Detection and characterization of individual endocytosis of AMPA-type glutamate receptor around postsynaptic membrane
Detection and characterization of individual endocytosis of AMPA-type glutamate receptor around postsynaptic membrane
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DOI：
10.1111/gtc.12493
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发表时间：
2017-06-01
期刊：
影响因子：
2.1
通讯作者：
Hirano, Tomoo
中科院分区：
文献类型：
--
作者：
Fujii, Shumpei;Tanaka, Hiromitsu;Hirano, Tomoo
突触可塑性如长时程抑制（LTD）被认为是学习和记忆的细胞机制。LTD通过突触后AMPA型受体（AMPAR）数量的减少来表达。虽然内吞作用是已知的突触后膜上的AMPAR的减少中发挥重要作用，难以检测单个的内吞事件阻碍了突触周围的AMPAR动力学的澄清。在此之前，我们开发了一种在玻璃表面诱导形成突触后样膜（PSLM）的方法，并利用全内反射荧光显微镜观察了PSLM周围的荧光素标记的AMPAR。通过这种方法，在PSLM和非PSLM中记录了AMPAR-荧光素的单独胞吐。在其他研究中，内吞囊泡含有pHluorin标记的受体可视化通过改变细胞外pH值。在这里，我们结合了PSLM形成方法和快速pH值变化方法，并检测个别的内吞事件的AMPAR周围PSLM具有高的空间和时间分辨率。通过与转铁蛋白受体的内吞事件的比较，表征了AMPAR的内吞事件。与转铁蛋白受体不同，AMPAR的组成性内吞不依赖于网格蛋白和发动蛋白。然而，由LTD诱导的刺激引发的AMPAR内吞作用是网格蛋白和动力蛋白依赖的。
Synaptic plasticity such as long-term depression (LTD) has been regarded as a cellular mechanism of learning and memory. LTD is expressed by the decrease in number of postsynaptic AMPA-type receptor (AMPAR) at glutamatergic synapses. Although endocytosis is known to play an essential role in the decrease in AMPAR on postsynaptic membrane, the difficulty to detect individual endocytic events hampered clarification of AMPAR dynamics around synapses. Previously, we developed a method to induce formation of postsynaptic-like membrane (PSLM) on the glass surface and observed pHluorin-tagged AMPAR around PSLM with total internal reflection fluorescence microscopy. By this method, individual exocytosis of AMPAR-pHluorin was recorded in both PSLM and non-PSLM. In other studies, endocytic vesicles containing pHluorin-tagged receptors were visualized by changing extracellular pH. Here, we have combined PSLM formation method and rapid pH change method, and detected individual endocytic events of AMPAR around PSLM with high spatial and temporal resolutions. Endocytic events of AMPAR were characterized by comparison with those of transferrin receptor. Constitutive endocytosis of AMPAR was not dependent on clathrin and dynamin in contrast to that of transferrin receptor. However, AMPAR endocytosis triggered by LTD-inducing stimulation was clathrin- and dynamin-dependent.