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首页> 外文期刊>The European Journal of Neuroscience >Cell-type-specific visualisation and biochemical isolation of endogenous synaptic proteins in mice
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Cell-type-specific visualisation and biochemical isolation of endogenous synaptic proteins in mice

机译：小鼠内源突触蛋白的细胞型特异性可视化和生化分离

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In recent years, the remarkable molecular complexity of synapses has been revealed, with over 1,000 proteins identified in the synapse proteome. Although it is known that different receptors and other synaptic proteins are present in different types of neurons, the extent of synapse diversity across the brain is largely unknown. This is mainly due to the limitations of current techniques. Here, we report an efficient method for the purification of synaptic protein complexes, fusing a high-affinity tag to endogenous PSD95 in specific cell types. We also developed a strategy, which enables the visualisation of endogenous PSD95 with fluorescent-protein tag in Cre-recombinase-expressing cells. We demonstrate the feasibility of proteomic analysis of synaptic protein complexes and visualisation of these in specific cell types. We find that the composition of PSD95 complexes purified from specific cell types differs from those extracted from tissues with diverse cellular composition. The results suggest that there might be differential interactions in the PSD95 complexes in different brain regions. We have detected differentially interacting proteins by comparing data sets from the whole hippocampus and the CA3 subfield of the hippocampus. Therefore, these novel conditional PSD95 tagging lines will not only serve as powerful tools for precisely dissecting synapse diversity in specific brain regions and subsets of neuronal cells, but also provide an opportunity to better understand brain region- and cell-type-specific alterations associated with various psychiatric/neurological diseases. These newly developed conditional gene tagging methods can be applied to many different synaptic proteins and will facilitate research on the molecular complexity of synapses.
机译：近年来，揭示了突触的显着分子复杂性，突触蛋白质组中鉴定出超过1,000种蛋白质。虽然已知不同的受体和其他突触蛋白存在于不同类型的神经元中，但大脑过度的突触多样性在很大程度上是未知的。这主要是由于当前技术的局限性。这里，我们报告了纯化突触蛋白复合物的有效方法，将高亲和力标记融合到特定细胞类型中的内源PSD95。我们还开发了一种策略，它能够在Cre-Reobominase的细胞中与荧光蛋白标签进行内源性PSD95的可视化。我们证明了突触蛋白复合物蛋白质组学分析和这些特定细胞类型的可视化的可行性。我们发现从特定细胞类型纯化的PSD95复合物的组合物不同于从组织中提取的那些具有多种细胞组合物的组合物。结果表明，在不同脑区中PSD95复合物中可能存在差异相互作用。通过比较来自全海马的数据集和海马的CA3子场的数据集，我们检测到差异相互作用的蛋白质。因此，这些新颖的条件PSD95标记线路不仅可以用作精确地解剖特定脑区和神经元细胞亚群的突触分集的强大工具，而且还提供了更好地理解与之相关的脑类型和细胞类型的改变的机会各种精神病/神经疾病。这些新开发的条件基因标记方法可以应用于许多不同的突触蛋白，并有助于研究突触的分子复杂性。

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