Investigating the Molecular Mechanisms that Drive Electrical Synapse Development

项目摘要 电突触是复杂的细胞和生化结构，在健康和疾病中具有重要作用。 它们由神经元缝隙连接组成，通过通道连接突触神经元的细胞质 由跨膜连接蛋白（Cx）组成。然而，在知识方面存在着惊人的差距， 围绕非连接蛋白电突触蛋白的鉴定和 驱动电突触形成的分子机制。电子显微图像首次揭示了 化学突触的分子组装得到充分表征，显示出大的电子致密区域 在突触前和突触后膜下面，现在称为活动区（AZ）和突触后密度 （私营部门司）。在神经元缝隙连接处也观察到类似的胞质电子致密区，这表明 调节电突触的额外机制的存在。米勒实验室最近将ZO 1b鉴定为 是斑马鱼Mauthner中Cx定位和电突触功能的必要和充分条件 神经回路ZO 1b是一种多结构域分子支架，已知用于组织胞质和跨膜 上皮紧密连接处的蛋白质。有趣的是，紧密连接处的ZO蛋白显示出迷人的 生物化学性质称为液-液相分离（LLPS），允许它们产生非- 细胞内的膜结合区室和浓缩结合伴侣以构建流体分子 组件.事实上，ZO 1b的化学突触类似物PSD 95，以及AZ的其他突触支架， 和PSD也被认为通过LLPS组织化学突触结构。然而，在这方面， 由于缺乏可评估的模型，LLPS在体内的功能表征一直是困难的 系统该建议使用蛋白质动力学、结合测定和结构/功能突变体的组合 在细胞培养中，首先确定涉及Cx-支架和ZO 1b LLPS的功能结构域，然后 将这些发现在体内转化为光学透明的，遗传上易处理的Mauthner细胞回路。一起 这些结果将为电突触发育所需的分子提供基础模型， 驱动它的生物化学相互作用，以及对申请人在神经发育方面的加速培训， 蛋白质生物化学和斑马鱼遗传学。

PROJECT SUMMARY Electrical synapses are complex cellular and biochemical structures with important roles in health and disease. They are composed of neuronal gap junctions that link the cytoplasm of synapsing neurons through channels composed of transmembrane Connexin proteins (Cx). However, there are striking gaps in knowledge surrounding the identification of non-Connexin electrical synapse proteins and the characterization of molecular mechanisms driving electrical synapse formation. Electron micrograph images first revealed the now well-characterized molecular assemblies of the chemical synapse, showing large electron dense regions beneath pre- and postsynaptic membranes now known as the Active Zone (AZ) and the Postsynaptic Density (PSD). Similar cytoplasmic electron dense regions have been observed at neuronal gap junctions, suggesting the presence of additional machinery regulating electrical synapses. The Miller lab recently identified ZO1b as being necessary and sufficient for Cx localization and electrical synapse function in the zebrafish Mauthner neural circuit. ZO1b is a multidomain molecular scaffold known for organizing cytosolic and transmembrane proteins at epithelial tight junctions. Interestingly, ZO proteins at tight junctions display the fascinating biochemical property known as liquid-liquid phase separation (LLPS) allowing them to create a non- membrane-bound compartments within the cell and concentrate binding partners to build fluid molecular assemblies. Indeed, ZO1b’s chemical synapse analog PSD95, as well as other synaptic scaffolds of the AZ and PSD, have also been suggested to organize chemical synapse architecture through LLPS. However, functional characterization for LLPS in vivo has been difficult due to the absence of an assessable model system. This proposal uses a combination of protein dynamics, binding assays, and structure/function mutants in cell culture to first determine the functional domains involved in Cx-scaffolding and LLPS of ZO1b and then translates those findings in vivo to the optically transparent, genetically tractable Mauthner cell circuit. Together the results will provide a foundational model for the molecules required for electrical synapse development and the biochemical interactions that drive it, as well as accelerated training for the applicant in neurodevelopment, protein biochemistry, and zebrafish genetics.