Molecular Mechanisms for Co‐Assembly of Endocytic and Exocytic Machineries at a Synapse

项目总结 在突触前神经末梢，两个蛋白质机器的精确纳米级定位，活动区 和内吞细胞器，是高保真突触传递的关键。活动区域，它生成 突触囊泡的释放部位与突触后受体对齐，以确保有效的信号传递。 释放后恢复囊泡的内吞装置被组装在活动区附近。多数 这些机器的部件与大脑疾病有关，强调了它们对大脑的重要性 功能。这项拨款产生了对分子机制的基本洞察，这些分子机制将 脊椎动物突触活动区旁边的内吞器官。 突触前支架Liprin-α是在空间上组织这两个机器的理想候选者 理由。首先，Liprin-α广泛的突触相互作用体和消融引起的广义表型 单一的无脊椎动物基因预测，它组织了多个突触前隔室。第二，我以前的 工作和初步数据表明，四种脊椎动物Liprin-α蛋白的一个子集的缺失已经导致 活动区和内吞成分的错误定位，这表明脊椎动物的共同组织作用 利普林-α。最后，Liprin-α经历了相分离，这是一种潜在的突触组织机制，以及 我正在进行的工作建立了活动区和内吞蛋白都被招募到Liprin-α时相 凝析油。因此，我假设Liprin-α将内吞细胞器定位在活动区旁边 脊椎动物的突触。 我将使用新产生的Liprin-α四重突变体来测试，在这些突变体中，四个Liprin-α基因都可以缺失 这个假说。目标1(培训)将剖析Liprin-α在活动区组织中的角色，方法是将纳米 兴奋性Liprin-α四次消融后活动区蛋白质的标度定位和活动区功能 在培养的神经元和Hold氏囊内有抑制性突触。目标2(培训)将确定 Liprin-α通过使用相同的方法来组织内吞机构。这两个目标将相辅相成 通过在细胞系中的实验来评估Liprin-α招募内吞和活动区蛋白的能力。这就做 我还做了结构-功能实验来剖析介导Liprin-α的蛋白质序列的相关性 突触前组装中的相分离。这些目标将确立Liprin-α在组织中的作用 活动区和内吞集合体，并将提供必要的培训经验的研究 并使用超分辨率显微镜评估其体内组织结构。目标 3(独立)将检验内吞细胞器调节空间的互补模型 这些机器的组织。特别是，我将剖析Dynamin的结构角色，这是基本的 用于突触内吞作用，也经历了时相分离。我将使用动力素三重突变体并在此基础上 在(培训)期间实施的方法目标1和2来确定这些角色。

Project summary At presynaptic nerve terminals, the precise nano-scale positioning of two protein machineries, the active zone and the endocytic apparatus, is critical for high fidelity synaptic transmission. The active zone, which generates release sites for synaptic vesicles, is aligned to postsynaptic receptors to ensure efficient signal transmission. The endocytic apparatus, which restores vesicles after release, is assembled adjacent to active zones. Most components of these machineries are associated with brain disorders, underlining their importance for brain function. This grant generates fundamental insight into the molecular mechanisms that position the endocytic apparatus next to the active zone at vertebrate synapses. The presynaptic scaffold Liprin-α is an ideal candidate to spatially organize these two machineries for three reasons. First, the broad synaptic interactome of Liprin-α and the generalized phenotypes caused by ablating the single invertebrate gene predict that it organizes multiple presynaptic compartments. Second, my previous work and preliminary data show that deletion of a subset of the four vertebrate Liprin-α proteins already causes mislocalization of active zone and endocytic components, which suggests co-organizing roles for vertebrate Liprin-α. Finally, Liprin-α undergoes phase separation, which is a potential synaptic organizing mechanism, and my ongoing work establishes that both active zone and endocytic proteins are recruited to Liprin-α phase condensates. Therefore, I hypothesize that Liprin-α positions the endocytic apparatus next to active zones at vertebrate synapses. I will use newly-generated Liprin-α quadruple mutants, in which all four Liprin-α genes can be deleted, to test this hypothesis. Aim 1 (training) will dissect roles for Liprin-α in active zone organization by comparing the nano- scale positioning of active zone proteins and active zone function after Liprin-α quadruple ablation in excitatory and inhibitory synapses in cultured neurons and at the Calyx of Held. Aim 2 (training) will determine roles for Liprin-α in organizing the endocytic apparatus by using the same approaches. Both aims will be complemented with experiments in cell lines to assess the ability of Liprin-α to recruit endocytic and active zone proteins. I will also do structure-function experiments to dissect the relevance of the protein sequences that mediate Liprin-α phase separation in presynaptic assembly. These aims will establish the roles of Liprin-α in the organization of active zones and endocytic assemblies, and will provide an essential training experience in the studies of the endocytic machinery and in the assessment of their in vivo organization using super-resolution microscopy. Aim 3 (independent) will test the complementary model that the endocytic apparatus mediates the spatial organization of these machineries. In particular, I will dissect structural roles for Dynamin, which is fundamental for synaptic endocytosis, and also undergoes phase separation. I will use Dynamin triple mutants and build upon the methodology implemented during (training) aims 1 & 2 to determine these roles.