使用普洛林来评估阿克森指导受体在细胞培养和小鸡胚胎中的动力学
Summary
我们在这里描述了使用pH敏感的绿色荧光蛋白变种，pHluorin，来研究在细胞表面贩运的轴向制导受体的间歇性时间动力学。pHluorin标记的受体在细胞培养和 体内都表达，使用小鸡胚胎的电波。
Abstract
在开发过程中，轴突制导受体在调节轴突对吸引力和排斥性暗示的敏感性方面起着至关重要的作用。事实上，激活制导受体是信号机制的第一步，允许轴尖，生长锥，对配体作出反应。因此，在细胞表面调节它们的可用性是参与设置生长锥敏感性的机制之一。我们在这里描述了一种方法，以精确可视化在体外和体内发育的小鸡脊髓的轴向导受体的间子-时间细胞表面动力学。我们利用绿色荧光蛋白 （GFP） 变种的 pH 依赖荧光特性，专门检测用于等离子膜的轴向制导受体的分数。我们首先描述了这种pH依赖结构的体外验证，我们进一步详细说明了它们在体内的使用，在小鸡脊髓和弦，以评估轴向导受体的兴趣的间断-时间动力学。
Introduction
在导航过程中，轴突集成了多个环境提示，引导他们朝着目标前进。这些线索激活轴龙终端表面的制导受体，即生长锥体，进而启动适当的信号通路。因此，受体细胞表面分布的时间和空间调节对于确定生长锥体1的灵敏度至关重要。在此背景下，中线交叉通过通信轴突是研究受体细胞表面水平调节的极好模型。在正在发育的脊髓中，小卖部轴突最初被吸引到穿过中线的腹腔底板上。穿越后，他们失去了对地板吸引器的响应，并获得对地板排斥反应，以便他们能够退出地板，导航到他们的最终目的地在神经系统的对立面2，3。调节生长锥表面的受体可用性是响应性切换到中线提示4，5的机制之一。因此，选择性地监测生长锥体等离子膜中的受体至关重要。我们在这里描述了一种基于绿色荧光蛋白（GFP）变种的pH依赖荧光特性的方法，以具体可视化在 体外 和 体外血浆膜的轴向导受体，在发育中的小鸡脊髓中。
罗斯曼和他的同事设计了点突变pH敏感变种的GFP，包括黄道pHluorin6。黄道磷脂素在接触酸性pH（<6）时具有无荧光特性，同时在中性pH处具有荧光。这允许区分细胞内酸性隔间（即内分泌体，贩运卵泡）与荧光受体中加入等离子膜的无荧光受体，从而暴露在细胞外中性pH7中。我们利用这一点来监测普莱辛A1的等离子膜本地化，这是一种轴向制导受体，对中线驱虫剂血红素3B5（图1A）的生长锥反应进行调停。我们在这里描述了pHluorin-plexinA1构造的体外特征，以及在发育中的小鸡脊髓中这种构造的ovo电波8-10，然后是低温剖腹产的微观分析，从而能够以空间和时间分辨率在体内跟随轴向受体动力学。
Protocol
1. 克隆策略，以标记普莱辛A1受体与普洛林 选择适当的表达载体作为骨干（例如小鼠受体plexinA1表达载体，安德烈亚斯·普舍尔博士11的一种礼物）。注意：此普莱辛A1向量设计为实现在等离子膜中高效插入HA-或VSV标记受体。 通过 PCR 放大黄道 pHluorin 编码序列，使用足够的质粒作为模板（例如，使用 pHluorin 标记的 GABA 受体，Jacob2博士的一种礼物）。?…
Representative Results
图1。A. 在细胞环境中的普洛林-普莱辛A1荧光特性的方案。PHluorin 在 pH 是酸性 （<6） 的细胞内隔间中是无荧光的，例如在贩运静脉或内分泌体中，当暴露在 pH 是中性的细胞外介质中时，它是荧光的。这只能可视化普洛林-普莱辛A1受体?…
Discussion
该协议提供了一个分步程序，以遵循轴向导受体在细胞培养和小鸡胚胎脊髓发育环境中的动态。
要设计一种 诺沃 普洛林标记蛋白，需要考虑两点关于克隆策略。首先，pHluorin 标签应暴露在酸性内生体的流明中，因此应暴露在细胞外隔间，以便可视化等离子膜受体池。因此，pHluorin序列对受体序列的正确定位直接取决于所研究的受体类型（即受体的N端或C端部分?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢霍迈拉·纳瓦比、弗雷德里克·莫雷特和伊莎贝尔·三亚的帮助。这项工作得到了 CNRS、协会特许经营协会（AFM）、ANR YADDLE、拉贝克斯·德维坎、拉贝克斯·皮层、ERC YODA 到 V .C的支持;C.D-B和A.J分别得到拉利格癌症和拉贝克斯·德韦坎奖学金的支持。
Materials
| COS7 cells | ATCC | CRL-1651 | |
| DMEM GlutaMAX | GIBCO | 61965-026 | |
| Sodium pyruvate | GIBCO | 11360-039 | |
| Amphotericin B | Sigma | A2942 | |
| Fetal bovine serum | GIBCO | 10270-106 | |
| Penicillin/Streptomycin | GIBCO | 15140-122 | |
| Exgen500 reagent | Euromedex Fermentas | ET0250 | |
| PBS -Ca2+ -Mg2+ | GIBCO | 14190-094 | |
| Fast green dye | Sigma | F7252 | |
| 32% Paraformaldehyde aqueous solution | Electron Microscopy | 15714-S | Dilute extemporaneously in PBS to achieve a 4% solution |
| Gelatin from cold water fish skin | Sigma | G7041 | |
| Sucrose | Sigma | S0389 | |
| Cryomount | Histolab | 00890 | |
| Hoechst 34580 | Invitrogen | H21486 | |
| Mowiol 4-88 | Fluka | 81381 | |
| Consumables | |||
| Bottom-glass 35 mm dish | MatTek | P35G-1.5-14-C | |
| 5 ml Syringe | Terumo | SS-05S | |
| Needles 0.9 mm x 25 mm | Terumo | NN-2025R | |
| Capillaries | CML | PP230PO | capillaries are stretched manually in the flame |
| Superfrost Plus Slides | Thermo Scientific | 4951PLUS | |
| Material | |||
| Curved scissors | FST | 129-10 | |
| Microscalpel | FST | 10316-14 | |
| Forceps | FST | Dumont #5 REF#11254 | |
| Equipment/software | |||
| Time lapse microscope | Zeiss | Observer 1 | |
| Temp module S | PECON for Zeiss | ||
| CO2 module S | PECON for Zeiss | ||
| Metamorph software | Metamorph | ||
| Eggs incubator | Sanyo | MIR154 | |
| Electroporator apparatus | Nepa Gene CO., LTD | CUY21 | |
| Electrodes | Nepa Gene CO., LTD | CUY611P7-4 | 4 mm platinum electrodes |
| Fluorescence stereomicroscope | LEICA | MZ10F | |
| Cryostat | MICROM | HM550 | |
| Confocal microscope | Olympus | FV1000, X81 | |
| Fluoview software | Olympus | ||
| CLC Main Workbench software | CLC Bio | ||
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Citar este artigo
Delloye-Bourgeois, C., Jacquier, A., Falk, J., Castellani, V. Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo. J. Vis. Exp. (83), e50883, doi:10.3791/50883 (2014).