成像pHluorin标记插入到质膜受体原代培养的小鼠神经元
Summary
通过标记与superecliptic pHluorin膜受体的胞外域的，并通过成像这些融合在培养的小鼠神经元受体，我们可以直接可视化个别水泡插入事件到质膜的受体。这种技术将有助于阐明受体插入到质膜的分子机制。
Abstract
质膜（PM）和细胞内受体之间贩运的机制更好地理解需要具有出色的空间分辨率和时间分辨率的实验方法。此外，这种方法还必须有分辨能力定位于PM在细胞内的受体。最重要的是，检测的受体，在一个单一的囊泡需要优异的检测灵敏度，因为每个囊泡进行只有一小数量的受体。检查受体活动的标准方法，包括表面生物素的生化检测，缺乏必要的空间和时间的决议;和荧光显微镜检查的immunolabeled表面的受体，这需要细胞化学固定，因此缺乏足够的时间分辨率1-6。为了克服这些限制，我们和其他人开发的，采用了一种新STRAtegy，使可视化动态插入的受体出色的空间分辨率和时间分辨率7-17 PM。该方法包括pH敏感的GFP，superecliptic的pHluorin 18的N-末端胞外结构域的受体的标记。 superecliptic pHluorin荧光在中性pH值和非荧光在酸性pH值（pH值<6.0）的独特属性。因此，标记受体的非荧光内酸性细胞内转运囊泡管腔或内体舱室时，它们变得容易地可视化，只有当暴露于细胞外的中性pH值环境上的外表面的PM。因此，我们的战略使我们能够区分PM表面受体，在细胞内囊泡。为了达到足够的空间和时间分辨率，以及学习所需的动态贩卖受体的敏感性，我们采用全内反映离子荧光显微镜（TIRFM），这使我们能够实现光学成像（〜170 nm）的最佳的空间分辨率，时间分辨率的视频率显微镜（30帧/秒），且灵敏度来检测单个GFP的荧光分子。成像pHluorin标记的受体TIRFM下，我们能够直接可视个人到PM培养的神经元受体插入事件。这种成像方法可能被应用于任何膜蛋白的胞外结构域，可以标记superecliptic pHluorin，并且将允许的密钥的详细机制不同的膜蛋白（受体，离子通道，运输等）插入到夹层PM。
Protocol
1。准备鼠神经胶质细胞培养的神经元培养空调涂有胶原蛋白溶液（1:3稀释Purecol DDH 2 O中）的T75烧瓶。烧瓶一身正气，干通宵的组织文化引擎盖。 夹层缓冲液（人工脑脊液：119 mM氯化钠，5 mM KCl中，1毫摩尔MgCl 2和30mM葡萄糖，25mM的HEPES，pH值7.4，无钙）和神经胶质细胞介质（DMEM培养液与10％胎牛血清，10单位/ ml青霉素，10 μg/ ml链霉素，和Glutamax）的制备和贮存在4℃下?…
Discussion
不知什么原因，小鼠神经元总是很难培养的大鼠神经元。在我们的经验中，混合培养的神经元和神经胶质细胞的原代培养的小鼠神经元。然而，这样的混合培养TIRF成像实验是不适合的，在这种类型的培养的神经元和其流程往往顶部的神经胶质细胞上生长的情况，神经元的胞体和树突状进程鞭长莫及TIRF显微镜。因此，较低的密度盖玻片上少数神经胶质细胞的神经元培养TIRF成像的理想选择。我们首?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是从杰克逊实验室的启动资金支持。
Materials
| Name of Reagent | Company | Catalogue Number | Comments |
| purified bovine collagen solution (Purecol) | Advanced Biomatrix | 5005-B | |
| Hank’s Balanced Salt Solution (HBSS) | GIBCO | 14185-045 | |
| penicillin-streptomycin (Pen Strep) | GIBCO | 15140-122 | |
| sodium pyruvate | GIBCO | 11360-070 | |
| DMEM High Glucose | GIBCO | 10313-021 | |
| fetal bovine serum (FBS) | |||
| GlutaMAX | GIBCO | 35050-061 | |
| papain | Worthington Biochemical Corp. | LS003126 | |
| Deoxyribonuclease I from bovine pancreas (DNase I) | SIGMA | DN25-10MG | |
| Dulbecco’s Phosphate Buffered Saline (DPBS) | GIBCO | 14190-144 | |
| 0.05% trypsin | GIBCO | 25300-054 | |
| poly-l-lysine hydrobromide | SIGMA | P2636-1G | |
| boric acid | Fisher-Scientific | BP 168-500 | |
| Neurobasal Medium | GIBCO | 21103-049 | |
| B-27 Serum-Free Supplement | GIBCO | 17504-044 | |
| heat inactive horse serum | GIBCO | 26050-070 | |
| Lipofectamine 2000 | Invitrogen | 11668 019 | |
| HEPES | Fisher-Scientific | BP310-500 | |
| Culture Insert | Millipore | PICM03050 |
References
- Thomas, G. M., Hayashi, T., Chiu, S. L., Chen, C. M., Huganir, R. L. Palmitoylation by DHHC5/8 targets GRIP1 to dendritic endosomes to regulate AMPA-R trafficking. Neuron. 73, 482-496 (2012).
- Anggono, V., Huganir, R. L. Regulation of AMPA receptor trafficking and synaptic. Curr. Opin. Neurobiol. , (2012).
- Makuch, L., Volk, L., Anggono, V., Johnson, R. C., Yu, Y., Duning, K., Kremerskothen, J., Xia, J., Takamiya, K., Huganir, R. L. Regulation of AMPA receptor function by the human memory-associated gene KIBRA. Neuron. 71, 1022-1029 (2011).
- Thomas, G. M., Lin, D. T., Nuriya, M., Huganir, R. L. Rapid and bi-directional regulation of AMPA receptor phosphorylation and trafficking by JNK. EMBO J. 27, 361-372 (2008).
- Lin, D. T., Huganir, R. L. PICK1 and phosphorylation of the glutamate receptor 2 (GluR2) AMPA receptor subunit regulates GluR2 recycling after NMDA receptor-induced internalization. J. Neurosci. 27, 13903-13908 (2007).
- Hayashi, T., Rumbaugh, G., Huganir, R. L. Differential regulation of AMPA receptor subunit trafficking by palmitoylation of two distinct sites. Neuron. 47, 709-723 (2005).
- Lin, D. T., Makino, Y., Sharma, K., Hayashi, T., Neve, R., Takamiya, K., Huganir, R. L. Regulation of AMPA receptor extrasynaptic insertion by 4.1N, phosphorylation. 12, 879-887 (2009).
- Araki, Y., Lin, D. T., Huganir, R. L. Plasma membrane insertion of the AMPA receptor GluA2 subunit is regulated by NSF binding and Q/R editing of the ion pore. Proc. Natl. Acad. Sci. U.S.A. 107, 11080-11085 (2010).
- Li, Y., Roy, B. D., Wang, W., Zhang, L., Zhang, L. F., Sampson, S. B., Yang, Y. P., Lin, D. T. Identification of Two Functionally Distinct Endosomal Recycling Pathways for Dopamine D2 Receptor. Journal of Neuroscience. , (2012).
- Yu, Y. J., Dhavan, R., Chevalier, M. W., Yudowski, G. A., von Zastrow, M. Rapid delivery of internalized signaling receptors to the somatodendritic surface by sequence-specific local insertion. J. Neurosci. 30, 11703-11714 (2010).
- Yudowski, G. A., Puthenveedu, M. A., von Zastrow, M. Distinct modes of regulated receptor insertion to the somatodendritic plasma membrane. Nat. Neurosci. 9, 622-627 (2006).
- Yudowski, G. A., Puthenveedu, M. A., Leonoudakis, D., Panicker, S., Thorn, K. S., Beattie, E. C., von Zastrow, M. Real-time imaging of discrete exocytic events mediating surface delivery of AMPA receptors. J. Neurosci. 27, 11112-11121 (2007).
- Ashby, M. C., De La Rue, S. A., Ralph, G. S., Uney, J., Collingridge, G. L., Henley, J. M. Removal of AMPA receptors (AMPARs) from synapses is preceded by transient endocytosis of extrasynaptic AMPARs. J. Neurosci. 24 (AMPARs), 5172-5176 (2004).
- Kopec, C. D., Real, E., Kessels, H. W., Malinow, R. GluR1 links structural and functional plasticity at excitatory synapses. J. Neurosci. 27, 13706-13718 (2007).
- Makino, H., Malinow, R. AMPA receptor incorporation into synapses during LTP: the role of lateral movement and exocytosis. Neuron. 64, 381-390 (2009).
- Makino, H., Malinow, R. Compartmentalized versus global synaptic plasticity on dendrites controlled by experience. Neuron. 72, 1001-1011 (2011).
- Wang, Z., Edwards, J. G., Riley, N., Provance, D. W., Karcher, R., Li, X. D., Davison, I. G., Ikebe, M., Mercer, J. A., Kauer, J. A., Ehlers, M. D. Myosin Vb mobilizes recycling endosomes and AMPA receptors for postsynaptic plasticity. Cell. 135, 535-548 (2008).
- Miesenbock, G., De Angelis, D. A., Rothman, J. E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature. 394, 192-195 (1998).
- Kaech, S., Banker, G. Culturing hippocampal neurons. Nat. Protoc. 1, 2406-2415 (2006).
- Sekine-Aizawa, Y., Huganir, R. L. Imaging of receptor trafficking by using alpha-bungarotoxin-binding-site-tagged receptors. Proc. Natl. Acad. Sci. U.S.A. 101, 17114-17119 (2004).
Tags
PHluorin-tagged Receptor InsertionPlasma MembranePrimary Cultured Mouse NeuronsReceptor TraffickingSpatial ResolutionTemporal ResolutionVesicle Detection SensitivitySurface BiotinylationBiochemical DetectionFluorescence MicroscopyImmunolabeled Surface ReceptorsDynamic Insertion VisualizationSuperecliptic PHluorinPH-sensitive GFP
Cite This Article
Li, Y., Roy, B. D., Wang, W., Zhang, L., Sampson, S. B., Lin, D. Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons. J. Vis. Exp. (69), e4450, doi:10.3791/4450 (2012).