通过近距离标记监测活动驱动的内源性突触蛋白迁移。

IF 9.8 1区 生物学 Q1 Agricultural and Biological Sciences PLoS Biology Pub Date : 2024-10-28 eCollection Date: 2024-10-01 DOI:10.1371/journal.pbio.3002860
Carlos Pascual-Caro, Jaime de Juan-Sanz
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引用次数: 0

摘要

为了能够在大脑中传递信息,突触小泡与突触前膜融合,释放其内容物,并使无数囊泡跨膜蛋白短暂暴露。然而,由于突触小泡循环的快速动态性,很难在这种短暂的表面暴露过程中特异性地分离出贩运蛋白,因此目前还没有多功能的方法来量化神经元活动过程中内源性蛋白的突触转移。在这里,我们开发了一种新方法,利用突触裂隙接近标记来捕获和量化活动驱动的突触内源性突触蛋白的贩运。我们的研究表明,加速突触裂生物素化时间以匹配囊泡外排的快速动态,可以捕获神经活动期间在突触表面短暂暴露的内源性蛋白质,从而首次实现了对几乎所有内源性突触蛋白质转运的研究。作为概念验证,我们进一步应用这项技术,获得了非规范贩运蛋白(如 ATG9A 和 NPTX1)表面转运的直接证据,这些蛋白曾被认为在活动期间进行贩运,但尚未得到直接证明。本文介绍的技术进步将促进未来的研究,剖析活动过程中突触外排蛋白的分子特征,帮助确定维持哺乳动物大脑神经递质的分子机制。
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Monitoring of activity-driven trafficking of endogenous synaptic proteins through proximity labeling.

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.

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来源期刊
PLoS Biology
PLoS Biology BIOCHEMISTRY & MOLECULAR BIOLOGY-BIOLOGY
CiteScore
15.40
自引率
2.00%
发文量
359
审稿时长
3-8 weeks
期刊介绍: PLOS Biology is the flagship journal of the Public Library of Science (PLOS) and focuses on publishing groundbreaking and relevant research in all areas of biological science. The journal features works at various scales, ranging from molecules to ecosystems, and also encourages interdisciplinary studies. PLOS Biology publishes articles that demonstrate exceptional significance, originality, and relevance, with a high standard of scientific rigor in methodology, reporting, and conclusions. The journal aims to advance science and serve the research community by transforming research communication to align with the research process. It offers evolving article types and policies that empower authors to share the complete story behind their scientific findings with a diverse global audience of researchers, educators, policymakers, patient advocacy groups, and the general public. PLOS Biology, along with other PLOS journals, is widely indexed by major services such as Crossref, Dimensions, DOAJ, Google Scholar, PubMed, PubMed Central, Scopus, and Web of Science. Additionally, PLOS Biology is indexed by various other services including AGRICOLA, Biological Abstracts, BIOSYS Previews, CABI CAB Abstracts, CABI Global Health, CAPES, CAS, CNKI, Embase, Journal Guide, MEDLINE, and Zoological Record, ensuring that the research content is easily accessible and discoverable by a wide range of audiences.
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