通过实验在体内测量。
Abstract
蛋白质稳定性影响生物学的许多方面，并测定蛋白质的清除动力学可以提供重要的见解的生物系统。在FDAP实验中，生物体内蛋白质的间隙可以被测量。感兴趣的蛋白质将被标上photoconvertible荧光蛋白，在体内表达和光转换，并降低在光转换信号随时间被监测。然后，数据被配备有适当的间隙模型，以确定该蛋白的半衰期。重要的是，蛋白群的有机体的不同区室的清除动力学可以分别通过施加房室掩模检查。这种方法已被用于斑马鱼发育期间，以确定细胞内和细胞外的半衰期分泌信号蛋白。在这里，我们描述了一种协议，用于FDAP实验在斑马鱼胚胎。它应该是可能使用FDAP确定的间隙动力学任何taggable蛋白在任何光学访问的有机体。
Introduction
蛋白质在细胞和生物体的水平由生产和清除它们的速率来确定。蛋白质的半衰期的范围可以从几分钟到几天1-4。在许多生物系统，关键蛋白的稳定或间隙对细胞活性有重要影响。所需的细胞周期进展5,6，发育信号7-9，细胞凋亡10，和正常功能和维持神经元11,12的细胞内蛋白质稳定性调制。胞外蛋白的稳定性影响的分布和分泌的蛋白质，如形态发生13,14的可用性，组织内。
在过去的几十年中，蛋白质稳定性已经主要在细胞培养用放射性脉冲标记或放线菌酮追踪实验15评估。在这样的脉冲追踪实验，将细胞或者瞬时暴露于一个“脉冲”放射性氨基的酸前体掺入到新合成的蛋白质，或它们暴露于放线菌酮，抑制蛋白质的合成。培养的细胞，然后在不同时间点收集，并且任一免疫沉淀，随后进行放射自显影（放射性脉冲追踪实验）或免疫印迹（在放线菌酮的实验）被用来量化蛋白质的间隙随时间。
常规的蛋白质稳定性试验有几个不足之处。首先，在这些测定蛋白质通常不表达在其内源性的环境中，而是在组织培养，有时在来自不同物种的细胞。对于蛋白质的稳定性是依赖于上下文的，这种方法是有问题的。其次，它是不可能跟随蛋白清除在个体细胞或生物体随着时间的推移，而从这些分析中的数据反映了在不同时间点的平均细胞的不同种群。由于单个细胞可能已经开始用不同量的蛋白质，也可能采取了放射性标记或酮在不同的时间，或可具有不同的间隙动力学，例如聚合数据可能是误导性的。最后，在放线菌酮的追踪实验的情况下，另外的蛋白质合成抑制剂的可能产生意想不到的生理效果，可以人为地改变蛋白质稳定性16-18。这些缺点，可避免使用荧光衰减后光转化（FDAP），其利用photoconvertible蛋白质来动态测量在活生物体19-25蛋白清除的技术（见讨论用于FDAP技术的限制）。
Photoconvertible蛋白的荧光蛋白的激发和发射特性暴露于光26特定波长后更改。一种常用的变体是Dendra2，“绿色到红色”photoconvertible蛋白最初具有前引文和发射性质类似于绿色荧光蛋白，但暴露于UV轻“光转化”后-其激发/发射性能变得类似于红色荧光蛋白23,27。重要的是，光转化后产生的新的蛋白质将不会有相同的激发/发射性质的光转换蛋白，允许生产和清除的经光转化解耦光转换蛋白质的仅一个池和观察。标记与photoconvertible蛋白兴趣蛋白质从而提供了一个方便的方法来进行脉冲标记蛋白在完整的，光学访问的活生物体。
在FDAP测定（ 图1A）中，感兴趣的蛋白被标记为一个photoconvertible蛋白和表达在生物体（ 图1B）。该融合蛋白是光转换，并降低在光转换信号随着时间的推移由fluorescen监视CE显微镜（ 图1C）。然后，数据被装有合适的模型，以确定融合蛋白（ 图1D）的半衰期。
此处所描述的测定法FDAP被设计在早期胚胎发育19来确定外半衰期在斑马鱼胚胎分泌信号蛋白。但是，这种方法可以适用于任何透明模型生物体容忍实时成像，并且可以被用于监控任何taggable胞内或胞外蛋白的清除。此处所描述的技术的变型中培养的细胞20,23和22 果蝇和小鼠胚胎21已被执行。
Protocol
1.生成Photoconvertible融合构建和注射Dechorionated斑马鱼胚胎产生含有融合到绿色到红色photoconvertible蛋白（见讨论 ）感兴趣的蛋白质的功能性构建体，然后用体外转录以产生mRNA的加帽的融合蛋白编码如米勒等人，2012 19。 用链霉蛋白酶，从约30斑马鱼胚胎取出chorions在单细胞阶段。另外，手动dechorionate使用镊子28胚胎。 注：胚胎必须dechoriona…
Representative Results
FDAP已被用来确定在斑马鱼胚胎19的半衰期外信号蛋白。胚胎34时其中的一个蛋白质，斜视，诱导mesendodermal基因的表达。斜视Dendra2激活mesendodermal基因的表达，在类似的未标记斜视的水平，就证明了定量RT-PCR和原位杂交检测19。胚胎被共注射Alexa488葡聚糖和mRNA编码斜视-Dendra2并经受FDAP测定。在细胞外光转换的信号强度的降低随时间是明显的（ 图4A）。使用…
Discussion
一个FDAP实验的成功依赖于官能photoconvertible融合蛋白的产生。标记的蛋白质可以影响其生物活性和/或生物物理特性，包括其局部化，溶解性和稳定性36-41。准备测试，以几个不同的融合构建活动找到一个处于活动状态。我们已发现，改变的蛋白相对于所感兴趣的蛋白的photoconvertible的位置或使用更长的接头（ 例如 ，使用的氨基酸序列LGDPPVAT 19）可增强融合蛋白的活性。在信?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
笔者想感谢杰弗里·法雷尔，詹姆斯加格农和詹妮弗伯格曼对稿件的意见。 KWR是在FDAP法的发展过程中得到了国家科学基金会研究生研究奖学金计划。这项工作是由美国国立卫生研究院，以AFS赠款和由德国研究基金会（埃米诺特计划），马普学会和人类前沿科学计划（职业发展奖），以PM资助。
Materials
| PyFDAP (download from the following website: http://people.tuebingen.mpg.de/mueller-lab) | Install and operate using the instructions provided on the PyFDAP website; PyFDAP is compatible with Linux, Mac, and Windows operating systems. | ||
| mMessage mMachine Sp6 Transcription Kit (Life Technologies, AM1340) | To generate capped mRNA for injection into embryos | ||
| Alexa488-dextran conjugate, 3 kDa (Life Technologies, D34682) | Co-inject with mRNA to create intracellular and extracellular masks | ||
| 6-well plastic dish (BD Falcon) | Incubate embryos in agarose-coated wells until ready for mounting | ||
| Embryo medium | 250 mg/l Instant Ocean salt, 1 mg/l methylene blue in reverse osmosis water adjusted to pH 7 with NaHCO3 | ||
| Protease from Streptomyces griseus (Sigma, P5147) | Make a 5 mg/ml stock and use at 1 mg/ml to dechorionate embryos at the one-cell stage | ||
| 5 cm diameter glass petri dish | For embryo dechorionation | ||
| 200 ml glass beaker | For embryo dechorionation | ||
| Microinjection apparatus | For injection of mRNA and dye into embryos at the one-cell stage | ||
| Stereomicroscope | For injecting and mounting embryos | ||
| 1x Danieau's medium | Dilute low melting point agarose and perform imaging in this medium; recipe: 0.2 mm filtered solution of 58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.3 mM CaCl2, 5 mM HEPES pH 7.2 | ||
| UltraPure low melting point agarose (Invitrogen, 16520-100) | For mounting embryos; use at a concentration of 1% in Danieau's medium: add 200 mg to 20 ml Danieau's medium, microwave until dissolved, then aliquot 1 ml into microcentrifuge tubes; aliquots can be stored at 4 °C, re-melted at 70 °C, and cooled to 40 – 42 °C when ready to use | ||
| Glass Pasteur pipette (Kimble Chase (via Fisher), 63A53WT) | For mounting embryos; flame the tip to prevent jagged edges from injuring embryos | ||
| Metal probe | For positioning embryos during mounting | ||
| Glass bottom dishes (MatTek, P35G-1.5-14-C) | Use the appropriate cover glass thickness for your objective; part number listed here is for cover glass No. 1.5 | ||
| 15 ml tube (BD Falcon) filled with ~5 ml embryo medium | For rinsing residual agarose from the Pasteur pipette | ||
| Inverted laser scanning confocal microscope | A mercury arc lamp, 488 nm laser, 543 nm laser, and the appropriate filter sets are required | ||
| Heated stage | To maintain embryos at the optimal temperature of 28 °C during the experiment | ||
| Confocal software capable of time-lapse imaging | Must be able to define multiple positions and automatically image them at defined intervals | ||
| 25x or 40x water objective | Objective for imaging | ||
| 10x air objective | Objective for photoconversion | ||
| Immersion oil | Immersion oil with the same refractive index as water |
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Diesen Artikel zitieren
Rogers, K. W., Bläßle, A., Schier, A. F., Müller, P. Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP). J. Vis. Exp. (95), e52266, doi:10.3791/52266 (2015).