Mapping the Proteome of the Synaptic Cleft through Proximity Labeling Reveals New Cleft Proteins.
Mapping the Proteome of the Synaptic Cleft through Proximity Labeling Reveals New Cleft Proteins.
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DOI：
10.3390/proteomes6040048
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发表时间：
2018-11-28
期刊：
影响因子：
3.3
通讯作者：
Biederer T
中科院分区：
文献类型：
--
作者：
Cijsouw T;Ramsey AM;Lam TT;Carbone BE;Blanpied TA;Biederer T
突触是一种特殊的神经元细胞-细胞接触，是哺乳动物大脑中网络通信的基础。在神经元群体和回路中，利用了一组不同的突触，它们的分子组成不同，以实现异质连接模式和功能。除了突触前和突触后的特化，突触间隙现在被理解为突触的一个组成部分，有助于它们的结构和功能组织。本研究采用过氧化物酶介导的邻近标记方法，将兴奋性突触细胞粘附蛋白SynCAM 1与辣根过氧化物酶（HRP）融合，在培养的皮层神经元中作为报告基因，对裂隙蛋白质组进行定位。该报告者标记兴奋性突触，如通过共聚焦显微镜所测量的，并靶向突触间隙的边缘区，如使用3D dSTORM超分辨率成像所确定的。用膜不渗透的生物素-苯酚化合物限制标记到细胞表面的邻近标记，和无标记定量（LFQ）质谱结合膜与突触表面蛋白的比率HRP标记用于鉴定兴奋性裂隙的蛋白质组学内容。新的裂缝候选人进行了鉴定，受体型酪氨酸蛋白磷酸酶zeta的选择和成功验证。这项研究支持了过氧化物酶介导的邻近标记用于突触间隙蛋白质组学的强大适用性及其在理解健康中的突触异质性和疾病（如精神疾病和成瘾）变化方面的潜力。
Synapses are specialized neuronal cell-cell contacts that underlie network communication in the mammalian brain. Across neuronal populations and circuits, a diverse set of synapses is utilized, and they differ in their molecular composition to enable heterogenous connectivity patterns and functions. In addition to pre- and post-synaptic specializations, the synaptic cleft is now understood to be an integral compartment of synapses that contributes to their structural and functional organization. Aiming to map the cleft proteome, this study applied a peroxidase-mediated proximity labeling approach and used the excitatory synaptic cell adhesion protein SynCAM 1 fused to horseradish peroxidase (HRP) as a reporter in cultured cortical neurons. This reporter marked excitatory synapses as measured by confocal microcopy and was targeted to the edge zone of the synaptic cleft as determined using 3D dSTORM super-resolution imaging. Proximity labeling with a membrane-impermeant biotin-phenol compound restricted labeling to the cell surface, and Label-Free Quantitation (LFQ) mass spectrometry combined with ratiometric HRP tagging of membrane vs. synaptic surface proteins was used to identify the proteomic content of excitatory clefts. Novel cleft candidates were identified, and Receptor-type tyrosine-protein phosphatase zeta was selected and successfully validated. This study supports the robust applicability of peroxidase-mediated proximity labeling for synaptic cleft proteomics and its potential for understanding synapse heterogeneity in health and changes in diseases such as psychiatric disorders and addiction.