Live-cell imaging of receptors around postsynaptic membrane formed on adhesion-protein-coated glass
Live-cell imaging of receptors around postsynaptic membrane formed on adhesion-protein-coated glass
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粘附蛋白涂层玻璃上形成的突触后膜周围受体的活细胞成像
DOI：
10.1038/nprot.2013.171
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发表时间：
2014
期刊：
影响因子：
14.8
通讯作者：
T.
中科院分区：
文献类型：
--
作者：
Tanaka;H.;Fujii;S. & Hirano;T.
该方案描述了如何成像周围的兴奋性突触后膜形成粘合蛋白涂层玻璃表面谷氨酸受体的运输。该方案是为了阐明受体如何在突触可塑性的诱导过程中移动。分离的神经元被培养在涂有neurexin的盖层上，这种盖层诱导在玻璃表面形成突触后膜样结构。然后将带有荧光蛋白标记的谷氨酸受体转染到神经元中，并用全内反射荧光显微镜观察。整个过程大约需要3个星期。神经元活动引起的细胞表面受体数量的变化可以被量化，并且在伪突触后膜周围可以清楚地观察到受体的个别胞吐事件。该方案在研究膜蛋白在细胞膜其他特殊区域（如抑制性突触后膜、突触前膜或免疫突触）周围的运动方面具有潜在的应用价值。
This protocol describes how to image the trafficking of glutamate receptors around excitatory postsynaptic membrane formed on an adhesion protein–coated glass surface. The protocol was developed to clarify how receptors move during the induction of synaptic plasticity. Dissociated neurons are cultured on a coverslip coated with neurexin, which induces the formation of postsynaptic membrane-like structures on the glass surface. A glutamate receptor tagged with a fluorescent protein is then transfected into neurons, and it is observed with total internal reflection fluorescence microscopy. The whole process takes about 3 weeks. Changes in the amount of cell-surface receptors caused by neuronal activities can be quantified, and individual exocytosis events of receptors can be clearly observed around the pseudo-postsynaptic membrane. This protocol has potential applications for studies of movements of membrane proteins around other specialized regions of the cell membrane, such as the inhibitory postsynaptic membrane, the presynaptic membrane or the immunological synapses.