Paris Brain Institute (ICM). Sorbonne University, Inserm, CNRS, APHP, Hôpital de la Pitié Salpêtrière, Paris, France.
PLoS Biol. 2024 Oct 28;22(10):e3002860. doi: 10.1371/journal.pbio.3002860. eCollection 2024 Oct.
To enable transmission of information in the brain, synaptic vesicles fuse to presynaptic membranes, liberating their content and exposing transiently a myriad of vesicular transmembrane proteins. However, versatile methods for quantifying the synaptic translocation of endogenous proteins during neuronal activity remain unavailable, as the fast dynamics of synaptic vesicle cycling difficult specific isolation of trafficking proteins during such a transient surface exposure. Here, we developed a novel approach using synaptic cleft proximity labeling to capture and quantify activity-driven trafficking of endogenous synaptic proteins at the synapse. We show that accelerating cleft biotinylation times to match the fast dynamics of vesicle exocytosis allows capturing endogenous proteins transiently exposed at the synaptic surface during neural activity, enabling for the first time the study of the translocation of nearly every endogenous synaptic protein. As proof-of-concept, we further applied this technology to obtain direct evidence of the surface translocation of noncanonical trafficking proteins, such as ATG9A and NPTX1, which had been proposed to traffic during activity but for which direct proof had not yet been shown. The technological advancement presented here will facilitate future studies dissecting the molecular identity of proteins exocytosed at the synapse during activity, helping to define the molecular machinery that sustains neurotransmission in the mammalian brain.
为了实现大脑中的信息传递，突触小泡融合到突触前膜，释放其内容物，并短暂暴露无数的囊泡跨膜蛋白。然而，由于突触囊泡循环的快速动力学使得在这种短暂的表面暴露期间特异性分离运输蛋白变得困难，因此，仍然缺乏用于定量检测神经元活动期间内源性蛋白突触易位的通用方法。在这里，我们开发了一种使用突触裂邻近标记的新方法，以捕获和定量研究在突触处活动驱动的内源性突触蛋白的运输。我们表明，加速裂邻近标记时间以匹配囊泡胞吐的快速动力学，允许捕获在神经活动期间在突触表面短暂暴露的内源性蛋白，从而首次能够研究几乎每一种内源性突触蛋白的易位。作为概念验证，我们进一步应用这项技术获得了非典型运输蛋白（如 ATG9A 和 NPTX1）表面易位的直接证据，这些蛋白在活动期间被提出进行运输，但尚未有直接证据证明。这里提出的技术进步将有助于未来研究在活动期间突触处胞吐的蛋白质的分子特征，有助于定义维持哺乳动物大脑中神经传递的分子机制。
