Proteomic analysis of the electrical synapse

电突触的蛋白质组学分析

• 批准号: 10042722
• 负责人: Adam C Miller
• 金额: $ 40.56万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-15 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10042722
• 关键词: Affect; Affinity; Animal Model; Animals; Behavior; Biochemical; Biochemistry; Biological; Biological Assay; Biotin; Brain; Cell Culture Techniques; Cells; Chemical Synapse; Chemicals; Clustered Regularly Interspaced Short Palindromic Repeats; Communication; Complex; Connexins; Development; Disease; Dissection; Electrical Synapse; Escherichia coli; Escherichia coli Proteins; Feeling; Fostering; Foundations; Future; Gap Junctions; Genes; Genetic; Genome engineering; Goals; Grant; Human; Immunohistochemistry; In Vitro; Individual; Label; Ligase; Link; Macromolecular Complexes; Mass Spectrum Analysis; Membrane; Methods; Modeling; Molecular; Molecular Structure; Motor; Nervous system structure; Neurodevelopmental Disorder; Neuromodulator; Neurons; Neurotransmitters; Pathway interactions; Perception; Problem Solving; Property; Proteins; Proteome; Proteomics; Regulation; Resistance; Role; Route; Sensory; Set protein; Side; Signal Transduction; Stains; Structure; Synapses; Synaptic Transmission; System; Tight Junctions; Validation; Work; Zebrafish; autism spectrum disorder; base; connectome; gap junction channel; in vivo; insight; neural circuit; neurotransmitter release; novel; postsynaptic; protein protein interaction; scaffold; synaptogenesis; targeted treatment; trafficking; unpublished works

The nervous system uses two forms of fast synaptic transmission, chemical and electrical, that both contribute to the dynamic computations that create thought, feelings, and actions. Chemical synapses are well studied, and the biochemical mechanisms by which neurotransmitter is released and received are well understood. By contrast, we know relatively little about the macromolecular complex of the electrical synapse. Electrical synapses are made from tens to thousands of gap junction channels that create direct, low-resistance routes of cytoplasmic communication between neurons. They contributed to sophisticated function in neural circuit computation, they display plasticity through a number of short- and long-term mechanisms, and their assembly is regulated during development. Together, this all suggest a complex macromolecular structure that controls their formation and function, yet a critical barrier to progress in the field remains in the identification of the proteins of the electrical synapse. The overarching goal of the proposal is to establish zebrafish electrical synapses as a model to understand their proteomic diversity. Aim1 will demonstrate that electrical synapse proteins can be identified using genome engineered zebrafish that express electrical synapse proteins tagged with TurboID. TurboID is an evolved E.coli protein that allows for in vivo, proximity-depending labeling of proteins with biotin. Such biotinylated proteins can then be efficiently isolated from the animal and analyzed using mass spectrometry. Aim2 will then assess the biochemical interactions and cellular localization of the identified proteins using expression systems for protein-protein interactions and in vivo immunohistochemistry in zebrafish. If successful, this grant will fundamentally shift the understanding of electrical synapses, revealing proteins involved in trafficking pathways, synaptic structure, and functional regulation. The proposed studies will provide novel insight into the molecular complexes of the electrical synapse in a model vertebrate, providing a foundation for the identification of targets for therapy of neurodevelopmental disorders.

神经系统使用两种形式的快速突触传递，化学的和电的，这两种形式都有助于 创造思想、情感和行动的动态计算。化学突触已得到充分研究， 神经递质释放和接收的生化机制已广为人知。经过 相比之下，我们对电突触的大分子复合物知之甚少。电气 突触由数十到数千个间隙连接通道组成，这些间隙连接通道创建直接、低电阻的路径 神经元之间的细胞质通讯。它们对神经回路的复杂功能做出了贡献 通过计算，它们通过许多短期和长期机制表现出可塑性，并且它们的组装 在开发过程中受到监管。总之，这一切表明一个复杂的大分子结构控制着 它们的形成和功能，但该领域取得进展的一个关键障碍仍然是确定 电突触的蛋白质。该提案的总体目标是建立斑马鱼电学 突触作为理解其蛋白质组多样性的模型。 Aim1 将证明电突触 可以使用表达标记的电突触蛋白的基因组工程斑马鱼来识别蛋白质 与 TurboID。 TurboID 是一种进化的大肠杆菌蛋白，可在体内根据邻近度标记 含有生物素的蛋白质。然后可以有效地从动物中分离出此类生物素化蛋白质并进行分析 使用质谱法。然后 Aim2 将评估生物化学相互作用和细胞定位 使用表达系统进行蛋白质-蛋白质相互作用和体内免疫组织化学鉴定蛋白质 在斑马鱼中。如果成功，这笔资助将从根本上改变对电突触的理解，揭示 参与运输途径、突触结构和功能调节的蛋白质。拟议的研究 将为模型脊椎动物中电突触的分子复合物提供新的见解， 为确定神经发育障碍治疗靶标奠定基础。

项目成果

Transformation of an early-established motor circuit during maturation in zebrafish.

斑马鱼成熟过程中早期建立的运动回路的转变。

• 发表时间: 2022-04-12
• 影响因子: 8.8
• 作者: Pallucchi, Irene;Bertuzzi, Maria;Michel, Jennifer Carlisle;Miller, Adam C;El Manira, Abdeljabbar
• 通讯作者: El Manira, Abdeljabbar

The disconnect2 mutation disrupts the tjp1b gene that is required for electrical synapse formation.

disconnect2 突变破坏了电突触形成所需的 tjp1b 基因。

• 发表时间: 2022
• 作者: Michel, Jennifer Carlisle;Lasseigne, Abagael M;Marsh, Audrey J;Miller, Adam C
• 通讯作者: Miller, Adam C